GENOMIC BREEDING OF BLACKBERRY FOR IMPROVED FIRMNESS AND POSTHARVEST QUALITY

GENOMIC BREEDING OF BLACKBERRY FOR IMPROVED FIRMNESS AND POSTHARVEST QUALITY

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1018578

Grant No.

2019-67013-29196

Cumulative Award Amt.

$430,000.00

Proposal No.

2018-06274

Multistate No.

(N/A)

Project Start Date

Apr 15, 2019

Project End Date

Apr 14, 2025

Grant Year

2019

Program Code

[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Horticulture

Non Technical Summary
The U.S. fresh-market blackberry industry has experienced rapid growth and change during the past 15 years driven by increased consumer demand, new cultivars, advanced production methods, and year-round product availability. The entire berry industry is expanding, and blackberries in particular have been driving this growth with an increase in sales of 14.7% between 2015 and 2016. Fresh-market blackberries accounted for $549 million in sales during 2016 and are currently the fourth-most important domestic berry crop, behind strawberries, blueberries, and raspberries (CA Strawberry Commission, 2017).Despite recent growth, the blackberry industry continues to be plagued by short shelf life and loss of fruit quality during shipping. Blackberries are considered one of the most difficult fruits to ship due to their susceptibility to many conditions that reduce marketability, shelf life, and fruit quality including decay, juice leakage, berry softening, and weight. Red drupelet reversion is a particularly problematic postharvest physiological disorder affecting fresh market blackberries. Blackberries harvested when completely black and full ripe often develop red discoloration after they are returned to room temperature following cold storage. This discoloration limits fruit marketability as consumers perceive it as unattractive. Furthermore, excessive reversion can result in rejection of whole shipments of berries as lots of blackberries with greater than 10% of fruit lacking full black coloration are not considered permissible by USDA marketing standards (USDA-AMS 2016). Red drupelet reversion is likely a form of chilling injury experienced as rapid temperature changes weaken cell walls and membranes and the anthocyanins that give berries their black color are degraded in a less acidic environment.The University of Arkansas System Division of Agriculture (UA) is home to the largest public-sector fresh-market blackberry breeding program in the US. Since its inception in 1964, the UA blackberry breeding program has publicly released 16 cultivars that have transformed the industry. Two major achievements have been the development of erect, thornless cultivars and primocane-fruiting cultivars that produce a fall crop on first year canes. The improvement of fruit firmness is an important objective in the UA blackberry breeding program, as firm fruit generally perform better for many postharvest quality parameters. Significant improvements in firmness have been made through classical breeding, with substantial variation in firmness observed among UA selections and cultivars. A dramatic advance in firmness was achieved with the discovery of a very distinct 'crispy' texture in a thorny, floricane fruiting selection. Crispy selections are considerably more firm than other UA breeding selections and cultivars at harvest and after seven days of cold storage and experience significantly less red drupelet reversion. Crispy-textured blackberries have the potential to increase the profitability of the US blackberry industry because of their superior postharvest performance and resistance to red drupelet reversion. However, the underlying biology of crispy texture is not understood and the trait has been difficult to recover in crosses between crispy and non-crispy selections in the UA breeding program. Genes and enzymes associated with cell wall metabolism and softening during ripening in other fruits are well understood and diagnostic markers associated with firmness have been identified and implemented in other fruit crops including apple and peach. The development of genetic markers for crispy texture in blackberry would greatly expedite breeding for this trait and facilitate the stacking of genes for crispy texture, thornlessness, and primocane fruiting in elite cultivars. The implementation of molecular breeding in blackberry has been delayed by complications including polyploidy, multisomic inheritance, and heterozygosity. However, recent advances in computational tools for polyploids and development of genomic resources for related species have removed many of these barriers.Our long-term goal is to develop new crispy blackberry cultivars with enhanced firmness and superior postharvest performance. The primary objective of this proposal is to implement marker-assisted selection for texture in the UA fruit breeding program to reduce breeding cycle time and expedite the process of combining crispy texture with other desirable traits. Based on preliminary results in blackberry and studies in other fruit crops, we hypothesize that variation in fruit firmness in fresh-market blackberries is determined by mutations in a few gene(s) related to ripening and cell walls. The specific objectives of our proposal are:Objective 1. Measure fruit firmness and red drupelet reversion in a panel of 400 breeding selections from the UA blackberry breeding program for fruit firmness and red drupelet reversion and genotype the panel to identify molecular markers for firmness and red drupelet reversion.Objective 2. Study the biology behind softening during ripening in blackberries with soft, firm, and crispy texture by measuring the cell wall components at different stages of fruit development.Objective 3. Develop a mapping population segregating for crispy texture that will be evaluated for texture and red drupelet reversion and genotyped to identify molecular markers for firmness and red drupelet reversion.The novel crispy blackberry phenotype results from a naturally occurring genetic mutation (non-GMO) and is likely to be deployed in farmers' fields, potentially extending the shelf life of fresh-market blackberries and reducing losses from red drupelet reversion. This proposal will have substantial impact for the US blackberry industry because molecular markers developed in this project will be implemented in applied breeding programs and will expedite the development of elite crispy cultivars.

Animal Health Component

70%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1129	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1129 - Berries and cane fruits, general/other;

Field Of Science
1081 - Breeding;

Keywords

blackberry

rubus

marker assisted selection

postharvest

red drupelet reversion

texture

Goals / Objectives
Our long-term goal is to develop new crispy blackberry cultivars with enhanced firmness and superior postharvest performance. The primary objective of this proposal is to implement marker-assisted selection for texture in the UA fruit breeding program to reduce breeding cycle time and expedite the process of combining crispy texture with other desirable traits. Based on preliminary results in blackberry and studies in other Rosaceae fruit crops, we hypothesize that variation in fruit firmness in fresh-market blackberries is determined by mutations in a few gene(s) related to polysaccharide-modifying enzymes. We further hypothesize that crispy texture may be recessively inherited, with four copies of a non-functional allele at a gene coding for a polysaccharide-modifying enzyme, such as polygalactonurase (PG), necessary to recover crispy texture. Allele dosage at this gene may contribute other quantitative variation in flesh firmness seen in the UA blackberry breeding program. We will leverage the new tools we have developed including two diploid reference genomes, a tetraploid blackberry resequencing panel, and a high throughput phenotyping protocol for red drupelet reversion to test these hypotheses and understand the underlying genetics controlling texture variation in cultivated blackberries.

Project Methods
Objective 1. Genome-wide association mapping of fruit firmness and red drupelet reversion in blackberry selections from the UA breeding program. 1.1 Germplasm and harvest. The GWAS panel will consist of approximately 400 UA blackberry breeding selections and cultivars selected between 1967 and 2017. The cultivars and selections are maintained in 6 m plots at the UA Fruit Research Station in Clarksville, AR. The panel will be evaluated for two years (2019-2020) for firmness and red drupelet reversion. Two half pint clamshells per genotype will be filled with fruit harvested at the shiny-black stage of ripeness and free of defects. Each genotype will be harvested on two separate dates, approximately one week apart. Fruit will be stored for seven days in cold storage at 5 ºC prior to evaluations.1.2 Red drupelet reversion and image analysis. After seven days of cold storage, fruit will be removed from cold storage and returned to room temperature. Fruit will be arranged in a single layer on a green plastic cutting board and photographed in photo box. Digital images will be analyzed in ImageJ with color thresholds (CIEL*a*b*) set to measure the proportion of red drupelet area to total berry area.1.3 Firmness measurement. Following image analysis, five randomly selected berries per clamshell will be weighed and assessed for firmness using a Stable Micro Systems TA.XT.plus Texture Analyzer (Texture Technologies Corporation, Hamilton, MA). Fruit compression will be performed by placing individual berries horizontally on a flat surface using a cylindrical plane probe of 7.6 cm diameter at a rate of 2 mm/s with a trigger force of 0.02 Newtons (N).1.4 Genotyping with Capture-Seq. The GWAS panel will be genotyped with RAPiD Genomics Capture-Seq technology. We will use previously generated resequencing data from a panel of 24 diverse blackberry cultivars and selections to design 20,000 probes distributed across the blackberry genome. Probes will be designed to capture polymorphisms within genes that have been implicated in cell wall metabolism in other fruits such as polygalactonurase, pectinmethylesterase, pectin lyase, and expansin. DNA libraries will be prepared and enriched using the targeted probe set in the RAPiD Genomics automated laboratory and sequenced with Illumina HiSeq to achieve at least 80x coverage per marker on average.1.5 Data analysis. We will require a minimal read depth of 60x and a genotyping quality (GQ) scored of at least 26 to assign a genotype call. This will result in nulliplex (aaaa), simplex (aaab), duplex (aabb), triplex (abbb), and quadruplex (bbbb) genotype calls relative to the diploid reference sequence. Genome wide association analysis will be performed in GWASpoly.Objective 2. Measure the temporal sequence of cell wall disassembly events and changes in gene expression in developing blackberries with soft, firm, and crispy texture.2.1 Germplasm and sample collection. Three genotypes chosen to represent the range of textures present in UA blackberry breeding germplasm will be harvested from two replicate plots for this study during 2019 and 2020. Fruit from each replicate plot will be harvested at five different growth stages: large green, full red, 50% black, full shiny black (ripe), and dull black (overripe). Ten berries from each sample will be immediately flash-frozen in liquid nitrogen and transported to the laboratory for storage at -80 °C for gene expression studies. The remaining harvested fruit will be transported to the laboratory and processed immediately. Ten berries per genotype, replication, and growth stage will be assessed for firmness (see section 1.3)2.3 Isolation and fractionation of cell walls. Approximately 20 g of tissue (exocarp and mesocarp) for each developmental stage will be powdered while frozen and homogenized in 100 mL of ice-cold 80% ethanol using a Polyton homogenizer. Insoluble material will be washed with ice-cold 80% ethanol, stirred in TRIS-buffered phenol, precipitated with ethanol, washed with 95% ethanol, stirred in chloroform:methanol (1:1, v/v), and washed with acetone. Aliquots (100 mg) of acetone-insoluble cell walls will be sequentially extracted with the chelating agent trans-1,2-diaminocyclohexaneN,N,N9,N9-tetraacetic acid (CDTA) (for 24 h, twice) and with Na2CO3 containing 0.1% NaBH4 (24 h, twice), to produce extracts enriched in ionically bound and covalently bound pectin, respectively.2.4 Size exclusion chromatography. Na2CO3 extracts will be neutralized with glacial acetic acid, and then CDTA and Na2CO3 extracts will be dialyzed extensively against deionized water. After dialysis, uronic acid and neutral sugar contents will be quantified. Volumes containing 1 mg uronic acid will be freeze-dried and redissolved in 1.6 mL of 0.4 mg ml-1 imidazole then neutralized by the addition of 0.4 mL of 1 M ammonium acetate, pH 5.0. Samples will be chromatographed on a column and extracts will be dialyzed extensively against deionized water, then uronic acid and neutral sugar contents will be quantified as above.2.5 Candidate gene expression analyses. Total RNA will be extracted from flash frozen berries. Candidate gene(s) identified in objective 1 based on their involvement with fruit cell wall modification in other fruits and/or significant association with firmness and/or red drupelet reversion in GWAS analysis will be selected for gene expression analysis. PCR primers and probes for real-time quantitative PCR (RT-qPCR) will be designed for candidate and reference genes and tested in silico for polymorphisms in annealing regions or potential cross reactions to other gene sequences. RT-qPCR will be performed and repeated measures analysis of variance will be used to test for significant changes in gene expression across fruit development stages in the three blackberry genotypes representing the range of fruit textures.Objective 3. QTL mapping fruit firmness and red drupelet reversion in a biparental population segregating for crispy texture.3.1 Population development and micropropagation. The UA breeding selection A-2652T was derived from a cross between the non-crispy advanced selection A-2428T and the crispy parent A-2453T. We propose to backcross A-2652T to its crispy parent, A-2453T, during spring 2019. Three clonal replicates of the parents and approximately 200 progeny will be established in the field in a randomized complete block design with three-foot spacing between plants in spring 2020.3.2 Phenotyping the biparental mapping population for texture and red drupelet reversion. Ripe fruit at the shiny black stage will be harvested from each plot and phenotyped for firmness and red drupelet reversion (see sections 1.2 and c.1.3) during June 2021 and 2022. The Pearson correlation coefficient between firmness and red drupelet reversion and the broad sense heritability of each trait will be calculated in SAS 9.4.3.3 Genotyping with Capture Seq. Young leaf tissue from the progeny and parents will be collected and extracted during spring 2020. The population will then be genotyped with the Capture-Seq probe set (see section 1.4).3.4 Development of linkage map and QTL mapping. Bioinformatics and genotype calling will be performed as described in section 1.5. A dense linkage map of the population will be constructed using TetraploidSNPMap (Hackett et al., 2017). QTL mapping for firmness and red drupelet reversion will also be conducted in TetraploidSNPMap. Significant QTL will then be tested with simple models, including simplex (Aaaa x aaaa), duplex (AAaa x aaaa), and double simplex (Aaaa x Aaaa), to test additive and dominant effects and determine the best fitting model following Hackett et al. (2014).

Progress 04/15/23 to 04/14/24

Outputs
Target Audience:Raspberry and blackberry research community: Results from research supported by this grant were presented in three posters and one oral presentation at the International Society for Horticultural Sciences Rubus and Ribes Symposium in July 2023. Research supported by this grant was also presented in an invited oral presentation in the Fruit and Nut section in Plant Animal Genome conference in 2024and in an a presentation at the American Society for Horticultural Science meeting in 2023. Private and public sector blackberry breeders: the UADA Fruit Breeding Program regularly hosts blackberry growers and shipper/marketers from around the world during the growing season in June and July. We also shared results through informal discussions with representatives of breeding and shipper/marketer companies at industry meetings like the North American Raspberry and Blackberry Association conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PhD student Alexander Silva and undergraduate student Isabella Vaughn have been conducting research related to flowering in blackberries using the datasets generated in this project. How have the results been disseminated to communities of interest?Results from research supported by this grant were presented in three posters and one oral presentation at the International Society for Horticultural Sciences Rubus and Ribes Symposium in July 2023. Research supported by this grant was also presented in an invited oral presentation in the Fruit and Nut section in Plant Animal Genome conference in 2024and in an a presentation at the American Society for Horticultural Science meeting in 2023. We also shared results through informal discussions with representatives of breeding and shipper/marketer companies during their visits to our field site during June and July and at industry meetings like the North American Raspberry and Blackberry Association conference. What do you plan to do during the next reporting period to accomplish the goals?During the next cycle we intend to focus on preparing manuscripts for publication and developing genomic selection models to predict low- and medium-heritabilty traits in blackberry. We intend to submit manuscripts describing GWAS for sweetness and acidity, plant height, thorns, and primocane-fruiting during the next reporting period. We also intend to submit a paper describing RNA-seq analysis related to differentially expressed genes in soft and crispy fruited blackberries. We are also finalizing the functional annotation of the R. ulmifolious reference genome and we intend toconduct comparative analysis of the R. ulmifolius and R. argutus genomes to explore potential candidate genes forprimocane-fruiting and thornlessness. We intend to continue developing better datasets on fruit firmness and red drupelet reversion and to use these phenotypic and genotypic datasets to train genomic selection models that target the improvement of postharvest quality during the next project cycle in collaboration with PhD student Alexander Silva.

Impacts
What was accomplished under these goals? During this cycle we published an article in Frontiers in Plant Sciencedescribing image analysissoftware developed to quantify red drupelet reversion in blackberry titled 'ShinyFruit: interactive fruit phenotyping software and its application in blackberry.' We also continued work on gene expression and enzyme activity in soft and 'crispy'-fruited blackberries to identify potential candidate genes.Results from this study suggest that a PG gene (Ra_g27485) and a PME inhibitor gene (Ra_g888) may be associated with crispy texture as they were differentially expressed between the studied cultivars and were located in significant peak regions identified in the GWAS study. The genotype data generated for this project has also been leveraged to identify QTL and candidate genes associated with flowering time, annual fruiting, and fruit quality traits.

Publications

Type: Journal Articles Status: Published Year Published: 2023 Citation: Chizk, T.M., J.A. Lee, J.R. Clark, and M.L. Worthington. 2023. ShinyFruit: interactive fruit phenotyping software and its application in blackberry. Front. Plant Sci. 14: 1182819. doi: 10.3389/fpls.2023.1182819
Type: Journal Articles Status: Published Year Published: 2024 Citation: Worthington, M., Chizk, T.M., Johns, C.A., Nelson, L.D., Silva, A., Godwin, C. and Clark, J.R. (2024). Advances in molecular breeding of blackberries in the Arkansas fruit breeding program. Acta Hortic. 138: 85-92. doi: 10.17660/ActaHortic.2024.1388.12
Type: Journal Articles Status: Published Year Published: 2023 Citation: Vaughn, I., A. Silva, C. Johns, L. Nelson, and M. Worthington. 2023. Validation of a diagnostic marker for primocane-fruiting in blackberry. Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences. 24: 60-65. https://scholarworks.uark.edu/discoverymag/vol24/iss1/13/
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Johns C, M Worthington, Chizk TM, Silva A, Nelson L, Hardigan M, Thompson E, Clark J. 2023. Association analysis and marker development for prickle-free blackberry canes. XIII International Rubus and Ribes Symposium. Poster Presentation. July 20, 2023. Portland Oregon
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Worthington M, Chizk TM, Wang YJ, Clark JR. 2023. The potential role of polygalacturonase and pectin methylesterase in blackberry fruit softening patterns. XIII International Rubus and Ribes Symposium. Poster Presentation. July 20, 2023. Portland Oregon
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Vaughn, I., A. Silva, C. Johns, L. Nelson, and M. Worthington. 2023. Validation of a diagnostic marker for primocane-fruiting in blackberry. HortScience 58(9) Supplement 2023 ASHS Annual Conference. S102
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Silva, Alexander, L. Nelson, C. Johns, E. Thompson, M. Hardigan, J.R. Clark, M. Worthington. Genetic control of primocane fruiting in tetraploid blackberry. https://pag.confex.com/pag/31/meetingapp.cgi/Paper/53780

Progress 04/15/22 to 04/14/23

Outputs
Target Audience:Rosaceae genomics community: The Rosaceae genomics community benefited from the publication and release of a new reference genome for Hillquist blackberry (R. argutus), which is available on theGenome Database for Rosaceae (https://www.rosaceae.org/Analysis/13328362) under the accession number tfGDR1056. We also made important progess on the annotation of a second blackberry genome and thegenetics of fruit firmness and red drupelet reversion as well as other major traits segregating inthe UA blackberry breeding program (i.e. thornlessness, plant architecture, primocane fruiting) during this reporting period that will be disseminated tothe broader research community during the next reporting cycle. Fruit breeders: We reached fruit breeders and the Rosaceae research community throughpresentations atthe annual meeting of the Toolsfor Polyploids SCRI project. We also shared results through informal discussions withrepresentatives Pairwise Plants, Hortifrut, Pacific Berry Breeding, Edward Vinson LTD, Driscolls, Mountain Blue, and others. We worked with partners at Hortifrut and the USDA-ARS breeding program to validate new genetic markers for primocane fruiting and thornlessness during this cycle and intend to publish these results and share them with the breeding community at Plant Animal Genome and the International Society for Horticultural Science Rubus Ribes meeting during the next cycle. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student (Mason Chizk) and one MS student (Carmen Johns) successfully defended their graduate work related to this project during the previous reporting period. Dr. Chizk is now employed as a consulting scientist with AbacusBio in Edinburgh Scotland and Ms. Johns has been promoted to Assistant Fruit Breeder in the Arkansas Fruit Breeding Program. A third graduate student working on this project (Carly Godwin) successfully defended her thesis and graduated with her MS in December 2021. Ms. Godwinis now employed as an assistant tomato breeder with Plenty in Laramie, WY. Alexander Silva is continuing his work as a doctoral student on this project. He has conducted research on the genetic control of primocane fruiting and phenological traits in blackberry and will begin developing genomic selection models based on his datasets and those created by Dr. Chizk, Ms. Johns, and Ms. Godwin during the next project cycle. Undergraduate studentIsabella Vaughn also assisted the graduate student team working with this project and conducted an independent research project validating a new KASP marker for primocane fruiting in a segregating biparental population. Dr. Chizk was given the University of Arkansas Dept. Horticulture Outstanding PhD student award and the J. Moore Plant Breeding Fellowship during the 2022/23 academic year. Ms. Johnswas also chosen as the University of Arkansas Dept. Horticulture Outstanding MS student award during the past cycle. How have the results been disseminated to communities of interest?Results from this project were disseminated through exit seminars and theses/dissertations from Ms. Johns and Dr. Chizk during this reporting cycle. Dr. Worthington described the preliminary results of this project in quarterly meetings of the USDA-SCRI tools for polyploids project. Dr. Chizk also presented a poster with his preliminary results at the National Association of Plant Breeders meeting.Results have also been disseminated to key stakeholders through informal discussions and visits with other breeders, growers, and shipper/marketers at the University of Arkansas System Division of Agriculture Fruit Research Station. Lastly, we have shared results throught two open-access publications (one published and one under review).We anticipate that the 2023/2024 cycle will yieldseveral open-access peer reviewed publications and presentations at the International Society for Horticultural Science Rubus Ribes, American Society for Horticultural Science,and Plant Animal Genome conferences. What do you plan to do during the next reporting period to accomplish the goals?During the next cycle we intend to focus on preparing manuscripts for publication and developing genomic selection models to predict low- and medium-heritabilty traits in blackberry. We intend to submit manuscripts describing GWAS for sweetness and acidity, plant height, thorns, and primocane-fruiting during the next reporting period. We also intend to submit a paper describing the ShinyFruit phenotyping app and RNA-seq analysis related to differentially expressed genes in soft and crispy-fruited blackberries. We are also finalizing the functional annotation of theR. ulmifoliousreference genome and we intend to conduct comparative analysis of theR. ulmifoliusandR. argutusgenomes to explore potential candidate genes for primocane-fruiting and thornlessness.We intend to continue developing better datasets on fruit firmness and red drupelet reversion and to use these phenotypic and genotypic datasetsto train genomic selection models that target the improvement of postharvest quality during the next project cycle in collaboration with PhD student Alexander Silva.

Impacts
What was accomplished under these goals? The first major accomplishment of the previous reporting period was the publication and release of the newRubus argutusreference genome assembly and annotation. This assembly and annotation enabled us to develop a custom Capture-Seq probe set that resulted in65,995 polymorphic SNPs with an average read depth of216 across 300 genotypes used in our GWAS study for fruit firmness and red drupelet reversion. The genome also allowed us to identify plausible candidate genes near significant marker-trait associations with functions related to cell wall dissassembly and to conduct RNA-seq analyses with firm and soft berries at green, red, and black developmental stages. The new genome assembly and annotation are now publicly available to the broader research community at the Genome Database for Rosaceae. PhD student Mason Chizk completed his dissertation research on thegenetic control of red drupelet reversion and fruit firmness in blackberry during this period. He successfully defended his dissertation in February 2023 and submitted a paper to Frontiers in Plant Sciences describing major results from his GWAS analysis in March 2023.In this study we performedGWAS of fruit firmness and RDR measured in 300 tetraploid fresh-market blackberry genotypes from 2019-2021 with 65,995 SNPs concentrated in genic regions of the R. argutus reference genome.Three variants on homologs of polygalacturonase (PG), pectin methylesterase (PME), and glucan endo-1,3-β-glucosidase explained 27% of variance in fruit firmness and were located on chromosomes Ra06, Ra01, and Ra02, respectively. Another PG homolog variant on chromosome Ra02 explained 8% of variance in RDR, but it was in strong linkage disequilibrium with 212 other RDR-associated SNPs across a 23 Mb region. A large cluster of six PME and PME inhibitor homologs was located near the fruit firmness quantitative trait locus (QTL) identified on Ra01. Although we orginally anticipated that fruit firmness was controlled by mutations in just a few genes, our work demonstrates that postharvest quality traits in blackberryare likely controlled by many small-effect QTLs. We intend to continue developing better datasets on fruit firmness and red drupelet reversion and to use these phenotypic and genotypic datasetsto train genomic selection models that target the improvement of postharvest quality during the next project cycle in collaboration with PhD student Alexander Silva. Dr. Chizk also completed analyses related to validation of ShinyFruit, a user-friendlyShinyApp forfor image-based phenotyping of size, shape, and color-related qualities in fruits and vegetables. He also conducted a studylayering data from RNA sequencing, enzyme activity, and fruit softening patterns over three stages of berry development to investigate the potential roles of pectin methylesterase (PME) and polygalacturonase (PG) in mediating differential softening patterns between the soft fruited 'Black GemTM' and crispy-fruited A-2453T. Results from this study suggest that a PG gene (Ra_g27485) and a PMEinhibitor gene (Ra_g888) may be associated with crispy texture as they were differentially expressed between the studied cultivars and were located in significant peak regions identified in the GWAS study. We intend to submit both these publications during the next project period. In addition, MS studentCarmen Johns defended her thesis on the genetic control of primocane height and thorn density using genotype data generated using funds from this project. We intend to submit her work and the thesis work from graduated MS student Carly Godwin on genetic control of sweetness and acidity during the next project reporting period. PhD student Alexander Silva has joined our team and generated data on flowering time and primocane fruiting for GWAS analyses. He has also worked with our team to develop and validate diagnostic KASP markers for primocane fruiting and thornlessness. These are the first molecular markers associated with traits of economic importance in blackberry. We intend to publish primer sequences and results for these markers during the next reporting cycle.

Publications

Type: Journal Articles Status: Published Year Published: 2022 Citation: Br?na, T., R. Aryal, O. Dudchenko, D.J. Sargent, D. Mead, M. Buti, A. Cavallini, T. Hyt�nen, J. Andr�s, M. Pham, D. Weisz, F. Mascagni, G. Usai, L. Natali, N. Bassil, G.E. Fernandez, A. Lomsadze, M. Armour, B.A. Olukolu, T.J. Poorten, C. Britton, J. Davik, H. Ashrafi, E.L. Aiden, M. Borodovsky, M.L. Worthington*. 2022. A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. Hillquist). Genes Genet. Genomes. 13:jkac289. https://doi.org/10.1093/g3journal/jkac289
Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Chizk, T.M., J.R. Clark, C. Johns, L. Nelson, H. Ashrafi, R. Aryal, and M.L. Worthington. 2023. Genome-wide association identifies key loci controlling blackberry postharvest quality. Front. Plant Sci. 14:1182790. doi: 10.3389/fpls.2023.1182790
Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Johns, C. 2022. Genetic control of prickles and plant height in blackberry. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4527

Progress 04/15/21 to 04/14/22

Outputs
Target Audience:Rosaceae genomics community: The Rosaceae genomics community will benefit from the development of genomic resources for tetraploid blackberry that can be used for synteny analyses. Information about the genetics of fruit firmness and red drupelet reversion as well as other major traits segregating in the UA blackberry breeding program (i.e. thornlessness, plant architecture, primocane fruiting) will be useful for the broader research community. Fruit breeders: New genetic markers for crispy texture that are identified in this project can be deployed for marker-assisted selection for crispy texture in applied blackberry breeding programs. These markers will be used to plan crosses and enrich seedling populations for progeny with crispy texture. We reached fruit breeders and the Rosaceae research community through poster presentations at the National American Society for Horticultural Science (ASHS) meeting, the Southern Region American Society for Horticultural Science (SR-ASHS) meeting, and the annual meeting of the Tools for Polyploids SCRI project. We also shared results throughinformal discussions with representatives Pairwise Plants, Hortifrut, Pacific Berry Breeding, Edward Vinson LTD, Driscolls, Mountain Blue,and others. Blackberry growers and marketers: Crispy textured blackberries have the potential to revolutionize the blackberry industry, potentially extending shelf life and reducing the incidence and severity of red drupelet reversion. Selection for this trait has been slow using classical recurrent mass selection strategies, but the use of genomic breeding will expedite the process and allow breeders to stack this trait with other desirable characteristics. New cultivars developed using these markers will be grown in producers' fields across the US (particularly in the Southeast and California, where the fresh-market blackberry industry is concentrated). The UA blackberry breeding program has an outstanding track record of cultivar development and other breeding programs in the public sector and private sector are likely to adopt the technology as well. We reached growers and marketers through the annual Arkansas Blackberry Growers' Association field day as well as visits to the station by 13 different commercial shipper/marketers of blackberries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Mason Chizk was hired as a PhD student working on the project in May 2019. Mr. Chizk presented his work at theSouthern Region American Society for Horticultural Science meeting, the National American Society for Horticultural Science (ASHS) meeting, and the annual meeting of the Tools for Polyploids SCRI project. His poster on 'Rapid digital phenotyping of red drupelet reversion and fruit morphology in blackberry' was awarded first place in the Southern Region ASHS graduate student poster competition in 2022.Two other MS students on departmental assistantships (Carly Godwin and Carmen Johns) also worked on phenotyping the blackberry GWAS panel for other traits including thorn density, primocane fruiting, berry size, sweetness, acidity, internode length, and seediness. Carly Godwin successfully defended her thesis and graduated with her MS in December 2021 and is now employed as an assistant tomato breeder with Plenty. Carly was also awarded first prize in theASHS Scholars Ignite Competition at the American Society for Horticultural Sciences Annual Conference in August 2021 for her work on this project. Carmen Johns is preparing to defend her thesis in May 2022. A second PhD student, Alexander Silva Cordoba, started in January 2022 and is using the marker data generated in this project to validate a candidate gene for primocane fruiting, develop diagnostic markers for primocane fruiting and thornlessness, and build genomic selection models for fruit firmness and other quantitative traits.Undergraduates Hannah Mather and Isabella Norsworthy also assisted the graduate student team working with this project and gained valuable laboratory and field breeding skills. How have the results been disseminated to communities of interest?Results from GWAS analyses were presented by graduate during the national ASHS meeting in August 2021, the annual Tools for Polyploids SCRI project meeting in January 2022, andthe Southern Region ASHS meeting in February 2022. Carly Godwin has also published her thesis and presented an exit seminar to University of Arkansas colleagues and breeders from Hortifrut. Dr. Worthington described the preliminary results of this project in an invited seminars at the Michigan State University Plant Breeding Symposium.Results have also been disseminated to key stakeholders through informal discussions and visits with other breeders, growers, and shipper/marketers at the University of Arkansas System Division of Agriculture Fruit Research Station and through our collaborative research with Pairwise plants and other public sector caneberry breeders. We anticipate that the 2022/2023 cycle will yield one MS thesis, on PhD dissertation, and several peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?In the final year of project funding we intend to submit at least four publications based on the phenotyping methods developed for berry image analysis andGWAS analyses that graduate students have conducted as a part of this project. These analyses have been completed and the final step is manuscript preparation. PhD student Mason Chizk will also complete his project on cell wall fractionation, polygalacturonase activity, and differential gene expression in soft and crispy blackberry genotypes. Mason Chizk intends to defend in December 2022 and he will be preparing his dissertation and associated analyses. PhD student Alexander Silva Cordoba, who is funded through the SCRI Tools for Polyploids project, will use the data generated by other graduate students in this project as a training population to develop genomic selection models in tetraploid blackberry.

Impacts
What was accomplished under these goals? PhD student Mason Chizk collected a third year of data on fruit firmness and red drupelet reversion during 2020, evaluating 205 genotypes over two harvest dates. Mr. Chizk also worked on fine-tuning the image analysis protocol for assessing red drupelet reversion, berry length and width, and drupelet number and collected a third year of data for his methods paper on this topic. In addition, two MS students funded through other sources (Carmen Johns and Carly Godwin) collected data on primocane height, thorn density, primocane fruiting, soluble solids content, acidity, and seed size for the genotypes in the GWAS panel. Graduate students Chizk, Godwin, and Johns all worked together to calculate trait heritabilities, generate BLUPs, curate Capture-Seq marker data, and perform GWAS analyses for a range of traits using GWASpoly. Significant marker-trait associations were discovered for fruit firmness, red drupelet reversion, primocane fruiting, thornlessness, plant height, soluble solids content, and acidity. PI Worthington also worked to finish writing a manuscript describing the assembly and annotation of the diploid blackberryRubus argutusduring this period. Mr. Chizk worked with program associate Lacy Nelson to extract RNA from crispy and soft-fruited blackberries during this period. RNA sequencing was performed at RAPiD genomics, with results expected to be returned in May 2022.

Publications

Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Godwin, Carly. 2021. Genetic Control of Sweetness, Acidity, and Seediness in Blackberry. Thesis, MS in Horticulture, University of Arkansas, Fayetteville.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Chizk, T.M., M. Worthington, R. Threlfall, H. Ashrafi, R. Aryal, J.R. Clark. 2021. Genome wide association study (GWAS) in tetraploid blackberry for improvement of fruit morphological and shipping characteristics. 2021 ASHS Annual Meeting. Denver, CO. Aug. 5-9, 2021. HortScience 56(9) Supplement - P. S163.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Godwin, C., M. Worthington, R. Aryal, H. Ashrafi, R. Threlfall, J.R. Clark. Genetic control of sweetness and acidity in blackberry. 2021 ASHS Annual Meeting. Denver, CO. Aug. 5-9, 2021. HortScience 56(9) Supplement - P. S226.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Johns, C., M. Worthington, and J.R. Clark. 2021. Genetic control of internode length in blackberry. 2021 ASHS Annual Meeting. Denver, CO. Aug. 5-9, 2021. HortScience 56(9) Supplement - P. S65.

Progress 04/15/20 to 04/14/21

Outputs
Target Audience:Rosaceae genomics community: The Rosaceae genomics community will benefit from the development of genomic resources for tetraploid blackberry that can be used for synteny analyses. Information about the genetics of fruit firmness and red drupelet reversion as well as other major traits segregating in the UA blackberry breeding program (i.e. thornlessness, plant architecture, primocane fruiting) will be useful for the broader research community. Fruit breeders: New genetic markers for crispy texture that are identified in this project can be deployed for marker-assisted selection for crispy texture in applied blackberry breeding programs. These markers will be used to plan crosses and enrich seedling populations for progeny with crispy texture. We reached fruit breeders and the Rosaceae research community through poster presentations at the National American Society for Horticultural Science (ASHS) meeting and throughinformal discussions with representatives Pairwise Plants, Hortifrut, Pacific Berry Breeding, Edward Vinson LTD, Driscolls, and others. Blackberry growers and marketers: Crispy textured blackberries have the potential to revolutionize the blackberry industry, potentially extending shelf life and reducing the incidence and severity of red drupelet reversion. Selection for this trait has been slow using classical recurrent mass selection strategies, but the use of genomic breeding will expedite the process and allow breeders to stack this trait with other desirable characteristics. New cultivars developed using these markers will be grown in producers' fields across the US (particularly in the Southeast and California, where the fresh-market blackberry industry is concentrated). The UA blackberry breeding program has an outstanding track record of cultivar development and other breeding programs in the public sector and private sector are likely to adopt the technology as well. We reached growers and marketers through poster presentation and discussions at the Southeast Fruit and Vegetable Growers Conference in January 2021. We also reached many growers and marketers during visits to the UA Fruit Research Station during June 2020, though we had fewer visitors than usual because of COVID restrictions Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Mason Chizk was hired as a PhD student working on the project in May 2019.Mr. Chizk presented his work at the North American Raspberry and Blackberry Association conference and Southern Region American Society for Horticultural Science meeting, and the National AmericanSociety for Horticultural Science (ASHS) meeting. His poster on developing phenotyping protocols for red drupelet reversion earned second place in the ASHS PhD student poster competition in 2020.Two other MS students on departmental assistantships also worked on phenotyping the blackberry GWAS panel for other traits including thorn density, primocane fruiting, berry size, sweetness, acidity, internode length, and seediness.Rishi Aryal, the postdoctoral researcher in Co-PI Ashrafi's lab worked on CaptureSeq probe design and developed new bioinformatics skills. How have the results been disseminated to communities of interest?Results were disseminated through poster presentation at the National Amercian Society for Horticultural Science conference.Results have also been disseminated to key stakeholders through informal discussions and visits with other breeders and growers at the University of Arkansas System Division of Agriculture Fruit Research Station and through our collaborative research with Pairwise plants and other public sector caneberry breeders. We anticipate that the 2021/2022 cycle will yield one MS thesis and many more presentations and papers. What do you plan to do during the next reporting period to accomplish the goals?Dr. Worthington and her students will continue to explore the GWAS dataset during the next reporting period. We intend to collect one more year of phenotypic data in June 2021 and to calculate broad sense heritabilities for each of the traits we measured. We also hope to measure linkage disequilibrium and evaluate population structure in the panel. As we refine the GWAS results for each of the traits, we intend to look for likely candidate genes. We also intend to finish RNA extractions and submit samples for RNASeq projects related to fruit firmness and primocane fruiting. MS student Carly Godwin intends to defend her MS in December 2021and Carmen Johns plans to defend in May 2022. We intend to present our findings at several national meetings during the next reporting period and to begin manuscript preparation for submission to peer reviewed journals

Impacts
What was accomplished under these goals? PhD student Mason Chizk collected a second year of data on fruit firmness and red drupelet reversion during 2020, evaluating 235 genotypes over two harvest dates. Mr. Chizk also worked on fine-tuning the image analysis protocol for assessing red drupelet reversion, berry length and width, and drupelet number and collected a second year of data for his methods paper on this topic. In addition, two MS students funded through other sources (Carmen Johns and Carly Godwin) collected data on primocane height, thorn density, primocane fruiting, soluble solids content, acidity, and seed size for the genotypes in the GWAS panel. DNA from 500 blackberry selections was submitted to RAPiD genomics in Fall 2020 and genotyped with 35,000 CaptureSeq probes designed using the Rubus argutus reference genome. Marker data was returned in March 2021. Mr. Chizk converted VCF data to tetraploid allele dosage calls using the program UpDog. We have run preliminary analyses with the marker dataset (ranging from 80,000 to 125,000 markers depending on QC thresholds). Students are gearing up to present their findings at the 2021 ASHS National Meeting and to prepare writing manuscripts and thesis/dissertation chapters. We also leveraged this dataset by participating as a breeding program in the new Tools for Polyploids SCRI project led by David Byrne from TAMU. Dr. Worthington is a co-PI in that project and has received funding to recruit a new PhD student to develop genomic selection models using the data collected from the GWAS panel.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chizk, T.M., M. Worthington, K.D. Buck, and J.R. Clark. 2020. ImageJ phenotyping of red drupelet reversion in blackberry. 2020 ASHS Annual Meeting. Virtual meeting. Aug. 10-13, 2020. HortScience 55(9) Supplement P. S93.

Progress 04/15/19 to 04/14/20

Outputs
Target Audience:Rosaceae genomics community: The rosaceae genomics community will benefit from the development of genomic resources for tetraploid blackberry that can be used for synteny analyses. Information about the genetics of fruit firmness and red drupelet reversion as well as other major traits segregating in the UA blackberry breeding program (i.e. thornlessness, plant architecture, primocane fruiting) will be useful for the broader research community. Fruit breeders:New genetic markers for crispy texture that are identified in this project can be deployed for marker-assisted selection for crispy texture in applied blackberry breeding programs. These markers will be used to plan crosses and enrich seedling populations for progeny with crispy texture. We reached fruit breeders and the Rosaceae research community through poster presentations at the National Association of Plant Breeders Meeting and the North American Raspberry and Blackberry Association conference, and informal discussions with representatives Pairwise Plants, Hortifrut, Pacific Berry Breeding, Edward Vinson LTD, Driscolls, and others. Blackberry growers and marketers: Crispy textured blackberries have the potential to revolutionize the blackberry industry, potentially extending shelf life and reducing the incidence and severity of red drupelet reversion. Selection for this trait has been slow using classical recurrent mass selection strategies, but the use of genomic breeding will expedite the process and allow breeders to stack this trait with other desirable characteristics. New cultivars developed using these markers will be grown in producers' fields across the US (particularly in the Southeast and California, where the fresh-market blackberry industry is concentrated). The UA blackberry breeding program has an outstanding track record of cultivar development and other breeding programs in the public sector and private sector are likely to adopt the technology as well. We reached growers and marketers through poster presentation and discussions at the North American Raspberry and Blackberry Association conference and during the USDA-NIFA-SCRI funded US Blackberry Stakeholder Planning Meeting that PI Worthington organized in March 2020. We also reached many growers and marketers during visits to the UA Fruit Research Station during June 2019. Changes/Problems:Because of COVID-19 social distancing restrictions, we have been unable to propagate our biparental mapping population (objective 3). We have decided to pursue genomic selection as a new objective instead of mapping in a biparental population. We will collect tissue from new selections made during 2019 and 2020 and genotype them with our CaptureSeq probes in addition to the GWAS panel. The 2019 and 2020 data from the GWAS panel will be used as a training population to predict firmness and red drupelet reversion in newer selections. PI Worthington is interested in implementing genomic selection in the cultivar development program, and we ultimately feel that this change will be beneficial for the project. What opportunities for training and professional development has the project provided?Mason Chizk was hired as a PhD student working on the project in May 2019. Mr. Chizk worked on soybean breeding at NCSU for his MSc, so he learned a lot about fruit production and breeding in clonally propagated crops during his first year. He also learned a lot about food science and biochemistry through his coursework and independent research. Mr. Chizk presented his work at the North American Raspberry and Blackberry Association conference and Southern Region American Society for Horticultural Science meetings. Two other MSstudents on departmental assistantships also worked on phenotyping the blackberry GWAS panel for other traits including thorn density, primocane fruiting, berry size, sweetness, acidity, internode length, and seediness. The undergraduate research assistant who helped Mr. Chizk with data collection in 2019 has now joined our program as an MS student working on muscadine grapes. Rishi Aryal, thepostdoctoral researcher in Co-PI Ashrafi's lab worked on CaptureSeq probe design and developed new bioinformatics skills. How have the results been disseminated to communities of interest?Results have been disseminated through poster presentations at the North American Raspberry and Blackberry Association conference, National Association of Plant Breeders conference, and Southern Region ASHS conference. Results have also been disseminated to key stakeholders through informal discussions in the US Blackberry Stakeholder Strategic Planning meeting organized by PI Worthington (funded by USDA-NIFA-SCRI) and through visits with other breeders and growers at the University of Arkansas System Division of Agriculture Fruit Research Station and other sites What do you plan to do during the next reporting period to accomplish the goals?We plan to collect the second year of phenotype data for the blackberry GWAS project, submit the GWAS panel for CaptureSeq genotyping, and begin data analysis during the next reporting period. We also intend to submitgreen, red, and blackberries from firm, soft, and intermediate textured genotypesfor RNAseq analysis and begin our cell wall fractionation study. Last, we intend to collect DNA from new breeding selections made during 2019 and 2020 growing seasons. We intend to use the 2019 and 2020 data from the GWAS panel as a genomic selection training population to predict firmness and red drupelet reversion in these new selections.

Impacts
What was accomplished under these goals? During 2019 Mr. Chizk (PhD student funded by this project), PI Worthington, program associate Lacy Nelson, and undergraduate researcher assistants collected the first year of firmness and red drupelet reversion phenotype data on the blackberry GWAS project.Other graduate students also worked on phenotyping the blackberry GWAS panel for other traits including thorn density, primocane fruiting, berry size, sweetness, acidity, internode length, and seediness. PI Worthington worked with Co-PI Ashrafi and his postdoctoral researcher analyzed blackberry resequencing data and identified polymorphisms for CaptureSeq probe design. DNA was extracted from approximately 300 genotypes in the blackberry GWAS panel during the first year of funding. Mr. Chizk also collected green, red, and black berries from firm, soft, and intermediate textured genotypes for RNAseq analysis and cell wall fractionation.

Publications