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
NEW
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, 2023
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 09/01/20 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.