Source: NEW MEXICO STATE UNIVERSITY submitted to NRP
TREES FOR THE FUTURE: COORDINATED DEVELOPMENT OF GENETIC RESOURCES AND TOOLS TO ACCELERATE BREEDING OF GEOGRAPHIC AND CLIMATE ADAPTED PECAN TREES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029191
Grant No.
2022-51181-38332
Cumulative Award Amt.
$6,017,917.00
Proposal No.
2022-05253
Multistate No.
(N/A)
Project Start Date
Sep 15, 2022
Project End Date
Sep 14, 2025
Grant Year
2024
Program Code
[SCRI]- Specialty Crop Research Initiative
Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239
Performing Department
(N/A)
Non Technical Summary
Pecan (Carya illinoinensis) is a large deciduous native North American tree grown commercially in 22 US states by about 25,000 pecan farmers for its delicious and highly nutritious nutmeats. Pecan orchards can continue to produce for 100 years or more and have great economic impact in many rural communities across the US. The genetic diversity of pecan is the foundational resource for breeders to improve nut production across the broad climatic range of pecan cultivation, which spans the desert southwest to humid southeastern USA. Each region poses distinct horticultural challenges that constrain pecan production, including alternate bearing, disease pressure, salinity and drought stress and these are all exacerbated by a changing climate. To address these constraints this collaborative project integrates inquiries into the molecular and physiological basis of high-value plant traits, tests for differential adaptation across environments, and ultimately identifies loci for genomic and marker-assisted selection frameworks. These data will allow the development of vital genetic tools necessary for increasing understanding of regional adaptation, promoting resource conservation, and selecting improved cultivars/rootstocks for all major pecan regions. We will specifically address key traits necessary for geographical adaptation of pecan such as timing of bud break, rootstock-scion interactions, salinity tolerance, microbiome composition, disease resistance, insect tolerance, and size control options. The scientific results from this effort will be used in a National Education Campaign to educate the public on the importance of plant genetics and changing climates using interactive media.
Animal Health Component
20%
Research Effort Categories
Basic
65%
Applied
20%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1021211106015%
1111211108010%
1321211108015%
2011211108015%
2021211108110%
2061211108010%
7011211101010%
9031211303015%
Knowledge Area
206 - Basic Plant Biology; 102 - Soil, Plant, Water, Nutrient Relationships; 111 - Conservation and Efficient Use of Water; 132 - Weather and Climate; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 903 - Communication, Education, and Information Delivery; 701 - Nutrient Composition of Food;

Subject Of Investigation
1211 - Pecan;

Field Of Science
1060 - Biology (whole systems); 1081 - Breeding; 1010 - Nutrition and metabolism; 1080 - Genetics; 3030 - Information and communication;
Goals / Objectives
1. Leveraging pecan genetics towards breeding pecan trees for climate adaptation.Historic adaptation to climates from southern Mexico to Illinois has shaped the genetic diversity of native pecan. We will generate molecular markers that predict suites of adaptive traits to accelerate breeding for both scion- and rootstock-related traits. Sub-Objective 1A. Generating a more complete database of pecan genomic diversity.Specifically, we will (1) generate a new genome assembly and annotation for the northern wild 'Colby' genotype, (2) update the 'Oaxaca' genome with HiFi sequencing to bring it in line with the state-of the art genomes, and (3) build ade novogenome annotation and assembly for the 'Success' cultivar, which is highly diverged from the core set of pecan genotypes.Sub-Objective 1B. Genetic mapping of genotype-by-climate interactions for phenology.Specifically,we will evaluate the climate dependence of phenological (bud-break, flowering where available, prevalence of vivipary, etc.) characters among a subset of the GWAS panel across pecan orchards in six states (CA, AZ, NM, OK, TX, and GA) using ground truth and time lapse cameras. Genetic mapping will be accomplished to explicitly model the genotype by environment interactions. The effect of local climate among years will be modeled. Finally, we will define sequences for marker assisted selection as large insertion-deletion polymorphisms proximate to QTL peak positions.Sub-Objective 1C. Elucidation of the molecular mechanisms of vivipary.Genetic networks that lead to differential shuck splitting and seed dormancy we seek to fill this knowledge gap and provide breeders with crucial molecular targets to breed vivipary-resistant cultivars.Sub-Objective 1D. Targeting climate matched wild relatives for pecan improvement.Targetedintrogressions fromclimate-or trait-matchedCarya specieswill be evaluated for their use for the could improvement of pecanproduction in novel regions. Species will be collected and grown at the USDA Pecan germplasm repository and subsequently sequenced.2.Describing physiological differences of water relations among pecan genotypes to improve water use efficiency.Water is a precious resource in all US pecan growing regions. We will probe the genetic basis of rootstock and scion water-use efficiency, and salinity tolerance to generate molecular targets for breeding under stressful environmental conditions.Sub-objective 2A. The interaction of pecan rootstock genotypes with scion.We will challenge existing and newly generated scion-rootstock combinations with water deficit in field conditions usingan existingmicro-sprinklersystem.This crossed experimental design will allow for explicit modeling of the physiological dynamics of interactions between scion and rootstock germplasm. Sub-objective 2B. The interaction of hickory rootstocks with pecan scions.We will evaluate water and nutrient relations in pecan scions as a function of rootstock species diversity of pecan cultivars on 12 Carya species. Nutrient status of the scion (to evaluate nutrient acquisition) and nut quality measures will be evaluated on these grafted trees and on the same cultivars on pecan rootstocks. Sub-objective 2C. Salinity tolerance pecan rootstock genotypes. Clonal Elliott rootstocks will be examined for their salinity tolerance. Sub-objective 2D. Characterizationof pecan Late Embryogenesis Abundant (LEA) proteins for putative drought tolerance.We will test the temporal dynamics of LEA gene families across a range of desiccation treatments to define and potentially target increased desiccation tolerant alleles.3.Characterizing interactions of pecan trees with beneficial and detrimental organisms for improved tree health, reduced pesticide application, and increased production.Diseases caused by fungal and bacterial organisms (as well as insect pests present enormous challenges to pecan growers. We will experimentally manipulate and evaluate host performance, microbiome diversity, and pest and pathogen damage to dissect the genetic basis of pecan yield in the face of these pressures.Sub-Objective 3A. Genetic diversity, quantitative and molecular genetic tests of the basis of resistance to pecan scab.Phenotypes of the mapping population trees will be characterized and inheritance of resistance to scab will be identified providing a basis for improved resistance breeding. Sub-Objective 3B.Climatic, environments, and management impacts on Pecan Bacterial Leaf Scorch caused byXylellafastidiosa.We willdetermine the presence of PBLS in different pecan-producing areas and correlateincidenceto climatic conditions. Management of PBLS by hedge pruning will also be evaluated.Sub-Objective 3C.Unraveling the spatial, temporal, and host genotype influences of the pecan microbiome. Theleaf, root,and rhizospheremicrobiomes of 'LxO' plantings in three different locations will be evaluated forcomposition and structurebased on location. Collection of phenotypic data and tissue nutrient concentrations on these plants will allow us to characterize associations of microbiome composition with plant nutrient uptake and with the potential for pecan tree adaptation to different/changing environmental conditions.4.Elucidating pecan gene networks and markers that control reproductive traits such as flowering, nut size, and kernel biochemical composition.We will further elucidate gene networks that control pecan phenology to mitigate the impacts of alternate bearing. We will also pursue the molecular mechanisms that influence nutrition and allergens associated with pecan nutmeats. Sub-Objective 4A. Characterization of kernel and nut morphological diversity. This project will develop marker-assisted selection tools allow pecan breeders to cull progeny with predicted poor nut qualities before maturity, simultaneously accelerating genetic gain and reducing the cost of pecan breeding programs. Sub-Objective 4B. Isoform regulatory networks underlying reproductive traits. Analyses of gene networks will determine if there are altered transcript isoforms, regardless of significant changes in total read counts, expectedly leading to new knowledge of gene regulation involved within genotypes.5. Leveraging pecan genetics for determining markers for pecan tree architecture and growth.Tree architecture and phenology represent two crucial determinants of orchard productivity. We will examine the genetic control of canopy size, density, and structure. Smaller tree canopies are desired, and we will evaluate pecans and otherCaryafor the genetics controlling tree vigor and height. Sub-Objective 5A. Tree architecture and size control for pecan. The native and experimental populations of pecan germplasm will be evaluated to determine trade-offs between true size control and lack of vigor. 'Cheyenne' and 'Cordele Dwarf' (unknown parentage) both have compact growth compared to other known pecan genotypes and are good candidates for size control. Our multivariate genetic mapping efforts will provide molecular targets for accelerated improvement of size control by leveraging beneficial alleles in these cultivars.
Project Methods
We will use trans-disciplinary approaches to continue to build and employ the genetic tools that the pecan industry recognizes as essential for their success. Our group includes the following expertise: plant genomics, plant molecular biology, plant tissue culture, plant pathology, pecan breeding, pecan horticulture, soil science, agricultural economics, statistics, computer science, and outreach/extension. We have a diverse advisory group. Specific methodology for this effort includes the following. 1. Genome sequencing. Genome assembly and annotation will follow our established protocols, which produced one of most complete outbred diploid plant genomes to date (Pawnee v1). Our genome assembly pipeline combines 25-30X/haplotype PacBio Hi-Fi reads to build contigs, 50X/haplotype HiC reads for chromosome integration, and sequence polishing from 60-80X 2x150bp Illumina reads. Our genome annotation pipeline combines 2x150 Illumina RNA-Seq reads from multiple tissues, a pooled PacBio ISO-Seq library, and homology support. 2. Genome wide association studies. We will conduct GWAS on a genetically dispersed subset of our existing diversity panel of nearly 507 genotypes. We will evaluate the climate dependence of phenological (bud-break, flowering where available, prevalence of vivipary, etc.) characters among a subset of the GWAS panel across pecan orchards in six states (CA, AZ, NM, OK, TX, and GA) using ground truth and time lapse cameras. Genetic mapping will be accomplished to model the genotype by environment interactions and to test how alleles differentially affect multiple traits and years and climates. 3. Vivipary studies. Phenotypic data will be mined from our existing databases USDA-ARS pecan databases on the genotypes that have full shuck split and those that do not. GWAS will be applied to shuck splitting rate. Replicated RNA-Seq experiments to determine genes driving the two likely processes that cause vivipary: shuck splitting and seed dormancy/germination. Differential expression patterns of each genotype will be statistically compared 4. Carya wild relatives sequencing and population collection. We will generate a single new de novo genome reference and annotation for two broadly distributed non-pecan Carya species that have hybridized and introgressed with cultivated pecan. We will establish range-wide collections of the four Carya species in a permanent orchard and phenotype juvenile growth traits. Polymorphisms will be analyzed. 5. The interaction of pecan rootstock genotypes with scion. We will challenge existing and newly generated scion-rootstock combinations with water deficit in field conditions using an existing micro sprinkler system. This experimental design will allow for explicit modeling of the physiological dynamics of interactions between scion and rootstock germplasm. 6. The interaction of hickory rootstocks with pecan scions. We will evaluate water and nutrient relations in pecan scions as a function of rootstock species diversity. Pecan cultivars 'Desirable' and 'Stuart' were grafted onto 12 Carya species in 1986. 7. Salinity tolerance pecan rootstock genotypes. Divergent genotypes from the five-year study will be micropropagated and evaluated for their salinity tolerance. Leaf tissue sodium and chloride, as well as other essential nutrients such as potassium, will be evaluated to model genotype-specific relationships of stress indicators to leaf concentrations. 8. Characterization of pecan Late Embryogenesis Abundant (LEA) proteins for putative drought tolerance. We will test the temporal dynamics of LEA gene families across a range of desiccation treatments to define and potentially target increased desiccation tolerant alleles using existing pecan transcriptome studies (and those developed under other objectives here) to determine the timing and tissue specific expression of pecan LEA proteins. 9. Genetic diversity, quantitative and molecular genetic tests of the basis of resistance to pecan scab. The QTL for scab resistance in the 'LxO' population grown in two sites (TX and GA) and the scab-segregating populations of 'Elliott' x 'VC1-68' planted in Somerville, TX and 'Pawnee' x 'Elliott' population in Tifton, GA will be mapped. Discriminant metabolites of resistant and susceptible reactions will be extracted and analyzed in order to find biomarkers that can differentiate host reactions to pathogenic and nonpathogenic scab isolates. Analyses will be performed using standard population genetic analysis packages. 10. Climatic, environments, and management impacts on Pecan Bacterial Leaf Scorch caused byXylellafastidiosa. The presence of PBLS in different pecan-producing areas (NM, AZ, CA, OK, TX, and GA) will be collected and incidence correlated to climatic conditions. The spatial distribution of XF within the pecan seed's cellular environment will be monitored using FISH. Hedge pruning will also be performed. 11. Unraveling the spatial, temporal, and host genotype influences of the pecan microbiome. The leaf, root, and rhizosphere microbiomes of 'LxO' plantings in three different locations will be evaluated for composition and structure based on location. Tree phenotypic and nutrient data will be collected. Total genomic DNA will be extracted from ('LxO') from 3 locations and sequenced using amplicon Illumina MiSeq technology for bacterial (16S rRNA gene) and fungal (ITS 1 gene) compositions. Data will be analyzed using the Qiagen CLC Microbial Genomics Module. 12. Elucidation of expanded physiological roles of pecan allergen proteins. Monoclonal antibodies specific for pecan allergens Car i 1 and 2 will be generated to evaluate translation, modification, accumulation and degradation of pecan allergens using Western blot assays. An assay will also be performed to determine if over-expression of these allergen genes have an anti-fungal or anti-aphid effect using a transient expression method in detached leaves. 13. Aphid resistance of pecan. Honeydew production by aphids will be rated on field-grown USDA Byron mapping population trees, in situ, using water-sensitive cards. We will determine aphid species composition by sampling and identifying all aphids from 10 compound leaves per tree. The data obtained will be used for QTL analysis on this mapping population. 14. Characterization of kernel and nut morphological diversity. We will integrate the multi-year phenotypic datasets with molecular data to map loci correlated with phenotypes in diversity panels (GWAS), make causal inference via linkage mapping, and accelerate selection via genomic prediction. We will also develop new methods to characterize nut and kernel morphology. The genetic loci discovered by these efforts will serve as factors in genomic prediction and marker assisted selection models. 15. Isoform regulatory networks underlying reproductive traits. We will use our statistical pipeline to test for differential isoforms first maps RNA-Seq reads across contrasting experimental conditions mapped to a reference genome. 16. Leveraging pecan genetics for determining markers for tree architecture and growth. Pecan trees on their own roots and Carya relatives located within the USDA Provenanceorchards will be measured for height and trunk diameter relative to the age of the tree and planting location including soil chemistry. These phenotypic and age comparisons will be integrated with new and existing molecular data to develop statistical associations better genetic markers and phenotypes including size and growth. In addition, the 32-year-old 'Ch x CD' mapping population planted at the USDA in Byron, GA will be evaluated to develop causal loci for marker assisted selection. The mapping population will be included in a resequence panel as will the 'Cordele Dwarf' tree.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:The target audience included different segments of the pecan industry including pecan growers, nursery people, and pecan processors. US Pecan growers and other pecan industry people were reached at grower meetings including Western Pecan Grower Association; Arizona Pecan Grower Meeting, Oklahoma Pecan Growers Association, Texas Pecan Growers Association, Northern Nut Growers Meeting, Tri-State Pecan Growers Meeting, Southeastern Pecan Growers Association (Alabama), North American Nut Growers Conference, and Georgia Pecan Growers Association. Information was disseminated in trade magazines such as The Pecan Grower and Pecan South that reaches large audiences within the pecan industry. The scientific community was also updated by peer-reviewed published scientific articles in scientific journals. The journals Plant Disease, Frontiers in Plant Science, HortScience, Journal of Forestry Research, BMC Genomics, and Food and Bioprocess Technology. In addition, the scientific community was also updated at National and International conferences including but not limited to the American Society of Horticulture Science; Plant and Animal Genomics Conference (PAG), International Society of Horticulture Science (International Symposium on Walnut and Pecan), and American Society of Phytopathology. The pecan website (pecantoolbox.nmsu.edu) was also updated to give relevant 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 provided opportunities for training of undergraduate and graduate students and professional development of scientists during the past year. Several undergraduate and graduate students were taught and assisted with data collection, data input, data analyses, grafting, and phenological data collection, pecan orchard management, and green house management. Undergraduate and graduate students were also trained in molecular techniques such as DNA extraction, RNA extraction, quantitative reverse transcriptase (QRT-PCR) PCR, gene synthesis design, downstream applications including primer and probe design, gene synthesis design, plant tissue culture propagation, genome analyses, genome annotation, RNA-Seq analyses, microbiome analyses. Graduate students and postdoctoral associates were trained to conduct chemical analysis (metabolomics) for studying pecan scab resistance and postharvest pecan color stability; they were also trained in using LC-MS for root exudate analyses, ICP analyses for zinc and nutrient analyses, and protein separations and identification. Graduate students and postdoctoral associates were also trained in horticulture practices, molecular biological/life science analysis, cloning, plant transformation, and plant tissue culture.Training also included software development and bioinformatic skills including genome comparisons, isoform analyses, RNA-Seq data analysis, annotations, statistical applications and R programming, and microbiome analysis. Undergraduate, Graduate Students, and Post-doctoral associates presented their research at local, regional, national, and international meetings. Specific trainings for scientists included but were not limited to PI and grant management, workshops on mentoring undergraduate, graduate students, and post-docs. How have the results been disseminated to communities of interest?Dissemination of grant objectives and results were accomplished using several avenues during this grant year. Presentations included oral presentations, poster presentations, video presentations, trade journal articles, press releases, and peer reviewed publications. The pecan research website (pecantoolbox.nmsu.edu) allows users to know the latest research information by linking them to specific articles, websites, information, genetic tools, pecan phenotypic data, videos, and genomic data. Pecan research results for this grant were disseminated at pecan grower meetings as well as national and international conferences that specifically target the pecan industry and include growers, nurserymen, shellers, and other processors. The pecan grower conferences included Western Pecan Growers Association, Oklahoma Pecan Growers Association, Tri-state pecan growers association, Arizona Pecan Growers association, and Northern Nut Growers Association. Scientific conferences included the Plant and Animal Genomics Conference (International), The American Society of Horticultural Sciences Annual Conference, American Phytopathological Society (APS) Plant Health Conference, International Symposium on Walnut and Pecan, and the National association of plant breeders. In addition, articles were published in trade magazines targeting information dissemination to the grower community. These included the West Coast Nut Magazine, Ivanhoe Sol and Exeter Sun Gazette, the Pecan Grower, and Pecan South Magazine. Peer-reviewed manuscripts of research findings were submitted and published in various scientific journals. These included: Plant Disease, Frontiers in Plant Science, HortScience, Journal of Forestry Research, BMC Genomics, and Food and Bioprocess Technology. The 'Trees for the future' USDA advisory board met for its annual meeting in April 2024 online. A presentation was given on the current research updates and the advisory board gave feedback on current and future research plans. What do you plan to do during the next reporting period to accomplish the goals?The newly constructed pecan genomes will have completed annotations and will be available through Phytozome and the pecantoolbox.nmsu.edu. In addition, a pangenome construction will take place for the new and existing genomes. Candidate gene discovery including the presence and absence of genes will be analyzed from the different genotypes. The genomes will be used for gene discovery to determine what controls traits of interest. Carya species will be sequenced and used for mapping hickory introgressions in pecan. These resources are essential for developing marker assisted breeding in pecans aimed at climate adaptation. Phenotypic data for pecan trees throughout the geographic span of the USA will continue to be collected and analyzed. Following analyses from the 2024 data collection it will be determined if protocols for phenotypic collection need to be further optimized to ensure that collection is standardized. Cameras will be deployed in each location, as was done in Arizona, for further refinement of phenotypic collection. Analyses of the 'L×O' mapping population phenotypic data collected in 2024 will be completed and further phenotypic data will be collected in Spring 2025. This will allow for fine genetic mapping to explicitly model the genotype by environment interactions and to test how alleles differentially affect multiple traits across years and varying climates. The effect of local climate and geography over the years will be modeled within a repeated measure mixed effect logistic regression. Finally, we will define polymorphisms that can be used for marker assisted selection. Vivipary analyses will continue with the collection of green shucks from three different time points from 'Pawnee' (known susceptibility to vivipary) and 'Sioux' (rare vivipary) and RNA will be extracted for RNA-Seq analyses. RNA-Seq will be completed for the RNA from shuck tissue and from the seed treatments. To avoid potential mapping biases reads will be mapped and counted against annotated 'Oaxaca' genome which is genetically equidistant to both 'Sioux' and 'Pawnee'. Differential expression patterns of each genotype will be statistically compared through linear repeated-measures models and co-expression networks. The sampling of native Carya will be pursued and adjusted for year 3 sampling across the midwestern region of the USA. Work will continue to look at water relations between rootstock and scion of pecan trees. A water deficit study is in progress with the grafted clonal pecan trees. Gas exchange and water use efficiency will be measured. Tissue from the scion material will also be collected from this study, RNA will be extracted and used for RNA-Seq analyses. Comparisons will be made between the control conditions and the water deficit study and between the different rootstocks and scion combinations. Clonal pecan rootstock trees will be used in a greenhouse salinity study followed by a field salinity study. In addition, RNA-Seq analyses will be performed to evaluate/validate genes involved in salinity tolerance. Additionally, assays on drought and high salt media will be performed with the E.coli engineered with selected LEA pecan genes. In addition, phenotypic data will continue to be collected for the hickory rootstock/pecan scion trees. Phenotypic data on scab severity, and insect populations among the pecan provenance collections and the 'L×O' mapping population will continue. This data will be used to evaluate resistance genes for scab and aphids to be utilized in future breeding efforts. Further analyses will be performed analyzing the potential QTL on Chromosome 5 that was implicated in scab resistance. Continued experimentation on PBLSsymptom development of pecan bacterial leaf scorch due to the interaction of Xylella fastidiosa and environmental conditions will continue as will histology locating PBLS during seed development. Microbiome analyses will continue and MiSeq will be completed for the 'L×O' mapping populations located in TX, and two locations in GA. Additionally, microbiome analyses will be completed to determine the changes that occur between potted plants and when they are planted in an orchard setting. The role that pecan allergen proteins such as Car i 1 and Car i 2 play in scab resistance and insect resistance will continue. Additionally, genes that code for pollen allergens will be further studied using RNA-Seq and qRTPCR. Integration of automation for phenotyping nutshell color and measurements will continue. Isoform analyses will be continued for several gene networks including flowering. The genes involved in pecan reproduction including the genes and splice variants involved in pollen germination will continue. Analyses of RNA-Seq expression and isoform analyses will be completed. In addition, ZIP expression for zinc uptake will be measured and compared between tissue culture shoots vs roots. Analyses are in progress to determine if micropropagated tissues zinc channels work the same way in soil based potted plants. This will also be translated to the field grown pecans. Collected tissue from the mother trees of the 'Cordele dwarf' will also be introduced into tissue culture for micropropagation to be utilized in further experiments. The 'Cheyenne' x 'Cordele Dwarf' nuts from crosses will be stratified and introduced into tissue culture. Trees from the mapping population will continue to be propagated in tissue culture. The 'Cheyenne' x 'Cordele Dwarf' mapping population will continue to be evaluated and measurements taken for further characterization. These trees will continue to be micropropagated to create several clones for further analyses. In addition to the science, outreach efforts will continue with trade articles and a social media campaign targeting the US pecan industry. Videos will be recorded and available on the pecantoolbox.nmsu.edu to explain peer reviewed papers. Models mapping climate, genetics, and phenotypic traits will be built. Educational modules on genetics and climate will begin to be generated that will target the general audience along with K-12 and undergraduate students.

Impacts
What was accomplished under these goals? The second year of the grant focused on coordinated experimentation and data collection between the collaborating institutions and stake holders. Objective 1 Genomic sequencing using the PacBio-HiFi platform was completed for select pecan cultivars and genomes were assembled (87Mx3-2.11 ('Oaxaca'), 'Lakota', 'Elliott', 'Schley', and 'Success'). RNA-Seq analyses is in progress and annotation of these genomes will follow. The new genomes will be accessible on Phytozome and the pecantoolbox.nmsu.edu. Phenotypic descriptors were refined for collection and analyses foryear 2 collection. These descriptors include bud break timing, flowering, disease(s) progression, and vivipary. Refined phenotypic data was collected from multiple pecan orchards in CA, NM, AZ, OK, TX, and GA. Cameras were deployed in four orchards in AZ on three different cultivars. Pictures were captured every 15 minutes to allow for phenotypic observations through the canopies. The phenotypic and climate data from the Spring 2024 collection is currently being analyzed. These data will be used to develop a simulation where genetics, climate, and growth parameters are modeled. Progress was made in elucidating the genetic networks that control germination and dormancy. Seeds obtained from 'Sioux' and 'Pawnee' cultivars were subjected to the following treatments 1. Dormant, 2. Stratified, 3. Germinated. Seed tissues were collected following each treatment and RNA was extracted. QRT-PCR was performedfor specific genes involved in germination anddifferences in gene expression in dormant seeds vs germinated seeds was observed.Objective 2. Water use efficiency experimentson grafted pecan clonal rootstock trials at NMSU. These trees were used to measure gas exchange, including intrinsic water use efficiency. Microscopy was conducted to compare anatomy/morphology of leaf surface and mesophyll. Leaf tissues were analyzed for carbon isotope (C13) discrimination which is another indicator of intrinsic water use efficiency. Tissues from the scion materials were collected andwill be used for RNA-Seq. A water deficit study is in progress with these trees. Water use efficiency and gene expression will be measured during water deficit conditions. Pecan cultivars 'Desirable' and 'Stuart' were grafted on 12 species of hickory at USDA (SEFTNRS), Byron,GA to establish graft compatibility. Agronomic and horticultural traits were evaluated for year 2. Measurements collected included tree height, trunk diameter, nut load, yield, and nut quality characteristics. The impact of different species of hickory rootstock and their influence on tree yield and nut quality of the two common pecan cultivars will be determined. The study will provide information on whether non-pecan hickories may be beneficial rootstocks to improve pecan production. Salinity tolerance for pecan rootstock is important for pecan trees in the Western US. Elliot trees from the salinity study in Safford, AZ and open-pollinated seedlings were successfully micropropagated. A salinity assay was conducted at various time points (24 hours, 48 hours, 1 week, and 1 month) at concentrations (0 mM, 25 mM, 50 mM, 60 mM, and 75 mM NaCl2) under tissue culture conditions. Preliminary gene expression profiling was initiated on plants subjected to salinity stress. These clonal plants are currently being acclimatized in a greenhouse for further assays. In-silico analyses of the four reference pecan genomes identified 332 LEA genes that are important for abiotic stresses such as drought. These were categorized into sub-groups based on their characteristic domains and mapped onto chromosomes. An in-silico analysis was performed to determine tissue-specific expression patterns of LEA genes in catkins, roots, swelling buds, leaves, pistillate flowers, and dormant buds. Primers for select LEA genes were developed for further expression profiling using quantitative PCR (qPCR). Selected LEA genes were also introduced into an E.coli expression vector and expression analyses are in progress. Objective 3. Pecan scab (caused by V. effuse) is a costly and limiting factor for production in the Eastern US. Multiple avenues of experimentation are being evaluated to find resistance in pecan. The provenance orchard (300 pecan trees) and the mapping populations ('L×O; 'P×E'; ) were evaluated for scab severity. QTL analysis of the 'P×E' mapping population was performed for scab resistance and a major QTL for resistance was mapped to chromosome 5 of Elliott. A metabolomics platform for pecan scab resistance research was developed and scab resistance and scab susceptible samples were analyzed. This included primary and secondary metabolites, that covered a wide range of plant growth and defense signaling and mechanisms. Our new standard area diagram for assessing leaf scab severity in pecans was published and will be used across the diverse regions of pecan cultivation by both scientists and growers alike. PBLS symptoms were evaluated in CA, AZ, NM, OK, TX, and GA. A simple diagram was made to assist researchers/lay persons in determining PBLS symptoms and percent incidence. An experiment looking at the effects of heat and drought stress of pecan seedlings infected with Xylella fastidiosa was performed and there was a significant effect of high heat on PBLS symptom expression. In addition, experiments are underway to determine the impact of hedging pecan trees with PBLS. Microbiome analyses is in progress. Rhizoplane, rhizosphere, and leaf samples for microbiome analyses of the 'LxO' mapping populations at three orchard locations were collected as were clonal trees that were in potting soil and planted in an orchard. Samples are being processed for MiSeq analyses. A fungal survey was conducted in pecan orchard soilsacross the western US and a manuscript was submitted for peer-reviewed publication. All trees in the 'LxO' population in Byron, GA, and the Provenance collection were evaluated during year 2 for aphid species abundance, pecan phylloxera, leaf miner species, and beneficial insects. Objectives 4 and 5. Pecan nut shape is highly heritable, which will enable improvement through breeding. Nut samples were collected from the 'Pawnee' x 'Elliot' and 'Elliot' x 'VC168'and digitally analyzed for nut shape in preparation for QTL analysis. Pathway-based metabolomics with machine learning algorithms were employed to identify key metabolites of postharvest pecan color stability. Machine learning models were used and compared to find relevant biomarkers of pecan color phenotypes The results were submitted for publication and disseminated to stakeholders. RNA splicing patterns in several different gene networks including flowering and nutrient channels have been analyzed. We found several genes that present significant alternative splicing patterns associated with dichogamy genotypes. This study provides evidence for the prevalence of alternative RNA splicing in the transcription regulation of pecan dichogamy. Genes that are involved in zinc uptake were investigated. Gene expression levels of zinc regulated transport-like (ZIP-IRT) from pecan were measured in 'Wichita' during different zinc treatments. A mapping population of 'Cheyenne' x 'Cordele Dwarf' planted in Byron, GA in 1989 to study traits related to size control continued to be evaluated. Height and caliper measurements were conducted during year 2 on these trees to evaluate the heritability of reduced tree size and the effectiveness of seedling selection on mature tree size. Efforts are underway to increase the population by performing additional controlled crosses. This population was introduced into tissue culture and micropropagation of the population is ongoing for further evaluation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Hilton A, Wang X, Jo YK, Conner P, Randall J, Chatwin W, Bock C. Standard Area Diagrams for Pecan Leaf Scab: Effect of Rater Experience, Location, and Leaf Size on Reliability and Accuracy of Visual Estimates. Plant Dis. 2024 Jun;108(6):1820-1832. doi: 10.1094/PDIS-09-23-1947-RE. Epub 2024 May 31. PMID: 38277651.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Ogundipe, S.O., Usack, J.G., Pegg, R.B., Suh, J.H., 2024. Thermal and non-thermal processing on the physical and chemical properties of tree nuts: A review. Food and Bioprocess Technology, in press, https://doi.org/10.1007/s11947-023-03314-8
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Bhattarai, G., Conner, P., Randall, J.J., Shadgou Rhein, H., 2024. Transcriptome analysis under pecan scab infection reveals the molecular mechanisms of the defense response in pecan. Acta Horticulturae, accepted.
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Wang, X., Kubenka, K., Hilton, A., Chatwin, W., Pisani, C., Bock, C., Heerema, R.J., Tondre, B., Randall, J.J., 2024. Influences of rootstocks on pecan scion growth in a replicated test orchard. Acta Horticulturae, accepted.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Hilton, A., Wang, X., Young-Ki, J., Conner, P., Randall, J.J., Chatwin, W., Bock, C.H. 2024. Development and validation of a standard area diagram set to aid the assessment of severity estimates of pecan leaflet scab and the effects of evaluator experience and location, and leaf size Plant Disease https://doi.org/10.1094/PDIS-09-23-1947-RE.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Kaur A., Zhang L, Maness N., Ferguson L., Graham C., Sun Y., Panta S., Pokhrel N., Yang M., Moss J. 2024. Dormant carbohydrate reserves enhance pecan tree spring freeze tolerance: controlled environment observation. Frontiers in Plant Science. 15:1393305. https://doi.org/10.3389/fpls.2024.1393305
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Zhang L., Kaur A., Sun Y., Ferguson L. 2023. Do non-structural carbohydrates contribute to pecan (Carya illinoinensis) secondary budburst? HortScience. 58(10):11921193. https://doi.org/10.21273/HORTSCI17335-23 ho
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Kaur A., Zhang L., Yang M., Maness N., Graham C.J., Kumari R., Sun Y., Panta S., Ferguson L. 2023. Evaluation of natural spring freeze tolerance of five pecan scionrootstock combinations. HortScience. 58(10):11391148. https://doi.org/10.21273/HORTSCI17258-23
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Pisani, C., Bock, C.H, and Randall J. 2023. Visual rating and the use of image analysis for assessing canopy density in a pecan provenance collection during leaf fall. Journal of Forestry Research, 34(6): 1843-1854.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Brungardt J, Alarcon Y, Shiller J, Young C, Monteros MJ, Randall JJ, Bock CH. 2024. Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection. BMC Genomics: 5;25(1):180. doi: 10.1186/s12864-024-10010-0
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Cervantes, K., Hereema, R.J., Randall, J.J, 2024, Microbiome observations of fully-watered and deficit-watered micropropagated rootstock pecan trees. Acta Horticulturae, accepted.
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Kang, M.J., Pegg, R.B., Kerr, W.L., Wells, L., Conner, P.J., Suh, J.H., 2024. Metabolomic analysis combined with machine learning algorithms enables the evaluation of postharvest pecan color stability. Food Chemistry, under revision.
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Stock, M.L., Heerema, R.J., Randall, J.J., Pietrasiak, 2024. Preliminary survey of fungal diversity in pecan orchards of the Southwestern USA. Acta Horticulturae.
  • Type: Journal Articles Status: Other Year Published: 2024 Citation: Hilton, A., Bock, C. and Chen, J. (2024). A Genome Resource for Xylella fastidiosa subsp. multiplex Strain Causing Pecan Bacterial Leaf Scorch in the Southeastern United States. Phytopathology. In Preparation.
  • Type: Other Status: Published Year Published: 2024 Citation: Fichtner, E.J., Bhandari, S., Randall, J., Heerema, R. 2024. Walnut and Pecans: Related Crops with Different Bud Break Bloom Habits. West Coast Nut Magazine, UCCE In a Nutshell Newsletter, UCCE Blot
  • Type: Other Status: Published Year Published: 2024 Citation: Fichtner, E. 2024. Pecans: The native nut produced in Tulare County. Ivanhoe Sol and Exeter Sun Gazette. Published in English and Spanish
  • Type: Other Status: Published Year Published: 2024 Citation: Zhang L. 2024. Exploring Pecan Secondary Buds as a Spring Freeze Crop Recovery Method. Pecan South. 57(1): 2426.
  • Type: Other Status: Published Year Published: 2024 Citation: Angelyn Hilton, Warren Chatwin, and Xinwang Wang. Selecting the Perfect Pecan. Pecan South Magazine. July 2023. https://www.pecansouthmagazine.com.
  • Type: Other Status: Published Year Published: 2024 Citation: Stock, M. 2024. From Mushrooms to Microbes: How Fungi Fine-Tune Orchard Health. Pecan South 57(4): 6-12. [Trade Magazine]
  • Type: Other Status: Published Year Published: 2024 Citation: Angelyn Hilton. Meet the New USDA-ARS Plant Pathologist. Pecan South Magazine. January 2024. Meet the New USDA-ARS Plant Pathologist - Pecan South magazine
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sahithi Reddy Pulicherla, Chris Mattison, Doris Alexa Arnedo, Jennifer Randall. (2024) "Unraveling the role of LEA(Late embryogenesis abundant) genes in pecan stress resilience." American Society of Horticultural Sciences 2024 Annual Conference. (Oral presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Anna Harmon, Jennifer Randall "Nothing to Sneeze at: Elucidating the Role of Allergen Genes in Carya illinoinensis" - Presentation (Research & Creativity Week) New Mexico State University Feb 29, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Anna Harmon, Hormat Shagdou Rhein Jennifer Randall "Elucidating the Role of Allergen Genes in Pecan" - Poster (ACES Open House) NMSU. April 6, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sahithi Pulicherla, Jennifer Randall(2023) "Characterization of Late embryogenesis abundant (LEA) genes in Pecan (Carya illinoinensis)". Graduate research and arts symposium, NMSU. (Poster presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sahithi Pulicherla, Jennifer Randall (2024) "Role of late embryogenesis abundant (LEA) genes in stress regulation of pecan (Carya illinoinensis)". Research and creativity week, NMSU. (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sahithi Pulicherla, Jennifer Randall (2024) "Characterization of Late embryogenesis abundant (LEA) genes in Pecan (Carya illinoinensis)". ACES open house, NMSU. (Poster presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Wang, X., Kubenka, K., Hilton, A., Tondre, B., Chatwin, W., Pisani, C., Bock, C., Heerema, R. (Presenter), Randall, J. J., International Symposium on Walnut and Pecan, International Society for Horticultural Science, Grenoble, France, "Influences of rootstocks on pecan scion growth in a replicated test orchard", Scope: International, published in proceedings, Invited or Accepted? Accepted. (June 13, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stock, M. (Presenter), Heerema, R., Randall, J. J., Pietrasiak, N., International Symposium on Walnut and Pecan, International Society for Horticultural Science, Grenoble, France, "Surveying fungal diversity in pecan orchards of the Southwestern USA", Scope: International, published in proceedings, Invited or Accepted? Accepted. (June 14, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Heerema, R. (Presenter), Arizona Pecan Growers Conference, Arizona Pecan Growers Association, Tucson, AZ, "Getting to the root of pecan production", Scope: Regional. (August 25, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Bracamontes, S. (Presenter), R. Heerema, Western Pecan Growers Association Conference and Tradeshow, Las Cruces, NM, In a Nutshell: The Inner Workings of a Pecan Leaf (March 4, 2024)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Heerema, R. (Presenter), Stock, M., Randall, J. J., Pietrasiak, N., New Mexico Pecan Growers Association Conference, NMPGA, Las Cruces, NM, "Beneficial fungi on and around pecan roots", Scope: State. (October 27, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Heerema, R. (Presenter), Simposio Internacional de Nogal Pecanero, INIFAP, Hermosillo, Sonora, Mexico, "Getting to the root of pecan production", Scope: International, published in proceedings. (September 14, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stock, M. (Presenter), Heerema, R., Randall, J. J., Pietrasiak, N., Western Pecan Growers September Webinar, Western Pecan Growers Association, Zoom (presented live), "Soil Fungi in Pecan Orchards", Scope: Regional. (September 12, 2023).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jennifer Randall The Half Circle of Life! The genetics involved in the development of flowers, pollen, and nuts in pecan trees'. The International Plant and Animal Genomics Conference, San Diego, January 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Marcus,M., Andazola,A., Cervantes,K., Harmon,A., Heerema, R.J., and Randall, J.J., 2023. The impact of water restriction on pollen viability and pollen tube formation of the pecan cultivar 'Pawnee'. ASHS annual conference, Orlando, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dena,F., Harmon,A., Andazola,A., Cervantes, K., Madison, M.,Heerema,R.J., Belteton,S., Randall,J.J., 2023. Pollen characteristics between protandrous and protogynous pecan cultivars grown in New Mexico.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jennifer Randall, Trees for the future Western Pecan webinar November 14, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paul Gabriel, Andra Andazola, Jennifer Randall 'Nutrient Uptake Channels/Genes of Pecans Allow Crosstalk Between Pecan and Its Immediate Environment.' 58th Annual Western Pecan Growers Association Conference, March 3-5th, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paul Gabriel, Andra Andazola, Jennifer Randall 'Nutrient Uptake Channels/Genes of Pecans Allow Crosstalk between Pecan and Its Immediate Environment.' American Society for Horticultural Science, August 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Madison Marcus, Andra Andazola, Kimberly Cervantes, Anna Harmon, Richard J. Heerema, Jennifer J. Randall (2024) Does water deficit conditions impact pollen viability and pollen tube formation of the pecan cultivar Pawnee? Research AND Creativity Week, NMSU, Las Cruces, NM.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jennifer Randall(Presenter) USDA NIFA Project Director Training.Interdisciplinary: Coordinated development of genetic tools for pecans, March 9, 2023.


Progress 09/15/22 to 09/14/23

Outputs
Target Audience:The target audience included different segments of the pecan industry including pecan growers, nursery people, and pecan processors. US Pecan growers and other pecan industry people were reached at grower meetings including Western Pecan Grower Association; Arizona Pecan Grower Meeting, Oklahoma Pecan Growers Association, Texas Pecan Growers Association, Northern Nut Growers Meeting, Tri-State Pecan Growers Meeting, Southeastern Pecan Growers Association (Alabama), North American Nut Growers Conference, and Georgia Pecan Growers Association. Information was disseminated in trade magazines such as The Pecan Grower and Pecan South that reaches large audiences within the pecan industry. The scientific community was also updated by peer reviewed published scientific articles in scientific journals. The journals included PLOS One, BMC Genomics, Plants, Frontiers, Journal of Agriculture and Food Chemistry, Food and Bioprocess Technology, Journal of Forestry Research. In addition, the scientific community was also updated at National and International conferences including but not limited to the American Society of Horticulture Science; Plant and Animal Genomics Conference (PAG), International Society of Horticulture Science (International Symposium on Walnut and Pecan), and American Society of Phytopathology. The pecan website (pecantoolbox.nmsu.edu) was also updated to give relevant 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 provided opportunities for training of undergraduate and graduate students and professional development of scientists during the past year. Several undergraduate students were trained in several disciplines including bioinformatics, plant sciences, plant biology, and economics. This training specifically included 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, gene synthesis design, and quantitative reverse transcriptase (RT-PCR) analysis, and Western Analysis. 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 gene comparisons, 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 rRNA microbiome and 18S rRNA microbiome analyses. Undergraduate, Graduate Students, and Post-doctoral associates presented their research at both local, regional, national, and international meetings. Specific trainings for scientists included but were not limited to PI and grant management, workshops on mentoring undergraduate, graduate students, and post-docs. Trainings also included the use of cameras and digital means for phenotype data collection. How have the results been disseminated to communities of interest?Dissemination of grant objectives and results were accomplished using several avenues during this grant year. Presentations included oral presentations, poster presentations, trade journal articles, press releases, and peer reviewed publications. The pecan research website (pecantoolbox.nmsu.edu) allows users to know the latest research information by linking them to specific articles, websites, information, genetic tools, pecan phenotypic data, videos, and genomic data. Pecan research plans for this grant and results were disseminated at pecan grower meetings that specifically target the pecan industry and include growers, nurserymen, shellers, and other processors. These included the Arizona Pecan Grower Association, Georgia Pecan Grower Association, Oklahoma Pecan Grower Association, Texas Pecan Grower Association, Northern Nut grower meeting, Southeastern Pecan Grower Association, and Western Pecan Grower Association. In addition, a few articles were published in trade magazines for future communication with the pecan industry and scientific community. These included both The Pecan Grower by Georgia Pecan and Pecan South. The 'Trees for the future' USDA advisory board was established in Fall of 2022 and the board met for their first meeting in March 2023. The advisory board received a copy of the 'Trees of the future' grant and timeline along with a presentation to answer questions and to discuss approaches. Peer reviewed manuscripts of research findings were submitted and published in a variety of scientific journals. These included: Euphytica, Plants, Journal of Agricultural and Food Chemistry, Food and Bioprocess Technology, Journal of Forestry Research, BMC Genomics, and PLOS Biology. Results were communicated at National and International scientific conferences. These Included, the American Society of Horticulture Science, American Society of Phytopathology, Plant and Animal Genomics Conference (PAG), and International Science of Horticulture for Walnuts and Pecans (Grenoble France Summer 2023). What do you plan to do during the next reporting period to accomplish the goals?High molecular weight DNA extraction will be completed for the pecan cultivars and ten wild Carya species. This DNA will be sequenced using the HudsonAlpha pipeline and RNA-Seq will be completed for genome annotations. Further optimization and annotation of existing resequencing data will occur. Continued phenotypic analyses from orchards in CA, AZ, NM, OK, TX, and GA will occur for bud break timing, flowering, vivipary, and diseases (scab and pecan bacterial leaf scorch). These phenotypic analyses will be combined to evaluate phenotypes and bud break timing through the U.S. on several pecan cultivars. Phenotypic analysis will also continue in the 'L x O' mapping populations in GA and Texas. Vivipary analyses including RNA-seq will be continued to determine genes that are involved in germination. Collection of pecans in shuck material will be collected in late Fall for further analyses. Grafted clonal rootstock trees located at NMSU will be further studied for their water use efficiency and the role the rootstock and their genetics play on this composite tree. Wild Carya seed collections will begin in Year 2 in the Southeastern United States for establishment in the USDA Pecan Repository in Texas. Phenotypic data will continue to be collected on the hickory rootstock with pecan scion trees to further determine the impact of rootstock on pecan scions. The surviving trees from the Safford, Arizonasalinity study will continue to be micropropagated in tissue culture for further analyses. The role of LEA genes in drought tolerance will continue to be evaluated in pecan tissues with and without drought conditions. The role of plant health with pathogens and insects on pecans will continue. Phenotypic observations for scab will continue along with culturing scab isolates from both Texas and Georgia. We will conduct chemical analysis (metabolomics) for the collected pecan scab-related samples to find biomarkers for pecan scab resistance. There will be repeat assessments of all trees in the L x O mapping population for scab severity and phenotypic traits. To explore interactions between pathogen diversity and host genotype/geographic source, scab populations in the 19 pecan provenances in the provenance collection will be sampled and subject to population genetic analysis. Scab resistance QTLs will be evaluated in the 'Pawnee' x 'Elliot' mapping population using both field and controlled environment resistance data. Final analysis of the scab resistance transcriptome experiment will be completed and published. Xylella experiments will continue with determining the impact of growing conditions on symptomology in addition to localization of XF in nut tissues. Microbiome samples will be taken in the 'LxO' orchards and samples extracted for NGS. Repeat assessments of all trees in the 'L x O' mapping population and pecan provenance collection for aphid abundance. Continued development and analysis of anti-pecan allergen antibodies, native and recombinant pecan allergen purification, and characterization of pecan allergens and the effects of processing on pecan allergens and their impact on insect and fungal pests. Nut shape and composition analyses will continue. Image analysis methods will continue to be developed and refined. We plan to use our high throughput digital nut morphology phenotyping pipeline to begin analyzing nuts after they are harvested this coming fall. GWAS analysis of nut shape in the provenance collection will be completed and it will be determined if suitable molecular marker data is obtained. Also, metabolomic analyses and color will be continued. We will continue to highlight alternative spliced genes using the 'Pawnee' reference genome and expand the effort to new RNA-seq data to be generated on the project. Analyses of genes involved in pollen tube germination and pistillate flower fertilization with pollen will continue. The 'Cheyenne' x 'Cordele Dwarf' mapping population will continue to be evaluated and measurements taken for further characterization. These trees will continue to be micropropagated to create several clones for further analyses. In addition to the science, outreach efforts will continue with trade articles targeting the US pecan industry. Development of pecan budgets that can be used to explore the economic value of developing disease-resistant pecan varieties (vivipary, scab, etc.). Social media will also be an avenue for communicating with the general pecan industry about research progress. Videos for growers will be generated from published scientific papers by the authors and uploaded onto the 'pecantoolbox' for further dissemination of the research. Educational modules on genetics and climate will begin to be generated that will target the general audience along with K-12 and undergraduate students.

Impacts
What was accomplished under these goals? The first year of the grant was focused on coordination between the institutions and stake holders across the United States in several aspects. Phenotypic descriptors were collected and agreed upon and field orchard management was synchronized for specific orchards to allow for data collection with limited variables. Objective 1. Tissue collections for genome sequencing for three pecan cultivars and ten wild Carya species was accomplished for high molecular weight DNA extraction. RNA was extracted from collected tissues (leaves, swelling buds, dormant buds, flowers) for RNA-Seq analyses for annotation purposes. Phenotypic descriptors were developed for bud break timing, flowering, disease(s) progression, and vivipary. Phenotypic data was collected from multiple orchards in CA, NM, OK, TX, and GA. Climate stations for each orchard site was listed so that climate between geographical areas can be ascertained. Orchard maintenance and phenotypic collection also occurred for the mapping population 'Lakota' x 'Oaxaca' ('L x O') that is available in three different locations (Sommerville, TX; Byron, GA; and Tifton, GA). In addition, image analysis-based methods were compared to physical measurements. These included tree heights, trunk diameters, canopy width, bud break timing, flowering timing, leaf nutrient status, leaf scab, and leaf measurements . These data are the basis to begin environment x genotype studies, to provide an understanding of inheritance of different phenotypic traits. The different climates in TX and GA provide a population for screening of horticultural traits. The study on vivipary began with seed collection and RNA extraction of seeds at different stages of germination for RNA-Seq analysis. Specific genes were evaluated for their expression in dormant and germinated seeds. Objective 2. Water use efficiency experiments in NM began by maintaining clonal rootstock trials previously planted at NMSU and grafting scions to these trees. Tree measurements including photosynthesis were collected. The cultivars Desirable and Stuart were grafted on 12 species of hickory at the Southeastern Fruit and Tree Nut Research Station (SEFTNRS), Byron, Georgia in 1986 to establish graft compatibility. Agronomic and horticultural traits were assessed on these pecan trees grafted onto hickory rootstocks. Measurements collected included tree height, trunk diameter, nut load, yield, and nut quality characteristics. The aim now is to determine whether different species of hickory rootstock influences tree yield and nut quality of the two common pecan cultivars. The study will provide information on whether non-pecan hickories may be beneficial rootstocks to improve pecan production. Salinity tolerance for pecan rootstock is important for pecan trees in the Western US. Graft wood from trees in a previous salinity study in Safford AZ were collected and grafted onto potted trees in NM. Several of these trees were introduced into tissue culture for propagation. Additionally, approximately 50 Elliot seeds were germinated and introduced into tissue culture for salinity evaluation. The presence of LEA genes were evaluated through the four complete pecan genomes to determine if they play a role in drought tolerance. Objective 3. Pecan scab (caused by V. effuse) is a costly and limiting factor for production in the Eastern US. Multiple avenues of experimentation are being evaluated to find resistance in pecan. The 'LxO' mapping population was assessed for scab severity. Leaf and nut scab ratings were collected. This data will be used for QTL mapping. 'Oaxaca' nuts exhibiting scab were sent to the University of Georgia for isolation for the casual V. effuse strain. Pecan tissue was also collected for metabolomics analysis for scab resistance. Seeds from nine cultivars that tested positive for Xylella fastidiosa (XF), causal agent for pecan bacterial leaf scorch, were harvested in 2022 and propagated in the greenhouse. XF diagnostics using qPCR are in progress to determine transmission rates and identify XF-positive seedlings. The seedlings will then be moved into growth chambers to continue the experiment. Developing nutlets from two cultivars, including 'Cape Fear', were sampled throughout the season for Fluorescent In-Situ Hybridization (FISH) to detect XF in developing nuts. Microbiome analyses will be accomplished for the 'L x O' mapping populations that are in TX and GA. Orchard conditions between the three orchards were synchronized and soil nutrient data was collected for the trees. The Car i 1 and Car i 2 proteins in pecan were identified as main allergens in nuts. Further analyses of these genes are on-going to determine if they have a role against insect or fungal pests. Protein from these genes were purified and these genes were synthesized and placed into a binary vector for further analyses in plants. Insect pests including aphids impedes pecan production. All trees in the 'LxO' population in Byron, GA and the Provenance collection were evaluated for aphid species abundance, pecan phylloxera, leaf miner species, and beneficial insects. Objective 4. Nut samples from different mapping populations were collected for shape and quality analyses. Collected nut samples from 2022 ('Elliot' x 'VC168' population) were analyzed for in-shell measurements using physical and image analyses based techniques. Batch processing of kernel morphological traits like dorsal groove width using machine learning methods was explored and are in progress. Variability between kernels from the same tree is high, causing difficulties in the automated detection of kernel grooves. In addition, nuts were collected for metabolomics to look at pecan color stability and kernel biochemical composition. Gene alternate splicing was evaluated across pecan cultivars and tissue types. Preliminary work on isoforms was accomplished with flowering RNA-seq datasets. In addition, pistillate flowers were pollinated on a 'Western' tree with 'Wichita' pollen and collected over a time course for RNA-Seq experiments. A mapping population of 'Cheyenne' x 'Cordele Dwarf' was created and planted in Byron, GA in 1989 to study traits related to size control. Leaves and budwood were collected from these hybrids as well as pollen from the 'Cordele Dwarf' mother tree and sent to collaborators at NMSU where micropropagation will take place and propagate for a rootstock trial in future studies. Height and caliper measurements were conducted on eight of these trees to evaluate the heritability of reduced tree size and effectiveness of seedling selection on mature tree size. Budwood from 'Cheyenne x Cordele Dwarf' was grafted onto potted trees at NMSU and these grafts have and will continue to be introduced into tissue culture for better evaluation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lovell, J. Integrating comparative and quantitative genetics for crop improvement, North American Plant Breeders Conf. (Greenville, SC)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lillywhite, J.M. A changing market environment: Potential impacts for pecans. Western Pecan Growers Association 2023 Conference. Las Cruces, NM. March 8, 2023.
  • Type: Journal Articles Status: Published Year Published: 2023 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. 2023. Comparative transcriptome analyses reveal insights into catkin bloom patterns in pecan protogynous and protandrous cultivars, PLOS Biology, https://doi.org/10.1371/journal.pone.0281805
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Wang, X., Kubenka, K., Hilton, A., Chatwin, W., Pisani, C., Bock, C., Heerema, R.J., Tondre, B., Randall, J.J., 2023. Influences of rootstocks on pecan scion growth in a replicated test orchard. Acta Horticulturae, submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Stock, M. Finding Fungi: A study on the Fungal Communities in Pecan Orchard Soils in the Southwestern U.S. Western Pecan Growers Association 2023 Conference. Las Cruces, NM. March 8, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatwin, W. & Hilton, A. Picturing Pecans: Detecting and Predicting Pecan Tree Characteristics Western Pecan Growers Association 2023 Conference. Las Cruces, NM. March 8, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Bhattarai, G., Conner, P., Randall, J.J., Shadgou Rhein, H., 2023. Transcriptome analysis under pecan scab infection reveals the molecular mechanisms of the defense response in pecan. Plant and Animal Genomic Conference, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lovell, J. Integrating comparative and quantitative genetics for crop improvement, AGBT-AG (San Antonio, TX), 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Chatwin, W. (Invited, Author & Oral Presenter). "The USDA Pecan Breeding Program: Past, Present, and Future". Oklahoma Pecan Grower's September Seminar, Sapulpa, OK (October 22nd, 2022)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatwin, W., Udall, J. (Author & Poster Presenter). Two Phased Chromosome-Scale Assemblies of Pecan (Carya illinoinensis) Using Hi-C and PacBio HiFi Reads, PAG 30. Scherago International, San Diego, CA. (January 16th, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dena, F.J. , Harmon, A., Andazola, A., Cervantes, K., Marcus, M., Heerema, R.J., Belteton, S., and Randall, J.J. 2023. American Society of Horticulture Sciences Annual Conference, Orlando, FL, August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Bhattarai, G. and P. Conner. 2023. Understanding the genetics of scab resistance in pecan. Georgia Pecan Growers Assoc. Ann. Conf. March 29. Perry, Ga.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Chatwin, W., Hilton, A. (Invited, Author & Oral Presenter). "Picturing Pecans: Detecting and Predicting Pecan Tree Characteristics", 2023 Western Pecan Growers Association Conference and Trade Show, Las Cruces, NM (March 7th, 2022)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatwin, W. (Invited, Author & Oral Presenter). "Picturing Pecans: New Cultivar Releases and Modernizing Pecan Breeding at the USDA", Oklahoma Pecan Grower's Association Annual Convention & Trade Show, Tulsa, OK (June 2nd, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatwin, W., Hilton, A., Wang, X. (Invited, Author & Oral Presenter). Picturing Pecans: New Cultivars & Modernizing Pecan Breeding, Plant Breeding at USDA-ARS: Outstanding in the Field, Cornell University, Ithaca, NY (June 27th, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatwin, W. (Invited, Author & Oral Presenter). "Picturing Pecans: New Cultivar Releases and Modernizing the USDA ARS Pecan Breeding Program", Northern Nut Grower's Association Conference, Columbia, MO (July 25th, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Hilton, A. (Oral Presenter). The Modes of Transmission of Bacterial Pathogen, Xylella fastidiosa in Pecan (Carya illinoinensis), Entomological Society of America Southeastern Branch Meeting, Little Rock, AR (March 14th, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Hilton A., Bock, C., Wang, X., Jo, YK., Conner, P., Randall, J., Chatwin, W. (Author & Poster Presenter). The development and validation of a standard area diagram to rate severity of pecan leaflet scab, and the effect of rater experience, Plant Health 2023 Conference, Denver, CO (August 15th, 2023)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Conner, P. 2023. Genetics and breeding work in the UGA pecan breeding program. Georgia Pecan Growers Assoc. Research Committee Meeting. Feb. 2. Perry, Ga.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Marcus, M., Andazola, A., Cervantes, K., Harmon, A., Heerema, R.J., Randall, J.J., 2023. The Impact of Water Restriction on Pollen Viability and Pollen Tube Formation of the Pecan Cultivar Pawnee. American Society of Horticulture Sciences. Orlando, FL August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Wang, X., Stein, L., Black, M., Chatwin, W., Hilton, A., Kubenka, K., Klein, P., Randall, J.J., Grauke, L.J., 2023. GWAS of A Diverse Open-pollinated Pecan (Carya illinoinensis) Population, Plant and Animal Genomic Conference, San Diego, CA, 2023.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Brungardt, J., Alarcon, Y., Shiller, J., Young, C., Monteros, M.J., Randall, J.J., and Bock, C.H., 2023. Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection, BMC genomics, submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Randall, J., 2023. Invited to present Moving Forward with Pecan Genetics', Southeastern Pecan Growers Association, Alabama, February 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Zhang L. 2023. 'Bud break, soil nutrient, and mycorrhizal fungi in Great Buffalo Pecan Farm' 2023 Annual Convention & Trade Show, Oklahoma Pecan Growers Association
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Mattison, C. American Chemical Society, National Fall Meeting, 2022, poster presentation 'Heating Induces Chemical Modification and Variable Solubility of Pecan Allergens', Chicago IL, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Dupree, R. & Mattison, C. (Oral presentation) 'Mass spectrometric analysis of allergens from pecan protein extracts: influence of pecan heating on observable peptides and modifications', Indianapolis, IN American Chemical Society, National Fall Meeting, 2022
  • 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: Submitted Year Published: 2023 Citation: Bhattarai, G., Conner, P., Randall, J.J., Shadgou Rhein, H., 2023. Transcriptome analysis under pecan scab infection reveals the molecular mechanisms of the defense response in pecan. Acta Horticulturae, submitted.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Cervantes, K., Hereema, R.J., Randall, J., 2023, Microbiome observations of fully-watered and deficit-watered micropropagated rootstock pecan trees. Acta Horticulturae, submitted.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Cervantes, K. Velasco-Cruz, C., Grauke, L.J., Wang, X., Conner, P., Wells, L., Bock, C.H., Pisani, C., Randall, J.J., 2023. Influence of Geographical Orchard Location on the Microbiome from the Progeny of a Pecan Controlled Cross. Plants. 12(2):360.doi: 10.3390/plants12020360.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Clermont, K., Graham, C.J., Lloyd, S.W., Grimm, C.C., Randall, J.J., Mattison, C.P., 2023. Proteomic analysis of pecan (Carya illinoinensis) nut development, Journal of Agricultural and Food Chemistry, Foods. 2023 Feb; 12(4): 866; 10.3390/foods12040866.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Kumari R., Zhang L., Wang X., Maness N., and Pokhrel N. 2023. Influence of Non-Structural Carbohydrates on Dwarfing in Different Pecan Genotypes. American Society Horticultural Science (ASHS) annual meeting. July 30August 4. Orlando, FL.
  • Type: Other Status: Published Year Published: 2023 Citation: Lillywhite, J.M. Cycles and Seasonal Patterns in Pecan Prices. Pecan South Magazine. July 1, 2023.
  • Type: Other Status: Published Year Published: 2023 Citation: Lillywhite, J.M. Growing & Diversifying Pecan Production. Pecan South Magazine. March 1, 2023. https://www.pecansouthmagazine.com/pecan-south-magazine/?q=&y=&c=&a=340
  • Type: Other Status: Published Year Published: 2022 Citation: Lillywhite, J.M. The Connection Between Supply, Demand, and Pecan Prices. Pecan South Magazine. October 1, 2022. https://www.pecansouthmagazine.com/magazine/issue/2022-10/
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Ogundipe, S.O., Usack, J.G., Pegg, R.B., Suh, J.H., 2023. The effects of thermal and non-thermal processing on the physical and chemical properties of tree nuts: A review, Food and Bioprocess Technology, submitted.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Pisani, C., Randall, J.J., and Bock, C. 2023. Visual Rating and the Use of Image Analysis for Assessing Canopy Foliage Density in a Pecan Provenance Collection During Leaf Fall, Journal of Forestry. Research,.https://doi.org/10.1007/s11676-023-01635-0
  • Type: Other Status: Published Year Published: 2023 Citation: Randall, J.J., Mason, K., Fichtner, E.J. 2023. Trees of the Future Developing geographic and climate adapted pecan trees. The Pecan Grower.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2023 Citation: Scott, M.L., Heerema, R.J., Randall, J.J., Pietrasiak, 2023. Preliminary survey of fungal diversity in pecan orchards of the Southwestern USA. Acta Horticulturae submitted.