Brief Summary of Minutes of Annual Meeting

NCCC215 Breeding and Genetics Technical Committee Meeting

December 9-10, 2019

Chair: Dennis Halterman; Vice-Chair: Laura Shannon; Secretary: Dave Douches

Administrative Advisor: Ray Hammerschmidt

 Agenda

 Monday, December 9

1:00 – Welcome and Introductions

1:15 – Tribute to Creighton Miller

1:20 – 3:15 Research presentations

3:15 – 3:45 Break

3:45 – 5:30 Research presentations

Order of research presentations:

Michigan

Minnesota

North Dakota

Wisconsin

Other contributors

Tuesday, December 10

7:30 – 8:15 SCRI Diploid potato discussion

8:15 – 8:45 Potato pan-genome discussion

8:45 – 9:05 Approve minutes, new business, scheduling 2020 meeting, officer elections, announcements

9:05 – 9:15 Administrative Advisor report

9:15 – 10:30 Breeder presentation

10:30 – 11 Break

11 – 12 Breeder presentations

Minutes of meeting and Research Project report titles

 Michigan

• Sarah Lee – Douches – Overcoming self-incompatibility in diploid potatoes using CRISPR/Cas-9
• Paul Collins – Douches – Introgressing Self-compatibility to Solanum tuberosum dihaploids for diploid variety development
• Thilani Jayakody – Douches -- Gene editing in diploid potato
• Natalie Kaiser – Douches – Deploying durable Colorado potato beetle resistant diploid breeding lines
• Douches – Dihaploid Potato Production at Michigan State University; Application of molecular markers in marker assisted selection in Michigan State Breeding program; Certified Seed Minituber Production
• Will Behling – Interspecific crossing barriers and self-compatibility
• Genevieve Hoopes – Circadian rhythm in potato

 Wisconsin

• Maria Caraza-Harter – Genetics of Skin Set Red Potatoes
• Filipe Matias – FIELDimageR – A tool to analyze othologous data
• Lin Song – diploid potato breeding and the selection for self-compatibility
• Jansky (for Akito Nashiki) – Verification of cold stratification for TPS germination
• Shelley Jansky (for Jim Busse and Paul Bethke) – Fixing red color through inbreeding Red Norland
• Shelley Jansky (for Asma Aikhaja) – Creating dihaploids from Russet Nortotah
• Shelley Jansky – RILS and germplasm releases
• Dennis Halterman – Resistance breaking in PVY
• Dennis Halterman – infestans effector IPI-O1 disruption of RB gene
• Hari Karki – Halterman – Source of late blight resistance in Payette Russet
• John Bamberg – summary of USPG reported above

 Minnesota

• Heather Tuttle – Shannon – Diversity analysis of diploids and tetraploids using GBS data
• Husain Agha – Shannon – Dihaploid Seed Production – Using NIR spec to determine ploidy

North Dakota

• Felicity Merritt – Identification of genes involved in sugar end disorder in 4x potato

Maine

• Oluwafemi Alaba (with Han Tan) – Leveraging haploid induction in tetraploid potato

 Tuesday 12/10/19

Dennis Halterman called the meeting back to order and reminded us of the agenda.

 Shelley Jansky and Jeff Endelman– Diploid breeding grant update

• We were funded starting October 1, 2019
• The objectives were reviewed for the breeding and genetics community

.

Jeff Endelman – and Genevieve Hoopes – PanGenome update

• Genevieve gave a presentation on the assembly of the Atlantic and Castle Russet genomes

 Susie Thompson moved to approve the 2018 minutes and Dave Douches seconded. Unanimously approved.

 It was determined that NCCC215 will meet 12/7-8/2020, at the Hyatt Place, Chicago (same as 2019).

 Elections for secretary, call for nominations: Dennis Halterman nominated Josh Parsons. Josh accepted the nomination. Susie Thompson moved to close nominations. Josh Parsons was unanimously approved.  Laura Shannon will be chair and Dave Douches will be vice-chair.

Announcements

Administrative Advisor Ray Hammerschmidt– Noted that 2020 is the International Year of Plant Health.  He also gave a multistate committee update.  We are due for a mid-term review.  What is evidence of working together?  Collaborations, enhanced education of graduate students and post docs, leadership opportunities, huge attendance and participation at the technical meeting.  We should create an article for the potato industry popular press magazines (e.g. Spudman) describing the meeting.

Breeders Presentations

Jeff Endelman – Wisconsin

Susie Thompson – North Dakota

Laura Shannon – Minnesota

Dave Douches – Michigan

Isabel Vales – Texas A&M

Benoit Bizimungu – Agriculture and Agri-food Canada

Max Feldman – USDA/ARS Prosser, WA

Dennis closed the meeting at noon.

2019 Report for NCCC215

Accomplishments

Endelman (UW-Madison)

Variety development: In 2017, foundation seed of the fresh market russet variety W9133-1rus was released to the potato industry for commercial-scale evaluation. Based on the positive feedback from growers, this variety was named 'Plover Russet' in 2019. We also received notice from the USDA that our PVP application for Red Endeavor (W6002-1R) was approved. Certified seed acreage of the chip processing variety 'Hodag', which was released in 2015, exceeded 100 acres for the first time in 2019.

Research: A new diploid breeding project was initiated in 2016, and 2019 was the first field trial of over 200 dihaploid and F1 clones. QTL analysis allowed us to identify and select against "wild-type" (i.e., late) alleles at the CDF1 locus on Chromosome 5. 

Invited talks: Endelman gave seven invited research seminars in 2019, including at three international locations (Peru, Netherlands, United Kingdom). He also delivered 5 outreach presentations to the potato industry.

Grants: UW-Madison is the lead institution on the 2019 award of $3.0M from USDA-NIFA-SCRI for the project "A new paradigm for potato breeding based on true seed."

Shannon (UMN)

The Shannon lab completed the second field season of the reconstituted Minnesota potato breeding program in summer 2019. They are developing new Minnesota germplasm at the diploid and tetraploid level through crossing, dihaploid extraction, and selecting from unselected families from other programs. Additionally, they are evaluating the legacy material from the previous breeder, Dr. Christian Thill. All 30 remaining legacy cultivars have gone through anti-viral tissue culture and test negative for PVY. They will be evaluated in the 2020 field season. One legacy cultivar, MN13142, a dual-purpose long dormancy russet is in trials with industry this year.

            Shannon delivered two presentations at national scientific conferences and five talks targeted at growers and industry.  

Douches (MSU)

Understanding the components of self-compatibility in Solanum chacoense

The S. chacoense inbred line M6 has been used by a wide variety of potato breeding programs to introgress self-compatibility to diploid germplasm. It is hypothesized that self-compatibility in M6 is primarily conferred by the Sli locus on chromosome 12. We are utilizing a S. chacoense F2 population derived from a cross between M6 and a self-incompatible, but largely homozygous, S. chacoense line 80-1 to examine other genetic factors that may contribute to self-compatibility and examine the environmental stability of this self-compatibility. SNP genotyping of 325 self-compatible F2 individuals revealed distorted segregation not only on chromosome 12 but also on chromosome 1, which harbors the S-locus. An additional 200 unselected F2 individuals will be grown and selfed in two locations: Michigan State University and University Wisconsin Madison. Historically, fruit and seed set have been used as an indicator of self-compatibility. However, these phenotypes are confounded by other fertility traits. To directly examine and quantify the compatibility reaction, we will conduct stylar analysis of pollen tube growth using a protocol that has been optimized in the Douches lab. The fact that F5 lines derived from this F2 population are still segregating for self-compatibility is further evidence that multiple genes may be involved. Residual heterozygosity observed in the F5 generation, specifically enriched on chromosomes 12 and 8, suggests that heterozygosity at these loci may be necessary for survival. Thus, in addition to phenotyping, we have cloned and sequenced several candidate genes, including SRNase, hypothesized to be involved in self compatibility in the F2 population. Understanding the genetic landscape of self-compatibility in S. chacoense M6 will facilitate the efficient development of diploid inbred lines. 

Natalie Kaiser

The Implications of Endosperm Balance Number and Self-Compatibility on Inter specific Crosses.

Endosperm Balance Number cannot accurately categorize crossing behavior between species, as crossing behavior is species pair specific. The failure of species crosses has also been confounded by prezygotic barriers such as S-RNase, so S-RNase knock-outs could allow previously improbable crosses in the future. It is necessary to understand the exact specifics on why species crosses fail. EBN should not be used as a catch-all explanation of why interspecific incompatibility exists between species.

Will Behling

De Novo Assembly of DM1S1

Based on its high regeneration rate, self-compatibility and quality tuber traits, the diploid line DM1S1 from Virginia Tech University has ideal characteristics for use in genome editing. The next step is creating a genomic resource to assist in designing guides and screening for off target effects. To do this, I am using a hybrid assembly approach combining long DNA reads from Oxford Nanopore Technologies using the MinION and short reads using Illumina HiSeq4000.

Thilani Jayakody

Diploid Potato Breeding at MSU

Self-compatibility from diploid potatoes can be harnessed towards developing inbred lines to serve as parents for an F1 hybrid potato line development system. Work to introgress self-compatibility into breeding lines with good tuber traits and high yield was presented. A summary of the different types of crosses, and some of the best breeding lines was also presented. A section was presented which outlined the future plans for diploid potato breeding at MSU.

Paul Collins

Investigating the Role of the Circadian Clock in Potato Domestication. 
             

The circadian clock is composed of an endogenous oscillator which is regulated by environmental inputs which in turn drives the rhythmicity of molecular processes (e.g. gene expression over 24 hours) and corresponding physiological responses (e.g. leaf movement). No circadian period length difference was observed during potato domestication, in contrast to other crops analyzed, and we have developed a mapping population to assess the genetic architecture of the circadian period trait. We also developed time course transcriptomic data and luciferase reporter constructs to characterize downstream genes and pathways which are regulated by the circadian clock in potato. 

Atlantic and Castle Russet NRGene Genome Assembly QC.

Genome assemblies were generated by NRGene for two North American potato varieties, and basic quality control and preliminary anchoring of the scaffolds into haplotype resolved pseudomolecules has been conducted. While the gene content of both assemblies is nearly complete, many genomic regions only have 3 scaffolds instead of 4 scaffolds present. We’ve found that in many of these regions, the haplotypes were collapsed into a single scaffold potentially due to high nucleotide similarity between the haplotypes. Currently, we are working on anchoring the scaffolds into haplotype resolved pseudomolecules using phased GBS and HiC data. 
Genevieve Hoopes

Generation of Self-Compatible Diploid Potato Via CRISPR-Cas9 Genome Editing

Development of self-compatible diploid potato lines is useful to the potato community in that these lines could help fix desirable alleles and create inbred lines that could later be used to take advantage of heterosis. The Gametic Self-Incompatibility (GSI) model has been well established in Solanaceae and shows that S-RNase is a major factor in self-incompatibility in potato lines. We hypothesize that there are other major factors that contribute to self-incompatibility; therefore we are targeting two genes in concert with S-RNase with CRISPR-Cas9 to establish robust self-compatible lines. We are also working to understand why M6, a Solanum chacoense line, is naturally self-compatible. 

Sarah Lee

Jansky (USDA-ARS)

Germplasm Releases

USDA-Madison

Halterman (USDA-ARS)
Molecular markers linked to Verticillium wilt resistance in potato germplasm

Verticillium wilt (VW) of potato (Solanum tuberosum), caused by two different soil-borne fungi Verticillium albo-atrum R & B or V. dahliae Kleb., is a major limiting factor in potato production throughout North America. Yield losses in potato associated with the disease can reach up to 50% in severely infested fields. In tomato, resistance to race 1 of Verticillium dahliae is conferred by a dominant Ve gene that has been exploited in breeding programs from more than 50 years. However, previously developed markers within the Ve gene in potato are unreliable in predicting resistance. The goal of this project is to identify additional genomic regions that determine VW resistance in potato. An F2 mapping population was developed by selfing an F1 individual derived from two homozygous diploid parents, S. tuberosum DM1-3 (susceptible to VW) and S. chacoense M6 (resistant to VW). Using a rooted cutting protocol, the population was phenotyped and SNP genotyped. A major QTL in chromosome 1 was identified that explains 31% of the phenotypic variation. A total of 22 genes are located within the QTL region, and two genes have been selected for further functional validation studies. Using the sequence information of these two genes, are developing markers to distinguish between resistant and susceptible germplasm.  The marker information will be a valuable tool for potato breeders interested in selecting for VW resistance.

Molecular interactions that influence virulence contributions of the IPI-O family of Phytophthora infestans effectors

Phytophthora infestans, causal agent of potato late blight, is a destructive pathogen that is a frequently recurring problem worldwide. Several resistance genes exist in potato to counter against this pathogen, but the majority have been overcome after introgression into popular potato varieties. The RB gene, derived from Solanum bulbocastanum, has effector recognition specificity to members of the IPI-O family. Recognition of the IPI-O1 allele by RB elicits a hypersensitive resistance response while IPI-O4 can suppress this response. We have carried out several experiments to determine the virulence contributions of IPI-O1 and IPI-O4 during infection, and to identify host proteins involved in IPI-O recognition/suppression using co-immunoprecipitation and yeast two-hybrid. Our results indicate that both IPI-O1 and IPI-O4 contribute to P. infestans virulence, but their impact is influenced by the pathogen genotype. Protein interaction studies have identified both cytosolic- and membrane-localized host proteins that interact with IPI-O and will help to elucidate the function of these effectors in pathogen virulence. Together, we hope that our understanding of the function of the ubiquitous IPI-O effector will assist us in identifying or developing improved host resistance genes in potato. This work has been included in two publications (Chen and Halterman, 2017a; Chen et al., 2017b)

Foliar resistance to bacteria in potato

Solanaceous crops including tomato, pepper, and eggplant are susceptible to many foliar bacterial pathogens. However, cultivated potato is immune to most pathogenic Pseudomonas and Xanthomonas species. The purpose of this project is to understand the mechanisms involved in limiting infection of bacteria in the foliage of potato. We have previously found that many popular potato cultivars are immune P. syringae pv. tomato DC3000, while many wild species accessions of potato are susceptible or tolerant, suggesting that resistance to foliar pathogens may have been selected during cultivation of potato as a food crop. We have used various DC300 strains defective in pathogenesis. Populations between wild and cultivated potato have also been developed to map the resistance locus using SNP genotyping. The identification of novel genes involved in bacterial resistance will facilitate the development of new varieties of Solanaceous crops.

Development of diploid potato germplasm containing disease resistance

            Using a combination of diploid wild species hybrids and dihaploid cultivated germplasm from the programs of David Douches (Michigan State) and Shelley Jansky (USDA-ARS), we have developed populations segregating for resistance to potato late blight and early blight. Seventeen families from crosses between various parents were phenotyped using detached leaflet assays and whole plant inoculation assays (greenhouse) to identify individuals with increased resistance to either or both diseases. Three families contained individuals with high levels of late blight resistance and one family had individuals with increased early blight resistance. Selected individuals were grown in the field and assayed for agronomic characteristics including tuber size and shape, specific gravity, and chip quality. Three individuals were chosen for crossing with diploids containing resistance to other diseases, including PVY and Verticillium wilt.

Bamberg (USDA-ARS)

We are organizing to propose a grant for combining phenotype, metabolites, and genetic markers for segregating pops we made resulting from past CGC projects on Dickeya and Zebra chip.

A resource for the above is two CETS phytotron growth chambers for tuberizing we are installing at USPG.  In these we hope to propagate replicate genotypes for multifaceted evaluation in several locations without incurring the great effort and cost of in vitro maintenance, yet with very high phytosanitary security.  We expect these phytotrons to have many other applications that need uniform tuber samples for screening tuber traits.

We started a big archeology project with UT and OH collaborators.  Why should the genebank be involved in archeology?  USPG will benefit from a better understanding of factors that predict patterns of genetic diversity in the wild including the activities of ancient humans.  Collateral benefit is getting screening data on tuber size, frost tolerance, glycoalkaloids.

We previously reported a “MegaPop” for S.jamesii, a population from one place in the wild that captures most of the genetic diversity of the species.  That is the ultimate core subset!  Now we are pursuing identification of a parallel MegaPop for S. fendleri, the second species found in the US.

Using S. jamesii through S. verrucosum bridge crosses.  We took a good-flowering ver and made BC5 into tbr cytoplasm, selecting for self-incompatibility and good growth and flowers.  Now it crosses directly to S. jamesii without emasculation or double pollination with IvP.  We are going to repeat the whole experiment systematically this spring to confirm metrics of crossing success, including crosses with the other 1EBN species.

For the golden-fleshed specialty type Criolla, we created and selected S3 inbred families and a synthetic population both with high proportions of dark golden flesh and improved yield and type.  Preparing for S4 evaluation and selection next season.

Started core subset development of 92 populations of S. kurtzianum, including screening for differences in root vigor, fertilizer efficiency and drought tolerance.

Assessed inhibition of US native potatoes by invasive cheatgrass.  Preliminary results suggest cheatgrass inhibits sprouting of tubers, but not growth of potato shoots.  This study was first aimed at assessing how threatened USA native potato populations might be in the wild.  But if cheatgrass roots produce a safe, natural effective sprout inhibitor, it might have a valuable application for commercial storage.

Peru collaborative breeding and research continues.  Second named cultivar release pending.  New seedlots being bred and sent to Peru target high tuber calcium, frost and drought tolerance.   

Susie Thompson (North Dakota State University)

Seventy-six genotypes were used as parents in hybridizing efforts in 2019; 213 new families were created.  Parental germplasm included named cultivars and advancing selections.  Traits of focus included processing (chip and frozen), fresh market quality, PVY, late blight, Colorado Potato Beetle, and Verticillium wilt resistance.

In 2019, irrigated trial sites were at Inkster, Larimore and Oakes, ND, and at Park Rapids, MN.  Three trials were planted at Inkster.  The metribuzin sensitivity screening trial was conducted in collaboration with Dr. Harlene Hatterman-Valenti’s program.  Trial results are being used to validate the model developed by a previous graduate student.  The sugar end screening trial was the second year of Felicity Merritt’s thesis research.  A new trial in 2019 was in response to ND certified seed growers concern about efficient vine kill after repeated use of mineral oils in aphid management, in collaboration with Drs. Gary Secor and Andy Robinson.  Eight vine-kill scenarios were evaluated.  The Larimore trial site included the Processing Trial (20 selections, cultivars and industry standards), the National French Fry Processing trial (46 selections compared to Russet Burbank and Ranger Russet; six were NDSU advancing selections), the preliminary processing trial with 58 entries (advancing dual-purpose russet selections compared to industry standards), an irrigate preliminary chip process trial (106 genotypes), and maintenance of out-of-state selections.  We were unable to harvest this site due to heavy rains, snow and freezing temperatures in September and October.  Trials at Oakes were focused on fresh market selections and 16 promising dual-purpose russet selections compared to industry standards; common scab was not as prevalent in the fresh market genotypes as in previous years.  Trials at Park Rapids, MN, included a processing trial with 15 entries, the common scab screening trial with 64 entries across market types, and the replicated screening trial for Verticillium wilt resistance (25 genotypes across market types) conducted in collaboration with Dr. Neil Gudmestad’s program.  Bannock Russet and Dakota Trailblazer continue to be the most resistant genotypes to Verticillium based on colony forming units of stem tissue collected right before vinekill/harvest.  Promising advancing processing russet selections include ND12108CAB-3Russ, ND12109CB-2Russ, ND13103B-1Russ, ND13245C-4Russ, ND13252B=6Russ, and ND13252B-12Russ, amongst others.

Non-irrigated research sites included Crystal and Hoople ND.  The Fresh Market trial had 30 entries, while the preliminary fresh market trial included 90 entries.  Several fresh market selections look very promising, including ND1232B-2RY, ND1241-1Y, ND102663B-3R, ND081571-2R, ND081571-3R, ND102990B-2R, and ND113091B-2RY. Chip processing trials were located north of Hoople, and included the Chip Processing Trial included 22 advancing chip selections compared to chip industry standards.  The Preliminary chip processing trial evaluated 30 selections and industry standards, and the National Chip Processing Trial (NCPT), included 98 unreplicated selections (Tier 1) and 22 replicated entries (Tier 2) from US potato breeding programs, compared to five industry chip selections.  Outstanding chip selections coming through the program include ND7519-1, ND7799c-1, ND102642C-2, ND102922C-3, ND113307C-3, ND1221-1, ND12180ABC-8, ND13228AB-3, ND14348AB-1, ND14437CAB-1, ND14437CAB-2, and many others.  The non-irrigated sites were hampered by a lack of rainfall during summer 2019, with significant rain developing in early September.  Late blight screening trials at Prosper, ND conducted in collaboration with Dr. Secor’s program were drown out in early June by excessive rain.

The seedling nursery, seed maintenance plots, and increase lots were planted south of Baker, MN.  All lots were entered for certification with the Minnesota Department of Agriculture and passed certification; all were submitted for winter testing.  The seedling nursery included single hills from NDSU (115 families) and out of state cooperators; 724 single hills were selected.  Of 776 second year selections 233 were retained; 40 of 118 third and 146 of 252 fourth year and older selections were saved.  Production from the seed maintenance and increase lots is used to maintain the genotypes via phenotypic recurrent selection and is the seed source for our research and collaborative trials at NDSU, and research and industry collaborators in ND, MN, and beyond.  As in previous years, several Chilean selections from the INIA program at Osorno, Chile, were evaluated in collaboration with Drs. Gary Secor and Julio Kalazich.  Three hundred-one genotypes were submitted for SNP genotyping in 2019.

Grants awarded in 2019

Publications

Alsahlany, M., D. Zarka, J. Coombs, and D. Douches. 2019. Comparison of methods to distinguish diploid and tetraploid potato for applied diploid breeding. American Journal of Potato Research.  https://doi.org/10.1007/s12230-018-09710-7.

Caraza-Harter MV, Endelman JB (2019) Image-based phenotyping and genetic analysis of potato skin set and color. Crop Science, doi:10.2135/cropsci2019.07.0445

Enciso-Rodriguez F, Manrique-Carpintero NC, Nadakuduti SS, Buell CR, Zarka D and Douches D (2019) Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9. Front. Plant Sci. 10:376. doi: 10.3389/fpls.2019.00376.

Fulladolsa, A.C., A. Charkowski, X. Cai, J. Whitworth, S. Gray, and S.H. Jansky. 2019. Germplasm with Resistance to Potato Virus Y Derived from Solanum chacoense: Clones M19 (39-7) and M20 (XD3). American Journal of Potato Research. 96:390-395.

Jansky, S., Haynes, K. & Douches, D. Am. J. Potato Res. (2019) Comparison of Two Strategies to Introgress Genes for Resistance to Common Scab from Diploid Solanum chacoense into Tetraploid Cultivated Potato. https://doi.org/10.1007/s12230-018-09711-6.

Satya Swathi Nadakuduti, Colby Starker, Dae Kwan Ko, Thilani B. Jayakody, C. Robin Buell,

Daniel F. Voytas and David S. Douches (2019). Evaluation of methods to assess in vivo activity

of engineered genome-editing nucleases in protoplasts. Frontiers in Plant Science, 10: 110.

Satya Swathi Nadakuduti, Colby Starker, C. Robin Buell, Daniel F. Voytas and David S.

Douches (2019) “Genome editing in potato with CRISPR/Cas9”, Plant Genome Editing with

CRISPR Systems: Methods and protocols, Methods in Molecular Biology, Springer Nature

1917: 183-201 (Book chapter).