Progress 09/15/24 to 09/14/25

Outputs
Target Audience:Results on pod fiber and quality traitshave been communicated to scientific and industry audiences at several venues. Postdoc Burcu Celebioglu has presented our results at venues including the Plant and Animal Genome (PAG) conference (oral and poster, January 2025), the UC Davis Plant Breeding Retreat (oral and poster, December 2024), and the National Association of Plant Breeders meeting (poster presentation, October 2024). A preprint on this PvMYB26 pod fiber gene was also posted in April 2025. As of the end of this review period, the paper is in review at Molecular Plant (impact factor = 24.1, which is as high as Nature Plants + PNAS combined). Peer reviewer responses were returned immediately following this reporting period. This preprint and work towards publication in a high-impact journal will ensure that the work is impactful for broad scientific audiences. This preprint provides our submitted manuscript We also continue to engage with the private sector, such as with seed companies. We have had regular email correspondence with private sector plant breeders, and held Zoom meetings about the projects with Syngenta and KWS seeds in spring 2024. Changes/Problems:No changes since we made some updates early in the first year. What opportunities for training and professional development has the project provided?Postdoc Dr. Burcu Celebioglu has continued to build and hone her skills across a tremendous range of activities during the project. She has become quite a well-rounded scientist and breeder with broad skill sets, and will be a major asset in the national breeding community. These skills which she has developed and/or continued to sharpen during the reporting period include: Breeding program management: Crossing Generation advance Phenotyping DNA/RNA extractions (seed chip/NaOH, CTAB, kits) KASP/PACE Selections Breeding program data management Genotyping and sequencing Compare and contrast sequencing/genotyping options for project (e.g., BeadChip, Illumina WGS, Nanopore, high-quality assembly generation) Process whole genome sequencing data with shell scripting and R Visualize variant structures and export features of interest in IGV. Use of the NeighborNet algorithm, SplitsTree, and iTOL treesto characterize haplotype networks and historical patterns of trait evolution Soft skills Scientific oral presentation skills honed through several public presentations to large audiences Scientific writing skills honed through writing of preprint, several posters, several publications in drafting stage Networking with nation-wide plant breeders and geneticists at major meetings such as the National Association of Plant Breeders, Plant and Animal Genomes conference, and UC Davis Plant Breeding Retreat. How have the results been disseminated to communities of interest?During the reporting period, we drafted and submitted a research article that is in review at Molecular Plant, a high-impact journal (impact factor = 24). The paper is focused on a gene that reduces pod wall fiber deposition in common bean. By the end of the reporting period, we had received peer reviewer feedback, which we feel confident in addressing. Responses were submitted slightly after the end of the reporting period. The article was also submitted as a preprint and is freely available at:https://doi.org/10.1101/2025.05.15.654381 Results have been communicated at several venues. Postdoc Burcu Celebioglu has presented these at several academic meetings, including the Plant and Animal Genome (PAG) conference (oral and poster, January 2025), the UC Davis Plant Breeding Retreat (oral and poster, December 2024), and the National Association of Plant Breeders meeting (poster presentation, October 2024). These results have thus been disseminated to scientific audiences ranging from geneticists to breeders. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we have several major activities planned: RNA-seq of 1) a snap bean, 2) an isogenic flat/tough revertant, and 3) an isogenic string revertant at two developmental time points. qPCR of wall fiber/shape candidate genes in a series of nine round/tender types and corresponding isogenic flat/tough revertants for each, plus control lines. Whole genome shotgun sequencing of additional lines DNA extraction of 700 lines of MAGIC population and submission for 12K SNP Beadchip genotyping Extraction of SNP data from the now publicly available Andean Diversity Panel of dry beans (extracting SNPs from the same sites as published for the SnAP panel, for expanded set) Drafting of a manuscript on the genetic and developmental basis of pod wall fiber/shape and its reversion Drafting of manuscript on pod string and partial string Final revisions/responses to reviewers for our PvMYB26 paper Research presentations at additional scientific meetings, e.g., the Bean Improvement Cooperative meeting in 2025 and the National Association of Plant Breeders meeting in 2026.

Impacts
What was accomplished under these goals? Objective 1.Use cutting-edge genotyping and sequencing approaches to understand the genetic basis of environmental and genetic instability in contrasting environments. Generate high-quality genetic data on a panel of approximately 100 snap beans and dry bean controls, which vary in pod traits, using Illumina whole genome sequencing. Extract SNPs from this sequencing to make an expanded SnAP+ panel. The project has now run whole-genome shotgun sequencing and data processing of 74 bean genomes. During this reporting period we completed alignments and data mining of Illumina sequences of the initial panel of 50 bean varieties. In addition, a second round of 24 more samples were extracted, sequenced, aligned to reference genomes, and analyzed. These included the parents of a multi-parent advanced generation intercross ("MAGIC") population, with eight parents, as well as the parents of a biparental recombinant inbred population, populations which segregate for traits such as pod shape and partial string. For all samples, DNA was extracted using the Qiagen DNeasy kit. After nanodrop readings and gel electrophoresis, samples were sent to the UC Davis Genome Center for Illumina WGS at 20x depth. Samtools consensus was used to extract consensus sequence at features of interest. The sequencing data has been used to understand historical patterns of population structure, particularly for mutations of interest, described to activity 1.6. We are continuing to intensively mine this sequencing data for features of interest. We strongly believe that reversion is the result of unequal crossing over. This process should produce triplications at the same rate as reversions to single copies, and these triplicate types would likely have improved stability and improved quality. We developed code to mine copy number based on read depth, and have run it on all sequenced samples. We have not yet identified triplications but will soonscreen this in recently publicized WGS data in the species. Phenotype pod traits in the SnAP+ diversity panel and sequenced materials in cool environments (winter greenhouse) and warm environments (summer greenhouses and field trials), to characterize pod traits and identify patterns of genetic reversion and genotype x environment interaction. We previously reported on the identification of two independent mutations in a single gene (PvHDG12; Phvul.004G143500) that we believe conditions wall fiber/shape reversion. The structure of each mutation is based on tandem repeats, suggesting a mechanism for reversion (unequal crossing over). One mutation has very short repeats, which we hypothesized to be less susceptible to unequal crossing over than the very long repeats. To test this, we had generated clean seed stocks in 2024 at OSU, checking each plant.Seed from this field were then planted in June 2025 at OSU. In early September 2025,79,367 plants were checked for pod shape/fiber reversion by PI Parker and postdoc Celebioglu in field conditions at OSU. Among the79,367 screened plants, 50 revertants were found across six varieties. Among 27 lines with "Mutation 1" (expected to be less stable), we saw revertants in 8 lines (29.7%). Of 30 types with mutation 2 (predicted to be more stable), 0 had any revertants. The chi-squared test value for this isP=0.001, showing a highly significant difference in stability between alleles. We already developed a rapidly deployable genetic assay to screen for these two mutations, which could be almost immediately deployed to select for stability, reducingthe need for this kind of extensive phenotyping in the future. In addition, a 700-member MAGIC population descended from crosses between snap beans and dry beans was planted in the UC Davis greenhouse in July 2025. Pod string, fiber, and shape were evaluated. Traits will be genetically mapped in the upcoming year. Genetically map pod traits in the expanded SnAP+ populations through Genome-Wide Association Studies Leaf samples were taken from 700 lines of the MAGIC population. These leaves will be genotyped with the Illumina 12K BeadChip after DNA extraction. After genotyping, genetic mapping will be performed. Additionally, WGS was performed on the population'seight parents. Use Oxford Nanopore sequencing to generate long-read sequence data to identify structural variation in revertant / non-revertant pairs for each pod traits. We conducted Oxford Nanopore sequencing on eight varieties. Data has been aligned with three reference genomes and has clarified our understanding of the physical structure of the more stable pod wall fiber mutation. We also included an isogenic revertant, clarifying the genetic basis of reversion. This sequencing will also be valuable for understandingotherimportantsnap bean traits in the future. [Activity 5 has now been merged into an expanded activity 1, see other sections for details] Determine patterns of population structure and understand patterns of historical trait evolution through phylogenetic tree construction, haplotype gene network mapping, and comparison with phenotypic character state changes We have now deeply analyzed patterns of historical trait evolution inPvMYB26. We found that reduced pod fiber from this mutation evolved at least three times independently, twice in South America and once in ancient Mexico. Our results in Mexico show that Jalisco/Michoacan was an important center of origin for the crop. OneSouth American mutationarose in the genetic background of the "un-domesticated"debouckiigene pool, suggesting a possible third proto-domesticationin that gene pool. Similar detailed analyses of the geographic origin of wall fiber and pod stringhave been initiated and are ongoing. Objective 2.Characterize the developmental basis of pod quality traits Understand the anatomical basis of pod variation and stability with fluorescence microscopy of revertant / non-revertant and environmentally variable pairs In the winter of 2024, pod samples were taken from all available revertant lines and have sections 100 μm thick with a vibratome. Images of pod sections treated with Auramine O and Calcofluor were obtained by fluorescence microscopy. Understand transcriptional differences which may underlie pod variation using RT-qPCR of candidate genes in revertant / non-revertant pairs Pod samples of revertant/non-revertant pairs were obtained from both the Davis greenhouse in the late 2024 and from all revertant cultivars available in a field trial conducted at OSU in summer 2025. RNA extractions were performed from these pod samples using the Qiagen RNeasy kit, and RNA quantification was measured with Nano drop. After checking the quality of RNAs with bleach gel, cDNA synthesis was performed using the Qiagen cDNA synthesis kit. The 18 samples for RNA-seq all passed the QC process at the UCD Genome Center during the reporting period. cDNAs are ready to use for RT-qPCR and RNA seq, which will be performed in the upcoming reporting period. Objective 3.Develop genetic stocks with improved environmental stability and reduced rates of genetic reversion Conduct half-diallel crossing blocks between commercial varieties with contrasting levels pod trait stability We have now moved the more stable pod shape allele into two less stable commercial types. BC2F1 seeds were planted in the greenhouse and crossing is being done for BC3F1. In all generations obtained after F1, the seeds were tested with the PACE markers created for the pod shape/fiber mutations. BC2F1 seeds of the population prepared for partial string, similar to the pod fiber and shape, were obtained. Based on phenotyping results in the field and greenhouse, select families to advance to BC2F3 To be done next year. Genotype a plate of 96 BC2F3 breeding lines to identify those which have inherited stable alleles from donor parents To be done next year.

Publications

• Type: Other Status: Published Year Published: 2025 Citation: Celebioglu, B., Roy, J., Farmer, A., English, S., Yu, X., Xu, X., McClean, P.E., Gepts, P. and Parker, T., 2025. A domestication change at PvMYB26 in common bean sheds light on the origins of Middle American agriculture. bioRxiv, pp.2025-05.

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

Outputs
Target Audience:We have made a series of exciting discoveries which we have begun to share, including through scientific publications. Weare also still in the earlier stages of the project and are actively generating data to publish in the future. Target audiences so far have been scientists and breedersin academia and the private sector, such as at seed companies. We have been in direct contact with breeders from several companies, including Syngenta, HM Clause, Seneca Foods, Brotherton Seeds, and Crites, Pop Vriend, and van Waveren, and the University of Embu. We have intentionally only provided limited and private disclosures to them, as we generate data. Our closest collaboration over several years has been with Syngenta, with whom we have privately shared many of our major findings, due to their history of material and knowledge sharing with us. We believe the data will be of considerable commercial interest when it is released. A Record of Invention with the UC Davis Tech Transfer department was in drafting during the reporting period,(submitted after the reporting period). The Record of Invention covered the discovery of multiple alleles for the tender pod trait in snap (green) bean, as well as genetic tests to rapidly screen them. Changes/Problems:No major changes since we filed a project change reportin mid-2024. What opportunities for training and professional development has the project provided?The project hired a postdoc, Dr. Burcu Celebioglu, in January 2024. She has now beentrained on numerous methods by PI TravisParker. These include, but are not limited toanalyzing sequencing data using software such as IGVand 'omics databases (NCBI, LegumeInfo, Phytozome, etc.), designing PCR assays for a wide range of targets andpurposes, accessing germplasm from various sources including NPGS, performing RNA extractions and qPCR, fluorescence microscopy, use of geographic software such as QGIS, establishing her own crossing and selection program, and soft skills such as project management, presentation, and networking. In addition, eight interns have been trained by the PI and postdoc in diverse skills such as rearing of plant populations in the field and greenhouse, scientific data management, use of genomics and germplasm databases, screeningpod traits. A comprehensive presentation on both the genetic reversion mechanism for bean pods and the pod shattering mechanism was presented by PI Travis Parker at the International Legume Society meeting in Granada, Spain, and atthe Kirkhouse Trust annual meeting in Arusha, Tanzania.Travis Parker led a full-day training workshop for African breeders at the same meeting in Tanzania. A presentation onthe genetic reversion mechanism for snap bean pods was presented by postdoc Burcu Celebioglu at the UC Davis postdoctoral research symposium.An educational video on field label developmentwascreated by PI Travis Parker and shared on his YouTube channel. How have the results been disseminated to communities of interest?The results have been presented scientifically in the form of articles and presentations. Two articles were published in 2024. These include one peer-reviewed journal article published in Frontiers in Plant Science (IF = 7.3),titled "QTL mapping for pod quality and yield traits in snap bean (Phaseolus vulgaris L.)", in which PI Travis Parker was co-first author. Additionally, a report was published in the Annual Report of the Bean Improvement Cooperative titled "Loci mapped to Pv04 and Pv01 are required for round pod shape in snap bean (Phaseolus vulgaris)." PI Travis Parker again served as co-first author and postdoc Burcu Celebioglu served as middle author on this publication. In addition, results have been disseminated verbally at several internationally recognizedmeetings. They have also been shared privately in writing to private seed companies that have provided material and information in support to the project in the past. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, the lines who were submitted to Illumina WGS will be aligned with the G19833 and UI111 reference genomes, with features of interest compared and extracted in IGV. Pod traits will be evaluated in summer 2025in the greenhouse. A larger population will be subjected to revertant screening under field conditions in 2025 to better understand whether our two mutations differ in reversion rate. Similarly, the listed stability data from the PVPs will be compared. Long-read sequencing will help in resolving the complex structural features found in snap beans and comparable revertants, including screening for the possibility of varieties with triplications or other extreme genotypes. Candidate genes will be determined from the GWAS analysis results for pod traits from 2018-20, and will be published as a paper. Similarly, all pod traits obtained under UC Davis greenhouse conditions will be analyzed by GWAS and compared with those obtained under previous field conditions. SnAP population SNP information will be combined with those of the MDP and ADP population and structure, phylogenetic tree, and haplotype gene network mapping analyzes will be performed. Towards Objective 2, in the summerthe SnAP+ population will be grown in the greenhouse again and pod traits will be evaluated. Pod anatomy of revertant and non-revertant varieties will be examined with fluorescence microscopy and RT-qPCR will be performed on these pairs, and RNA in situ hybridization will be completed. For Objective 3,the BC1F1 population will be planted, and another backcross will be made to create the BC2F1 population. Then BC2F3 will be determined by testing under field conditions and marker-assisted selection. We are particularly interested in moving the pod fiber/shape allele we believe to be most stable into commercially important but unstable varieties, such as OSU 5630 (see previous) and Hystyle. Findings will be submitted as 2-3peer-reviewed articles at internationally recognized journals. Results will be presented at prestigious meetings, including at the International Plant and Animal Genome Conference (PAG32) in San Diego in January 2025. Shortly after the reporting period (October 2024), arecord of invention was also finalized for the pod shape/fiber gene and genetic screens to study it. This will be critical knowledge and selection resources for breeders working with a nutritious and broadly-consumed vegetable crop.

Impacts
What was accomplished under these goals? Objective 1.Use cutting-edge genotyping and sequencing approaches to understand the genetic basis of environmental and genetic instability in contrasting environments. 1. Generate high-quality genetic data on a panel of approximately 100 snap beans and dry bean controls, which vary in pod traits, using Illumina whole genome sequencing. Extract SNPs from this sequencing to make an expanded SnAP+ panel. Dark-grown leaves of 70 varieties of snap and dry beans with different pod traits were collected and DNA extraction was performed with the Qiagen DNeasy kit. These varieties contain two revertant/non-revertant pairs (five samples in total). After the Nanodrop readings and gel electrophoresis of the extracted DNA was performed, it was submitted to the UC Davis Genome Center for Illumina WGS. Library preparation and QC for these samples is now complete. Sequencing will be conducted at a depth of 15x (11 Gb total per accession). In light of our mapping results (see Activity 1.3), wealso mined Illumina sequencing data we generated in 2023 froma different study. We identified twomutations putatively controlling podfiber/shape, the most important of our studied traits:1) a 50 kb duplication which includesPhvul.004G143500 and no other gene models, and 2) a separate mutation with two 1-2 kbrepeats in the vicinity of the same gene model. Inrevertants, the repetitive structures are completely lost. This repetitive structures could explain reversiondue tounequal crossing over, as described in pod strings (Parker et al. 2022). We are now testing whether these two alleles revert at different rates, since unequal crossing over between long repeats happens at higher frequency than short repeats. To test this, we are developing large quantities of very high purity seed of varieties with these mutations(see below). 2. Phenotype pod traits in the SnAP+ diversity panel and sequenced materials in cool environments (winter greenhouse) and warm environments (summer greenhouses and field trials), to characterize pod traits and identify patterns of genetic reversion and genotype x environment interaction. Phenotyping the SnAP bean population and controls was completed atUC Davis (2024 - winter greenhouse). Three plants were planted for each variety and pod dimensions (length, weight, and height), string, fiber, and thickness evaluations were made on each of threepods. Data collection isnowcomplete. A similar experiment was set up in the greenhouse again for the summer in UC Davis. However, the greenhouse cooling fan had a electrical failure on twodates,with our temperature data loggerrecording maximum greenhouse temperatures of75°C, causing extreme plant stress andmortality. In a separate summer field experiment,clean seed stocks of approximately 100 varietieswere planted at OSU. At the end of August, string, fiber and reversionevaluations were made by postdoc Celebioglu. 4,446 total plants were screened. Reversion was found in sixplants in total, all in a singlevariety(OSU 5630, 7.6% of samples reverting for that variety). Full string formation was observed in fourplants (5.1%), and pod shape reversionwas identified in twoplants (2.5%). Harvested seed will be used for additional large-scale reversion screening in field trials in 2025. We are also mining PVP phenotype data from several hundred SnAP accessions with PVPs, as well as phenotype data generated by postdoc Celebioglu for string and wall fiber in 2018-2020. 3. Genetically map pod traits in the expanded SnAP+ populations through Genome-Wide Association Studies GWAS of string and fiber data collected at OSU between 2018-20 was performed using 60,000 SNPs in BLINK. SNPs were filtered for an MAF of 0.05 with a Bonferroni-corrected significance threshold of 0.01. Candidate gene analysis is ongoing. Similar analyses will be made for Davis winter greenhouse pod measurement data. We have also mined other data sets to map pod traits of interest. This includes accessing unpublished data from Haidar Arkwazee and Jim Myers at OSU and using it identify andmap two loci (on chromosomes Pv04 and Pv01) forpod shape. This work has now been published in the Annual Report of the Bean Improvement Cooperative. Similarly, we worked with Kenyan collaborators Serah Njau and Esther Arunga to use a recombinant inbred population they developed from a dry bean x wild bean cross to map 16 pod quality and productivity traits in snap bean, now published in Frontiers in Plant Science. 4. Use Oxford Nanopore sequencing to generate long-read sequence data to identify structural variation inrevertant / non-revertant pairs for each pod traits. Dark-grown leaves were taken from five isogenic linesdiffering inpod traits. The Promega Wizard HMW DNA extraction kit was used for DNA isolation. QCed DNA is ready to be sent to the UC Davis Genome Center. 5. [Activity 5 has now been merged into an expanded activity 1, see other sections for details] 6. Determine patterns of population structure and understand patterns of historical trait evolution through phylogenetic tree construction, haplotype gene network mapping, and comparison with phenotypic character state changes We used our rapid PCR screen for our Pv04 wall shape/fiber mutations to screen the entire SnAP panel of 378 accessions, based on the expectation that the two mutations vary in reversion rate. Of the accessions, 207 have characterized release dates. Aclear shift in frequency has occured, with a steady, decades-long trend away from the long-repeat mutation that we believe is less stable (at >90% frequency in the 1970s),until in the 2000s (the last decade with data available)it was at<50% frequency among new releases. This selection has historically been done painstakingly by breeders screening thousands oflines for stability. Our new rapid PCR screens make this analysis possible on large populations with same-day turnaround. Objective 2.Characterize the developmental basis of pod quality traits 1. Understand the anatomical basis of pod variation and stability with fluorescence microscopy of revertant / non-revertant and environmentally variable pairs We have begun fluorescence microscopy, using it to identify wall fiber variation corresponding to a mutation in a candidate gene on chromosome Pv05. Additional microscopy will continue in 2025. 2. Understand transcriptional differences which may underlie pod variation using RT-qPCR of candidate genes in revertant / non-revertant pairs We have begun using RT-qPCR, and have used it to identify variations in gene transcription related to the wall fiber variation and a deletionin a chromosome Pv05 candidate gene. Additional RT-qPCR will continue in 2025. We have also inspectedpreviously-generated RNA-seq data, and have found strongevidence supporting our Pv04 candidate gene for the control of pod shape/fiber. We are now workingwith Jack Xu at UC Davis to conduct RNA in situ hybridization for genes of interest affecting pod development and structure. Objective 3.Develop genetic stocks with improved environmental stability and reduced rates of genetic reversion 1. Conduct half-diallel crossing blocks between commercial varieties with contrasting levels pod trait stability We have used 48 accessions as parents in a crossing scheme to improve pod quality and stability. Of these,22characterized to havegood and stable qualityand were used as males, while 26 poor-quality varieties were used as females. From the first crosses, 61 F1s were obtained, whichwere replanted and BC1F1 was obtained with the relevant stable male (total 17) varieties, now ready for planting for the next backcross. We are particularly interested in moving the putatively more stable allele into two commercially important accessions with the unstable allele. 2. Based on phenotyping results in the field and greenhouse, select families to advance to BC2F3? 3. Genotype a plate of 96 BC2F3breeding lines to identify those which have inherited stable alleles from donor parents

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Njau SN, Parker TA, Duitama J, Gepts P and Arunga EE (2024) QTL mapping for pod quality and yield traits in snap bean (Phaseolus vulgaris L.). Front. Plant Sci. 15:1422957. doi: 10.3389/fpls.2024.1422957
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Parker, T. A., Arkwazee, H., Celebioglu, B., Gepts, P, and Myers, J. (2024). Loci mapped to Pv04 and Pv01 are required for round pod shape in snap bean (Phaseolus vulgaris). Annual Report of the Bean Improvement Cooperative. 67, 141-142.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Genetic basis of pod traits related to domestication and consumer preference in common bean. Presentation by Travis Parker at the International Legume Society Conference. Granada, Spain. September 2023.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: The genetic architecture of pod and seed traits in common bean. Invited presentation by Travis Parker at the Kirkhouse Trust Annual Combined Meeting. Arusha Tanzania, June 2024.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: "Understanding and reducing the genetic reversion mechanism for snap bean pods." Presentation by Burcu Celebioglu at the UC Davis Postdoctoral Research Symposium. March 2024.