DEVELOPMENT AND APPLICATION OF MOLECULAR MARKERS RELATED TO THE IMPROVEMENT OF POTATOES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006784
• Project Start Date: Jul 3, 2015
• Project End Date: Jun 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803

Performing Department
Plant Soil & Entomological Sci

Non Technical Summary
Research proposed here will develop and apply molecular markers in potato and wheat. Development includes identifying new markers and improving existing markers, as well as finding new uses for existing markers. New markers will be identified through mapping projects and through utilization of marker and sequence databases. Improvement of markers will attempt to modify existing protocols to increase efficiency and lower cost and in some cases identify alternative markers that might be more suitable. This process also includes testing of new protocols to improve functionality. One goal is to identify molecular markers that can take advantage of the polyploidy nature of potato and wheat. New technologies will be evaluated and utilized to increase the functionality of existing and new markers. Molecular markers can be used for many purposes and will be developed with these uses in mind. Markers can aid in the identification of plants with a particular gene of interest, and also identify plants with more than one gene. They also increase our understanding of complex traits. Ideally these applications will lead to better potato and wheat lines for growers.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1310	1080	100%

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

Subject Of Investigation
1310 - Potato;

Field Of Science
1080 - Genetics;

Keywords

potato

solanum tuberosum

wheat

molecular markers

gene

allele

mapping

resistance

germplasm

Goals / Objectives
1) Apply existing molecular markers for tagging valuable traitsMap and tag qualitative traits (potato, PVY resistance)*Map and tag quantitative traits (potato, G. pallida resistance)2) Develop new molecular markersDevelop user (laboratory) friendly markers (potato, PVY resistance)Identify markers with functionality in a wide diversity of germplasmIdentify tightly linked markers (potato, PLRV resistance)3) Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles (potato, Ryadg, Rysto, and Rx1/Gpa2)* Information in parenthesis indicates potential areas of research (crop, project)

Project Methods
Objective 1: Apply existing molecular markers for tagging valuable traits Objective 1a: Map and tag qualitative traitsThis objective pertains to mapping phenotypes of interest that have not been placed on a molecular linkage map. In the case of PVY resistance the population will be generated in house, while in the case of resistance to G. pallida the populations will be generated outside the University of Idaho (see objective 1b). In collaboration with the Karasev lab we will develop a Yukon Gem x Russet Norkotah mapping population to investigate Ny resistance genes recently identified in Yukon Gem. The hypersensitive response (HR), conferred by N genes in potato, is characterized by limited replication of a virus producing local necrotic lesions in an inoculated leaf. Individual seeds from a Yukon Gem by Russet Norkotah cross will be sterilized and put into tissue culture. Seedlings will be propagated and maintained in tissue culture so that a single population can be utilized when testing up to eight different PVY isolates, including but not exclusively PVYNA-N, PVYE, PVY-NE11, and PVYN-Wi. Phenotyping will be coordinated with the Karasev lab. DNA will be isolated from individual progeny and applied to Infinium arrays (SolCAP chip) which can be analyzed with Illumina iScan or HiScanSQ systems (for example at Michigan State University). Once raw data is generated, Illumina GenomeStudio software will be used to process the resulting intensity data for SNP calling. To determine SNP genotypes a cluster file will be developed by TraitGenetics (or similar software) and markers classes manually evaluated for accuracy. Multiple software packages will be used for mapping of the markers, e.g. JoinMap 4.0, Map Manager QTXb20 and MapChart 2.2. Segregation patterns will be verified prior to formation of linkage groups and positioning of markers on chromosomes. Potato-virus interactions will be placed on the map, and genomic regions evaluated for potential candidate genes. Objective 1b: Map and tag quantitative traits Preliminary results indicate the cultivar Eden is resistant to G. rostochiensis race Ro1, G. pallida race Pa2/3, and G. ellingtonae. An Eden x Western Russet cross (Dr. Rich Novy) has produced enough true seed for development of a population of at least 200 clones for mapping. Eden x Western Russet seed will be made available to the Kuhl lab for seed sterilization and establishment in tissue culture. Seedlings will be propagated and maintained in tissue culture so that a single population can be utilized when testing with all three Globodera species, G. rostochiensis, G. pallida, and G. ellingtonae. Tissue culture plantlets will be shipped to Oregon and New York for screening against G. ellingtonae, and G. rostochiensis, respectively, and will be made available to Dr. Dandurand at the University of Idaho for screening with G. pallida. DNA will be isolated from individual progeny and applied to Infinium arrays which can be analyzed with Illumina iScan or HiScanSQ systems (for example at Michigan State University). Linkage analysis will be conducted similar to that described in objective 1a. Depending on resources an alternative strategy will map quantitative trait loci using bulked segregant analysis using next-generation sequencing. For this strategy DNA from the most resistant and susceptible individuals, 15-20% of the total population, will be pooled for sequencing and later identification of quantitative trail loci.Objective 2: Develop new molecular markers Objective 2a: Develop user friendly markers In most cases the development of user friendly markers involves the use of a PCR-based approach, such that fragment variation can be identified on an agarose horizontal gel, i.e. fragment sizes in the range of 300-800 base pairs (bp). Primer design will take advantage of inherent sequence differences to generate a presence/absent phenotype, or amplify fragments of different sizes. In cases where fragment size is monomorphic, restriction enzymes may take advantage of sequence differences to cleave some fragments and leave others intact, thus generating fragments of different sizes. To this end, it may be useful to clone and sequence the parental markers and identify sequence differences. These differences can be taken advantage of by primer design or enzyme application. In cases where the conversion of existing markers fails it may be possible to utilize sequence from these markers to identify homologous regions in Solanum sequence data, i.e. tomato or potato genomic sequence. Once a homologous region is identified flanking sequence can be mined for possible marker development. Coding regions offer the most likely possibility for primer design, since these regions are most likely to be conserved. When possible, introns will be included in the amplicon to increase the likelihood of identifying polymorphisms. Objective 2b: Identify markers with functionality in a wide diversity of genotypes This objective may directly overlap with objective 2c. In many cases, the inability of a linked marker to be diagnostic for a particular phenotype is excessive recombination between the marker and the target locus. Sequence analysis similar to that in objective 2a may allow for genomic sequence to be recovered from ongoing sequencing projects in tomato and potato. Comparison of homologous regions from tomato and potato may suggest coding regions more likely to amplify from diverse germplasm. Lack of sequence data will require the screening of additional markers from the chromosome region in question (see objective 2c). Objective 2c: Identify tightly linked markers Many different factors may contribute to the inability to identify more closely linked markers, including the size and structure of the mapping population. For the purpose of identifying more closely linked markers bulk segregant analysis (BSA) has been shown to be an efficient way to streamline the process. Although many variations of BSA have been used, the basic concept involves pooling progeny with like phenotypes (resistance or susceptibility) for combined analysis. This might be done to streamline molecular marker screening to identify polymorphic or linked markers. By combining BSA with next generation sequencing it might be possible to rapidly identify markers linked to Rlretb conveying resistance in potato to PLRV (see objective 1b). In the case of Rlretb an alternative strategy will be to exploit genomic sequence data collected from bacterial artificial chromosomes (BACs) surrounding the Rlretb locus to develop more closely linked markers.Objective 3: Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status (≥2 alleles)Cleaved amplified polymorphic sequence (CAPS) markers will be identified in the literature linked to genes of interest in potato including, Ryadg, Rysto, and Rx1/Gpa2. These CAPS markers will be reproduced in a small number of lines and fragments will be cloned from resistant and susceptible lines. Approximately thirty cloned fragments will be sequenced from each marker. Particular attention will be paid to sequencing fragments from susceptible lines to capture as much genetic diversity as possible. Sequences will be aligned and analyzed with design software provided by Qiagen PyroMark to develop pyrosequencing assays to detect resistant alleles. The highest scoring assays will be selected and PCR primers ordered. Each primer set will be optimized to maximize fragment intensity and minimize/eliminate any secondary fragments. Those assays that meet these criteria will be further tested on progeny from a segregating population, and if possible several populations. Successful primers will be moved into the validation phase where pyrosequencing will be used to evaluated progeny with and without resistant alleles.

Progress 07/03/15 to 06/30/20

Outputs
Target Audience:The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems:COVID-19 has slowed research in the lab due to social distancing and limiting the number of lab personnel present at one time. What opportunities for training and professional development has the project provided?Attend Idaho Potato Conference, Pocatello, ID, January 2020 Attend The Potato Association of America 104th Annual Meeting, virtual, July 2020 How have the results been disseminated to communities of interest?Idaho Potato Conference, Pocatello, ID, January 2020 "Impact of PCN" "Pale cyst nematode research update" USDA, APHIS Potato Cyst Nematode review meeting, Pocatello, ID, January 2020 "GLOBAL breeding objective" "Immunity to PCN - part 2" "Breeding for PCN Resistance - Part 3" "Sterile Litchi Tomato" Potato Expo, Las Vegas, NV, January 2020 "Potato cysts nematode resistance: Efforts and successes of the GLOBAL grant working group" What do you plan to do during the next reporting period to accomplish the goals? Mapping of loci associated with G. pallida resistance in the Eden x Western Russet population will continue. This work should be completed during 2021. Additional progeny will be evaluated for PVY resistance in the Yukon Gem x Russet Norkotah population using different PVY isolates.

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits Numerous potato cultivars and breeding lines have been acquired with resistance to one or more Globodera species, potato cyst nematodes. Molecular marker 57R is being used to assess the presence of H1, which provides resistance to Ro1 and Ro4 pathotypes of G. rostochiensis. Contig 237 is being evaluated for the presence of GpaIVadg, which provides partial resistance to G. pallida. Molecular marker HC is being used to assess the presence of GpaV, which also provides resistance to G. pallida. Two additional markers on potato chromosome 5, SPUD 5000 and SPUD 1636, may be linked to Grp1, associated with resistance to both G. pallida and G. rostochiensis. Gro1-4, has also been applied, and is associated with partial G. rostochiensis resistance. In addition to breeding lines, markers have been applied to a number of populations including Eden x Western Russet, Performer x Basin Russet, NYW 69, NYW 72 and NYW 73. Objective 2. Develop new molecular markers. Mapping of PCN resistance in Eden x Western Russet will help identify QTL associated with partial resistance (Obj. 3). Genomic information from regions surround QTL will be used to develop molecular markers. ?Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. DNA from a tetraploid population segregating for G. pallida resistance has been extracted from 234 individuals and submitted for genotyping with the potato SNP chip version 3. DNA has also been extracted from 166 individuals in a population segregating for PVY necrotic lesion development and genotyped with the potato SNP chip version 3. Analysis of genotypic data is ongoing for both populations. SNP chip analysis allows, at many loci, to determine tetraploid segregation ratios. Association between resistance and marker data for G. pallida resistance is ongoing, while additional phenotypic data is being collected for PVY resistance.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wixom, A.Q., N.C. Casavant, T.J. Sonnen, J.C. Kuhl, F. Xiao, L-M. Dandurand, and A. Caplan. (2020) Initial responses of the trap-crop, Solanum sisymbriifolium, to Globodera pallida invasions. The Plant Genome. 13:e20016.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Levy, J.G., R. Gross, M.A.M. Herrera, X. Tang, K. Babilonia, L. Shan, J. Kuhl, M. Dibble, F. Xiao, and C. Tamborindeguy. (2020) Lso-HPE1, and effector of Candidatus Liberibacter solanacearum can repress plant immune response. Phytopathology. 110:648-655.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems:Loss of college funded technical staff scientist position (to another position), and subsequent withdrawal of permanent position funding. Another full-time position left for a career track job out-of-state. Both departures have slowed progress and how much can be achieved. What opportunities for training and professional development has the project provided?Attend Idaho Potato Conference, Pocatello, ID, January 2019 Attend The Potato Association of America 103rd Annual Meeting, Winnipeg, Manitoba, July 2019 How have the results been disseminated to communities of interest?Poster at The American Phytopathological Society Annual Meeting - Plant Health 2019, Cleveland, OH, August, 2019 "Molecular characterization of recombinant Potato virus Y (PVY) in potato from South Korea, PVYNTN strain" USDA, APHIS Potato Cyst Nematode review meeting, Pocatello, ID, March 2019 "PCN Immunity: Cloning & Testing of Selected Anti-Nematode Defense Genes" "Breeding for PCN Resistance" Globodera Alliance (GLOBAL) research meeting, Moscow, ID, October, 2019 "Breeding for PCN Resistance" Globodera Alliance (GLOBAL) Advisory Board meeting, Moscow, ID, October, 2019 "GLOBAL Breeding Objective" What do you plan to do during the next reporting period to accomplish the goals? Mapping of loci associated with G. pallida resistance in the Eden x Western Russet population will continue. This work should be completed during 2020. Additional progeny will be evaluated for PVY resistance in the Yukon Gem x Russet Norkotah population using different PVY isolates.

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits Numerous potato cultivars and breeding lines have been acquired with resistance to one or more species Globodera, potato cyst nematodes. Many of these lines have been screened to assess the presence/absence of previously identified molecular markers. Molecular marker 57R is being used to assess the presence of H1, which provides resistance to Ro1 and Ro4 pathotypes of G. rostochiensis. Contig 237 is being evaluated for the presence of GpaIVadg, which provides partial resistance to G. pallida. Molecular marker HC is being used to assess the presence of GpaV, which also provides resistance to G. pallida. Two additional markers on potato chromosome 5, SPUD 5000 and SPUD 1636, have been applied as well as molecular marker 221R on chromosome 12, all associated with G. pallida resistance. Gro1-4, has been applied, associated with partial G. rostochiensis resistance. In addition to breeding lines, markers have been applied to a number of populations including Eden x Western Russet, Performer x Basin Russet, NYW 69, NYW 72 and NYW 73. Objective 2. Develop new molecular markers. BAC sequence data from S. etuberosum is helping to define the physical map of the Rlretb region associated with PLRV resistance. Sixteen coding regions have been identified and their sequence verified. This data will help develop new molecular markers and identify candidate genes associated with Rlretb. ?Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. DNA from a tetraploid population segregating for G. pallida resistance has been extracted from 234 individuals and submitted for genotyping with the potato SNP chip version 3. DNA has also been extracted from 166 individuals in a population segregating for PVY necrotic lesion development and genotyped with the potato SNP chip version 3. Analysis of genotypic data is ongoing for both populations. SNP chip analysis allows, at many loci, to determine tetraploid segregation ratios. Association between resistance and marker data for G. pallida resistance is ongoing, while additional phenotypic data is being collected for PVY resistance.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kud, J., W. Wang, Y. Yuan, A. Caplan, J.C. Kuhl, L-M. Dandurand, and F. Xiao (2019) Functional Characterization of RING-Type E3 Ubiquitin Ligases in vitro and in planta. Journal of Visualized Experiments. 154:e60533. Dandurand, L.-M., I.A. Zasada, X. Wang, B. Mimee, W. DeJong, R. Novy, J. Whitworth, and J.C. Kuhl. (2019) Current Status of Potato Cyst Nematodes in North America. Annual Review of Phytopathology. 57:117-133. Kooliyottil, R., L-M. Dandurand, J.C. Kuhl, A. Caplan, F. Xiao, B Mimiee, and J. Lafond-Lapalme (2019) Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium. Scientific Reports. 9:13256. Kud, J., W. Wang, R. Gross, Y. Fan, L. Huang, Y. Yuan, A. Gray, A. Duarte, J.C. Kuhl, A. Caplan, A. Goverse, Y. Liu, L-M. Dandurand, F. Xiao (2019) The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling. PLOS Pathogens. 15:e1007720.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development.?The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?AttendIdaho Potato Conference, Pocatello, ID, January 2018 Attend10thWorld Potato Congress in Cusco, Peru, May 2018 Attend The Potato Association of America 102ndAnnual Meeting, Boise, ID, July 2018 How have the results been disseminated to communities of interest?Present posters at 10th World Potato Congress in Cusco, Peru, May 2018 "Globodera Alliance: (GLOBAL): Risk assessment and eradication of Globodera spp. in the U.S. Production of Potato" (lead author) "Seeking host resistance in potato to three Globodera species" (co-author) "Breeding and development of Globodera-resistant potato varieties with long tuber shape and russet skin for production in the western United States" (co-author) Present at The Potato Association of America 102nd Annual Meeting, Boise, ID, July 2018 "Use of Molecular Markers for Breeding PCN Resistance in the Russet Market Class" "Introgression of Globodera Resistance into the Russet Market Class" (co-author) "Early Defense Response of Solanum sisymbriifolium to Globodera pallida Infection" (co-author) "Identification of Molecular Strategies to Combat Pale Cyst nematode in Potato" (co-author) Idaho Potato Conference, Pocatello, ID, January 2018 "PCN Resistance" USDA, APHIS Potato Cyst Nematode review meeting, Pocatello, ID, January 2018 "Developing Resistance in U.S. Potato Cultivars" "Immunity to PCN - Part 2" "Sterile Litchi Tomato" What do you plan to do during the next reporting period to accomplish the goals?Now that BAC sequence data has generated a physical map both sequence and lab analysis will attempt to resolve the region near Rlretb. Depending of the extent of sequence coverage, additional BACs may be identified for sequencing. Sequence from S. etuberosum will be evaluated for evolutionary change by comparing it to potato and tomato sequence from homologous regions on chromosome 4. Additional markers might be developed and evaluated for linkage to Rlretb. Seven existing markers associated with Globodera resistance are currently being used, additional molecular markers will be identified and evaluated for potential use.

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits Numerous potato cultivars and breeding lines have been acquired with resistance to one or more species Globodera, potato cyst nematodes. Many of these lines have been screened to assess the presence/absence of previously identified molecular markers. Molecular marker 57R is being used to assess the presence of H1, which provides resistance to Ro1 and Ro4 pathotypes of G. rostochiensis. Contig 237 is being evaluated for the presence of GpaIVadg, which provides partial resistance to G. pallida. Molecular marker HC is being used to assess the presence of GpaV, which also provides resistance to G. pallida. Two additional markers on potato chromosome 5, SPUD 5000 and SPUD 1636, have been applied as well as molecular marker 221R on chromosome 12, all associated with G. pallida resistance. Gro1-4, has been applied, associated with partial G. rostochiensis resistance. Objective 2. Develop new molecular markers. Potato cultivars Yukon Gem and Russet Norkotah, as well as a small number of progeny, were evaluated for their allelic composition of eIF4E, which has been associated with recessive virus resistance. Unfortunately results suggest that eIF4E alleles do not appear to be play a significant role in PVY resistance, as expressed in these clones. BAC sequence data from S. etuberosum is helping to define the physical map of the Rlretb region associated with PLRV resistance. Sixteen coding regions have been identified and their sequence verified. This data will help develop new molecular markers and identify candidate genes associated with Rlretb. Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. DNA from a tetraploid population segregating for G. pallida resistance has been extracted from 234 individuals and submitted for genotyping with the potato SNP chip version 3. DNA has also been extracted from 166 individuals in a population segregating for PVY necrotic lesion development and genotyped with the potato SNP chip version 3. Analysis of genotypic data is ongoing for both populations. SNP chip analysis allows, for many loci, to determine tetraploid segregation ratios. Association between resistance and marker data will occur after more progeny have been evaluated.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Whitworth, J.L., R.G. Novy, I.A. Zasada, X. Wang, L-M. Dandurand, and J.C. Kuhl (2018) Resistance of potato breeding clones and cultivars to three species of potato cyst nematode. Plant Disease. 102:2120-2128.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chikh-Ali, M., M. Rodriguez-Rodriguez, K.J. Green, D-J. Kim, S-M. Chung, J.C. Kuhl, and A.V. Karasev (2019) Identification and molecular characterization of recombinant Potato virus Y (PVY) in potato from South Korea, PVYNTN strain. Plant Disease. 103:137-142.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Attend and participate in the Annual meeting of the Potato Association of America, in Fargo, North Dakota, July 2017, and WERA89 in San Diego, California. How have the results been disseminated to communities of interest? A talk was given at the Idaho Potato Conference, Pocatello, Idaho, in January 2017, titled Developing PCN resistance in US potato cultivar. Authors included J. Whitworth, R. Novy, J. Kuhl and W. DeJong. A poster was presented titled Screening potato for resistance to three potato cyst nematode species, at the 2017 Potato Expo in San Francisco, California. Authors included J. Whitworth, R.G. Novy, J.C. Kuhl, L-M. Dandurand, I. Zasada and X. Hong. (poster presented by M. Thornton) A talk was given at WERA89, San Diego, California, in March 2017, titled Response to PVY in a Segregating Population. Authors included J.C. Kuhl, A. V. Karasev, S. Struble, C.N. Funke, and M. Chikh-Ali. What do you plan to do during the next reporting period to accomplish the goals? Now that BAC sequence data has generated a physical map both sequence and lab analysis will attempt to resolve the region near Rlretb. Depending of the extent of sequence coverage, additional BACs may be identified for sequencing. Sequence from S. etuberosum will be evaluated for evolutionary change by comparing it to potato and tomato sequence from homologous regions on chromosome 4. Additional markers might be developed and evaluated for linkage to Rlretb. Four existing markers associated with Globodera resistance are currently being used, additional molecular markers will be identified and evaluated for potential use.

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits Numerous breeding lines have been acquired with resistance to one or more species of Globodera, potato cyst nematodes. These lines are currently being screened to assess the presence/absence of previously identified molecular markers. Molecular marker 57R is being used to assess the presence of H1, which provides resistance to Ro1 and Ro4 pathotypes of G. rostochiensis. Contig 237 is being evaluated for the presence of GpaIVadg, which provides partial resistance to G. pallida. Molecular marker HC is being used to assess the presence of GpaV, which also provides resistance to G. pallida. One additional marker, Gro1-4, is being investigated and is associated with G. rostochiensis resistance. Objective 2. Develop new molecular markers. BAC sequence data from S. etuberosum is helping to define the physical map of the Rlretb region associated with PLRV resistance. Sixteen coding regions have been identified and their sequence verified. This data will help develop new molecular markers and identify candidate genes associated with Rlretb. Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. DNA from a tetraploid population segregating for G. pallida resistance has been extracted from 234 individuals and submitted for genotyping with the potato SNP chip version 3. DNA has also been extracted from 166 individuals in a population segregating for PVY necrotic lesion development and genotyped with the potato SNP chip version 3. Analysis of genotypic data is ongoing for both populations. SNP chip analysis allows, for many loci, to determine tetraploid segregation ratios. Association between resistance and marker data will occur after more progeny have been evaluated.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience for PLRV, PVY, and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems:No major changes/problems to report. What opportunities for training and professional development has the project provided?Attend and participate in the Annual meeting of the Potato Association of America, in Michigan, July 2016, and the 13thAnnual Solanaceae Conference in Davis, California, September, 2016. How have the results been disseminated to communities of interest?Posters reporting results were presented at The Society of Nematologists and the Organization of Nematologist of Tropical America in Montreal, Quebec, Canada. July, 2016, the 13th Annual Solanaceae Conference in Davis, California, September, 2016, and the Parker Field Day in Moscow, Idaho, July, 2016. What do you plan to do during the next reporting period to accomplish the goals?Depending of the extent of sequence coverage surrounding Rlretb, additional BACs may be identified for sequencing. Sequence from S. etuberosum will be evaluated for evolutionary change by comparing it to potato and tomato sequence from homologous regions on chromosome 4. Additional markers might be developed and evaluated for linkage to Rlretb. Two tetraploid populations will continue to be phenotyped and genotyped. Eden x Western Russet segregating for resistance to G. pallida and Yukon Gem x Russet Norkotah for PVY necrotic response.

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits DNA from a tetraploid population segregating for G. pallida resistance has been extracted and a small number of individuals submitted for genotyping with the potato SNP chip. DNA has also been extracted from a population segregating for PVY necrotic lesion development and a small number of progeny genotyped with the potato SNP chip. Analysis of genotypic data is ongoing for both populations. Association between resistance and marker data will occur after more progeny have been evaluated. Objective 2. Develop new molecular markers. BAC sequence data from S. etuberosum is helping to define the physical map of the Rlretb region associated with PLRV resistance. This data will help develop new molecular markers and identify candidate genes associated with Rlretb. Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. As part of developing new assays for detecting allele dosage in tetraploid potato existing molecular markers were first applied for Rx1/Gpa2, Ryadg and Rysto. Sequence data generated from these markers were used to develop new pyrosequencing assays for detecting copy number. A protocol has been designed for Ryadg that appears to accurately detect dosage in a limited number of individuals, and a new strategy using nested PCR has been initiated for Rysto and Rx1/Gpa2.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kuhl, J.C., R.G. Novy, J.L. Whitworth, M.S. Dibble, B. Schneider, and D. Hall (2016) Development of Molecular Markers Closely Linked to the Potato Leafroll Virus resistance gene, Rlretb, for use in marker-assisted selection. American Journal of Potato Research. 93:203-212.

Progress 07/03/15 to 09/30/15

Outputs
Target Audience:The target audience for PLRV, PVY and PCN resistance research is primarily potato breeders, however this work will benefit in the long term potato growers and the larger potato industry by facilitating the incorporation of virus and nematode resistance into new potato varieties. Target audiences for marker dosage research are potato breeders and other potato researchers, who will be able to use these markers for cultivar development. Changes/Problems:Some Yukon Gem x Russet Norkotah lines were lost due to tissue culture contamination and will have to be replaced. Contamination was not due to human error but related to sterile hood failure, autoclave malfunction, and a thrips infestation. These issues are being addressed to prevent future loss. What opportunities for training and professional development has the project provided?Attend and participate in the Western Section of the American Society of Plant Biologists meeting in Pullman, WA in June 2015. At this conference a poster was presented and numerous seminars attended on the latest research in plant science. I attended and participated in the annual meeting of the Potato Association of America in Portland, ME in July 2014. How have the results been disseminated to communities of interest?A poster was presented at the Western Section of the American Society of Plant Biologists in Pullman, WA in June 2015, titled Using shoot organogenesis to generate tetraploid Solanum sisymbriifolium to produce hybrid triploid seed. The poster reported progress in developing seedless lines of litchi tomato for use as a trap crop in potato. What do you plan to do during the next reporting period to accomplish the goals?Establish in tissue culture ~200progeny of Yukon Gem x Russet Norkotah for screening for PVY resistance.Similarly establish in tissue culture ~200 individuals of A10915, Eden x Western Russet, for screening for G. pallida resistance. Utilizing the recently initiated nested PCR approach for Rysto and Gpa2/Rx1 new pyrosequencing assays will be developed and optimized.Validate these assays using segregating populations of individuals. Four progeny from the Yukon Gem x Russet Norkotah population, including three showing resistance to systemic spread, have been identified for additional hybridizations so that newly generated populations can be evaluated. Screen these populations for response to Potato Virus Y isolate ID20 (strain NE-11).

Impacts
What was accomplished under these goals? Objective 1. Apply existing molecular markers for tagging valuable traits Populations are being generated and placed into sterile tissue culture to later screening for resistance to PVY (Yukon Gem x Russet Norkotah) and to G. pallida (A10915, Eden x Western Russet). Objective 2. Develop new molecular markers. Genomic DNA from S. etuberosum BACs continues to be analyzed to determine if new markers more closely linked to Rlretb can be developed. Objective 3. Develop and apply molecular markers to tetraploid or hexaploid germplasm to determine allelic status, ≥2 alleles. As part of developing new assays for detecting allele dosage in tetraploid potato existing molecular markers were first applied for Rx1/Gpa2, Ryadg and Rysto. Various population have been generated that segregate for these genes and DNA extracted.

Publications