Source: UNIV OF WISCONSIN submitted to
CREATING A NEW PARADIGM FOR POTATO BREEDING BASED ON TRUE SEED
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1020255
Grant No.
2019-51181-30021
Project No.
WIS03024
Proposal No.
2019-03162
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2024
Grant Year
2019
Project Director
Endelman, J. B.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
HORTICULTURE
Non Technical Summary
Potato is the world's leading vegetable crop and its third most important food crop, but genetic improvement of potato is struggling to keep pace with industry and consumer demands. New potato varieties need to be resilient to a changing climate, amenable to sustainable production practices, profitable with reduced inputs, and responsive to heightened consumer expectations for wholesome nutrition. Because of its genetic structure, commercial potato can only be maintained and distributed asexually, using the potato tubers (the part that is eaten) as seed. For most other major crops, elite germplasm can be maintained sexually, using "true" (i.e., botanical) seed, which enables more efficient breeding based on inbred lines.This project will focus on four pillars needed to introduce the inbred-hybrid breeding system used in corn and many vegetable crops to potato. First, we will elucidate the genetics behind why some potatoes are able to overcome reproductive barriers to inbreeding (i.e., self-incompatibility), with the goal of introducing this trait more efficiently into breeding programs. Second, we will capture the existing genetic diversity of elite US potato, which istetraploid and recalcitrant to inbreeding, in a diploid condition that can be genetically purified more easily. Third, we will carry out recurrent selection using both genomic and phenotypic information, to develop improved, self-compatible, diploid germplasm for use in cultivar development. And, finally, we will evaluate methods for using true seed to produce a potato tuber crop and explore how true seed may impact the potato industry as a whole.The ultimate goal of this project is to replace the current, inefficient system of breeding in potato with an inbred-hybrid system. This paradigm shift, which has been called Potato 2.0, will dramatically increase the speed at which new varieties can be released with higher yield, better stress tolerance, and enhanced food quality. These genetic improvements will benefit the potato industry, consumers, and the environment.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011310108030%
2011310108150%
6011310301010%
2051310106010%
Goals / Objectives
Our long-term goal is to convert potato into a diploid crop in which inbred line-based F1 hybrid cultivars are grown from true potato seed. This will give potato breeders and the research community the tools needed to unlock the genetic potential of potato, enhancing the environmental and economic sustainability of the potato industry.Goal 1. Determine the genetic basis and environmental stability of self-fertility in potato.Objective 1a. Identify and validategenetic markers for self-compatibility.Objective 1b. Identify candidate genes for self-compatibility.Goal 2. Develop self-fertile, diploid germplasm that captures the genetic diversity of elite tetraploid potato.Objective 2a. Create and genotype a set of diploid haploids (i.e., dihaploids) that represent the round white, russet, and red germplasm groups in the US.Objective 2b. Select 20 diploid haploids for whole genome assembly to construct a haplotype database for potato.Objective 2c. Pollinate selected dihaploids with self-compatible donors, to create base populations for recurrent selection.Goal 3. Create partially inbred lines that are fixed (homozygous) for key traits.Objective 3a. Perform two cycles of recurrent selection for tuber and reproductive traits.Objective 3b. Create partially inbred lines that are homozygous for resistance genes for late blight, potato virus Y, and golden nematode.Goal 4. Develop agronomic and economic frameworks for incorporating true potato seed into potato production systems and assessing its impacts.Objective 4a. Conduct experiments to investigate the agronomic feasibility of different schemes for producing certified seed tubers from true potato seed.Objective 4b. Analyze the economic impacts of true potato seed from the perspectives of an individual producer (i.e., cost accounting) and the entire industry (i.e., microeconomic analysis).
Project Methods
Objective 1a. Identify and validategenetic markers for self-compatibility.For two diploid donors of self-compatibility (M6 and US-W4), we will develop F2 mapping populations and phenotype them for self-compatibility in replicated trials in greenhouses. Populations will be genotyped using a35K potato SNP array, and QTL analysis will be conducted using established methods. For fine mapping, we will score 2000 plants using a 384-well format with KASP markers to identify recombinants.Objective 1b. Identify candidate genes for self-compatibility.Candidate genes underlying the self-compatibility QTL will be predicted in silico using data from genome annotations, coupled with gene expression profiling (RNA-sequencing) of floral tissues. Based on high-resolution QTL mapping, expression profiling of floral organs, and bioinformatics/genomics, we anticipate identifying 2-3 candidate genes. We will confirm their function by generating knockout lines using CRISPR-Cas9.Objective 2a. Create and genotype a set of diploid haploids (i.e., dihaploids) that represent the round white, russet, and red germplasm groups in the US.Potato breeders from across the country will use the diploid clone IVP101 as a haploid inducer for their elite tetraploid clones. The ploidy of putative dihaploids will be confirmed with genotyping-by-sequencing, using established methods.Objective 2b. Select 20 diploid haploids for whole genome assembly to construct a haplotype database for potato.We will generate haplotype-resolved, whole genome assemblies using Oxford Nanopore Technology long read sequencing and scaffolding methods. Genome sequences will be annotated using established methods, with modifications to incorporate long-read cDNA sequences for transcript evidence. Linkage analysis from F1 populations will also be used to anchor and phase scaffolds into chromosome-scale molecules.Objective 2c. Pollinate selected dihaploids with self-compatible donors, to create base populations for recurrent selection.Self-compatible diploids will be used to pollinate dihaploids.Objective 3a. Perform two cycles of recurrent selection for tuber and reproductive traits.To initiate a recurrent selection (RS) cycle, 20 families of 50 plants each will be grown in the greenhouse as the S0 generation. Plants will be phenotyped for self-compatibility (SC), flowering time, pollen shed, vigor and senescence. At harvest, each SC plant will be scored for excessive stolon development (a negative trait), tuber size, shape, color, and yield. Selected S0 plants will be genotyped, and seeds will be sown to create an S1 population of 1000 plants. The process will be repeated for two more generations, creating S3 populations. S3 individuals will be genotyped and selected for intermating. The greenhouse generations will generate tubers, which will be used for field trials to collect additional data for genomic selection models.Objective 3b. Create partially inbred lines that are homozygous for resistance genes for late blight, potato virus Y, and golden nematode. KASP markers linked to resistance genes for these traits will be used to screen populations during inbreeding and select homozygous individuals.Objective 4a. Conduct experiments to investigate the agronomic feasibility of different schemes for producing certified seed tubers from true potato seed.True potato seed lots generated in Objectives 2 and 3 will be used to test different treatments for their ability to promote rapid and uniform germination. These include optimizing the mother plant environment, proper seed extraction and drying, seed priming, treatments to overcome dormancy, and appropriate germination conditions. Field trials will be conducted to compare direct seeding with transplant-based production. Each year, in-season evaluations will include stand counts and rate of canopy development based on remote sensing imagery. Tubers will be harvested with a mechanical digger and post-harvest evaluations will include total tuber weight, tuber number and tuber size distribution.Objective 4b. Analyze the economic impacts of true potato seed from the perspectives of an individual producer (i.e., cost accounting) and the entire industry (i.e., microeconomic analysis).A partial budget analysis will focus on the activity and input differences between the status quo and alternative production schemes based on true potato seed. Economic estimates of the broader societal benefits will be determined using social surplus in an equilibrium displacement analysis. The cost and any productivity changes will shift the supply curve for potato seeds/seed tubers, and the new equilibrium price and changes in producer and consumer surplus will be estimated. Existing seed industries for different crops will be reviewed to assess the positive and negative aspects of other potential market models.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Potato growers, agronomists, researchers, and other stakeholders were reached. Changes/Problems:Objective 1. Greenhouse conditions became too hot to reliably phenotype a mapping population for self-compatibility, so the experiment will be repeated in Fall 2023. In the fine-mapping experiment for cytoplasmic male sterility, no genomic interval was fully consistent with the phenotype data, most likely due to a recording or phenotyping error. Objective 2. Field phenotype data will not be available for all 100 dihaploids due to material limitations or inherent genetic weaknesses. Objective 3. The discovery of disease-infected source material necessitated the destruction of breeding populations in two programs. Poor male fertility in a promising breeding lineage was discovered to be the result of sterile cytoplasm, for which there is no known restorer gene; the populations were therefore discarded. Objective 4. Several rows in our transplant field suffered severe die back around the time of hilling. The cause is not known, but we hypothesize the plants were damaged from the combined effects of fertilizer application and heat, highlighting the need for more research on field management of seedling transplants. What opportunities for training and professional development has the project provided?Nine undergraduate students, nine graduate students, and one postdoc were involved in project research activities. Students and staff had the opportunity to present their research at scientific meetings for the Potato Association of America and AGBT Ag. How have the results been disseminated to communities of interest?Project results were discussed with stakeholders at many events in Year 4, including summer field days and winter conferences in Maine, Michigan, Minnesota, Oregon, and Wisconsin. At the national Potato Expo in January 2023, outreach was conducted at the trade show and poster session, including a hands-on demonstration of diploid potato tubers. A full-day workshop was conducted with industry stakeholder participation at the Potatoes USA headquarters in June, to allow for in-depth discussion of project results and future research plans. Eight manuscripts were published in Year 4 based on project results, including in the industry magazine The Badger Common'Tater. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Phenotyping and genetic analysis of the F2 mapping populations segregating for self-compatibility will be completed: pop 1 is segregating for Sli and the SRNase-knockout, and pop 2 is segregating for stylar self-compatibility from S. verrucosum. For the study of cytoplasmic male sterility (CMS), we will develop and characterize F2 populations segregating for the restorer gene in T cytoplasm, to determine if it shows expression of the CMS trait in addition to the already established P cytoplasm. Objective 2. Several manuscripts are planned to disseminate results: (1) Description of the genetic and phenotypic diversity of the 100 dihaploids; (2) Population genetic study of selection, deleterious alleles, transposon frequency, and introgression in the dihaploids. (3) Investigation of whole-genome imputation accuracy from genotyping-by-sequencing and microarray technologies. Objective 3. New breeding populations will be generated and evaluated in 5 states: ME, MI, MN, OR, and WI. A manuscript describing the genomic selection model will be published. Objective 4. An experiment is planned to optimize greenhouse parameters for producing seedling transplants, including the timing of seed sowing and size of the plug tray. Our hypothesis is that the optimal stage of plant development has sufficient root mass to maintain mechanical integrity of the plug during transplanting but without triggering early tuberization due to container volume stress.

Impacts
What was accomplished under these goals? Objective 1. Our project has made progress toward understanding and exploiting the genetics of self-fertility on several fronts. Until now, inbreeding efforts have relied on the Sli gene to overcome the gametophytic self-incompatibility that naturally exists in cultivated potato, but the effectiveness of this approach varies with genetic background. The wild species S. verrucosum does not exhibit gametophytic self-incompatibility, and an interspecific mapping population with cultivated potato was characterized in a greenhouse experiment in Year 4 to better understand the genetics of this trait; analysis of the results is underway. Another accomplishment was to complete a fine mapping experiment (N=376) for a gene involved in cytoplasmic male sterility, which manifests as shrunken or missing anthers. This trait could be exploited to facilitate the production of hybrid seed and male sterility in hybrids, which may enhance tuber yields by eliminating true seeds as competitive "sinks" for nutrients. Objective 2. The development and genomic characterization of dihaploids (diploid haploids) from elite tetraploid germplasm creates a foundation for efficient diploid breeding. We have met our target of whole-genome sequencing for 100 dihaploids, generated from 60 different tetraploid clones spanning the major US germplasm groups (russet, chip, red). Confirming earlier studies, we found extreme genetic diversity in cultivated potato, with more than 20 variants per gene in the population on average. The sequencing data have been leveraged to design DNA markers for key traits, including maturity (CDF1) and tuber shape (OFP20). From the beginning, our goal has been to generate de novo, haplotype-resolved assemblies for 20 of the 100 dihaploids, which are now complete. Gene annotation is complete for 5 of the 20 so far, and we have identified 9,684 regions of perfect synteny between our dihaploids, the tetraploid Atlantic reference genome, and the DM reference genome. A multi-location (MN, MI, WI, ME) field trial of the dihaploids was conducted in 2023, to better characterize its phenotypic diversity. Objective 3. Dihaploids from Objective 2 have been used as founders for recurrent selection, to improve tuber and fertility traits. Elite diploids, selected at Michigan State University using similar techniques as a conventional tetraploid program, were evaluated in MI, NY, and WI in 2023. Total yields for several clones were comparable to elite tetraploids (although tuber appearance was not), providing further evidence that yield is not a limiting factor for diploids. The University of Maine and Oregon State University programs generated their first diploid breeding populations by crossing dihaploids with self-fertile diploids from the UW and MSU programs, bringing the total number of diploid breeding programs in the project to five (MI, WI, MN, ME, OR). Our first assessment of the accuracy of genomic selection for diploid potato was completed. Using a new genetic marker for maturity (CDF1), progress was made to develop diploid breeding populations fixed for different maturity variants, which is an essential step toward generating hybrid varieties with uniform maturity. Inbreeding experiments produced clones homozygous for the late blight resistance gene RB/Rpi-blb1 and the potato virus Y resistance gene Rychc. Objective 4. Our first agronomic experiment in 2023 was designed to study the influence of seed tuber size on yield components for a first-generation diploid hybrid. Seed tubers produced in 2022 from transplants were sorted into three size categories for the 2023 trial: small (~10 g), medium (~20 g) and large (~40 g). The large size is comparable to the size currently used for uncut tetraploid potato seed, while the small size is similar to hydroponically produced minitubers. Our key finding is that harvested tuber size was not affected by seed size, but the number of tubers and therefore total yield increased with seed size. A second field experiment was designed to validate hybrid maturity predictions based on the gene CDF1. Different hybrids were created with 0, 1, or 2 copies of the early variant CDF1.3 (in a wild-type genetic background), which showed progressively earlier flowering time and senescence in the field.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Endelman JB, Caraza-Harter MV, Shannon LM, Meng X, Douches D, Coombs J, Bethke PC, Hamernik AJ, Buell CR, Vaillancourt B, Tan EH, Parsons J (2023) Allelic diversity for maturity and skin color in dihaploids of potato. Plant and Animal Genome XXX, San Diego, CA. January 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Shannon LM (2023) Understanding Diversity in US Tetraploid Potato Cultivars to Inform Diploid Breeding. Plant and Animal Genome XXX, San Diego, CA. January 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Shannon LM, Mitchell P, Bethke P (2023) Potato 2.0  Update on Breeding, Agronomics and Industry Impacts. Potato Expo, Denver, CO. January 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Vaillancourt B, Mailloux K, Hamilton JP, Meng X, Bethke PC, Douches D, Jansky S, Tan EH, Shannon L, Endelman JB, Buell CR (2023) Haplotype Resolved Genome Assembly of Potato Dihaploids Capturing North American Allelic Diversity of Tetraploid Parents. AGBT Ag, San Antonio, TX. March 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Ames M, Halterman D, Hamernik A, Bethke P (2023) A survey of the Sli gene in wild and cultivated potatoes. 107th Annual Meeting of the Potato Association of America, PEI, Canada. July 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Caraza-Harter M, Christensen G, Endelman J (2023) Marker-assisted selection of partially inbred lines with different maturities. 107th Annual Meeting of the Potato Association of America, PEI, Canada. July 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Erickson M, Christensen G, Caraza-Harter M, Endelman J (2023) Investigating cytoplasmic male sterility in potato. 107th Annual Meeting of the Potato Association of America, PEI, Canada. July 2023.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Agha HI, Shannon LM, Morell P (2023) Unloading potatoes: Potato breeding moves forward with only half the genome. Cell Genomics 3:100343.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Agha HI, Schroeder L, Eikholt D, Schmitz Carley CA, Cavender-Bares J, Shannon LM (2023) Assessing the Effectiveness of Reflectance Spectroscopy Analysis to Determine Ploidy in Potato. American Journal of Potato Research 100: 135-141.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Bethke PC, Halterman DH, Francis DM, Jiang J, Douches DS, Charkowski AO, Parsons J (2022) Diploid potatoes as a catalyst for change in the potato industry. American Journal of Potato Research 99: 337357. https://doi.org/10.1007/s12230-022-09888-x.
  • Type: Other Status: Published Year Published: 2023 Citation: Bethke P, Hamernik A, Endelman J (2023) A new beginning - Producing diploid potato seed. The Badger CommonTater 75 (9):59-63.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Behling WL, Douches DS (2023) The effect of self-incompatibility factors on interspecific compatability in Solanum Section Petota. Plants 12:1709. https://doi.org/10.3390/plants12081709
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Lee S, Enciso-Rodriguez FE, Behling W, Jayakody T, Panicucci K, Zarka D, Nadakuduti SS, Buell CR, Manrique-Carpintero NC, Douches DS (2023) HT-B and S-RNase CRISPR-Cas9 double knockouts show enhanced self-fertility in diploid Solanum tuberosum. Frontiers in Plant Science 14:1151347.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Song L, Endelman JB (2023) Using haplotype and QTL analysis to fix favorable alleles in diploid potato breeding. Plant Genome e20339. doi:10.1002/tpg2.20339
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Sorensen PL, Christensen G, Karki HS, Endelman JB (2023) A KASP Marker for the Potato Late Blight Resistance Gene RB/Rpi-blb1. American Journal of Potato Research 100:240246. doi:10.1007/s12230-023-09914-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Williams N, Tan EH, Collins P (2023) Assessing potato haploid induction from diploid Solanum tuberosum selections. 107th Annual Meeting of the Potato Association of America, PEI, Canada. July 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Song L, Endelman J (2023) Exploring the potential of genomic selection for diploid potato. 107th Annual Meeting of the Potato Association of America, PEI, Canada. July 2023.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Potato researchers, growers, agronomists, and other industry representatives were reached through print, digital, and oral communications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has contributed to the training of four undergraduate students, six graduate students, and one postdoc, in plant sciences and agricultural economics. How have the results been disseminated to communities of interest?Results have been disseminated through a number of mechanisms, includingthree outreach publications in potato grower magazines and eight presentations at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Determine the genetic basis and environmental stability of self-fertility in potato. The F2 population segregating for S-RNase, HT-B, and Sli will be evaluated for self-fertility to compare the effects of these three genes and possible interactions. Mapping populations derived from the self-compatible wild species will be evaluated to potentially discover new loci affecting fertility traits. Objective 2. Generate and sequence dihaploids to capture the genetic diversity of North American germplasm. Sequencing will be completed for all 100 dihaploids, as well as haplotype-resolved and annotated assemblies for the 20 selected dihaploids. A practical haplotype graph will be generated from all available sequencing data and evaluated for its ability to impute haplotypes from skim sequencing. Objective 3. Develop improved inbreds through recurrent selection on tuber traits and true seed production. Diploid field trials will be conducted at four locations in 2023 (Maine, Michigan, Minnesota, Wisconsin), and genomic selection models will be created from SNP array data for traits such as yield and specific gravity. Multi-parental genetic mapping techniques will be used to characterize the effects of founder haplotypes for tuber shape and skin color. Objective 4. Conduct agronomic and economic studies about the introduction of true seed into the commercial seed system. A trial demonstrating the power of TPS for rapid seed multiplication will be completed. Starting with 8 plants each for 2 partially inbred lines, 12,000 seedlings were transplanted to fill a one-acre field in 2022. Yield, tuber number, and size distribution will be measured at harvest.

Impacts
What was accomplished under these goals? Objective 1. Determine the genetic basis and environmental stability of self-fertility in potato. Self-fertility is critical to developing inbred lines, but gametophytic self-incompatibility (SI) has historically been a barrier to achieving this goal in diploid potato. Natural or engineered variation at three genes has previously been shown to weaken or eliminate SI in potato: S-RNase, HT-B, and Sli. During Year 3, steps were taken to create a mapping population in which all three genes are segregating, for the purpose of comparing their effects and possible interactions in the same genetic background. Of the 40 F1 candidates, 19 inherited Sli and set fruit upon selfing in a greenhouse experiment. These 19 are being screened for the presence of the S-RNAse and HT-B knockouts, and F2 seeds from individuals with all three genes can then be used for the mapping study. The second major accomplishment in Year 3 was a screening study of wild potato germplasm to identify other potential sources of self-compatibility. Self-compatible accessions were identified in four wild species: S. pinnaseticum, S. polyadenium, S. kurtzianum, and S. verrucosum. Objective 2. Generate and sequence dihaploids to capture the genetic diversity of North American germplasm. The development and genomic characterization of dihaploids (diploid haploids) from elite tetraploid germplasm creates a foundation for efficient diploid breeding. By the end of Year 3, we have completed 20X whole-genome sequencing of 91 dihaploids, only 9 short of the original goal of 100. Confirming earlier studies, we have found extreme genetic diversity in cultivated potato, with approximately 1% heterozygous nucleotide frequency in each dihaploid and more than 10 alleles per gene in the population, on average. For 20 dihaploids selected for de novo, haplotype-resolved genome assembly, we have completed HiC and PacBio HiFi sequencing, as well as Oxford Nanopore sequencing of cDNA transcripts from leaf and tuber tissue. The first five assemblies (comprising 10 haplotypes) have excellent statistics, with complete BUSCO scores of 96.6-98.6% and N50 values of 60.5-67.1 Mb. Objective 3. Develop improved inbreds through recurrent selection on tuber traits and true seed production. Dihaploids from Objective 2 have been used as founders for recurrent selection, to improve tuber and fertility traits. Two main approaches are being used: 1. outbred recurrent selection, and 2. the development of partially inbred lines. Using similar field breeding techniques as a conventional tetraploid program, approach #1 is yielding dramatic results. In 2021, field trials at Michigan State University, many diploids outyielded the tetraploid checks in small plot (10-20 plant) trials. For approach #2, we have developed multiple lines with 40-50% homozygosity, including at key loci for maturity (CDF1.3 allele) and self-compatibility (Sli). A number of dihaploids have been identified with valuable disease resistance genes: 10 fertile dihaploids contain the extreme resistance gene Ryadg for potato virus Y (PVY), and three different R genes for late blight are distributed across 5 female-fertile dihaploids. Breeding populations have been enriched for these R genes using genetic markers. Objective 4. Conduct agronomic and economic studies about the introduction of true seed into the commercial seed system. New economic analysis has led to more specific predictions about how true potato seed (TPS) hybrid varieties may affect the seed supply chain for potato production. At present, the "seeds" for the first field year (FY1) of potato production are greenhouse-grown minitubers, for which a typical cost is $0.55/minituber. The availability of TPS would enable the use of seedling transplants instead of greenhouse minitubers as the planting stock for FY1. Seedling transplants are widely used in processing tomato production, and one published cost from UC Davis Extension is $0.20/seedling. Around half of this cost is the price of seed, and the other half is the cost of producing the transplant in a greenhouse. If potato yields and production costs from a transplant-based production system are similar to the use of minitubers, the cost of FY1 seed would be reduced by over 50% under these assumptions. Based on a replicated field trial (plot size 20 plants) in 2021 with three diploid F1 families, yields were indeed similar for seedling transplant vs. greenhouse minituber planting stock. In addition, different transplant methods (e.g., plugs vs. bare root) were evaluated.

Publications

  • Type: Other Status: Published Year Published: 2022 Citation: Mitchell P, Endelman J, Bethke P, Shi G. 2022. Impact of True Potato Seed on the Industry. Badger Common'Tater Vol 74, Issue 8:61-66.
  • Type: Other Status: Published Year Published: 2022 Citation: Bethke P, Jansky S. 2022. The Birds and the Bees...and the Potato. Potato Grower April 2022:25-26.
  • Type: Other Status: Published Year Published: 2021 Citation: Bethke P. 2021. Seeds of Change. Badger Common'Tater Vol 73, Issue 9:64-66.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Meng X, Buell CR, Bethke P, Douches D, Jansky S, Tan EH, Endelman J, Shannon SM. 2022. Genetic diversity of primary dihaploid potatoes from US cultivars. 106th Annual Meeting of the Potato Association of America, Missoula, Montana. July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Shaw K, Douches D. 2022. Dihaploid potato breeding at Michigan State University. 106th Annual Meeting of the Potato Association of America, Missoula, Montana. July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Caraza-Harter M, Endelman J. 2022. Diploid breeding for red potatoes. 106th Annual Meeting of the Potato Association of America, Missoula, Montana. July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sorensen P, Christensen G, Halterman D, Endelman J. 2022. Development and validation of a KASP assay for the late blight resistance gene RB. 106th Annual Meeting of the Potato Association of America, Missoula, Montana. July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Shannon LM. 2022. Diversity and admixture in cultivated potato. European Association for Potato Research, Krakow, Poland. July 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Shannon LM. 2022. Resequencing Potato Primary Dihaploids to Inform Diploid and Tetraploid Breeding. World Potato Congress, Dublin, Ireland. May 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Vaillancourt B, Mailloux K, Hamilton JP, Meng X, Shannon L, Buell CR. 2022. Haplotype Resolved PacBio HiFi Assembly of Potato Dihaploids Capturing North American Allelic Diversity of Tetraploid Parents. CROPS Conference, Hunstsville, AL. June 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Song L, Endelman JB. 2022. Utilization of Haplotype and QTL Analysis to Fix Favorable Alleles in Diploid Potato Breeding. 2022 Annual Meeting of the National Association of Plant Breeders. August 2022.


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

Outputs
Target Audience: Potato researchers, growers, agronomists, and other industry representatives were reached through both print and digital communications (examples given under Accomplishments). Changes/Problems:The COVID-19 pandemic created significant limitations to carry out research and outreach activities. As a result, we are behind schedule for expenditures and some milestones, but expect to catch up by the end of Year 3. What opportunities for training and professional development has the project provided?5 undergraduate students, 8 graduate students, and 3 postdocs contributed to the project in Year 2 and were mentored by key personnel. How have the results been disseminated to communities of interest?A number of publications and presentations have been made, inscientific journals and meetings, as well as industry publications and meetings. We have also utilized the project website, YouTube channel, and Twitter handle. Please see the Products for a complete list. What do you plan to do during the next reporting period to accomplish the goals?Objective 1.Besides the naturally occurring allele at Sli, alleles for SC have been created with gene editing techniques at the genes for S-RNase and HT-B, which are stylar proteins involved in SI. Our focus for Year 3 will be to create and characterize an F2 population segregating at all 3 loci, to study the relative influence and interaction of these genes on self-fertility. Objective 2. An additional 26 dihaploids will be sequenced in Tier 1 in the coming year, as well as 12 more dihaploids in Tier 2. Tier 1 sequencing data will be analyzed to assess genetic diversity, identify introgressions, and predict deleterious alleles. The first 8 genomes from Tier 2 will be completed. Objective 3.Dihaploids with the Ryadg gene for PVY resistance have been created, and we expect to create diploids homozygous for this gene in Year 3. Genetic mapping and genomic prediction studies of the 2021 field trial will be performed, and a coordinated field trial between the MSU and UW breeding programs will be planted in 2022. Objective 4.The 2021 seed source experiment will be completed and repeated in 2022. The economics of field production based on seedling transplants will be investigated. Economic analysis of the organization of the breeding and seed sectors will continue and expand to include international markets.

Impacts
What was accomplished under these goals? Objective 1.Self-fertility is critical to developing inbred lines, and one of the requirements for self-fertility is inactivation of the gametophytic self-incompatibility (SI) system present in many potato varieties and genetic resources. It had been hypothesized that a gene on chromosome 12, known as Sli, can inactivate SI in potato and confer self-compatibility (SC). This hypothesis was confirmed by Clot et al. (2020) in diploid breeding populations from the Netherlands, and although the molecular identity of Sli was not determined, they published a set of KASP DNA markers linked to the gene. During Year 2, we tested a subset of these KASP markers and confirmed their utility for breeding in North American germplasm. Michigan State University recently completed the fourth cycle of phenotypic recurrent selection for self-fertility, maturity, and tuber quality, and clones from each cycle had been maintained to enable a retrospective analysis (Kaiser et al. 2021). The results indicate a steady increase in the frequency of clones homozygous for the marker allele linked to Sli, and all clones with this genotype were SC. In contrast, having only one copy of Sli (i.e., the heterozygous genotype) was not always associated with SC, and similar observations of incomplete dominance were made in breeding populations at the University of Wisconsin. Objective 2.Diploid individuals called dihaploids are being created by haploid induction, which involves pollinating tetraploid varieties and breeding lines with certain Group Phureja clones. Research indicates the haploid inducer chromosomes are eliminated in the early stages of embryo development, so only the maternal genome is present in the offspring. All of the breeding programs participating in this project are creating dihaploids from their germplasm and nominating the best individuals for genome sequencing, which occurs in two stages or tiers. Tier 1 sequencing occurs at 20X coverage using Illumina short reads, with a goal of at least 100 dihaploids before the end of the project. We are nearly halfway to that goal, having sequenced 21 chip, 18 russet, 7 red, and 2 specialty clones. The goal of Tier 2 sequencing is to create a de novo haplotype-resolved assembly by combining 100X Illumina short reads with long reads (either ONT or PacBio HiFi) and proximity-by-ligation (HiC) libraries. Both leaf and tuber transcriptomes are also being sequenced in Tier 2. We have planned for 20 dihaploids in Tier 2 and made progress on 8 thus far, including completed assemblies for 2 dihaploids. Objective 3.Inbred lines are needed to generate hybrid varieties with homogeneous true potato seed (TPS), but vigorous inbreds are difficult to create from S. tuberosum dihaploids because of the large number of deleterious alleles in the genome. We are making slow but steady progress toward this goal by selfing or sib-mating for several generations, followed by intermating with other inbreds or "backcrossing" to other dihaploids, to begin the next breeding cycle. Haplotype analysis is being used to track identical-by-descent segments during inbreeding at key loci. For example, a number of F2 individuals with early maturity have been created that are homozygous at the CDF1 locus, and we are in the process of sequencing CDF1 amplicons to identify which alleles are present. Field trials of outbred F1 generations are being conducted in Wisconsin and Michigan to inform selection decisions, and the entries in the 2020 (N = 480) and 2021 (N = 432) trials were genotyped with the potato SNP array to enable genetic mapping and genomic prediction. Genome-wide association analysis of the data from uncrewed aerial surveys (UAS) of the 2020 field trial (i.e., using NDVI or other vegetation indices) identified a QTL on chromosome 2 for early vigor at 50 days after planting (DAP), which may be related to tuber dormancy. We also observed the effect of CDF1 during vine senescence at 100 DAP. Tuber total glycoalkaloid (TGA) levels (which ranged from 1 to 60 mg/g DW for the top 15 clones selected based on agronomic performance in Wisconsin in 2020) are being used for selection to eliminate unfavorable alleles inherited from the S. chacoense founders of our diploid breeding program. Objective 4. TPS will enable a new type of production system in potato, based on seedling transplants. A field trial was planted in Wisconsin in 2021 to study the impact of seed source (seedling transplant vs. greenhouse minituber vs. field tuber) on plant development, tuber number, and yield. Three diploid F1 populations are being used for this experiment, with two replicates per population per seed source and a plot size of 20 hills. Demonstration experiments were also planted to compare transplanting by hand vs. machine and the use of plugs vs. bare root. The prospect of diploid TPS varieties has already stimulated significant new investment in the potato breeding sector, and major changes are likely to occur in the future. To inform economic models of how the breeding and seed industries may reorganize, we have conducted interviews with breeders, minituber producers, and seed growers across the US. A conceptual framework for evaluating the distribution of social benefits among stakeholders is under development.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bethke PC, Jansky SH. 2021. Genetic and environmental factors contributing to reproductive success and failure in potato. American Journal of Potato Research 98:2441. doi:10.1007/s12230-020-09810-3
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kaiser N, Coombs JJ, Collins P, Alsahlany M, Jansky S, Douches D. 2021. Assessing the contribution of Sli to self-compatibility in North American diploid potato germplasm using KASP markers. American Journal of Potato Research 98:104-113. doi:10.1007/s12230-021-09821-8
  • Type: Other Status: Published Year Published: 2020 Citation: Bethke P, Jansky S. 2020. Building a better potato. Badger CommonTater 72:59-63.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Nashiki A, Jansky SH, Bethke PC. 2021. The effect of mother plant fertilization and stratification on the germination of true potato seed. American Journal of Potato Research 98:194-201. doi:10.1007/s12230-021-09830-7
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Busse JS, Jansky SH, Agha HI, Schmitz Carley CA, Shannon LM, Bethke PC. 2021. A high throughput method for generating dihaploids from tetraploid potato. American Journal of Potato Research 98:304-314. doi:10.1007/s12230-021-09844-1


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

Outputs
Target Audience:Potato breeders, growers, agronomists, and other industry representatives were reached through both print and digital communications (examples given under Accomplishments). Changes/Problems:The COVID-19 pandemic adversely affected the project in several ways: Objective 1. Laboratory work to validate genetic markers for self-incompatibility was delayed by several months due to cessation of non-essential research at Michigan State University (MSU). Objective 2. Tier 1 sequencing of dihaploid germplasm was delayed several months due to cessation of non-essential research at the University of Minnesota Genomics Center (UMGC). The UMGC has also become a major center for processing COVID-19 tests in Minnesota, which has prevented them from allocating resources for the Tier 2 sequencing. As a result, ONT sequencing is now being performed at MSU using reagents purchased by UMGC, and we are exploring options for Hi-C library construction in the private sector. Objective 3. Evaluation of several greenhouse populations for reproductive fertility was significantly curtailedbecause undergraduate students were barred from participating in research projects at the University of Wisconsin for several months. Objective 4. The scope of greenhouse and field trials wasreduced due to personnel limitations at the USDA-ARS. Outreach. The project field day planned for Summer 2020 was canceled. What opportunities for training and professional development has the project provided?In the first year, 6 undergraduate students, 7 graduate students, and 2 postdocs contributed to the project and received mentoring from project key personnel. How have the results been disseminated to communities of interest?1. A project website (https://potatov2.github.io/) was created to communicate information about project members, goals, and research progress, and the URL was distributed widely within the potato industry and academic communities. 2. At the January 2020 Potato EXPO, which is the largest trade show and education event for the US potato industry, an oral presentation about the project was made before the national audience. 3. Presentations about the project were also made at state and regional meetings of the potato industry, including the Pacific Northwest, North Dakota, Minnesota, Wisconsin, and Michigan. 4. Articles about the project targeted to the potato industry were published in the March 2020 issue of Spudman and distributed electronically to a mailing list of more than 1000 growers and industry representatives. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: 1. Results from the validation experiments for the Sli genetic markers will be compiled and presented to both project members and the wider potato breeding community, to enable more efficient selection for self-compatibility. 2.The genetic mapping populations to investigate other genes and environmental influences on self-fertility will be selected, genotyped, and characterized. Objective 2: 1. Results from the first set of 11 dihaploids sequenced at 20X will be compiled, and Tier 1 sequencing will be completed for 30 additional dihaploids. 2.Tier 2 sequencing (ONT, Illumina, and Hi-C) will be completed for the first 4 dihaploids, and data analysis will begin for the de novo assembly. We will initiate sequencing for the next set of 6 dihaploids for Tier 2. Objective 3: 1. Phenotypic and SNP array data for the 2020 field trials will be completed and analyzed. These data will be combined with information about reproductive fertility from the greenhouse trials to select clones to begin the next breeding cycle. 2. A 2021 field trial at two locations will be planted with over 500 unique clones, which will also be genotyped using the potato SNP array and markers for Sli. 3. Inbreeding will continue under greenhouse conditions, in conjunction with marker-assisted selection for disease resistance. Objective 4: 1. A field trial will be conducted to compare plant development in genetically similar material that starts from seed tubers versus transplants. 2. Interviews with potato industry stakeholders will be completed by the economics team and used to prepare a white paper describing the US seed potato industry structure and how it might change with advent of true potato seed.

Impacts
What was accomplished under these goals? Objective 1. Determine the genetic basis and environmental stability of self-fertility in potato. Activities completed: 1. After initiation of the project, a number of genetic (KASP) markers for the self-compatibility locus Sli on potato chromosome 12 were published by a Dutch research group (Clot et al. 2020). The publication came out right as the COVID-19 pandemic disrupted research operations at our universities, but we were able to order the reagents and have begun screening US germplasm to determine how well the markers predict self-compatibility. 2. Previous experiments had shown that, besides Sli, other genetic and environmental factors contribute to self-fertility in diploid potato. We have created 13 populations for genetic mapping of self-fertility traits, including pollen tube growth (via light microscopy), fruit production, and seed production. The parents of these populations include individuals with targeted knock-outs of S-RNase and other candidate genes, as well as individuals with Sli and other naturally occurring variants. We have begun screening a subset of each population to identify the most informative to fully characterize and use for genetic mapping. Objective 2. Generate diploids that capture the genetic diversity of elite tetraploid potato for the chip processing, russet and red markets. Activities completed: 1. Dihaploid extraction has thus far focused on 20 chip, 15 russet, and 10 red cultivars and elite clones. Ploidy of putative dihaploids has been confirmed in various ways, including chloroplasts per guard cell, genotyping, flow cytometry, and seed production following crosses to diploids. Confirmed dihaploids have been evaluated in the greenhouse for flower production and fertility, and some have been grown in the field to evaluate agronomic performance. 2.We are using a two-tier strategy for genomic characterization of dihaploids, with a budgeted goal of 100 dihaploids in Tier 1 (20X resequencing) and 20 in Tier 2 (de novo assembly) by the end of the project. For Tier 1, clones are sequenced using a short-read Illumina platform and aligned to the DM reference genome. Illumina libraries are currently being prepared for our first 11 dihaploids. 3.Tier 2 targets the female-fertile, tuber-bearing dihaploids that will become the founders of diploid breeding. In addition, clones are selected based on the uniqueness of the haplotypes identified in Tier 1 and confirmation of eudiploidy. Sequencing involves de-novo genome assembly using long reads from the Oxford Nanopore Technologies (ONT) platform plus error correction with Illumina short reads. We have already prepared high molecular weight DNA from four, well-characterized dihaploids for Tier 2 submission after their Tier 1 sequencing is complete. Objective 3. Develop improved inbreds through recurrent selection on tuber traits and true seed production. Activities completed: 1. Field trials of more than 500 new diploid clones were planted at two locations. Data to be collected includes vine maturity, total yield, tuber size, specific gravity, and chip fry color. These populations are segregating for the key traits we intend to fix during the project, including resistance to PVY, late blight, and golden nematode. Leaf tissue was collected and submitted for SNP array genotyping at 21K markers. 2.Greenhouse and field production of seed tubers for the 2021 field trial is underway. Over 1000 genetically distinct clones are under consideration. 3. To complementselection on agronomic and quality traits in the field, remnant seed tubers were planted in the greenhouse and either selfed or test-crossed as female parents to select for reproductive fertility. Objective 4. Create agronomic and economic insights to guide the introduction of true seed into the commercial seed system. Activities completed: 1. A field trial was planted using 48 different botanical seedlots to evaluate for plant development and uniformity. The same families are being grown in a greenhouse to produce tubers for a field trial in 2021 to compare plant development in genetically similar material that starts from seed tubers versus transplants. 2. A set of interview questions about the organization of potato breeding and technology transfer in the US was developed, and 12 breeders were surveyed by economists involved in the project. Results are being analyzed.

Publications

  • Type: Other Status: Published Year Published: 2020 Citation: Bethke, P, and Jansky, S. 2020. Are diploid potatoes in your future? Spudman, March 2020. (https://spudman.com/article/are-diploid-potatoes-in-your-future/)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bethke, P, and Jansky, S. 2020. Are hybrid potatoes in your future? Potato EXPO, Las Vegas, NV, Jan. 14, 2020.