Source: NEW MEXICO STATE UNIVERSITY submitted to
COORDINATED DEVELOPMENT OF GENETIC TOOLS FOR PECAN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1009971
Grant No.
2016-51181-25408
Cumulative Award Amt.
$4,404,284.00
Proposal No.
2016-04957
Multistate No.
(N/A)
Project Start Date
Sep 1, 2016
Project End Date
Aug 31, 2022
Grant Year
2021
Program Code
[SCRI]- Specialty Crop Research Initiative
Project Director
Randall, J.
Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239
Performing Department
EPPWS
Non Technical Summary
Pecan (Carya illinoinensis) is a large deciduous native North American tree grown in 27 US states by about 25,000 pecan farmers for its delicious and highly nutritious nutmeats; thus, it has great economic impact in rural communities across much of the U.S. It is considered a permanent crop, as trees bear for 300+ years. Pecan is recently domesticated, as commercial orchards were first planted in the late 1800s. It is successfully farmed in areas as diverse as the deserts of the southwestern US, the humid southeastern US, California's Central Valleys, the Southern Plains, and is substantially planted worldwide (Grauke et al., 2011). Growing regions within the U.S. are diverse, with farmers in each region facing major and unique horticultural issues. The horticultural issues to be addressed by genetic tool development include flowering, severe alternate bearing, disease susceptibility, salinity stress, drought stress tolerance, and micronutrient uptake insufficiencies. Project long-term goals are to increase pecan nutmeat yield and quality, optimize nutritional value, and subsequent stabilization of pecan markets by improving crop major limiting characteristics for all U.S. pecan farming regions. The objectives are focused to elucidate the genetics of these crop characteristics. Genetic variation within the species allows for differential adaptation in wide ranges of environments; thus, unraveling the genetics of several genotypes will allow for identification of genes controlling specific traits. Obtained data will allow development of vital genetic tools necessary for increasing understanding of regional adaptation, promoting conservation, and selecting improved cultivars/rootstocks for all major farming regions. All sectors of the industry will benefit--i.e., nurseries, producers, nutmeat marketers, consumers, and the interdisciplinary research community. The knowledge and tools generated will be available on public websites and disseminated to growers, nurseries, commercial marketers, consumers, and researchers.Pecan, a Native American Algonquin word describing a "nut requiring a stone to crack" (Trumball, 1872), is North America's most economically valuable native tree nut. Its cultivation impacts farmers and farm communities across the southern U.S. As an only-recently domesticated crop species, the development of genetic tools is essential for effectively addressing key constraints to stable and high quality nutmeat production. As the pecan industry has evolved since its inception in the late 1800s, trees have been introduced as an exotic crop to be farmed in relatively alien environments, and this has led to major stress-associated horticultural problems either directly or indirectly altering flowering and alternate bearing. The identification of genetic elements controlling key traits, such as efficient nutrient uptake/transport, scab disease resistance, salinity tolerance, drought tolerance, and nut quality need identification and integration to produce better adapted cultivars for cultivation. Identification of these underlying genetic elements and subsequent development of genetic markers associated with specific traits potentially influences the economics of pecan farming and marketing. Development of trees better suited for specific environments will yield quantitatively and qualitatively superior nutmeats and require less input by growers. This would increase the economic fitness of pecan growers while also enabling more environmentally friendly/sustainable pecan horticulture. Through our research and outreach efforts, the goal of this project is to discover and implement science-based solutions resulting in a stable supply of high-quality pecans over time. This will be of enormous benefit to all sectors of the U.S. pecan industry, from producers to processors and ultimately to consumers and rural communities throughout much of the US.We will use trans-disciplinary approaches to build genetic tools that the pecan industry recognizes as essential for their success. Research institutions include New Mexico State University (1862 land grant/Hispanic serving), University of Arizona, University of Georgia, The Samuel Roberts Noble Foundation, HudsonAlpha, USDA-ARS Southern Regional Research Center (New Orleans, LA), USDA-ARS Pecan Breeding and Genetics/National Collection of Genetic Resources for Pecans and Hickories (College Station, TX), and USDA-ARS Southeastern Fruit and Tree Nut Research Laboratory (Byron, GA). This trans-disciplinary research group contains the following expertise: plant genetics, plant molecular biology, pecan tissue culture, plant pathology, pecan breeding, pecan horticulture, soil science, agricultural economics, computer science, and outreach/extension. A panel advisory group has also been created that will include agricultural consultants, producers with large- and small-scale pecan orchards, pecan shellers, and nurserymen. The simplified goals of this proposal are 1. To evaluate the genetics that controls the diversity of native pecan. This will be done by obtaining genomic sequences, annotation of the genes, developing linkage maps and SNP markers; 2. Evaluate the molecular mechanisms of flowering, disease resistance, and tolerance to stresses; and 3. Build a designated database/website that will be available to share data and impact of the research with all sectors of the pecan industry.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20112111080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1211 - Pecan;

Field Of Science
1080 - Genetics;
Goals / Objectives
Complete genomic sequence of multiple reference pecan trees that captures the genetic diversity of the genome. The genomes will be annotated and the annotation will be made available on a publically accessible database.Development of linkage maps and dense SNP maps across multiple Carya populations. This will allow for identification of useful alleles for specific phenotypes.Phenotypic descriptor development, data collection, data entry and a website to house the information.Development of markers associated with pecan scab resistance for future marker-assisted breeding.Identification of molecular mechanisms, and the genetics that control horticultural traits such as flower initiation, nutrient transport, disease resistance, salinity tolerance, and nut quality.
Project Methods
OBJECTIVE 1. Genomic sequence of multiple reference pecan trees that capture the genetic diversity of the genome will be performed. The genomes will be annotated and the annotation will be made available on a publically accessible database. Sub-objective 1: Genome sequencing. The genotypes (87MX3-2.11, Pawnee, Lakota, and Elliott) we will undergo whole genome shotgun sequencing strategy. The pipeline for genome sequence consists of 1) High Molecular Weight (HMW) DNA isolation, 2) Initial survey sequencing with Illumina, 3) Large fragment collection on Pacific Biosciences RSII platform with fragment assembly, 4) Construction of a dense high resolution map with low-pass resequencing, 5) Integrating the fragment assembly 6) High accuracy gene annotation. Sub-objective 2. High accuracy gene annotation. For genome annotation, we will use homology from our high quality plant reference annotations, protein homology across the plant kingdom, and the RNA-seq generated. The JGI pipeline will be used for annotation.OBJECTIVE 2. Development of linkage maps and dense SNP maps across multiple Carya populations. This will allow for identification of useful alleles for specific phenotypes.Sub-objective 1: Development of shared segregating population using two reference genomes: 'Lakota' x 87MX3-2.11. Pollen (87MX3-2.11) and used to pollinate bagged clusters of 'Lakota' flowers targeting production of 1000 seed. At maturity, seed will be collected and characterized. Nuts will be numbered and planted or micro-propagated. The 188 seedlings and 8 grafted representatives of the two parents ('Lakota' and 87MX3-2.11) will be distributed to permanent orchard planting locations in US. DNA from 'Lakota' x 87Mx3.211 will be genotyped. That sequence will be aligned to each of the parental template genome sequences to produce dense linkage maps. Sub-objective 2: 'Pawnee' x 'Elliott' Mapping population. The mapping population consists of 120 progeny from 'Pawnee' × 'Elliott'. Phenotypes of several traits will be recorded for parent and progeny trees. Trees will be assessed for scab disease severity. DNA from progeny trees will be evaluated and for digital genotyping (DG). RAPD and AFLP markers from the previous linkage maps will also be included in map construction. 'Pawnee' and 'Elliott' linkage maps will be used for QTL interval mapping of phenotypic traits using MapQTL® 6. Sub-objective 3: 'Elliott' x VC1-68 Mapping block College Station, TX. The mapping population consists of 240 progeny obtained from reciprocal crosses. The progeny and parents will be sequenced by DG and the data will be analyzed.OBJECTIVE 3. Phenotypic descriptor development, data collection, data entry, and delivery. Sub-objective 1: Data entry of existing records into a relational database. A local relational database will be created to house the historic origination records that represent collections of NCGR Carya. Coordinate fields in the local database to receive information from orchards via collection devices and facilitate interoperability and movement of appropriate information from local database into GRIN-Global, the genomics databases, and a user-friendly grower website. Sub-objective 2: Standardized phenotype data collection from NCGR and provenance collection sister-sites. Develop Field Book digital collection devices with specific descriptors cooperatively developed for use in pecan that will allow orchard inventory recognition followed by digital recording of phenotypic descriptors.OBJECTIVE 4: Development of markers associated with pecan scab resistance for future marker-assisted breeding.Sub-objective 1. RNASeq contrasting for pecan scab. Two pecan cultivars, one susceptible to scab with significant yield loss ('Desirable') and one cultivar little yield loss ('Pawnee') will be used to monitor gene activity during the infection process using RNA seq and validated with qRT-PCR. Pawnee and Desirable will be inoculated at time '0'. Leaves sampled at multiple time-points (40 h: penetration, 72 h: subcuticular hyphal growth, 170 h: reproductive initials, and 240 h: symptom development). The samples will be analyzed by RNAseq analysis and qRT-PCR. Sub-objective 2. Pecan provenance collection screened for pecan scab segregation. The pecan provenance collection in Byron, GA comprises ~950 trees grown from seed sampled from 19 provenances covering the native range of pecan in the US and Mexico. Characterization of fruit and leaf scab ratings will be performed. Bulked segregant analysis will be used to construct six susceptible and six resistant bulks from different geographic regions and GBS will be used to genotype. These results will be used to identify genetic differences associated with tolerance to scab in the field.OBJECTIVE 5. Identification of molecular mechanisms, and the genetics that control horticultural traits such as flowering, nutrient transport, and nut quality.Sub-objective 1. Investigate the molecular mechanisms that control flower initiation and the effects of water deficit irrigation on flower initiation. The effects of water availability on carbohydrate status and floral initiation, blocks of Wichita and Western trees grown at the NMSU experimental station will be utilized. During year 2 and 3 irrigation status on 4 specific blocks of pecan trees will be reduced in comparison to control blocks. During years two and three, bud tissues from the treatments will be collected and high quality total RNA will be isolated and analyzed using RNAseq. The generated transcriptomes will also be aligned to the pecan genome.Transcript assembly and quantification will be compared between treatments. Validation of the RNAseq data will also be confirmed using qRT-PCR of floral genes using actin as an internal control. Sub-objective 2. Investigate the molecular mechanisms involved in differential acquisition of nutrients and their transport within the plant. Pecan seedlings utilized in both the eastern and western US were selected and grown in pots under three different soil lime treatments30% lime, 15% lime, and no added lime ("Control"). Differences in leaf nutrient analysis was measured and found to be different between cultivars. This study will be continued and RNAseq of root tissue from multiple plants within the different treatments will be performed.Sub-objective 3. Screening pecan genotypes for salinity tolerance in field conditions in Arizona. The field trial will be set up in a split plot design with irrigation treatments as the main plot and varieties as sub-plots, and with four replications. Irrigation: plots will be flood irrigated. There will be two water sources and three irrigation regimes that will provide three soil salinity levels low salinity (600 - 700 mg/L), high salinity approximately 2,000 mg/L, and well water. Irrigation regimes will be initiated in year 2 after establishment. Measurements will be collected and trees that are tolerant to high salinity soils will be analyzed by RNAseq. Sub-objective 5. Mining genomes and RNAseq data for allergens within pecan nuts. The pecan genomic and RNAseq sequencing projects from this proposal will be mined for pecan allergen sequences including the Car i 1, Car i 4, and the 7S viclin gene sequences. Protein analysis will also be done on selected cultivars. Sub-objective 6. Inferring molecular networks driving pecan trait and tissue specificity. We will integrate pecan genome, transcriptome, proteome, and GWAS to delineate a comprehensive interactome map that links molecule-molecule interactions and genotype-phenotype connections using available software tools. We will infer pecan gene networks at four scales: single trait-driver genes, gene ontology sets, functional pathways, and de novo networks.

Progress 09/01/16 to 08/31/22

Outputs
Target Audience:The target audience for this grant effort included different segments of the pecan industry including pecan growers, nursery people, and pecan processors. All segments of the US Pecan growers were reached at grower meetings through the five years of the grant. This included but were not limited to the following pecan grower association meetings: Western Pecan Grower Association; New Mexico Pecan Growers Association; Arizona Pecan Grower Meeting, Oklahoma Pecan Growers Association, Texas Pecan Growers Association, Northern Nut Growers Meeting, Tri-State Pecan Growers Meeting (Louisiana), Southeast pecan Growers Association; and Georgia Pecan Growers Association. Information was disseminated in trade magazines such as Pecan South and extension circulars that reach a large audience within the pecan industry. The scientific community was also updated by peer reviewed published scientific articles, genomic and phenotypic datasets were made available in databases, and bio-projects were made available in NCBI. The scientific community was also updated at National and International conferences including but not limited to the American Society of Horticulture Science, American Society of Plant Biology; Plant and Animal Genomics Conference (PAG), PACBIO Global summit, International Society of Horticulture Science, International Symposium on Walnut, Pecan, and Cashew, International Society for Horticultural Science, Pecan National Meeting (Noble Foundation), American Society of Phytopathology, Nature Conference-Harnessing the Plant Microbiome. The four chromosome genomes were made available on (Phytozome). The pecan website that was created due to these efforts (pecantoolbox.nmsu.edu) was also updated to give information to different segments of the pecan industry and scientific community. Changes/Problems:Due to the complications and delays caused by COVID-19, a no-cost extension was granted for this effort. What opportunities for training and professional development has the project provided?This project provided many opportunities for training of undergraduate and graduate students and professional development of scientists during the past five years. Twenty undergraduate students were trained in several disciplines including bioinformatics, plant sciences, and plant biology. This training specifically included experimental design for water deficit experiments, pecan fertilization experiments, clonal rootstock propagation, greenhouse management and data collection. Students were also trained in pecan horticulture techniques in the field and trained in collecting and recording phenotypic data including photosynthesis data. Students were also trained in molecular techniques such as DNA extraction, RNA extraction, and downstream applications including primer and probe design and qRT-PCR analysis. Several undergraduate students were trained in bioinformatics and learned gene alignments and promoter analyses. There were eleven graduate students that were trained in several disciplines during this grant effort. These disciplines included bioinformatics, plant sciences, plant biology, molecular biology, and economics. There were also five post-docs trained in plant sciences. Specific training included experimental design for several pecan studies, clonal rootstock propagation, greenhouse management, data collection, data analysis, statistics, writing, presentations. Graduate students and post-doc associates were also trained in pecan horticulture techniques in the field and trained in collecting and recording phenotypic data including photosynthesis data. Graduate students and post-docs were also trained in molecular techniques such as DNA extraction, RNA extraction, protein extraction, sequencing, library construction, and downstream applications including primer and probe design and qRT-PCR analysis, Western analysis, HPLC and using GC-MS instruments. Additional training included ThermoFisher Scientific and Agilent "Spectrum Mill" high-throughput processing of MS/MS and MS spectra for proteins and peptides; and Matrix Science "Mascot" software for identification, characterization, and quantitation of proteins and peptides using mass spectrometry data. Graduate students and post-doctoral associates were trained to use web-based programs for bioinformatics and in program coding and 'R' for bioinformatics analyses. Bioinformatic skills include organellar annotation, RNA-Seq data analysis, and microbiome analysis. Training on other bioinformatic software's included but were not limited to Qiagen CLC-Genomics workbench, Qiagen CLC-microbiome analyses, Geneious, PlantPan, Metagenomics, 16S microbiome analysis, MOTHUR, and etc. Training on building infrastructure for databases was done and included training to utilize Drupal and Tripal for website and database construction. Undergraduate, Graduate Students, and Post-doctoral associates presented their research at both local, regional, national, and international meetings. Five Ph.D. students completed their Ph.D. on projects associated with this grant effort (Dr. Nolan Bentley, Dr. Hormat Shadgou Rhein, Dr. Angelyn Hilton, and Dr. Kimberly Cervantes, Dr. Sajal Kumar). Three graduate students completed their M.S. degrees on projects associated with this grant effort. Professional development occurred during the five years for the scientists on this project. Specific trainings for scientists included but were not limited to PI and grant management, workshops on mentoring undergraduate, graduate students and post-docs, use of Drupal/Tripal for database construction, database entry and use; Linux command line, 'R' data analyses software, Bioinformatics analyses and statistics (in the form of workshops), Grin-global use for USDA entries, BG-base, phenotype data collection, genome wide association analyses, genome assembly, genome annotation, genome comparisons; and pangenome analyses. How have the results been disseminated to communities of interest?Results were disseminated using several avenues during this grant effort. Presentations included oral presentations, recorded video presentations, webinars, poster presentations, trade journal articles, press releases, and peer reviewed publications. The pecan research website (pecantoolbox.nmsu.edu) that was established during this grant effort was continually updated with research information for the pecan industry and scientific community. This webpage allows users to link to specific articles, websites, information, genetic tools, pecan phenotypic data, videos, and genomic data. Pecan research results and tools were disseminated at pecan grower meetings (total of 71 grower presentations) through the United States during the entirety of the grant effort including the Arizona Pecan Grower Association, California Pecan Grower Association, Georgia Pecan Grower Association, Oklahoma Pecan Grower Association, Texas Pecan Grower Association, Northern Nut grower meeting, Southeastern pecan association, Tri-State Pecan Grower Association, and Western Pecan Grower Association. Information was also disseminated in international forums including grower meetings in Mexico. These meetings and workshops target the pecan industry and include growers, nurserymen, shellers, and other processors. The results were presented both in person and using 'zoom and in written reports annually to the 'Coordinated development of genetic tools for pecan' advisory board that was established during the on-set of the grant effort. Peer reviewed publications (48 publications) were published (and submitted) in journals including but not limited to Nature Communications, Tree Genes and Genomes, Horticulture Science, Frontiers, PLOS, Acta Horticulture, European Plant Pathology, Plant Disease, Phytopathology, Crop protection, Horticulture Technology, CABI Ag and Bioscience, Euphytica, Journal of Agriculture and Food Chemistry, Forests, Bioinformatics, Plant Pathology, and Optical Review. Several articles were published in Pecan South and other trade magazines (total 26 trade articles) for further communication of scientific results to the pecan industry. Results were communicated at National and International scientific conferences (total of 68 scientific presentations). These included but were not limited to American Society of Horticulture Science, American Society of Phytopathology, American Society of Plant Biology, Plant and Animal Genomics Conference (PAG), International Horticulture Conference, and International Walnut, Pecan, and Cashew Horticulture Conference. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The pecan reference genomes of multiple pecan trees that capture the genetic diversity of the pecan species were completed and assembled to the chromosome level ( '87Mx-3-2.11', 'Lakota', 'Elliott', and 'Pawnee'). These genomes were annotated and deposited on the pecantoolbox.nmsu.edu and are available to the public on Phytozyome (https://phytozome-next.jgi.doe.gov). Analyses performed using these genomes included comparative genomics, gene ontology enrichment analysis, gene family expansions, and individual gene and promoter analysis. We constructed a 'pan-genome annotation' both within and among genomes to track the presence and DNA variation of genes. The genome and related studies were published in scientific journals and disseminated to growers using the pecantoolbox website, pecan conference talks, and grower magazines. In addition, several other sequencing efforts were applied that included long-read whole genome sequencing of four additional pecan cultivars ('Major', 'Mahan', 'Schley','Wichita'); a population genetic diversity re-sequence panel of 440 pecan cultivars and 67 Carya and related hybrid species; and additional 50X coverage sequencing of 380 pecan trees from the NCGR repository. The data obtained give us a foundation for analyzing the genetic diversity of pecan, understanding the genetic basis of traits in the pecan's crop-wild relatives; and for the identification of molecular-trait associations for use in breeding improved pecan cultivars and rootstock. The completed genomic sequences are an invaluable resource that will allow elucidation of pecan genetics and implementation of marker-assisted breeding. Several pecan mapping populations, including one population that was developed during this grant effort, were used for the development of linkage maps and SNP identification. Significant work was completed on development of linkage maps and identification of SNPs. From these efforts several Quantitative Trait Loci (QTLs) were identified and included bud-break, scab resistance, and Phylloxera resistance. These are significant QTL's that will be used in future pecan breeding and represent conserved mechanisms in plants. For example, the QTL for budbreak is syntenic to an identified major QTL in English Walnut and likely represents a conserved mechanism effecting bud-break. SSR markers were designed to amplify regions in linkage for these QTLs. Critical progress was made on pecan phenotypic descriptor development, data collection, data entry and a website to house the information. During this grant effort the pecantoolbox.nmsu.edu was established. This website includes information that is relevant to the pecan industry such as updates on pecan research efforts, links to the pecan genomes, links to searchable databases for pecan trees and their genetic and known phenotypic information, pecan disease management strategies, and tools for nurseries/other areas of the pecan industry that are interested in genetic identification of pecan trees. A local SQL database was built and implemented at the USDA Pecan Germplasm (TX) and data from 50 USDA pecan orchard systems from the past 30 years is now stored on this database. The database is structured to link data to individual pecan tree accessions and to interface with GRIN-Global, and a genomic database that links to the industry pecantoolbox website. This grant effort initiated and generated a new mapping population 'Lakota' x '87Mx3-2.11'. The progeny seedlings from this mapping population are planted in three different locations (Tifton, GA; Byron, GA; and College Station, TX). Collection of phenotypic data on the new mapping population and the other mapping populations was done and the data was entered into the new database for future retrieval and use. Multiple avenues for determining and evaluating disease resistance markers associated for pecan scab resistance included multiple genome-wide association studies (GWAS) of pecan trees with natural infection pressure and with in-vitro studies with inoculated scab assays followed by RNA-Seq analyses. These analyses resulted in identification of putative markers and specific loci that are involved in plant immunity and other metabolic processes. Markers associated with scab disease resistance are extremely valuable and are greatly needed for the pecan industry for future breeding/developing pecan trees with resistance to scab. Astounding progress was made on several fronts regarding the molecular mechanisms and genetics that control horticultural traits including pistillate and catkin flowering initiation, nutrient transport of essential micronutrients, salinity tolerance, allergen genetics, and initial microbiome analyses of pecan seedlings. Multiple types of analyses on the genetic signals for pecan floral initiation were performed. Identification of flowering genes involved in floral initiation and the timing of their activity was analyzed and proposed. The impact of phytohormones on pecan trees and their impact on specific flowering genes and alternate bearing were analyzed and reported. The genetic diversity of putative pecan allergen sequences were analyzed and the allergen sequence data indicate that the allergen proteins are highly conserved, under selective pressure, and serve physiologically important roles in the nut. A proteomic study determining protein accumulation in developing pecan nuts was conducted and the results of this investigation provide a foundation for more focused trait-specific proteomic analyses of pecans that may include reduced allergen content, improved polyphenol or lipid content, increased tolerance to salinity or other abiotic or biotic stresses, seed hardiness, and seed viability. Overall, 556 proteins were identified and the pecan allergens Car i 1 and Car i 2 were first observed to accumulate during the dough stage in late September. A seedling rootstock trial planted in a cold winter location in New Mexico was evaluated for survivorship and selected rootstocks were subsequently grafted to the scion 'Kanza'. Phenotypic observations and differences in the performance of different rootstocks in this cold environment were observed. Seedling rootstocks that originated from trees native to the warmer regions did poorly in this location. However, seedlings that originated from Northern cultivars/seedlings were better suited for the cold winters and late spring for this area. These data will allow pecan producers in colder regions to make better use of rootstock genetics. Micronutrient deficiencies are a major challenge for pecan production in the western US where soils are alkaline and calcareous. We tested a genetically diverse range of un-grafted pecan rootstocks in pots for their ability to take up micronutrients from high pH soils. The seedling rootstocks were grafted to a common 'Pawnee' scion to determine if the pattern of increased zinc uptake continues after the rootstocks have been grafted. The maternal seed source, 'Shoshoni' exhibited higher zinc levels in leaf tissues. Preliminary gene analysis of transporter genes from root tissues indicated that one pecan zinc transporter gene was associated with these increased Zn levels.Identification of a superior rootstock with zinc uptake would allow producers to reduce the number of foliar applied zinc treatments or eliminate the need for foliar zinc application. The impact of rootstock on micronutrient uptake in the scion were evaluated in an Arizona orchard with sodic soil with high levels of salinity. Salinity tolerance of pecan rootstocks 'Elliott' and 'Ideal' showed the least sensitivity to salt damage. Microbiome analysis of pecan seedlings indicated that there were microbial composition differences between pecan tissue types (leaves, stems, and roots) and between seedlings from different maternal cultivars.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bhattarai, G.,V. Bonhomme and P. Conner. 2022. Image-based morphometric analysis reveals moderate to highly heritable nut shape traits in pecan. Euphytica 218:102. Doi.org/10.1007/s10681-022-03049-1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Bhattarai, G., Connor, P., Randall, J.J., Shadgou Rhein, H., 2022. Transcriptome analysis under pecan scab infection reveals the molecular mechanisms of the defense response in pecan, PAG 2022.
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Clermont, K., Graham, C.J., Lloyd, S.W., Grimm, C.C., Randall, J.J., Mattison, C.P., 2022. Proteomic analysis of pecan (Carya illinoinensis) nut development, Journal of Agricultural and Food Chemistry, Submitted.
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Shadgou Rhein, H., Sreedasyam, A., Cooke, P., Velasco-Cruz, C., Grimwood, J., Schmutz, J., Jenkins, J., Kumar, S., Song, M., Heerema, R.J., Grauke, L.J., Randall, J.J. 2022. "Comparative transcriptome analyses reveal insights into catkin bloom patterns in pecan protogynous and protandrous cultivars", PLOS Biology, Submitted.
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Cervantes, K., Heerema, R.J., Randall, J.J., 2022. Insights into the Microbial Composition of Carya illinoinensis (Pecan) Seedlings from Different Maternal Pecan Cultivars Located in the Same Orchard, Frontiers in Microbiomes, Submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Cervantes, K., Heerema, R.J., and Randall J.J., 2022. Microbial Composition Analysis of Different Maternal Pecan Cultivar Progeny in the Same Orchard Location, Annual meeting of American Horticulture Horticulture Science; August 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bock, C.H., Frusso, E., Zoppolo, R., Ortiz, E.R., Shiller, J., Charlton, N.D., Young, C.A., and Randall., J.J. Population genetic characteristics and mating type frequency of Venturia effusa from pecan in South America. Phytopathology XX: xx. https://doi.org/10.1094/PHYTO-01-22-0031-R (accepted)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Bock, C.H., 2022. Interactions of Hickory Rootstocks with Pecan Scions Stuart and Desirable: A Nut Quality Study. Georgia Pecan Growers Association Annual Meeting, Perry, GA. 30 March 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Walworth, J.L. Performance of Pecan Rootstocks in Challenging Soil Conditions. XXI Simposio Internacional de Nogal Pecanero 2020, October 12  14, 2021. Hermosillo, Mexico.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Walworth, J.L. Performance of Pecan Rootstocks in Challenging Soil Conditions. Proceedings of the XXI Simposio Internacional de Nogal Pecanero 2020, October 12  14, 2021. Hermosillo, Mexico.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Litterman, R.A., Ott, B.M., Wen, J., Grauke, LJ, Schwartz, R.S., Handy, S.M., 2022. Reference-free discovery of nuclear SNPs permits accurate, sensitive identification of Carya (hickory) species and hybrids, Applications in Plant Science, https://doi.org/10.1002/aps3.11455
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang, X., Chatwin, W., Hilton, A., Kubenka, K., 2022. "Genetic diversity revealed by microsatellites in genus Carya Forests 2022, 13(2), 188; https://doi.org/10.3390/f13020188
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang, X., Kubenka, K., Chatwin, W., Thompson, T., Grauke, L.J., 2022. Pueblo Pecan: A compact cultivar for the western and central growing regions. HortScience V. 57, 7 https://doi.org/10.21273/HORTSCI16601-22
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Kumar, Sajal and M. Song, Overcoming biases in causal inference of molecular interactions. Bioinformatics, vol. 38, no. 10, pp. 28182825, 2022. doi:10.1093/bioinformatics/btac206
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Tenha, L. and Song,M. 2022. Inference of trajectory presence by tree dimension and subset specificity by subtree cover, PLoS Computational Biology, vol. 18, no. 6, pp. e1009829, 2022. doi:10.1371/journal.pcbi.1009829
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Sharma, Ruby, Zeinab Sadeghian Tehrani, Sajal Kumar and M. Song, Detecting genetic epistasis by differential departure from independence. Molecular Genetics and Genomics, vol. 297, no. 4, pp. 911924, 2022. doi:10.1007/s00438-022-01893-3
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Debnath, T. and Song, M. 2022. Fast optimal circular clustering and applications on round genomes, IEEE/ACM Transactions on Computational Biology and Bioinformatics, vol. 18, no. 6, pp. 20612071, 2021. doi:10.1109/TCBB.2021.3077573
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Cervantes, K., Hilton, A.E., Stamler, R.A., Heerema, R.J., Bock, C., Wang, X., Young-Ki, J., Grauke, L.J., Randall, J.J. 2022. Evidence for seed transmission of Xylella fastidiosa in pecan (Carya illinoinensis), Front. Plant Sci., Frontiers of Biology, 13, 780335, https://doi.org/10.3389/fpls.2022.780335. 2.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bock, C., Charlton, N.D., Shiller, J., Randall, J.J., Young, C.A. 2022. Population genetic diversity and structure of pecan scab pathogen, Venturia effusa, on cv. Desirable and native seedlings, and the impact of marker number, Plant Pathology, https://doi.org/10.1111/ppa.13551.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Bock, C., Frusso, E, Zoppolo, R., Ortiz, E.R., Shiller, J., Charlton, N.D., Young, C.A., Randall, J.J., 2022. Venturia effusa Population Genetic Characteristics from Pecan in South America APS, August 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hilton, A., Cervantes, K., Stamler, R., Heerema, R.H., Bock, C., Wang, X., Jo, Y.K., Grauke, L.J., Randall, J.J. 2022. Evidence for seed transmission of Xylella fastidiosa in pecan (Carya illinoinensis). APS August 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lillywhite, J.M. Supply Chain Woes and the Pecan Industry. Pecan South Magazine. May 1, 2022. https://www.pecansouthmagazine.com/magazine/article/supply-chain-woes-and-the-pecan-industry/.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lillywhite, J.M. The Ins and Outs of Consumer Behavior. Pecan South Magazine. February 1,2022. https://www.pecansouthmagazine.com/magazine/article/the-ins-and-outs-of-consumer-behavior
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lillywhite, J.M. Market and Strategy Tools for Pecan Stakeholders. Pecan South Magazine. October 2021. https://www.pecansouthmagazine.com/magazine/article/market-and-strategy-tools-for-pecan-stakeholders
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lillywhite, J.M. Supply Chain Disruptions: Effects on the Pecan Industry. Western Pecan Growers Association 2022 Conference. Las Cruces, NM. March 9, 2022.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: C. A. Smith, D. VanLeeuwen, R.J. Heerema, J. D. Sherman, M. J. Comeau, and J. L. Walworth. 2022. Zinc variability in pecan orchards: Implications for leaf sampling and nutrient recommendations. HortScience, 57(4), 550-557.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hilton, Angelyn, Cervantes, K., Randall, J., 2022. Pecan bacterial leaf scorch is transmitted from pecan nuts to rootstock. Pecan South June 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mattison, C. 2021. Food Allergen Preventive Controls presentation for Food Safety Preventive Controls Alliance (FSPCA) Preventive Controls of Human Food Course at LSU.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mattison, C., 2022. Heating induces chemical modification and variable solubility of pecan allergens, American Chemical Society, August 24, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hilton, Angelyn. 2022. "Pecan bacterial leaf scorch transmitted from seeds to seedlings". Texas Pecan Growers Conference, July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Randall, J.J., 2022. "Advances in Pecan Genetics". Georgia Pecan Growers Association, March 2022, Perry, GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Randall, J.J., 2022. "Updates on Pecan Genetic Research". Tri-State Pecan Growers Association, June 17, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Randall, J.J., 2022. "Seed transmission of Xylella fastidiosa in pecan". Tri-State Pecan Growers Association, June 17, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Randall, J.J., 2022. "Pecan Bacterial Leaf Scorch". Arizona Pecan Growers Association. August 26,2022.
  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Cervantes, K., 2022. Microbiome composition of Carya illinoinensis (pecan) seedlings and the effect of deficit watering on micropropagated pecan trees and their associated fungal communities.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Heerema, R., 2021. Protecting Pecan Orchards from Freeze Injury (oral), Arizona Pecan Growers Association Conference, Tucson, AZ, Aug. 27, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Heerema, R., 2021. Protecting Pecan Orchards From Freeze Injury (oral; presented remotely to conference via Zoom recording), New Mexico Pecan Growers Association Conference, Las Cruces, NM, Oct. 29, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Heerema, R., 2021. Pecan Rootstocks: How They Affect Pecan Tree Performance (oral; presented remotely to conference via Zoom recording), New Mexico Pecan Growers Association Conference, Las Cruces, NM, Oct. 29, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Heerema, R., 2021. Overview and Advances in Pecan Pest Management (oral), Luna County Pesticide Applicators Workshop, Deming, NM, Nov. 16, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Heerema, R., 2022. Pecan Varieties and Rootstocks for New Mexico (oral), New Mexico Crop Production Conference, Ruidoso, NM, Jan. 25, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Heerema, R., 2022. Whats Causing Leaf Scorch in my Orchards? (oral), Western Pecan Growers Association Conference and Tradeshow, Las Cruces, NM, March 9, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Heerema, R., 2022. Pecan Varieties and Rootstocks for California (oral), California Pecan Growers Association Conference, Yuba City, CA, April 6, 2022
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: R.J. Heerema. 2021. The importance of pecan rootstocks for semi-arid regions., Pecan South 54(7):28-30
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Heerema, R.J., 2022. Pecan root rhizosphere. Arizona Pecan Growers Association August 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Walworth, J., 2022. Zinc Variability. Arizona Pecan Growers Association, August 2022.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience during the fifth year of this grant effort included different segments of the pecan industry including pecan growers, nursery people, and processors at pecan grower meetings (online and in person). Grower meetings included the Arizona Pecan Grower Meeting (August 2021), Oklahoma Pecan Growers Meeting (May 2021), Texas Pecan Growers Meeting (July 2021), and Northern Nut Growers Meeting (on-line August 2021) and Arizona Pecan Growers Meeting.Information was disseminated in trade magazines such as Pecan South and extension circulars that reach a large audience within the pecan industry. The scientific community was also updated by peer reviewed published scientific articles, genomic and phenotypic datasets were made available in databases, and bio-projects were made available in NCBI. The scientific community was also updated at National and International conferences including the American Society of Horticulture Science (August 2021), and the PACBIO Global summit.The four chromosome genomes were made available on (Phytozome). The pecan website that was created due to these efforts (pecantoolbox.nmsu.edu) was also updated to give information to different segments of the pecan industry and scientific community. Changes/Problems:Due to the complications caused by COVID-19, a no-cost extension was granted for this effort. The grant will officially end February 28, 2022 and a final report will be submitted. What opportunities for training and professional development has the project provided?This project has provided many opportunities for training of undergraduate and graduate students and professional development of scientists. During this past year, fourteen undergraduate students were trained in several disciplines including bioinformatics, plant sciences, and plant biology. There were eight graduate students trained in several disciplines including bioinformatics, plant sciences, plant biology, molecular biology, and economics. There were also three post-docs trained in plant sciences. Specific training included experimental design for a number of pecan studies, clonal rootstock propagation, greenhouse management, data collection, data analysis, statistics, writing, presentations. Students were also trained in pecan horticulture techniques in the field and trained in collecting and recording phenotypic data including photosynthesis data. Students were also trained in molecular techniques such as DNA extraction, RNA extraction, and downstream applications including primer and probe design and qRT-PCR analysis. In regards to graduate students, the students have been trained in several genetic and plant molecular biology techniques. Students were trained to use web-based programs for bioinformatics. These bioinformatic skills include organellar annotation, RNA-Seq data analysis, microbiome analysis. Training on other bioinformatic softwares including Qiagen CLC-Genomics workbench, Geneious, PlantPan, and etc. Training to utilize drupal and tripal for website and database construction continued.There were limitations for students to present their data at meetings due to COVID19 but a few were able to present either virtually or in person at local, and national meetings. Specific training included the following. Kimberly Cervantes received training from on microbiome analyses including, Metagenomics, 16S microbiome analysis, MOTHUR, and she received further training on the Qiagen CLC workbench.Mr. Cyrus Smith earned a MS degree in Environmental Science while pecan rootstock salinity research. Dr. Hormat Shadgou Rhein completed her Ph.D. Fall of 2020 and received further training in RNA-seq analysis. Dr. Warren Chatwin participated in the pecan short course Texas A&M (June 7-10, 2021). )Dr. Kristen Clermont received training from Johns Hopkins University/Coursera "Introduction to Genomics Technologies" certificate; ThermoFisher Scientific and Agilent "Spectrum Mill" high-throughput processing of MS/MS and MS spectra for proteins and peptides; andMatrix Science "Mascot" software foridentification, characterization, and quantitation of proteins and peptides using mass spectrometry data. How have the results been disseminated to communities of interest?Several presentations including oral presentations, webinars, poster presentations, trade journal articles, press releases, and peer reviewed publications were made throughout the year to disseminate the current results and on-going research (listed under products). The pecan research website (pecantoolbox.nmsu.edu) that was established during this effort was also updated with new research information for the pecan industry and scientific researchers. This webpage allows the users to link to specific articles, websites, and information. Genome sequences were also linked to the pecantoolbox and the chromosome level genome sequences are available to the public through phytozome (https://phytozome-next.jgi.doe.gov).Recent pecan findings were disseminated at several pecan grower meetings including the Oklahoma pecan grower meeting, Georgia Pecan Grower Association meeting, Texas Pecan Grower Meeting, Northern Nut grower meeting, and Arizona pecan growers meeting. These meetings and workshops target the pecan industry and include growers, nurserymen, shellers, and other processors. Presentations were also made at National conferences such as the American Society of Horticulture Science (Denver 2021) and the American Phytopathology Society meeting 2021 (virtual). International conferences includedthe PACBIO Global summit. What do you plan to do during the next reporting period to accomplish the goals?The total genetic diversity panel analyses with the obtained sequences from the 820 pecan trees and 168 Carya and known hybrids will be continued and refined. These data sets will expand our knowledge of the genetic diversity within pecan and will enable development of genetic markers for future pecan breeding including developing pecan trees for specific regional climates. The additional whole genomes from 'Major', 'Mahan', 'Schley', 'Wichita', andCarya floridanawill be completed, annotated, and compared to the other genomes. The 'Lakota x 87Mx3-2.11' controlled cross test system will continue to be evaluated in its permanent locations in Texas and Georgia. The markers that have been associated with specific traits such as dichogamy, bud-break, and scab resistance will continue to be evaluated and refined. The remaining data 40% of the historical data at the NCGR and the newly acquired phenotypic data will continue to be inputted into the new SQL database which interfaces with Grin-global, and the pecantoolbox.nmsu.edu. Peer reviewed publications will be submitted to scientific journals on the work that is being completed including the molecular analysis of pecan mechanisms of flowering and alternate bearing, microbiome analyses, nutrient acquisition and transport of Zinc and Nickel, salinity tolerance, disease resistance, and allergen analysis. Further publications in trade journals of these topics will be prepared so that the grower and pecan industry base will also be notified of the progress on this research. The final advisory board meeting for this grant will take place at the end of September. All of the progress that has occurred on this grant will be listed on the pecantoolbox website so that it will be readily accessible to the industry and the scientific community.

Impacts
What was accomplished under these goals? The pecan reference genomes '87Mx-3-2.11', 'Lakota', 'Elliott', and 'Pawnee' were completed and assembled to the chromosome level. These genomes were annotated and deposited on the pecantoolbox.nmsu.edu and are available to the public on Phytozyome (https://phytozome-next.jgi.doe.gov). Analyses performed using these genomes included comparative genomics, gene ontology enrichment analysis, gene family expansions, and individual gene and promoter analysis.We constructed a 'pan-genome annotation' both within and among genomes to track the presence and DNA variation of genes. Gene variations were applied to the outbred Lakota genome, and the results lay a foundation for pecan crop improvement.The genome and related studies were published in high end journals and data is being made available in grower magazines.Long-read whole genome sequencing of four pecan cultivars ('Major', 'Mahan', 'Schley', and 'Wichita'), one hickory (Carya floridana), and one distantly related wild-relative (Rhoiptelea chiliantha)was completed in 2020. Continued analysis to process the raw sequences into chromosomes is on-going.In addition, a population genetic diversity re-sequence panel was chosen that included 440 pecan accessions and 67 Carya and other hybrid species. An additional 380 pecan trees from the National Clonal Germplasm Repository (NCGR-Carya) was sequenced at approximately 50X coverage and completed in July 2021.This additional sequencing endeavor included 101 hickory crop-wild relative accessions, historically important pecan cultivars and additional pecan trees representing furthergenetic diversity. These sequencing efforts will allow us to have a robust basis for analyzing the genetic diversity of pecan, understanding the genetic basis of traits in pecan's crop-wild relatives, and identifying molecular-trait associations for use in breeding improved pecan cultivars.These sequences are completed and analysis is currently underway to look at population and admix structure. These datasets are valuable in characterizing the genome-wide diversity of pecan which will be important for future selections for geographical regions. The completed genomic sequences are an invaluable resource that will allow elucidation of pecan genetics and implementation of marker-assisted breeding. Significant work continued on several pecan mapping populations and a number of QTLs were identified. These QTL's included bud-break, scab resistance, and Phylloxera resistance. SSR markers were designed to amplify regions in linkage for these QTLs. These QTLs were significant, for example, the QTL for budbreak is syntenic to an identified major QTL in English Walnut and likely represents a conserved mechanism effecting bud-break. Continued phenotypic data was collected on the 'Lakota' x '87Mx3-2.11' mapping population progeny seedlings. Microbiome analysis was performed on a subset of this mapping population and differences were observed among the microbiome with respect to the origin of the cross (GA and TX). Further work on the microbiome of seedlings from different cultivars wereanalyzed. Microbiome analyses are important in understanding the complete picture of disease responses and geographical adaptation of this complex tree system. A local SQL database was built and implemented at the USDA Pecan Germplasm (TX) and is being populated with data from USDA orchard systems. The database is structured to link data to individual pecan tree accessions and interfaces withGRIN-Global, and a genomic database that links to the industry pecantoolbox website. Multiple avenues for finding and evaluating disease resistance markers for pecan scab resistance continued including multiple genome-wide association studies (GWAS) of pecan trees in a natural infection and inoculated scab assays followed by RNA-Seq analyses. These analyses resulted in identification of putative markers and specific loci that are involved in plant immunity and other metabolic processes. Markers associated with scab disease resistance are extremely valuable for breeding/developing pecan trees with resistance to scab. The genetic diversity of putative pecan allergen sequences was analyzed.The allergen sequence data collected thus far show that the allergen proteins are highly conserved, under selective pressure, and serve physiologically important roles in the nut.Multiple analyses on flowering continued and included looking at genes that are involved in floral initiation and genes encoding for hormones and their impact on alternate bearing. Flowering genes involved in initiation of catkin and pistillate flowers were identified. Timing of initiation of flowering and the genes responsible were also analyzed. An ongoing seedling rootstock trial planted in a cold-winter location in New Mexico was evaluated for survivorship in 2019 and selected rootstocks were grafted to the scion 'Kanza'. Phenotypic observations and differences in performance in this cold environment among different rootstock seed sources grafted to a common scion were observed. These data will allow pecan producers in colder regions to make better use of rootstock genetics. Zinc micronutrient acquisition in a potted study with a genetic diverse range of pecan seedlings grafted to Pawnee in New Mexico high pH soils continued. The maternal seed source, 'Shoshoni' exhibited higher zinc levels in leaf tissues. Gene analysis of transporter genes was done and analysis indicates that one pecan zinc transporter gene was associated with this increased Zn level. Identification of a superior rootstock in regards to zinc would allow producers to reduce the number of foliar applied zinc or eliminate the need for foliar zinc application. The impact of rootstock on micronutrient uptake in the scion were evaluated in an Arizona orchard with sodic soil with high levels of salinity. Salinity tolerance of pecan rootstocks from geographically distinct origins with 'Elliott' and 'Ideal' showed the least sensitivity to salt damage. The trees from this study will be cloned and continued to be studied.Objective 1.Complete genomic sequence. The genomes of Carya illinoinensis '87MX3-2.11', 'Lakota', 'Elliott', and 'Pawnee' were completed using a number of sequencing platforms including PacBio single-molecule real-time (RS II, and SEQUEL I) and Illumina (HiSeq X Ten and 2500). Pangenome analysis was performed and introgressions from wild Carya were identified.Objective 2.Mapping Populations. Phenotypic characteristics including trunk diameter, plant height, scab disease expression, and bud-break were collected for the 'Lakota' and 87Mx3-2.11 mapping population progeny that are located in College Station, TX, Tifton, GA, and Byron GA.Objective 3.Phenotypic data. A SQL relational database was constructed and is being populated with data from 50 orchard systems for nearly 30 years. An estimated 60% of all data has been integrated to date. Also, hand-held data tablets were purchased for USDA-TX and the data obtained from these can be uploaded into the database.Objective 4.Development of scab markers. Analysis of differential gene expression data from RNA-Seq and GBS continued. A population of 288 seedlings from a segregating population were phenotyped for scab for further mapping.Objective 5.Horticultural traits. RNA-Seq data sets were analyzed to elucidate genes and gene-networks that are important for flowering and dichogamy. Microbiome analysis indicated that differences were observed among the overall pecan microbiome in both cultivars and tissue types. Putative transporter genes were screened in a potted rootstock trial to determine if there was an association between gene expression and zinc accumulation from rootstocks. Preliminary analysis identified gene expression/association of one zinc transporter in pecan.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Bentley, Nolan B., L. J. Grauke, Erin Ruhlman, Robert R Klein, Keith Kubenka, Xinwang Wang, and Patricia Klein. 2020. 'Linkage mapping and QTL analysis of pecan (Carya illinoinensis) full-siblings using genotyping-by-sequencing', Tree Genetics & Genomes, 16: 1-20.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bhatarrai, G., V. Bonhomme, and P. Conner. 2021. Image-based morphometric analysis revealed moderate to highly heritable nut shape traits in pecan. Amer. Soc. Hort. Sci. Ann. Conf., Aug 2021 . Denver, CO.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bock, C.H., Alarcon, Y. Conner, P.J., Young, C.A., Randall, J.J., Pisani, C., Grauke, L.J., Wang, X., and Monteros, M.J. 2020. Foliage and fruit susceptibility of a pecan provenance collection to scab, caused by Venturia effusa. CABI Agriculture and BioScience 1:19. https://cabiagbio.biomedcentral.com/articles/10.1186/s43170-020-00020-9
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bock, C., Charlton, N., Shiller, J., Randall, J.J., Young, C.A., 2021, Effect of number of microsatellite markers on estimates of population genetic diversity and structure in pecan scab pathogen, Venturia effusa, American Society of Phytopathology Annual Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Clermont, K., Mattison, C. 2021, "Pecan Allergen Accumulation and Peptide Monitoring" American Chemical Society Spring 2021 meeting, Biological Chemistry Division, April 14, 2021, online.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lovell, J.T., Bentley, N., Bhattarai, G., Jenkins, J.W., Sreedasyam, A. Alarcon, Y. Bock, C.H., Boston, L.-B., Carlson, J., Cervantes, K., Clermont, K., Krom, N., Kubenka, K., Mamidi, S., Mattison, C., Monteros, M., Pisani, C., Plott, C. Rajasekar, S., Rhein, H.S., Rohla, C., Song, M., St. Hilaire, R., Shu, S., Wells, L., Wang, X., Webber, J., Heerema, R.J., Klein, P., Conner, P., Grauke, L.J., Grimwood, J., Schmutz, J., Randall, J.J. Four de novo genomes to accelerate breeding in the outbred and diverse tree nut crop, pecan. Nature Communications 12: 4125. https://www.nature.com/articles/s41467-021-24328-w
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lovell, J., 2021 Pan-genome annotations to connect genetic variants de novo assemblies, JGI.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Lovell, J., 2021, Building and studying complex plant genomes for crop improvement, Colorado State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mattison, C. 2021. Food Allergen Preventive Controls For Human Food" Invited online/virtual oral presentation, Louisiana State University, Baton Rouge, LA, February 24, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Conner, P. 2021. Pecan tree breeding at the University of Georgia. Amer. Soc. Hort. Sci. Ann. Conf. Aug. 7, 2021. Denver, CO.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Debnath, Tathagata and M. Song. Fast optimal circular clustering and applications on round genomes, IEEE/ACM Transactions on Computational Biology and Bioinformatics. doi:10.1109/TCBB.2021.3077573
  • Type: Other Status: Published Year Published: 2021 Citation: Heerema, R. 2021. Pecan Cultivars for New Mexico. NMSU Cooperative Extension Service Guide H-659. https://aces.nmsu.edu/pubs/_h/H659.pdf. (submitted Nov. 2020; published June 2021)
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hilton A, Jeong M, Hsu J-H, Cao F, Choi W, Wang X, et al. 2021. Thermal treatment using microwave irradiation for the phytosanitation of Xylella fastidiosa in pecan graftwood. PLOS ONE 16(1): e0244758. https://doi.org/10.1371/journal.pone.0244758
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Mattison, C. 2021. "Pecan Allergen Accumulation and Peptide Monitoring" Online/virtual poster presentation American Chemical Society Spring 2021 meeting, Biological Chemistry Division, April 14, 2021
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Mattison, C. 2021. Characterization of Food and Arthropod Allergens" Invited online/virtual oral presentation at the Department of Cell and Molecular Biology, School of Biological, Earth, and Environmental Sciences. University of Southern Mississippi, Hattiesburg, Mississippi, November 6th, 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Randall, J.J., 2020, Genetic Tools for Pecans, XXI Simposio Internacional de Nogal Pecanero, On-line.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sharma, Ruby, Sajal Kumar, and M. Song, 2021, Fundamental gene network rewiring at the second order within and across mammalian systems, Bioinformatics. doi:10.1093/bioinformatics/btab240
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Schmutz, J., 2020, Applying advanced genomic techniques to build research infrastructure for crop improvement, Bayer, invited talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Schmutz, J., 2020, HiFi: Amazing applications for genomic research, PACBIO Global Summit, November 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Song, M. and H. Zhong, Efficient weighted univariate clustering maps outstanding dysregulated genomic zones in human cancers, Bioinformatics, vol. 36, no. 20, pp. 50275036, 2020. doi:10.1093/bioinformatics/btaa613
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Smith, C.A., J.L. Walworth, M.J. Comeau, R.J. Heerema, J.D. Sherman, and R. Norton. 2021. Impacts of maternal genotype on pecan seedling performance in an alkaline, saline-sodic soil. HortScience. https://doi.org/10.21273/HORTSCI15922-21.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: St. Hilaire, L., Rhein, H., Mason, K., Heerema, R., Randall, J.J., 2021, Characterization of putative flowering genes located on chromosome 4 in Carya illinoinensis, ASHS August 2021, Denver, CO.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Thompson, M., Randall, J.J., Heerema, R.J.,2021. Differential Expression of Key Floral Initiation Genes in Response to Plant Growth Regulator Application and Alternate Bearing in Pecan Journal of the American Society for Horticultural Science. (Online publish ahead of issue; https://doi.org/10.21273/JASHS04954-20).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Walworth, JL. 2020. Performance of Pecan Rootstocks in Challenging Soil Conditions. Presented online at the XXI Simposio Internacional de Nogal Pecanero 2020, October 12  14, 2020. Hermosillo, Mexico.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wang, Jiandong, Sajal Kumar and M. Song, Joint grid discretization for biological pattern discovery, Proceedings of the 11th ACM International Conference on Bioinformatics, Computational Biology and Health Informatics (BCB 20), Article No.: 57, pp. 110, Virtual Event, USA, September 2124, 2020. doi:10.1145/3388440.3412415
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Yamagata, H., Noda, K., Randall, J.J., Kamiya, H., Oki, K. A consecutive monitoring method for pecan orchards and a discovery of a mysterious circle in a pecan orchard using UAV. Optical Review.
  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Rhein, Hormat, PECAN FLOWER CLOCKS: RNA-SEQ PROFILING OF FLORAL INITIATION AND DEVELOPMENT IN PROTOGYNOUS AND PROTANDROUS PECAN TREES, Ph.D. Dissertation NMSU.
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Shadgou Rhein, H., Lovell, J., Sreedasyam, A., Jenkins, J., Song, M., Li, Y., Bentley, N., Klein, P., Wang, X., Heerema, R.J., Grimwoood, J., Schmutz, J., Grauke, LJ, Randall, J.J., 2021, Identification of floral specific genes in Carya illinoinensis through RNA-Seq. PLOS ONE, submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Randall, J., 2021, 'Advancements in Genetic Tools for the Industry', Arizona Pecan Growers Association oral presentation. August 27th, Tucson, AZ.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Randall, J.J., 2021, "Pecan Genetics-Shaping the future of pecan". Oklahoma Pecan Growers Annual Conference, June 2021, virtual/oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Randall, J.J., 2021. "Shaping the future of the pecan industry through genetics", Texas Pecan Growers Conference, July 2021, San Marcos, Texas.
  • Type: Other Status: Published Year Published: 2021 Citation: https://www.hudsonalpha.org/using-genomics-to-improve-our-future-pecan-trees/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Randall, J.J., Cervantes, K., Ray,D.K., Sanchez, A., Mason, K., Fisk, J.N., Soneji, J.R., Sanchez, L., Grauke, L.J., Wang, X., 2021. Insights into the impact of geography and genetics on the microbiome of Carya illinoinensis ISHS 2021. DOI 10.17660/ActaHortic.2021.1318.34. Pgs. 235-240.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Grauke, LJ. 2021. 'Carya Vulnerability', nutgrowing.org. Oral/Virtual presentation August 2021.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rhein, H.S., Barnes, S., Van Scyoc, B., Fisk, J.N., Song, M., Thompson, M., Heerema, R.J., Randall, J.J., 2020. RNA-Seq analysis of genes involved in pistillate flower initiation in Carya illinoinensis. Acta Hortic. 1318. ISHS 2021. DOI 10.17660/ActaHortic.2021.1318.24. Pgs. 157-163.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ishimori, M., Takanashi, H., Fukami, K., Cervantes, K., Nagano, A.J., Kajiya-Kanegae, H., Grauke, L.J., Tsutsumi, N., Randall, J.J., Iwata, H., 2021. Genome-wide RAD-Seq analysis revealed subpopulation structures of the pecan (Carya illinoinensis) germplasm collection and their relationship to geographical distribution patterns. ISHS 2021. DOI 10.17660/ActaHortic.2021.1318.26. Pg. 177-183.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience: The target audience during the fourth year of this grant effort included different segments of the pecan industry including pecan growers, nurserymen, and processors at pecan grower meetings. Information was disseminated in trade magazines such as Pecan South and Pacific Nut Producer that reaches a large audience within the pecan industry. The 'Coordinated development of genetic tools for pecan' Advisory Board meeting was held in May 2020 which updated the board on the results from the grant effort. Grower meetings included Arizona Pecan Growers Meeting (August 2020), Western Pecan Growers Conference (March 2020), American Pecan Congress, Southeastern Pecan Growers Conference and the University of Georgia Pecan Cultivar Tour (September 2019). The scientific community was also updated by peer reviewed published scientific articles, genomic and phenotypic data-sets that were made available in databases. The scientific communithy was also updated at National and International conferences including the American Society of Horticulture Science, Plant Science Symposium, Horticulture Industry Show, and Plant and Animal Genomics Conference, San Diego (January 2020). The pecan website (pecantoolbox.nmsu.edu) was also updated to give information to different segments of the pecan industry and scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided many opportunities for training of undergraduate and graduate students and professional development of scientists. In regards to pecan horticulture, at least ten undergraduate students were trained this past year. This training included learning experimental design for a number of pecan studies, clonal rootstock propagation, greenhouse management, data collection, data analysis, and statistics. Students were also trained in pecan horticulture techniques in the field and trained in collecting and recording phenotypic data including photosynthesis data. Students were also trained in molecular techniques such as DNA extraction, RNA extraction, and downstream applications including primer and probe design and qRT-PCR analysis. In regards to graduate students, the students have been trained in several genetic and plant molecular biology techniques. Students were trained to use web-based programs for bioinformatics. These bioinformatic skills include organellar annotation, RNA-Seq data analysis, microbiome analysis. Professional staff attended a virtual bioinformatic training given by the National Center for Genomic Research in August 17-28, 2020. Training on other bioinformatic softwares including Qiagen CLC-Genomics workbench, Geneious, PlantPan, and etc. Training to utilize drupal and tripal for website and database construction continued. Implementation and training on BG Base also occurred. The students presented their data at local, national, and international meetings. BG-base database training was given to by Mike O'Neal to Ellen Provin, Keith Kubenka, Xinwang Wang, Clive Bock, Cristina Pisani, Warren Chatwin, and Jennifer Randall. Kimberly Cervantes received training from NCGR on RNA-Seq, Metagenomics, 16S microbiome analysis and she received further training on the Qiagen CLC workbench. How have the results been disseminated to communities of interest?Several presentations were made throughout the year to disseminate the current results and on-going research. These included oral presentations, webinars, poster presentations, trade journal articles, and peer reviewed publications. The pecan research website (pecantoolbox.nmsu.edu) that was established during this effort was also updated with new research information for the pecan industry and scientific researchers. Recent pecan findings were disseminated at several pecan grower meetings and workshops including: Arizona pecan growers meeting, and Western pecan growers meeting, and the University of Georgia Pecan Cultivar Tour. These meetings and workshops target the pecan industry and include growers, nurserymen, shellers, and other processors. The 'Coordinated development of genetic tools for pecan' advisory board and scientific team met via 'zoom' for a research update. Presentations were also made at National conferences such as the American Society of Horticulture Science (given virtually during 2020) and international conferences including the Plant and Animal Genomics Conference (January 2020). What do you plan to do during the next reporting period to accomplish the goals?The genetic diversity panel analyses with the obtained sequences from the 440 pecan trees and 65 Carya and known hybrids will be continued and refined. These data sets will expand our knowledge of the genetic diversity within pecan and will enable development of genetic markers for future pecan breeding including developing pecan trees for specific regional climates. Potential genetic introgressions of crop wild relatives will be analyzed in the pecan diversity panel. The 'Lakota x 87Mx3-2.11' controlled cross test system will continue to be evaluated in its permanent locations in Texas and Georgia. The markers that have been associated with specific traits such as scab resistance will continue to be evaluated and refined. The remaining data will continue to be inputted into the new SQL database which interfaces with BG Base, Grin-global, and the pecantoolbox.nmsu.edu. Phenotypic data that is actively acquired will also be populated into the new SQL/BG-Base databases. Analysis of RNA-Seq, GBS, GWAS, and other molecular analysis of pecan mechanisms involved in disease resistance, flowering, alternate bearing, nutrient acquisition and transport, salinity tolerance, and allergen analysis will continue and publications that are in progress will be completed and submitted for publication. We are also planning a 'Pecan conference' in Spring or Summer 2021 that will bring together the pecan industry and the scientific team to show our findings and how this will help/enhance/improve the pecan industry.

Impacts
What was accomplished under these goals? The pecan reference genomes '87Mx-3-2.11', 'Lakota', 'Elliott', and 'Pawnee' were completed and assembled to the chromosome level. These four genomes were annotated and deposited on the pecantoolbox.nmsu.edu and we anticipate they will be available on Phytozyome. Analyses were performed with these genomes including comparative genomics, gene ontology enrichment analysis, gene family expansions, pangenome analysis, and individual gene and promoter analysis. These genomes have been used as a reference genome for mapping reads obtained from numerous experiments utilizing GBS or RNA-Seq libraries. In addition, a population genetic diversity re-sequence panel was chosen that included 440 pecan accessions and 67 Carya and other hybrid species. These sequences are completed and analysis is currently underway to look at population and admix structure. These datasets are valuable in characterizing the genome-wide diversity of pecan which will be important for future selections for geographical regions. The completed genomic sequences are an invaluable resource that will allow elucidation of pecan genetics and implementation of marker-assisted breeding. Significant work continued on several pecan mapping populations. GBS analysis of 151 progeny from the 'Elliott' x 'Vc-168' cross was used to create high-density genetic linkage map. Quantitative trait locus (QTL) analyses revealed 1 major and 2 minor bud-break QTLs and the major bud-break QTL is syntenic to an identified major QTL in English Walnut and may represent a conserved mechanism effecting bud-break. Further phenotypic data was collected on the 'Lakota' x '87Mx3-2.11' mapping population progeny seedlings and a subset of the seedlings were re-sequenced. QTL mapping revealed a major effect locus associated with the severity of pecan Phylloxera infection. Microbiome analysis was performed on a subset of this mapping population and differences were observed among the microbiome with respect to the origin of the cross (GA and TX). Microbiome analyses are important in understanding the complete picture of disease responses and adaptation of this complex tree system. A local SQL database was built and implemented at the USDA Pecan Germplasm (TX) and is being populated with data from USDA orchard systems. The database is structured to link data to individual pecan tree accessions and interfaces with BG-Base, GRIN-Global, and a genomic database that links to the industry pecantoolbox website. Multiple avenues for finding and evaluating disease resistance markers for pecan scab resistance continued including multiple genome-wide association studies (GWAS) of pecan trees in a natural infection and inoculated scab assays followed by RNA-Seq analyses. These analyses resulted in identification of putative markers and specific loci that are involved in plant immunity and other metabolic processes. Markers associated with scab disease resistance are extremely valuable for breeding/developing pecan trees with resistance to scab. The genetic diversity of putative pecan allergen sequences was analyzed. Two allergen proteins Car-i-1 Car-i-2 were essentially identical among all cultivars tested with minor amino acids substitutions observed in the Car-i-2 allergen sequence. Multiple analyses on flowering continued and included looking at genes that are involved in floral initiation and genes encoding for hormones and their impact on alternate bearing. Work to date, suggests that gibberellins and auxins play an important role in flowering and alternate bearing. Flowering genes putatively involved in initiation of catkin and pistillate flowers as found by differential gene expression studies were further analyzed for their specific roles. An ongoing seedling rootstock trial planted in a cold-winter location in New Mexico was evaluated for survivorship in 2019 and selected rootstocks were grafted to the scion 'Kanza'. Differences in performance in this cold environment among different rootstock seed sources grafted to a common scion will observed. These data will allow pecan producers in colder regions to make better use of rootstock genetics. Zinc micronutrient acquisition in a potted study with a genetic diverse range of pecan seedlings grafted to Pawnee in New Mexico high pH soils continued. The maternal seed source, 'Shoshoni' exhibited higher zinc levels in leaf tissues and using qRT-PCR expression of one pecan zinc transporter gene was associated with this increased Zn level. The impact of rootstock on micronutrient uptake in the scion were evaluated in an Arizona orchard with sodic soil with high levels of salinity. Salinity tolerance of pecan rootstocks from geographically distinct origins continued and 'Elliott' and 'Ideal' showed the least sensitivity to salt damage. The significant relationships between leaf potassium:sodium ratio and observed salt injury and tree growth rate suggest that this measure could be used to predict salt tolerance in short-term evaluations. Identification of a superior rootstock in regards to zinc would allow producers to reduce the number of foliar-applied zinc or eliminate the need for foliar zinc application. Objective 1. Complete genomic sequence. The genomes of Carya illinoinensis '87MX3-2.11', 'Lakota', 'Elliott', and 'Pawnee' were completed using a number of sequencing platforms including PacBio single-molecule real-time (RS II, and SEQUEL I) and Illumina (HiSeq X Ten and 2500). The resulting contigs were oriented, ordered and joined together into 16 chromosomes using syntenic markers, as well as the Hi-C data. Heterozygous single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) phasing errors were corrected using the raw PACBIO data and homozygous SNPs and INDELs were corrected using Illumina reads. The final versions of reference assemblies range in the size from 649-669 Mb with contig N50 of 3.7-4.4 Mb. The number of annotated genes ranged from 31,042 - 31,911 and the annotation was updated with the DOE JGI Plant Annotation Pipeline. Objective 2. Mapping Populations. Phenotypic characteristics including trunk diameter, plant height, scab disease expression, blotch miner (Cameraria caryaefoliella Clemens), black pecan aphid (Melanocallis caryaefoliae Davis), and Phylloxera infestations were collected for the 'Lakota' and 87Mx3-2.11 mapping population progeny. The seedlings were removed from their juvenile nursery and 482 were transplanted into their permanent spacing at the USDA repository in College Station, Texas, worksite in January 2020. Another set of 482 seedlings were planted in Bryon, Georgia. Objective 3. Phenotypic data. A SQL relational database was constructed and is being populated with data from 50 orchard systems for nearly 30 years. An estimated 20% of all data has been integrated to date. Objective 4. Development of scab markers. DGE data from a RNA-Seq experiment where pecan trees were inoculated with scab isolates from 'Desirable', 'Pawnee', or a water control found that at 48 hours post-infection there were a higher number of upregulated genes expressed in the resistant reaction when compared to susceptible reaction. Gene ontology enrichment indicated genes in defense pathways were upregulated in the resistant and the genes for photosynthesis and cutin biosynthesis were among those downregulated in the susceptible reaction. Objective 5. Horticultural traits. Tissue was collected and RNA was extracted) from six cultivars ('Apache', 'Mahan', 'Elliott', 'Lakota', 'Major', and 'Pawnee'). RNA-Seq libraries were sequenced on an illumina platform. The data is currently being processed and will help elucidate and confirm data on dichogamy.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bhattarai, G. and P. Conner. 2019. Construction of high-density genetic maps of pecan (Carya illinoinensis) with genotyping by sequencing. UGA Plant Center Fall Retreat Oct. 25, Helen, GA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Bhattarai, G., S. Cao, and P. Conner. 2020. QTL Mapping for Pecan Scab (Venturia effusum) Resistance Based on High-Density Genetic Maps in Pecan (Carya illinoinensis). Plant & Animal Genome Conference, Jan. 12, San Diego, CA. (Poster)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Bhattarai, G., V. Bonhomme, and P. Conner. 2020. Morphometic Analsysis of Nut Shape of Commercial Pecan Cultivars. Univ. Of Georgia Inst. of Plant Breeding, Genetics, and Genomics Annual Retreat. July 30, Athens, GA. (Poster)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Conner, P. 2019. Pecan tree breeding at UGA. Nat. Assoc. Plant Breeders Ann. Conf. Aug. 29, Pine Mountain, GA (Oral)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Cervantes, K., Grauke, L.J., Wang, X., Conner, P.C., Bock, C.H., and Randall, J.J. 2020. Pecan seedling microbiome composition may be influenced by geographical location and host genetics. XXVIII Plant and Animal Genome Conference. January 11-15, 2020.
  • Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Bentley, N.B. (2020). New Genomic Resources for Pecan (Cayra illinoinensis). Ph.D. dissertation, Texas A&M University. OakTrust.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Bentley, N.B., Grauke, L.J., & Klein, P.E. (2020). Genomic Resources for Pecan. Pecan Growers Advisory Board, Webinar.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Rohla, C., and Graham, C. 2020. Pecan Topics for June. Oklahoma State University Zoom meeting, 69 participants.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Bentley, N.B., Grauke, L.J., & Klein, P.E. (2020). Linkage Mapping and QTL Analysis of Pecan Scab Susceptibility and Spring Emergence from Genotyping by Sequencing (GBS) of a Full-Sibling Pecan Population. Poster presented at Plant and Animal Genome, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Rohla, C., and Graham, C. 2020. Pecan Topics for May. Oklahoma State University Zoom meeting, 56 participants.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Rohla, C., and Graham, C. 2020. Pecan Topics for July. Oklahoma State University Zoom meeting, 60 participants.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Rohla, C. (2020). Equipment needs and tips for planting pecans. Horticulture Industry Show
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Mattison,Chris. 2020. Proteomic Analysis of Developing Pecan Nuts - South Eastern Pecan Growers Association Sandestin Florida February 22-23, 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Mattison, Chris. 2020. Food Allergen Preventive Controls For Human Food - Food Safety Preventive Control Alliance (FSPCA), Louisiana State University, Baton Rouge LA, November 6, 2019.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Walworth, J., C. Smith, R. Heerema, J. Sherman. 2020. Update on Rootstock Salinity Study Western Pecan Growers Association Conference. March 1  3, Las Cruces, NM.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Song, J. 2019.Model-free methods for functional pattern discovery. Workshop on Big Data Applications, Challenges, and Techniques, New Mexico State University, Las Cruces, NM. November 22, 2019. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: St. Hilaire, R. 2020. What do innovations in plant sciences have to do with orchard management? 54th Annual Western Pecan Growers Association Conference, March 2, 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Randall, J., Rhein, H., 2020, Alternate bearing and flowering gene networks in Carya illinoinensis (pecans), Plant and Animal Genomics Conference, San Diego, Jan. 2020 (oral).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Sanchez, A., Cervantes, K., and Randall J.,2020. Microbiome and phenotypic effects of antibiotics on micropropagated Carya Illinoinensis (pecan), ASHS annual conference, August 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Mason, K., Twitchell, A., Rhein, H., Randall, J., 2020. Battling alternate bearing: promoter analysis of flowering genes in pecan. NMSU URCAS April 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: St. Hilaire, Diamanti, F., Rhein, H., Randall, J.,2020. Characterization of putative flowering genes located on Chromosome 4 in Carya illinoinensis. Carya illinoinensis ASHS annual conference August 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Mason, K., Twitchell, A., Shadgou Rhein, H., Randall, J., 2020 Promoter Motif Analysis of Putative Flowering Genes in Carya illinoinensis. ASHS conference August 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Randall, J. 2020, 'Genetic tools for pecans', Arizona Pecan Growers Virtual Conference, August 28,2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nguyen, Hien H., Hua Zhong and M. Song. Optimality, accuracy, and efficiency of an exact functional test. Proceedings of the Twenty-Ninth International Joint Conference on Artificial Intelligence, Main track. pp. 26832689, Kyoto, Japan, January 5-10, 2020. https://doi.org/10.24963/ijcai.2020/372
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kumar, Sajal and M. Song. Statistical Inference of Discrete Combinatorial Functional Dependency in Biological Systems. Proceedings of the 14th Machine Learning in Computational Biology (MLCB) Meeting}, Vancouver, Canada, December 13-14, 2019. https://mlcb.github.io/mlcb2019_proceedings/papers/paper_24.pdf
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Eliaka Dvo?�kov�, Sajal Kumar, Ji?� Kl�ma, Filip }elezn�, Karel Drbal, Mingzhou Song. Evaluating model-free directional dependency methods on single-cell RNA sequencing data with severe dropout. Proceedings of International Conference on Bioinformatics Research and Applications (ICBRA). pp. 5562, Seoul, South Korea, December 19-21, 2019. https://doi.org/10.1145/3383783.3383793
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Willingham, Jessica Solving the Jefferson Tree Mystery. Noble Legacy, spring 2020, www.noble.org.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shadgou Rhein, H., Li, Y.,Kumar, S.,Song, J., Heerema, R., Schmutz, J., Jenkins, J., Grimwood, J., Grauke, LJ., Randall, J.J., 2020, DEGs involved in formation of pecan (Carya illinoinensis) pistillate and staminate flowers. XXVIII Plant and Animal Genome Conference. January 11-15, 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Randall, J., 2020, Pecan Genetic Improvement, Western Pecan growers conference March 2020.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Monteros, M.J. More efficient plants ahead, thanks to genomic technologies. Noble News and Views. Tech Issue. Vol 37. No. 8. https://www.noble.org/news/publications/ag-news-and-views/2019/august/more-efficient-plants-ahead-thanks-to-genomic-technologies/
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Rohla, C. (2020). The woe of winter in the heat of the summer. Pecan South, https://www.pecansouthmagazine.com/magazine/article/
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Rohla, C. (2020). Before you fertilize, understand what your trees need. Pecan South, https://www.pecansouthmagazine.com/magazine/article/
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Graham, C. (2020). Protecting our crop, protecting our fungicides. Pecan South, https://www.pecansouthmagazine.com/magazine/article/
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Graham, C. (2020). IDing varieties & examining nut abnormalities. Pecan South, https://www.pecansouthmagazine.com/magazine/article/
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hilton, Angelyn and Randall, J., 2020, Improved detection of Xylella fastidiosa in pecan. Pecan South August 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Grauke, LJ, 2020, Dennis Ring, Pecan South
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Grauke, LJ, December 2019, Speaking of family trees,Pecan South.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Grauke, LJ, December 2019, Family trees, the next generation Pecan South.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Grauke, LJ, September 2019, Family trees, generations and propagations. Pecan South.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Thompson, M., Randall, J.J., Heerema, R.J.,2020. Differential Expression of Key Floral Initiation Genes in Response to Plant Growth Regulator Application and Alternate Bearing in Pecan Journal of the American Society for Horticultural Science. (Accepted).
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Bentley, N.B., Grauke, L.J., Kubenka, K., Klein, R.R., Wang, X., Ruhlman, E. & Klein, P.E. (2020) Linkaage Mapping and QTL Analysis of Pecan (Carya illinoinensis) Full-Siblings Using Genotyping-by-Sequencing. Tree Genetics & Genomes. Accepted.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Boyer, K. S., Fairbanks, W. S., Rohla, C. T. & Webb, S. L. (2020). Surficial soil damage by wild pigs (Sus scrofa) decreases pecan harvest efficiency. Crop Protection, 128, https://doi.org/10.1016/J.CROPRO.2019.104992.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ouedraogo, F. B., Brorsen, B. W., Biermacher, J. T. & Rohla, C. T. (2020). Effects of Pruning at Planting on Pecan Trunk Development and Total Shoot Growth. HortTechnology, 30 (2), 248-250 doi: https://doi.org/10.21273/HORTTECH04535-19.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Bock C.H., Y. Alarcon, PJ, Conner, CA Young, U Stokes, JJ Randall, C Pisani, LJ Grauke, X Wang, and MJ Monteros. Foliage and fruit susceptibility of a pecan provenance collection to scab, caused by Venturia effusa. Submitted to CABI Ag and Biosci
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hilton, A., Wang, X., Zhang, M., Cervantes, K., French, J., Randall, J.J., Bock, C., Grauke, LJ., Jo, Y.K. 2020. Improved methods for detecting Xylella fastidiosa in pecan and related Carya species. European Plant Pathology,. 157:899918 (https://doi.org/10.1007/s10658-020-02050-5).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, X., Shadgou Rhein, H., Jenkins, J., Schmutz, J., Grimwood, J., Grauke, LJ, Randall, J.J., 2020. Chloroplast genome sequences of Carya illinoinensis from two distinct geographic populations. Trees and Genomes, 16:48. (https://doi.org/10.1007/s11295-020-01436-0 )


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:The target audience during this third year included different segments of the pecan industry including pecan growers, nurserymen, and processors at pecan grower meetings. Grower meetings included Georgia Pecan Growers Conference (March 2019), Arizona Pecan Growers Meeting, New Mexico Pecan Growers Meeting, Western Pecan Growers Conference (March 2019), Oklahoma Pecan Growers Association (June 2019), Texas Pecan Growers Meeting (July 2019),Tri-state pecan growers meeting, Western Pecan Production Short-course, Native pecan field day (Oklahoma), Clay County Pecan Field Day (TX), and the University of Georgia Pecan Cultivar Tour.The scientific community was also updated at National and International conferences including the American Society of Horticulture Science, American Society Plant Biology, Plant and Animal Genomics Conference, San Diego (January 2019).The pecan website (pecantoolbox.nmsu.edu) is also available to give information to different segments of the pecan industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided many opportunities for training of undergraduate and graduate students and professional development of scientists. In regards to pecan horticulture, at least eight undergraduate students were trained this past year. This training included learning experimental design for water deficit experiments, pecan fertilization experiments, clonal rootstock propagation, greenhouse management and data collection. Students were also trained in pecan horticulture techniques in the field and trained in collecting and recording phenotypic data including photosynthesis data. Students were also trained in molecular techniques such as DNA extraction, RNA extraction, and downstream applications including primer and probe design and qRT-PCR analysis. In regards to graduate students, the students have been trained in several genetic and plant molecular biology techniques. Students were trained to use web-based programs for bioinformatics. These bioinformatic skills include organellar annotation and RNA-Seq data analysis. This includes alignment of RNASeq to genomes and determining differential gene expression analysis. Graduate students and professional staff attended bioinformatic training at the National Center for Genomic Research in Santa Fe. Training on other bioinformatic softwares also occured including Qiagen CLC-Genomics workbench, Geneious, and etc. Training to utilizedrupal and tripal for website and database construction has also occurred. Implementation and training on BG Base and Asset Base also occurred. The students presented their data at local, national, and international meetings. YaninaAlarcón a research scientist with Noble Foundation received the following trainings: 1.2018 Oklahoma Pecan Management, (February-October, 2018) Oklahoma State University. 2. Noble Research Institute training: Hypothesis testing (April 30, 2019), 3. HPC Onboarding-Taurus high-performance computing cluster (March 25, 2019), 4. Linux Command Line (March 18, 2019), 5. Plotting in R (July 31, 2018), 6. Data Carpentry for Genomics (June 5-6, 2018), 7. Intro to R (April 26, 2018). 1. Dr. LJ Grauke and Mike O'Neal (BG-Base) received training in the use of the digital asset manager AssetBank from Stewart Oak. Emphasis was on organization and uploading of image files from ARS-TX into AssetBank, and assignment of accession information prior to incorporation into BG-Base.Training was followed by successfully uploading hundreds of nut images (On 14 May, 2019). 2. Dr. LJ Grauke visited the offices of Mike O'Neal (BG-Base developer) in Addison, Texas and worked to coordinate linkage of passport and phenotypic information in that database with images in the digital asset manager AssetBank, and with spatial coordinates describing source germplasm and living inventories maintained in an ArcGIS geodatabase (July 16-17, 2019). 3. Dr. Xinwang Wang received training at the National Center for Genome Resources Summer Bioinformatics Intensive, Santa Fe, New Mexico.Training covered the fundamentals of UNIX followed by in-depth concepts, workflows and bioinformatic tools used for genomic sequence data analysis (July 1-26, 2019). 4. Hormat Shadgou Rhein received training at the National Center for Genome Resources for RNA-Seq bioinformatics training from November 12-17, 2018. How have the results been disseminated to communities of interest?Multiple types of presentations have been made throughout the year to disseminate the current results and on-going research. These include oral presentations, poster presentations, trade journal articles, and peer reviewed publications. Also, a pecan research website (pecantoolbox.nmsu.edu) has been established that gives updates on pecan research. Word has been disseminated at several pecan grower meetings and workshops including: Georgia Pecan Growers, the Oklahoma Pecan Growers meetings, Tri-state pecan growers meeting, Arizona pecan growers meeting, and Western pecan growers meeting, Western Pecan Production Short-course, Native pecan field day (Oklahoma), Clay County Pecan Field Day (TX), and the University of Georgia Pecan Cultivar Tour. These meetings and workshops target the pecan industry and include growers, nurserymen, shellers, and other processors. The advisory board for this grant met in person in Las Cruces, New Mexico on March 3, 2019 for a research update. Local presentations were also made at the Texas A&MPlant Breeding Symposium, Noble Foundation and at NMSU. Presentations were also made at National conferences such as the American Society of Plant Biologists and the American Society of Horticulture Science and international conferences including the Plant and Animal Genomics Conference (January 2019). A website for Development of Pecan Genetic Tools was launched that links a tripal database which is housing the pecan genomics data. This website will also link to BG Base for phenotypic data retrieval. This website will also serve as a means for disseminating results to the pecan and other interested communities. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we plan to complete the structural genome annotations of the 'Lakota' and 'Elliot' pecan genomes. Annotation of the pecan genomes will be deposited into the pecantoolbox website and public databases. We will continue sequencing the 'Pawnee' genome to have a chromosome level build as the other three pecan genomes. We willsequence and analyze an additional 480 pecan trees for their genetic diversity.With these data sets we will be able to comparethe cultivars used by the industry with the native diversity that is available for pecan. This will enable the development of markers for future breeding purposes to develop trees for specific regional climates.The 'Lakota x 87Mx3-2.11' progeny will continued to be evaluated. Markers associated with phenotypes such as scab susceptibility/resistance will be refined. The progenies are currently planted in GA and TX orchards and part of the population may be transplanted in different geographical areas such as NM, AZ, and OK. The tissue culture cloned progeny of 'Lakota X 87Mx3-2.11' will continued to be propagated and transitioned to soil in anticipation field planting. Phenotypic data will continue to be populated in the local BG base database with implementation of further phenotypic data acquired from participating locations with active pecan trials. The necessary data from this database will also be transferred into the Grin- global system. This database will be linked to the new pecantoolbox website. The phenotypic data and genomic data will be deposited within the databases. Asset Bank, the newly established local database for the USDA Pecan repository, will continue to be populated with acquired pictorial documentation and historical records at the USDA Pecan Germplasm repository (TX). Scab evaluations will continue to be made on the pecan trees in the Provenance collection.An imaging software WinFOLIA will be evaluated for its potential to quantify scab lesions on pecan trees with scab symptoms. The Genome Wide Association Study with the Provenance collection will be refined to look forSNPs in candidate genes that conferred resistance across all four years. Analysis will also include evaluation of SNPs that are year-specific due to climatic reasons and may fluctuate due to Genome X Environment interactions within the natural scab populations and pecan trees.Marker analysis for resistance to scab will continue and be refined with the mapping populations. Also, RNA-Seq data will be collected for the experiment on 'Desirable' and analyzed for differential gene expression patterns. Experimental analysis of pecan mechanisms involved in flowering, nutrient transport, and salinity rootstocks will be continued with more molecular work performed in the next year. Publications are currently in progress on the complete genomes, and mechanisms in floral initiation in pecan.

Impacts
What was accomplished under these goals? The annotation of the '87Mx3-2.11' pecan reference genome has been completed and assembled to the chromosome level. This annotated genome is on the pecantoolbox.nmsu.edu and we anticipate that it will be available on Phytozyome. The 'Lakota' and 'Elliott' genomes are completed, assembled to the chromosome level and are being annotated. An unusual genome organization was found during analysis of the 87Mx3.211 genome. An ancient whole gene duplication event estimated to be around 50 to 100 Mya was found during analysis.The sixteen chromosomes split into two subgenomes of 8 chromosome each, nearly identical in gene structure, but retain no identifiable shared sequence beyond these retained genes.The completed genomic sequence is an invaluable resource that will allow for understanding the genetics that control traits and implementation of marker assisted breeding.The mapping population 'Lakota x 87Mx3-2.11' progeny seedlings were evaluated for their susceptibility/resistance to scab. Phenotypic data was collected and statistically evaluated with respect to the origination of the cross (GA or TX) to scab susceptibility.This differential response of the seedlings to scab will aid in determining genetic markers associated with resistance to this disease.The 'Lakota X 87Mx3-2.11' mapping population were planted in orchards in both Tifton, GA and at the USDA Pecan Germplasm repository in Texas. The tissue culture cloned progeny of 'Lakota X 87Mx3-2.11' continued to be propagated and their nutrient needs evaluated. Pecan tissue was collected and DNA extracted for sequencing from an additional 480 accessions from the USDA Pecan germplasm collection.This dataset is valuable in characterizing the genome-wide diversity of pecan which will be important for future selection for geographical regions.A local BG database was implemented at the USDA Pecan Germplasm (TX) and phenotypic data from the Pecan Germplasm repositories in TX and the Provenance orchard in Byron GA are being populated within the database. Asset Bank is being implemented to archive images and historical documents.The databases will allow for transfer into Grin-global and will link to the industry pecantoolbox website. This website will allow for industry access to the data and information. Multiple avenues for finding markers associated with scab resistance have continued including genome wide association studies (GWAS), detached leaf scab assays, and attached scab assays followed by RNA-Seq analyses. The GWAS analyses with three years of scab data evaluations resulted in putative markers scab resistant markers. Differential gene expression analyses of 'Western' and 'Wichita' buds through a time course resulted in identification of putative genes involved specifically in catkin and pistillate flower initiation.This is important for the understanding of flowering and alternate bearing in pecan.Evaluation of micronutrient transport continued in a potted study with a genetically diverse range of pecan seedling rootstocks grafted to Pawnee in high pH soils. The maternal seed source, 'Shoshoni' had higher zinc in leaf tissues than any other seed source within the study and the data suggests that maternal genotypes influences zinc uptake. Root samples are being analyzed for transporter gene activity. Identification ofa superior rootstock in regards to zinc would allow producers to reduce the number of foliar-applied zinc applied or eliminate the need for foliar zinc application.Further studies with the impact of rootstock on micronutrient uptake in the scion were evaluated in an Arizona orchard.Leaf nutrient concentrations exhibited a high degree of variability that may be related to the genetic variability of open pollinated seed rootstock.Salinity tolerance of pecan rootstocks from geographically distinct origins continued and Elliott and Ideal showed the least sensitivity to salt damage.Objective 1. Complete genomic sequence.The genomeCarya illinoinensis87MX3-2.11 was improved by integrating Hi-C and the latest map build. Version 1.0, includes 647MB of sequence with 98.0% organized into 16 chromosomes and a contig N50 of 4.4MB. The annotation was updated with the DOE JGI Plant Annotation Pipeline. This annotation includes 31,911 genes and 36,250 alternative transcripts. The 'Lakota' genome was improved using Hi-C data integration and using synteny with the completed 87Mx3-2.11 v. 1.0 for validation. The assembly includes 667MB of sequence with 96% organized into 16 chromosomes and a contig N50 of 3.7MB. The 'Elliot' genome was completed using HudsonAlpha's MECAT/CANU pipeline, integrated Hi-C data and synteny with 87Mx3-2.11 V.1.0. This assembly includes 653MB of sequence with 95.4% organized into 16 chromosomes and a contig N50 of 4.4MB.Objective 2. Mapping Populations.'Lakota' and 87Mx3-2.11 controlled cross nuts were planted and some introduced into tissue culture. Seedlings were screened for scab resistance/susceptibility. DNAwas extracted from the 1062 mapping population and parentage was confirmed in 82% of the seedlings. DNA from 144 confirmed progeny representing phenotypes from pecan scab were sequencedusing Illumina generating a total of 1.5Tb of sequence.The generated linkage maps will facilitate linkage QTL mapping of seedling characteristics. These trees are planted at the USDA Pecan germplasm repository. GBS of a 156 F1 mapping population of 'VC1-68' and 'Elliott' progeny was done and QTLs related to vigor, pecan scab susceptibility, and bud-break are being analyzed.C. Plastid and DNA fingerprinting.MoreSSR and SNP markers were identified for pecanDNA fingerprinting for cultivar identification.Objective 3. Phenotypic data.Remote sensing was used to georeference trees in the USDA TX Repository for spatial visualization within an ArcGIS geodatabase. Aerial and ground images were collected using a drone. Images were processed to generate an orthomosaic of the orchard. A database of tree locations, attributes and coordinates are being integrated into BG-Base. A prototype linking relational and image data to spatial maps was developed.Objective 4.Development of scab markers.A.RNA-Seq experiment was established by inoculating 100 'Desirable' potted trees in an indoor controlled environment. Trees were inoculated with scab isolates from 'Desirable', 'Pawnee', and a water control. Inoculations were confirmed using microscopic analysis. Leaf samples were collected 6, 12, 24, 48, 72, 96, and 144 hours post inoculation and RNA was extracted from the leaves for RNA-Seq analysis. B. GWAS of the USDA provenance collection (871 trees) with scab phenotypic data identified 117 SNPs in putative candidate genes that may be involved in resistance.Objective 5. Horticultural traits.A.RNA-Seq and DEG analysis of 'Western' and 'Wichita' buds identified genes involved in pistillate and catkins initiation and putative gene networks for floral development in pecan. B. Eight maternalgenotypes across the pecan native range were used as rootstocks to study Zn uptake in alkaline soils. 'Shoshoni' seedlings had up to 150% higher Zn concentrations. RNA was extracted from roots to measure gene expression of putative transporter genes responsible for Zn uptake. An Arizona orchard is being utilized to determinethe contributions of soil versus tree variability on foliar nutrient concentrations. The rootstocks are open-pollinated seed originating from 'Ideal' maternal inheritance. Leaf nutrient concentrations exhibited variability with foliar Zn concentrations ranged from 11.8 to 31.9 mg/kg. Soil zinc ranged from 0.9 to 11.8 mg/kg, available soil zinc and foliar zinc concentrations are not closely related.Variation in foliar concentration may be related to the variable genetics of open pollinated rootstock.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Alarcon, Y., Bock, C.H., Steele, J., Tang, Y., Wood, B.W., Rohla, C., and Monteros, M.(2019). Genome-Wide Association Study (GWAS) of Pecan Scab Resistance in a Provenance Collection. Plant and Animal Genome Conference, San Diego, CA, 2019.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Alarcon, Y. Bock, C.H., Steele, J., Tang, Y., Wood, B.W., Rohla, C., and Monteros, M.(2019). Genome-Wide Association Study (GWAS) of Pecan Scab Resistance in a Provenance Collection. National Association of Plant Breeders Annual Meeting, Pine Mountain, Georgia, U.S.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bentley, N. B., Grauke, L. J., & Klein, P. E. (2019). The Development of Linkage Maps for Pecan from GBS Data and Their Application to Refine the Next Pecan Reference Genome. Poster presented at Plant and Animal Genome, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Bentley, N. B., Grauke, L. J., Conner, P. & Klein, P. E. (2018). Genetic Linkage Maps for Pecan from Biparental Mapping Population GBS Data. Seminar presented at the Plant Breeding/Genetics Circle, Texas A&M University, College Station, TX.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Bentley, N. B., Grauke, L. J., & Klein, P. E. (2018, November 16). Development of Genetic Linkage Maps Synteny between Pecan and Walnut Identification of Trait Associated Loci. Seminar presented at the Institute for Plant and Genomics & Biotechnology, Texas A&M University, College Station, TX.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Cervantes, K., (2018). The influence of the pecan microbiome on pecan adaptation. New Mexico State University Molecular Biology seminar series.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Cervantes, K., (2019). The influence of drought stress on pecan physiology. New Mexico State University Molecular Biology seminar series.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cervantes, K., (2019). Insights into the impact of geography and genetics on the microbiome of Caryay illinoinensis. New Mexico State University Graduate Student Research Council Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cervantes, K., Grauke, L.J., Wang, X., Conner, P.J., Bock, C.H., and Randall, J.J. (2019). Influence of geographical location and host genotyype on pecan seedling microbiome composition. American Society of Plant Biologists, San Jose, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Grauke, L.J., Ishimori, M., Iwata, H., Randall, J. J. (2019). GWAS and Genomic prediction for evaluating breeding populations and germplasm collection: case studies in Japanese Pear, Apple, and Pecan. Plant and Animal Genomics Conference 2019.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Grauke, L.J. (2019) Pecan Breeding: The Next Generation. TriState Pecan Conference, New Roads, Louisiana.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Grauke, L J (2019). Dynamic use of the National Collection of Genetic Resources for Pecans and Hickories. Invited oral presentation in Workshop Fruit and Nut Germplasm Collections: Treasuries of Genetic Diversity American Society for Horticultural Sciences Annual Conference, Las Vegas, Nevada.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Heerema, R., (2018). Advancements in pecan rootstocks. New Mexico Pecan Growers Association Conference, Las Cruces, NM.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Heerema, R., (2018). Pecan cultivars and rootstocks. Western Pecan Production Short Course, Las Cruces NM.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Heerema, R., (2018). Pecan rootstocks for the southwestern states. Arizona Pecan Growers Association Conference, Tucson, AZ.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Heerema, R., Sherman, J., and Randall, J. (2018). Pecan Diseases. Western Pecan Production Short Course, Las Cruces, NM.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ishimori, M., Takanahi, H., Fukami, K., Cervantes, K., Grauke, L., Tsutsumi, N., Randall, J. J., Iwata, H. (2019). Genome-environment associations suggest local climate adaptation in native Carya Populations. Plant and Animal Genomics Conference 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lowe, K.S., Shadgou Rhein, H., Heerema, R.J., Randall, J.J., St. Hilaire, R., (2019). Environmental, Epigenetic, and Physiological Approaches to Decipher Alternate Bearing in Pecans, Annual meeting of American society of Horticultural Science, Las Vegas 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Randall, J., (2018). Genetics and the future of the pecan industry. Western Pecan Production Short Course.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Randall, J., (2018). Pecan rootstocks. Western Pecan Production Short Course.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Randall, J., (2019). Pecan tree genetics. Biology Department Seminar, New Mexico State University, Las Cruces, NM.
  • Type: Other Status: Other Year Published: 2019 Citation: Randall, J., (2019). Coordinated development of genetic tools in pecan, Advisory Board update, Las Cruces, NM. March 3, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shadgou-Rhein, H., Schmutz, J., Grimwood, J., Jenkins, J., Song, M., Heerema, R.J., Grauke, L.J., Randall, J.J., (2019). DEG studies of flower initiation genes in type I and type 2 pecan varieties. PAG 2019. San Diego.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shadgou-Rhein, H., Schmutz, J., Grimwood, J., Jenkins, J., Song, M., Heerema, R.J., Grauke, L.J., Randall, J.J., (2019). DEG studies of flower initiation genes in protandrous and protygnous pecan trees. National Center of Genomic Research, Santa Fe, NM.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Shadgou Rhein, H., Schmutz, J., Grimwood, J., Jenkins, J., Song, M., Heerema, R.J., Grauke, L.J., Randall, J.J., (2019). Unmasking the mysteries of Catkin floral initiation in Carya illinoinensis through RNA-Seq analysis, Annual meeting of the American society of plant biologists.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Shadgou Rhein, H., (2019). Molecular mechanisms of Carya illinoinensis (Pecan) Flower Induction. New Mexico State University Molecular Biology Seminar Series.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Song, M., (2019). Model-free Methods for Inferring Networks in Molecular Biological Systems. Faculty of Science, Charles University, Prague, Czech Republic.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Song, M. (2019). Model-free Methods for Inferring Networks in Molecular Biological Systems. Central European Institute of Technology (CEITEC), Brno, Czech Republic.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Walworth, J.L., R. Heerema, and J. Sherman. Leaf Nutrients: Tree to Tree Variability.2019 The Western Pecan Growers Association annual conference, March 3-5, 2019, Las Cruces, NM.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Walworth, J.L., Pecan rootstock salinity study in Arizona. Coordination of development of genetic tools for pecan Advisory Board meeting March 3, 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Monteros, M.J. (2019) More efficient plants ahead, thanks to genomic technologies. Noble News & Views. Vol 37: issue 8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bentley, N., Grauke, L. J., & Klein, P. (2019). Genotyping by sequencing (GBS) and SNP marker analysis of diverse accessions of pecan (Carya illinoinensis). Tree Genetics & Genomes, 15(1), 8.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Grauke, L J. (2019). Family Trees: Looking back, going forward. Pecan South 52(1):42-49,50-53.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Grauke, L J. (2019). Family trees: Roots and resilience. Pecan South 52(4):12-15, 17,18,20-21.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Grauke, L J. (2019). Family trees: Generations and Propagations. Pecan South 52(6): (in press).
  • Type: Websites Status: Published Year Published: 2019 Citation: Young, C. and N. Charlton. How to identify and control pecan scab. Available at: https://www.noble.org/globalassets/images/news/noble-news-and-views/2018/04/pdf/how-to-identify-and-control-pecan-scab.pdf
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Randall, J.J., (2019) Powering Pecans, leveraging genetics to defend crops against disease and weather, Retaking the Field: Science Breakthroughs for thriving farms and a healthier nation. Volume 4, pg. 14-15; March 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tuskan, G., Grover, A. T., Schmutz, J., DiFazzo, S., Myburg, A., Grattapaglia, D., Smart, L., Yin, T., Aury, J.-M., Kremer, A., Leroy, T., LeProvost, G., Plomion, C., Carlson, J. E., Randall, J. J., Westbrook, J., Grimwood, J., Muchero, W., Jacobson, D., Michener, J. K. (2018). Hardwood tree genomics: Unlocking woody plant biology. Frontier Plant Science, 9. www.frontiersin.org/article/10.3389/fpls.2018.01799
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Grauke, LJ, Wang XW. (2019). Considerations related to the designation of a Core subset for Carya. Acta Horticulturae (International society of Horticulture Science). In Press.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Wang, XW, Grauke LJ. SNP discovery and SSR mining from Carya illinoinensis RAD sequences. . Acta Horticulturae (International Society of Horticulture Science), In Press.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Rhein, H., Barnes, S., VanSyoc, B., Fisk, J.N., Song, M., Thompson, M., Heerema, R.J., Randall, J.J.. RNA-Seq analysis of genes involved in pistillate flower initiation in Carya illinoinensis. Acta Horticulturae (International Society of Horticulture Science), In Press.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Thompson, M. Y., Randall, J. J., Heerema, R., VanLeeuwen, D. (2019). Exogenous Plant Growth Regulators Show Promise for Management of Alternate Bearing in Pecan. HortScience 54(7) pgs. 1204-1207.
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Wang, X., Shadgou Rhein, H., Jenkins, J., Schmutz, J., Grimwood, J., Grauke, L.J., Randall, J.J. (2019) Chloroplast genome sequences of Carya illinoinensis from two distinct geographic populations, Tree genetics and genomes.
  • Type: Other Status: Other Year Published: 2019 Citation: Workshop: Managing Your Pecan Orchard. January 17, 2019 at Noble Research Institute.
  • Type: Other Status: Other Year Published: 2019 Citation: Workshop: Controlling Pests in Pecan Orchards. April 18, 2019 at Noble Research Institute.
  • Type: Other Status: Other Year Published: 2019 Citation: Workshop: Clay County Pecan Field Day. May 2, 2019 at Montz Pecan Company Charlie, TX.
  • Type: Other Status: Other Year Published: 2019 Citation: Workshop: Native Pecan Field Day. April 25 at 5 Star Pecan and Cattle Ranch Okemah, OK.
  • Type: Other Status: Other Year Published: 2018 Citation: Workshop, "University of Georgia Pecan Cultivar Tour", Tifton and Ray City, GA (September 20, 2018).
  • Type: Other Status: Other Year Published: 2018 Citation: Western Pecan Production Shortcourse Workshop. October 15-October 17, 2018. 170 participants including pecan growers, processors, nurseries, and researchers.


Progress 09/01/17 to 08/31/18

Outputs
Target Audience:The target audience during this second year included different segments of the pecan industry including pecan growers, nurserymen, and processors at pecan grower meetings. The scientific community was also updated at National and International conferences including the American Society of Horticulture Science (September 2017); International Symposium on Walnut, Pecan and Cashew, Santiago, Chile (December 2017); Plant and Animal Genomics Conference, San Diego (January 2018); Georgia Pecan Growers Conference, Tifton, Georgia (March 2018), National Pecan Scientist Meeting in Texas (July 17, 2018); American Society of Horticulture Science,Washington, D.C. (August 2018)and International Society Horticulture Science (Istanbul, Turkey August 2018). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided many opportunities for training of undergraduate and graduate students. In regards to pecan horticulture, two undergraduate students were trained this past year on caring for clonal pecan rootstock trees in a greenhouse including learning experimental design for pecan fertilization experiments and collecting data. In regards to graduate students, the students have been trained in several genetic and plant molecular biology techniques. Students have been trained on using web-based programs for bioinformatics. These bioinformatic skills include organellar annotation and RNA-Seq data analysis. This includes alignment of RNASeq to genomes and determining differential gene expression analysis. Students have also had more training in qRT-PCR analysis. Training of using Drupal and Tripal for website and database construction has also occurred. The students include presenting their data at local, national, and international meetings. Dr. Jennifer Randall attended USDA NIFA PI training at the national ASHS 2017 meeting. Dr. LJ Grauke and Keith Kubenka attended the Plant Germplasm Operations Committee meetings in Washington DC June 18-22, 2018.Meetings were held with the developer of the GRIN-Global database, BG-Base developer, and Stefan Lura of US National Arboretum. These workshop meetings concerning using BG-Base in conjunction with ArcGIS geodatabase as a local database, linking georeferenced, inventory specific records and images to passport records, and providing them to GRIN-Global and other web nodes (including genomic and industry information websites). On 26-27 July 2018, Mike O'Neal (BG-Base developer) visited the ARS Somerville worksite and worked with LJ Grauke to coordinate entry of passport information into that database.The team continued work with Stefan Lura and Kathleen Emerson-Dell (US National Arboretum) in refining methods of image storage and retrieval using Asset Bank software. Dr. Grauke received training in GRIN-Global development team on August 22nd. Dr. Grauke and Dr. Randall attending Grin-global workshop at the national ASHS meeting held August 3, 2018 in Washington DC. How have the results been disseminated to communities of interest?Multiple oral and poster presentations have been made throughout the year to disseminate the research and current results. Pecan grower meetings include the Georgia Pecan Growers and the Oklahoma Pecan Growers meetings. These meetings target the pecan industry and include growers, nurserymen, shellers, and other processors. The advisory board for this grant met via 'Zoom' on May 8, 2018 for a research update. Other communities of interest include Pecan Scientists at the National Pecan Scientist Meeting that occurred in conjunction with the Texas Pecan Growers Meeting. Local presentations were also made at the Texas A&MPlant Breeding Symposium. Presentations were also made at National conferences such as the American Society of Plant Biologists (September 2017 and August 2018). Presentations were also made at international conferences including the VIII International Symposium on Walnut, Cashew and Pecan in Santiago, Chile (December 2017), the Plant and Animal Genomics Conference (January 2018)and the International Society of Horticulture Science in Istanbul Turkey (August 2018). A website for Development of Pecan Genetic Tools was launched that links a tripal database which is housing the pecan genomics data. This website will also link to BG Base for phenotypic data retrieval. This website will also serve as a means for disseminating results to the pecan and other interested communities. What do you plan to do during the next reporting period to accomplish the goals?Within in the next reporting period we plan to have annotation of 87Mx3.211 and 'Lakota' genomes at the chromosome level. The 'Elliot' genome will be assembled and annotated. We will continue sequencing the 'Pawnee' genome. Annotation of the pecan genomes will also be done and as they are completed they will be deposited into the pecantoolbox and public databases. Linkage maps from the established mapping populations will also be continued. The 'Lakota x 87Mx3-2.11' progeny will continued to be evaluated and will begin to be planted in different geographical areas. The tissue culture cloned progeny of Lakota X 87Mx3-2.11 will be transitioned to soil in anticipation field planting. Geo-referencing of the Pecan Germplasm repositories in Texas and the Provenance orchard in Byron Georgia will be completed and the data will be housed in in the local BG database that is being implemented. The data from this database will also be transferred into Grin-global. This database will be linked to the new pecantoolbox website. Phenotypic descriptors for pecan trees in all locations will be measured. The phenotypic data and genomic data will be deposited within the databases. Continued scanning of historic documentation at the Carya Germplasm repositiory will be continued this year and stored in the new database system. Marker analysis for resistance to scab will continue with described methods. Experimental analysis of pecan mechanisms involved in flowering, nutrient transport, and salinity rootstocks will be continued with more molecular work performed in the next year.

Impacts
What was accomplished under these goals? A Drupal based website was launched for housing pecan researchhttp://pecantoolbox.nmsu.edu. This website links to a Tripal database that houses genomic data generated for pecan. The website will also link to a local BG base database that is being implemented for pecan phenotypic data. The BG base database will incorporate data into the Grin-global database. A high-quality annotation for the pecan 87MX3.211 that incorporates the latest improvements in genome sequencing was completed and is available on the new website. Hi-C chromatin mapping was collected for both 87Mx2.11 and the Lakota genotypes and allowed for preliminary integration to produce chromosome builds for each genome. The'Pawnee' x 'Elliott' mapping population was genotyped using GBS. The segregation of alleles unique to 'Elliott' was analyzed and genetic linkage maps were generated. These maps were used to create a consensus map of linkage groups thought to represent pecan's 16 chromosomes. Continued analysis of 'Pawnee (scab susceptible) x Kanza' (scab tolerant) mapping population resulted in identification of possible scab resistance genes. These candidate genes within gene ontology categories were previously shown to play a role in fungal disease resistance mechanisms and were used to design molecular markers for genotyping. Progress was made with horticultural traits. RNA-Seq data was obtained and analyzed for differential gene expression of the genes involved in pistillate flower development.We investigated the interactive effects of pecan seedling maternal genotype on zinc uptake in alkaline soils. All maternal genotypes were deficient for zinc, however, 'Shoshoni' seedlings had 42% and 150% higher zinc concentrations. These data suggest that maternal genotype influences zinc uptake. We identified 21 putative genes from the ZIP family of metal transport genes in pecan.Salinity tolerance of 7 pecan rootstock genotypes from different geographical origins were evaluated in field conditions. Riverside, Elliot and Bradley show the least sensitivity to salt damage.Objective 1. Complete genomic sequence.A.Collected Illumina RNA-Seq for87Mx3.211(187M reads across 6 tissues), Lakota (410M reads across 11 tissues), Elliot (389M reads across 9 tissues), and Pawnee (409M reads across 12 tissues). B. Collected PACBIO ISO-Seq data to improve the annotation of87Mx3.211generating 535k FLcDNA gene sequences. PACBIO long read sequences were collected on two genotypes Lakota (87 chips on RS platform for ~100x sequence coverage) and Elliot (10 chips on Sequel platform for ~97x of sequence coverage). C. We collected Illumina resequencing coverage in 2x150 bp reads to 189x coverage for Lakota for genome polishing. We collected Hi-C chromatin mapping data for: 87MX3.2 (394Gb) and Lakota (250Gb). An annotation was completed for 87Mx3.211using the data collected for RNA-Seq (374M reads) and the processed CCS FLcDNA reads (535,000 genome corrected reads) with theDepartment of Energy Joint Genome Institute Plant Annotation Pipeline (Shu, unpublished). This assembly is in large contiguous PACBIO contigs (2.5 MB contig N50, 1,099 contigs, 705.6 MB assembled). We annotated 36,489 gene loci and additional 22,533 alternative transcripts identified. Progress on the 'Lakota' genome included an assembled initial contig version of the genotype 'Lakota' from PACBIO long read sequencing. We collapsed alternative haplotypes for 'Lakota' and corrected remaining PACBIO errors with high coverage Illumina data. We have achieved a contigN50 of 3.0MB with 1,623 contigs spanning 828.1MB of the genome. These are only preliminary results and we expect the final version to be closer to 700Mb of single haplotype data. Hi-C data was generated and analyzed. Hi-C is a technique for capturing longer chromosome links by fixing DNA coils that are nearby one another within the cell. Preliminary results indicate that we can order all 16 chromosomes for both 87Mx3.211 and 'Lakota'. Combined with the initial mapping efforts, we will be able to produce complete genomes for these two genotypes. The sequencing for the genotype 'Elliot' is completed (collected 120x coverage with PacBio reads) and are being assembled.Objective 2. Mapping populations.A.In 2016 and 2017 controlled crosses were made on 'Lakota' trees in multiple orchards in GA and TX using pollen from 87Mx3.211 for development of shared mapping populations. Nuts were harvested, numbered, and nut measurements were taken. These were planted in ARS greenhouses in GA or TX. Seeds were sent to NMSU for introduction into tissue culture. Seedling emergence was measured and samples in TX were collected for DNA identification. The 2016 'Lakota x 87Mx3.211'seedling population grown in GA were planted. Seedlings will be screened for scab disease resistance/susceptibility. GBS will be performed on the seedlings. B. 'Pawnee x Elliot' mapping population located in Tifton GA was evaluated for traits. DNA from these crosses were genotypes by GBS.The segregation of alleles unique to 'Elliot' were analyzed in both populations to generate separate genetic linkage maps. These maps were compared and used to create a consensus map of linkage groups to represent pecan's 16 chromosomes. C. Plastid and DNA fingerprinting. Chloroplast genomes for 87Mx3.211 and 'Lakota' have been annotated and analyzed. Genomes will be deposited in public databases and published.SSR markers and SNP markers were improved for pecanDNA fingerprinting for cultivar identification.Objective 3. Phenotype Data.A Drupal website was created and launched for the pecan industry. This website interfaces with a Tripal database that houses the pecan genomic data. A local database (BG Base) is being implemented at the USDA Pecan Repositoryto ensure inventory specific linkages to molecular identification, phenological records and images. This database will allow inclusion of phenotypic data including georeferenced data and historical records. The database will allow transfer from this local database into Grin-global.Objective 4. Markers for scab resistance.A.'Pawnee x Kanza' mapping population were analyzed by GBS and SNPs wereused to generate a draft genetic linkage map to identify genetic determinants for pecan scab response. Candidate genes for scab resistance were identified and markers were developed. B. Leaves from 871 trees from the pecan provenance collection were collected and DNA was extracted, quantified and submitted for GBS. GWAS models are being built by combining the SNP data with phenotypic scab ratings collected for three years.Objective 5. Horticultural traits.A.RNA-Seq was performed on buds from 'Western' trees to elucidate differential gene expression for pistillate flower formation.This data provides the first glimpse of gene networks and timing of the genes in pecan floral initiation.B.Eight maternalgenotypes across the native range of pecan were used to study the effects Zn uptake in alkaline soils. Seedlings from all maternal genotypes except 'Moore', 'Curtis', and 'Shoshoni' were deficient for zinc (<50 ppm) in 2015. All genotypes were deficient for zinc in 2016 and 2017, however, 'Shoshoni' seedlings had 42% and 150% higher zinc concentrations. These data suggest that maternal genotype influences zinc uptake.C.Salinity tolerance of 7 rootstock genotypes from different geographical origins rootstocks were evaluated in AZ field conditions. The trees were visually rated for salt damage.The pattern that has emerged to date is that Riverside, Elliot and Bradley show the least sensitivity to salt damage, show the most vigor in the field, and have the largest trunk diameters. Peruque and Giles are most sensitive to salt and least vigorous. It appears that salinity tolerance is loosely related to geographical origin.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Monteros, M. 2018. Opportunities for DNA fingerprinting in pecan and understanding the genetics of pecan scab resistance. Georgia Pecan Growers Conference. Tifton, GA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Monteros, M. 2018. Decoding pecan genetics and the potential impacts for pecan growers. Texas AgriLife Extension Horticulture Agents Training. Ardmore, OK.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Bentley, N. B., L. J. Grauke and P. E. Klein. 2018. Development of Genomic Resources for Pecan. Oral Presentation. Department of Horticultural Sciences Seminar Series, Texas A&M University, College Station, TX.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Bentley, N. B., L. J. Grauke and P. E. Klein. 2017. The Characterization of Pecan (Carya illinoinensis) Germplasm Using Genotyping by Sequencing (GBS). Oral Presentation. 2017 ASHS Annual Conference, Waikoloa, HI.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Randall, J.J., 2017. Pecan Genetics and clonal pecan rootstock. Oral Presentation. XVIII Simposio Internacional de Nogal Pecanero 2017, Hermosillo, Mexico.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Shadgou-Rhein, H., Barnes, S.D., Fisk, J.N., Song, M., Heerema, R.J., and Randall, J.J. 2018. Transcriptomic analysis of flower initiation genes in pecan. Plant and Animal Genomics Conference, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: French, J., Heerema, R. J. (presenter), and Flynn, R. 2018. Pecan Maternal Genotype: Implications for Nutrient Uptake in Alkaline Soils. Oral Presentation at Annual Conference of American Society for Horticultural Science, Washington, DC.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Herrera, C., Grauke, L. J., Randall, J. J. 2018. Coordinated Development of Genetic Tools for Pecan; linking the past, present, and future through databases. Oral presentation in Workshop Using the GRIN-Global Database as a Research and Breeding Tool. American Society for Horticultural Sciences Annual Conference, Washington DC.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Shadgou-Rhein, H., Song, M., Bently, N., Klein, P., Jenkins, J., Grimwood, J., Schmutz, J., Grauke, L.J., Randall, J.J. 2018. Gene expression analysis of pecan (Carya illinoinensis) flower structures using RNA-Seq International Society of Horticulture Science. Istanbul, Turkey.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: French, J., Heerema, R. J., and Flynn, R. 2018. Pecan Maternal Genotype: Implications for Nutrient Uptake in Alkaline Soils. Abstract for Annual Conference of American Society for Horticultural Science, Washington, DC, 3 August, 2018. In Press (to be published in an online supplement to HortScience in the fall of 2018).
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Grauke, LJ, Wang XW. 2018. Considerations related to the designation of a Core subset for Carya. Acta Horticulturae.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Wang, XW, Grauke LJ. 2018. SNP discovery and SSR mining from Carya illinoinensis RAD sequences. Acta Horticulturae.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Tuskan, G.A., Groover, A.T., Schmutz, J., DiFazio, S.P., Myburg, A., Grattapagila, D., Smart, L., Yin, T., Aury, J.M., Kremer, A., Leroy, T., Le Provost, G., Plomion, C., Carlson, J.E., Randall, J.J., Westbrook, J., Grimwood, J., Muchero, W., Jacobson, D., Michener, J.K., 2018. Hardwood tree genomics: Unlocking woody plant biology. Frontiers of Biology.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Rhein, H., Barnes, S., VanSyoc, B., Fisk, J.N., Song, M., Thompson, M., Heerema, R.J., Randall, J.J. 2018. RNA-Seq analysis of genes involved in pistillate flower initiation in Carya illinoinensis. Acta Horticulturae.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Randall, J.J., Grauke, L.J., Wang, X. Developing genetic tools and the role of the national germplasm repositories. International Symposium on Walnut, Pecan, and Cashew, ISHS, Santiago, Chile.


Progress 09/01/16 to 08/31/17

Outputs
Target Audience: The target audience during this first year of effort included different segments of the pecan industry including pecan growers, nurserymen, and processors at pecan grower meetings. A workshop presentation was also given at the Western Pecan Short Course that wasdesigned to teach those interested in pecan agriculture. Also, the work was also shared in the scientific community both at the National Pecan Scientific Meeting that was held at the Noble Foundation and other National Conferences. Listed Below are the types of presentations to the target audience. Pecan Industry: Growers, Nurserymen, and Processors Invited Oral Presentations Randall, J. "Pecan Cloning Breakthrough and Pecan genetics."Arizona Pecan Growers Association Conference. Tucson, AZ. (August 26, 2016). Randall, J. "The future of pecan genetics."Western Pecan Production Short Course. Las Cruces, NM.(October 17, 2016). Randall, J. "Pecan Genetic Research and the future of the pecan industry."New Mexico Pecan Growers Association. Las Cruces, NM. (October 26, 2016). Randall, J.; "Pecan genetics and the future!"Georgia Pecan Growers Association.Tifton GA. (March 29, 2017). Monteros, M.J. et al. "Opportunities for DNA Fingerprinting in Pecan and Understanding the Genetics of Pecan Scab Resistance."Georgia Pecan Growers Conference. Tifton, GA. March 29, 2017. March 2nd2017.Monteros, M.J. et al. Decoding Pecan Geneticsand thePotentialImpacts forPecanGrowers?.Oklahoma Pecan Growers Association. Tulsa, OK. June 2, 2016. Poster Presentation Alarcon, Y. et al. DNA fingerprinting for pecan cultivar identification. 52nd Annual Georgia Pecan Growers Association (GPGA) Conference & Trade Show, Tifton, GA. March 2017. General Public: Publications Alarcon, Y., M.J. Monteros. 2016. Scientific advancements provide pecan cultivar identification tools. Ag News and Views. Vol. 34. No. 6. June 2016. Scientific Conferences and Symposiums Oral Presentations Bentley N. "The Characterization of Pecan Germplasm Using Genotyping by Sequencing (GBS)". Texas A&M Plant Breeding Symposium- The Vavilov Method: Using Genetic Diversity. College Station, TX Feb. 15-17, 2017. Grauke, LJ "Carya: The Next Generation". Texas A&M Plant Breeding Symposium- The Vavilov Method: Using Genetic Diversity College Station, TX. Feb. 15-17, 2017. Posters Alarcon, Y. et al. Identification of pecan scab resistance gene candidates in a pecan provenance collection using a combined bulked segregant analysis and genotyping by sequencing approach. American Society of Plant Biologists (ASPB) 2017. Honolulu, Hawaii June 24-28, 2017 Alarcon, Y. et al. Genomic resources for pecan and pecan scab resistance.National Pecan Scientists Meeting, Ardmore, OK. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided manyopportunities for training of undergraduate and graduate students. In regards to the science, one undergraduate student was trained in pecan tree pruning, irrigation systems, herbicide application, pecan fertilization, and pecan tree harvest. In regards to graduate students, the students have been trained in several genetic and plant molecular biology techniques. These include collections of pecan tissues into liquid nitrogen for RNA and DNA isolation. High quality DNA isolation for genome based sequencing. High quality RNA isolation and quantification for RNASeq library construction. Students have also been trained in creating linkage maps, and in bioinformatics of RNASeq data. This includes alignment of RNASeq to genomes and determining differential gene expression analysis. Another training opportunity that has been afforded to the students include presenting their data at local, national, and international meetings. How have the results been disseminated to communities of interest?Multiple oral and poster presentations have been made throughout the year to disseminate the research and current results. Pecan grower meetings include the Arizona Pecan Growers Meeting, New Mexico Pecan Growers, Western Pecan Growers, Georgia Pecan Growers Association, Oklahoma Pecan Growers, andTexas Pecan Growers. These meetings target the pecan industry and include growers, nurserymen, shellers, and other processors. Other communities of interest include Pecan Scientists. Presentations were made at the National Pecan Scientist Meeting that was held this year at the Noble Foundation in Ardmore, OK. Local presentations were also made such as theTexas A&M Plant Breeding Symposium- The Vavilov Method: Using Genetic Diversity.Presentations were also made at National conferences such as the American Society of Plant Biologists.Multiple abstracts have been submited and accepted for upcoming meetings including American Society forHorticulture Sciences and theVIII International Symposium on Walnut, Cashew and Pecan. Besides oral and poster presentations multiple trade articles have been published in Pecan South and Georgia Pecan in regards to the efforts and results. A website for Development of Pecan Genetic Tools has been created and is planning to be launched in October 2017. This website will also be a means fo disseminating results of the accomplished work to the pecan and other interested communities. What do you plan to do during the next reporting period to accomplish the goals?Within in the next reporting period we plan to continue pecan genome sequencing and sequencing analysis. This includes RNASeq library constructionand RNASeq analysis. Annotation of the pecan genomes will also be done and as they are completed they will be deposited into publically available genome databases.Linkage maps from the established mapping populations will also be continued. The Lakota x 87Mx3-2.11 progeny will be evaluated to ensure parentage and will begin to be planted in different geographical areas. The tissue culture cloned progeny of Lakota X 87Mx3-2.11 will be transitioned to soil in anticipation field planting in 2018-2019. Geo-referencing of the Pecan Germplasm repositories both in Texas and in Georgia will be completed and the data will be housed in databases that will be linked and accessible by the website we have developed and plan to launch in October 2017. Phenotypic descriptors for pecan trees in all locations will be deterined and measured. The data will be deposited within the database. Continued scanning of historic documentation at the Carya Germplasm repositiory will be continued this year and stored in the new database system that is under construction. Marker analysis for resistance to scab will continue with described methods. Experimental analysis of pecan mechanisms involved in flowering, nutrient transport, and salinity rootstocks will be continued with more molecular work performed in the next year.

Impacts
What was accomplished under these goals? Significant progress was accomplished. This includes the first complete pecan reference genome that will be available to other scientists and allow comparisons of tree species. Mapping populations were made and established populations were characterized for identification of disease resistance markers. A pecan website is about to be launched that will link databases that will house information critical for the pecan industry in regards to traits of pecan cultivars. Objective 1. Complete genomic sequence of multiple pecan trees Major activities and experiments conducted a. Pecan tissue from selected cultivars were collected for DNA and RNA isolation and sequencing. C. illinoinensis 87MX3-2.11 was sequenced using a whole genome shotgun sequencing strategy and standard sequencing protocols. Data Collected An assembly was generated using Illumina and PACBIO reads.This produced 3,016 contigs, and a total genome size of 863.1Mb. A second genome assembly was done that resulting in the primary assembly that consisted of 1,376 contigs, and a total genome size of 726.8Mb. Summary Statistics and discussion of the Results The total size of the pecan 87Mx3.211 genotype is 863.1Mb and assemblies are relatively complete. Key outcomes or other accomplishments realized The pecan genome 87Mx3-2.11 is nearly complete.This genome will be annotated and publically available. Objective 2. Development of linkage maps across Carya populations Major activities and experiments conducted Mapping population (Lakota x 87MX3-2.11) Crosses of Lakota x 87MX3-2.11 were made and nuts were non-destructively evaluated. Nuts were introduced into tissue culture or planted. Elliott x VC168 The mapping population consists of 157 progeny. Scab expression monitored and DNA was extracted for GBS analysis. Pawnee x Elliott Mapping population Pawnee X Elliott mapping populations were evaluated for budbreak and dichogamy type. Scab isolates were obtained and used for detached leaf assays. DNA was extracted from the progeny trees for GBS. DNA fingerprinting for cultivar identification Pawnee and Kanza pecan leaf tissue was harvested and used for RNAseq. DNA was extracted from 100 historical and commercial pecan cultivars and analyzed using GBS. Data Collected Mapping population Lakota X 87Mx3-2.11 was crossed during spring 2016 and 2017. Seeds collected during 2016 were measured non-destructively. Trees in the mapping population Elliott x VC168 were verified and GBS was performed. Scab analysis and GBS is in progress for Pawnee X Elliott population. DNA fingerprinting efforts identified 43,417 SNPs between Pawnee (scab susceptible) and Kanza (scab resistant). Ninety-six molecular markers were designed. Summary Statistics and discussion of the Results Seed resulting from Lakota x 87-Mx3-2.11 cross are being grown for data analysis. GBS analysis is in progress for the mapping population of Elliott X VC168. Scab analysis and GBS is in progress for the Pawnee X Elliott mapping populations. DNA fingerprinting resulted in sixteen SNPS that can be amplified with High Resolution Melting procedures to confirm identity in sixty pecan cultivars. Key outcomes or other accomplishments realized A mapping population from Lakota x 87Mx3-2.11 was generated and are being grown for future analysis. GBS was completed for Elliott x VC168 population. Linkage maps are being constructed and specific phenotypes and markers are being evaluated. Scab evaluations and GBS analysis are currently being done for the Pawnee X Elliott mapping population. DNA fingerprinting efforts resulted in the ability to identify 60 pecan cultivars using High Resolution Melting (HRM). Objective 3. Phenotypic descriptor development and data collection Major activities and experiments conducted Database development A local database will operate from an ArcGIS Enterprise relational geodatabase. A relational database is being restructured to ensure inventory specific linkages to molecular verification information, phenological records and images. Data entry Historic data is being scanned for archiving and prepared for entry into GRIN Global. Website A website is currently being created that will link to the databases. It should be available to the public by October 2017. Data Collected No new data to report. Summary Statistics and discussion of the Results No data collection to report. Key outcomes or other accomplishments realized Database construction is in progress that will hold phenotypic and genotypic data. Website development is anticipated to be available in 2017. Objective 4. Development of markers associated with pecan scab resistance for future marker-assisted breeding Major activities and experiments conducted Isolation of pecan scab pathogen for inoculation studies Pecan scab isolates from 63 trees were collected. Isolates were genotyped and the percentage of spore germination and penetration in pecan leaves are being evaluated. Screening the pecan provenance collection for pecan scab responses DNA bulks were created with equal amounts of DNA from three trees per bulk from samples corresponding to six pecan provenances. A scab resistant (R) and a scab susceptible (S) bulk were generated. The twelve bulks were genotyped based on pecan scab ratings. Primers developed targeting putative disease resistant genes. Molecular primers (oligos) targeting disease resistance genes were designed. Data Collected Scab isolates were collected. Screening of the scab resistant vs scab susceptible tree GBS data resulted in 3,570 SNPs that were polymorphic between resistant and susceptible bulks. SNPs (89) were located in candidate disease resistance genes. A total of twenty SNP targeting scab resistance genes were developed. Genetic differences between Pawnee (scab susceptible) and Kanza (scab resistant) are being identified. Summary Statistics and discussion of the Results. Scab isolates were collected and are being analyzed. Hundreds of SNPs were identified that may be used to identify genetic markers for scab resistance with several SNPs located in candidate disease resistance genes. Apple scab resistant genes including HcrVf2, HcrVf4, Rvi1were used to develop 20 SNPs. Key outcomes or other accomplishments realized Scab isolates were collected and are being analyzed. SNPs were identified that may be used to identify genetic markers for scab resistance. SNPs were located in known candidate disease resistance genes. SNPs targeting specific resistant genes were identified to determine genetic differences between scab resistant and susceptible cultivars. Objective 5. Identification of the genetics that control horticultural traits Major activities and experiments conducted Investigate the differential acquisition of nutrients and their transport within the plant Different pecan tree varieties were grown in pots under three different soil lime treatments.Leaf tissue analysis is underway.Putative zinc transporter genes (6) and iron transporter genes (3) were identified. Screening for salinity tolerance in field conditions. Pecan seeds of common used rootstocks were planted in Safford AZ in 2017.The potted trees were installed in a randomized plot design.The seedlings are being maintained with frequent irrigation for establishment. Data Collected Pecan seedlings grown in 3 different lime treatments were maintained. Nutrient analysis and tree measurements are ongoing. Putative pecan zinc and iron transporter genes were identified. Trees planted in AZ field for salinity trial. No data yet. Summary Statistics and discussion of the Results Preliminary data indicated that growth and nutrient acquisition differences observed. No data. Key outcomes or other accomplishments realized Cultivar differences in nutrient acquisition are being measured. Putative transporter genes for zinc and iron uptake were identified. No data yet available.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Alarcon, Y., M.J. Monteros. 2016. Scientific advancements provide pecan cultivar identification tools. Ag News and Views. Vol. 34. No. 6. June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bentley N. The Characterization of Pecan Germplasm Using Genotyping by Sequencing (GBS)". Texas A&M Plant Breeding Symposium- The Vavilov Method: Using Genetic Diversity. College Station, TX Feb. 15-17, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grauke, LJ Carya: The Next Generation. Texas A&M Plant Breeding Symposium- The Vavilov Method: Using Genetic Diversity College Station, TX. Feb. 15-17, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Alarcon, Y. et al. Identification of pecan scab resistance gene candidates in a pecan provenance collection using a combined bulked segregant analysis and genotyping by sequencing approach.�American Society of Plant Biologists (ASPB) 2017.�Honolulu, Hawaii June 24-28, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Alarcon, Y. et al. Genomic resources for pecan and pecan scab resistance.�National Pecan Scientists Meeting, Ardmore, OK.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Randall, J., Development of Genetic Tools for Pecan, American Society Horticulture Science 2017, Kona, HI September 18-22.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Randall, J., Cervantes, K., Ray, D., Fisk, J., Soneji, J., Sanchez, L., Grauke, LJ, Wang, X., Insights into the impact of geography and genetics on the microbiome of Carya illinoinensis, VIII International Symposium on Walnut, Cashew and Pecan, Santiago, Chile. November 29-December 2.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Randall, J., Rhein, H., Fisk, J., Barnes, S. Song, M., Thompson, M., Heerema, R.J., RNASeq analysis of genes involved in pistillate flower initiation in Carya illinoinensis, 2017, VIII International Symposium on Walnut, Cashew and Pecan, Santiago, Chile. November 29-December 2
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Grauke, LJ, Wang XW, Considerations related to the designation of a Core subset for Carya. 2017, VIII International Symposium on Walnut, Cashew and Pecan, Santiago, Chile. November 29-December 2.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Wang, XW, Grauke LJ., SNP discovery and SSR mining from Carya illinoinensis RAD sequences. 2017, VIII International Symposium on Walnut, Cashew and Pecan, Santiago, Chile. November 29-December 2.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Ishimori M, Fukami K, Cervantes K, Takanashi H, Nagano AJ, Kajiya-Kanegae H, Grauke LJ, Tsutsumi N, Randall J, Iwata H. Genome-wide RAD-Seq analysis revealed subpopulation structures of the pecan (Carya illinoinensis) germplasm collection and their relationship to geographical distribution patterns.2017. , VIII International Symposium on Walnut, Cashew and Pecan, Santiago, Chile. November 29-December 2.