GENOME-ENABLED GENETIC STUDY OF GRAPE BOTRYTIS BUNCH ROT RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1000810
• Grant No.: 2013-67014-21360
• Cumulative Award Amt.: $304,042.00
• Proposal No.: 2013-01900
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2018
• Grant Year: 2013
• Program Code: [A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production

Recipient Organization
MISSOURI STATE UNIVERSITY
901 S NATIONAL AVE
SPRINGFIELD,MO 65897-0027

Performing Department
Agriculture

Non Technical Summary
Grape bunch rot caused by the fungus Botrytis cinerea is one of the most destructive postharvest diseases in grapevines worldwide. Since successful control of Botrytis bunch rot can only be achieved through a combination of canopy management and heavily use of preventive fungicides, the identification of resistant germplasm could result in improved disease management. The Norton grape (Vitis aestivalis) is one of the very few commercial grape varieties that can be grown under elevated B. cinerea pressure, unlike the highly susceptible V. vinifera varieties, the European grape used for most winemaking worldwide, e.g., Cabernet Sauvignon. In view of this, we have developed a population of 94 F1 genotypes from a cross of V. aestivalis "Norton" with V. vinifera "Cabernet Sauvignon" which has yielded fruit for the past 2 years. A segregation analysis for B. cinerea resistance was performed in Fall 2012, resulting in 47 resistant and 45 susceptible hybrid genotypes, and fitting the expected ratio of 1:1. The data suggests that there is a heterozygous resistance quantitative trait locus (QTL) with a dominant resistant allele in Norton. This population also is being used to develop a linkage map with single nucleotide polymorphism (SNP) markers by using Genotyping-by-Sequencing (GBS) as well as with simple sequence repeat (SSR) markers. By removing time, financial and validation barriers to the development and application of molecular markers, this proposed project aims to accelerate the direct release of Norton-based new cultivars and improved efficiency of selection in subsequent generations.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1131	1081	100%

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

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1081 - Breeding;

Keywords

marker-assisted selection

norton grape

simple sequence repeat

single nucleotide polymorphism

Goals / Objectives
Goals The overall goals of this proposed project are to use genetic markers to rapidly deploy favorable alleles, accelerate breeding cycles for new cultivar release, train a new generation of plant breeders and attract new students to agricultural sciences. Objectives 1. Develop a Norton linkage map using genetic markers. 2. Identify genetic markers that are associated with Botrytis bunch rot resistance to use in futuremarker-assisted selection. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot. 4. Evaluate the viticultural and enological traits and determine the commercial potential ofNorton/V. vinifera F1 hybrids with Botrytis bunch rot resistance. . 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment andeducation.

Project Methods
Conduct multi-year segregation analyses with the existing population for resistance to Botrytis bunch rot. Localize the major QTL using tightly-linked genetic markers. Isolate the candidate gene by physical mapping of Norton bacterial artificial chromosome (BAC) clones. Evaluate the existing F1 population for adaptability, productivity and winemaking quality.

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:In this proposed project, using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-released, Norton-based cultivars.This research has led to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects.Research results have been presented at the XII International Conference on Grapevine Breeding and Genetics, 2018 Show Me State Grape and Wine Conference and Missouri Wine and Grape Research Board (MWGRB).In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. Changes/Problems:None What opportunities for training and professional development has the project provided?Linking theVitisGenI&II educational and research resources with theV. aestivalis-derived 'Norton' grape breeding program, this proposed work includes interdisciplinary training opportunities for students in plant breeding, genetics, genomics and plant pathology with a specific focus on viticulture.The following are the core competencies that have been provided. Build Research Capacity 1.Flower emasculation and pollination as well as mapping population generation 2.Vine pruning, leaf removal, cordon training and vineyard management 3.Linkage map construction using both SSR and SNP markers 4.Integration of a high-resolution linkage map for future Norton grape breeding program 5.Utilization of molecular markers to be applied for marker-assisted selection Build Educational Capacity 1.Comprehension of the basics and application of various molecular marker techniques 2.Familiarity with GBS protocols and their connection with statistical software 3.Implement GBS technology into teaching materials for students to understand the plant molecular breeding concept 4.Ability to make public presentations and write reports and manuscripts for publication Undergraduate Students: Karlene Negus and Vandiver (Summer Intern; June-August 2018) Key skills learned include emasculation of grapevine flowers, pollen preparation, pollination, seed germination, propagation of cuttings, assessment of disease index and berry quality as well as vineyard management; laboratory skills include DNA extraction, PCR, gel electrophoresis, analysis of SSRs via capillary electrophoresis and linkage map construction using JoinMap 4 software. Graduate Students: Rayanna Bailey (January 2018 - Present): Segregation analysis berry quality related traits Sadie Land (January 2018 - Present): Marker-assisted selection on downy mildew resistance locusRpv3in Chambourcin x Cabernet Sauvignon population Bryce Bentley (June 2018 - Present): Study of Chambourcin and Cabernet Sauvignon to Dicamba simulated drift Surya Sapkota (August 2013 - May 2018): Phenotyping of grape berry-B. cinereainteractions and integration of a high-resolution linkage map using both SSR and SNP markers Jacob Schneider (June 2016 - May 2018): Genetic study of rooting potential of hardwood cuttings Will Knuckles (January 2017 - May 2018): Constructing a mapping population of Jaeger 70 x Vignoles grapes via DNA markers Postgraduate Researcher: Li-Ling Chen (December 2013 - Present): TASSEL software and the integration of both Norton SSR and SNP maps Graduate Students Receiving degrees Surya Sapkota, Ph.D. degree, December 2017 Will Knuckles, MS degree, May 2018 Jacob Schneider, MS degree, May 2018 How have the results been disseminated to communities of interest?Oral/Poster Presentations Dr. Hwang a seminar entitled"Vitis aestivalis-derived 'Norton' Grape Breeding Progress Report from Missouri State University", 2017 North America Grape Breeding Conference, Davis, California, August 25 2017. Dr. Hwang gave a seminar entitled "Expanding Research on Berry and Juice Chromatographic Analysis to Expedite Grape Cultivar Improvement and Build Education Capacity", 2017 USDA-NLGCA Project Directors' Meeting, Morro Bay, California, October 3 2017. Dr. Hwang, Li-Ling Chen (Research Specialist) and two students (Will Knuckles and Jacob Schneider) attended the 25thMissouri State University Interdisciplinary Forum on May 05, 2018.Schneider presented a talk entitled "Genetic study of rooting potential of grape hardwood cuttings".Knuckles presented a poster entitled "The Identification of Interspecific Hybrids, Jaeger 70 x Vignoles Grapes Using SSR Markers". Dr. Hwang was invited to attend the Research Committee meeting of the Missouri Wine and Grape Board and reported the progress of the program to the Committee on October 17, 2017 and June 13, 2018. Dr. Hwang was invited to give a seminar entitled "QTL Mapping of Downy Mildew and Botrytis Bunch Rot Resistance in aVitis aestivalis-derived 'Norton'-based Population",XII International Conference on Grapevine Breeding and Genetics, July 15-20 in Bordeaux, France, July 18, 2018. What do you plan to do during the next reporting period to accomplish the goals?1.Maintain the existing mapping populations of Norton xCabernet Sauvignon and Chambourcin x Cabernet Sauvignon at Missouri State Fruit Experiment Station (MSFES). 2. Construct a high-resolution Norton linkage map based on both SSR and SNP markers using JoinMap 4 software 3. Identify Botrytis bunch rot resistant grape genotypes via marker-assisted selection 4. Continue to develop protocols for conducting segregation analysis of the mapping population for hardwood cutting rooting ability, sulfur sensitivity and cold hardiness 5. Combine the existing phenotyping and genotyping data and Identify markers tightly linked to the major quantitative trait locus (QTL) involved in the defense against Botrytis bunch rot 7. Continue to teach courses in "Plant Breeding and Genetics" and " Plant Breeding and Genetics Lab" with input from students 8. Implement GBS technology into the NSF-Funded Viticulture and Enology Science and Technology Alliance (VESTA) online courses 9. Submit the final report in December 2018

Impacts
What was accomplished under these goals? 1. Develop a high-density Norton linkage map using both SSR and SNP markers A mapping population with 182 genotypes was developed from a cross between 'Norton' andV. vinifera'Cabernet Sauvignon' from which a consensus map was constructed via 411 SSR markers on 19 linkage groups with a total distance of 1,678.6 cM.Genome-wide SNPs were identified using the TASSEL-GBS pipeline with the sequence data from both parents and 182 F1hybrids relative to the 12X.v2 ofV. viniferareference genome PN40024.A total of 43,971 polymorphic SNPs were identified and saved as a Variant Call Format (VCF) file.This file was uploaded into TASSEL 5.0 and filtered to 40,724 SNPs to create an hapmap file.Markers with >10% missing data across genotypes and those with >96% similarity were excluded, rendering 3,825 SNP markers.Combining these 3,825 SNP markers with 411 previously genotyped SSR markers yielded a total of 4,236 markers.A linkage map was constructed with 2,072 markers (407 SSRs and 1,665 SNPs) clustering into 19 linkage groups and spanning a genetic distance of 2,203.5 cM. 2. Botrytis bunch rot disease evaluation under a laboratory condition In years 2016 and 2017, the response toP. viticolain parents and 158 F1progenies was evaluated under the laboratory condition.Berry infection was evaluated based on the appearance of mycelium and conidia on berry surfaces.Berries were monitored for 10 days. Disease severity (proportion of each berry infected byB. cinerea) was assessed and evaluated as a visual semiquantitative scale of notation ranging from 1 to 5.1- no disease with no fungus establishment (highly resistant; Norton), 2- fungus establishment but growth restricted, 3- fungus growth with less than 1/3rd of the berries, 4- sufficient fungus growth with more than 50 % of a berry covered and 5- full growth of fungus and berries completely covered by fungal hyphae (highly susceptible; Cabernet Sauvignon).The Botrytis bunch rot phenotype segregated quantitatively in the mapping population with a continuous variation irrespective of the data from these two years. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot A significant QTL was identified on LG 2 flanked by SSR markers VMC6F1 and VMC3B10 at 4.2 cM apart.Using the general linear model in TASSEL, seven significant SNP-trait associations were detected between the two flanking SSR markers.However, only four SNPs were genetically positioned in the map constructed by JoinMap 4.1, further reducing the confidence interval to a genomic distance of 0.3-1.3 cM.It explained 18.4% of total phenotypic variation observed (R2) for Botrytis bunch rot with a maximum LOD score of 7.1, surpassing the permutation test significance threshold of 4.0.In addition, composite interval mapping in R/QTL confirmed these maker-trait associations.Flanking markers closely linked with the trait have been used for marker-assisted selection in the development of new cultivars with resistance to Botrytis bunch rot. 4. Evaluate the viticultural and enological traits and determine the commercial potential of Norton/V. viniferaF1hybrids with Botrytis bunch rot resistance In 2012, we made crosses between Norton and Vignoles (NxV), Norton and Point Noir (NxPN), Norton and Chardonnay (NxC) as well as Norton and Syrah (NxS).The initial harvest was obtained in 2016.Several of the F1hybrids from these crosses demonstrated strong resistance toB. cinereausing both marker and laboratory assays.We will continue to perform assays including the evaluation of cluster architecture, downy mildew resistance, dormant hardwood rooting ability, malic acid content and sulfur sensitivity to evaluate commercial potential. 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment and education Dr. Anson Elliott and Dr. Hwang continued to offer the Plant Breeding and Genetics course, which serves as a general introduction to the principles of classical breeding and biotechnology.The main purpose of this course is to examine concepts relevant to students in plant breeding, genetics, biotechnology, crop sciences and allied fields of plant pathology.The course content presented a broad range of applicable information pertaining to the modification and improvement of a wide range of crops, including both agronomic and horticultural crop species.Thirty-two students registered for this course in Spring 2018 and lectures on the GBS technology for SNP marker development, TASSEL (Trait Analysis by aSSociation Evolution and Linkage) pipeline and association mapping have been emphasized.Dr. Hwang also continued to offer the laboratory hands-on course (Plant Breeding and Genetics Lab, AGP 576/AGP 676) in Spring 2018 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) visualize DNA via electrophoresis, 4) perform polymerase-chain reaction (PCR) and 5) run DNA fragment analysis via capillary electrophoresis.The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers.Thirty-two (27 undergraduate and 5 graduate students) completed the course.Foruteen out of the 27 undergraduate students are from Ningxia University, China.They are part of one-year study away/exchange between Missouri State University and Ningxia University.Research Specialist, Li-Ling Chen, and graduate students, Rayanna Bailey, Will Knuckles, Sadie Land and Jacob Schneider served as teaching assistants for the class.The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Surya Sapkotas dissertation High Density Mapping and Quantitative Trait Loci Analysis for Fungal Diseases in Vitis aestivalis-derived Norton
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Will Knuckless dissertation The Identification of Intraspecific Hybrids Between Jaeger 70 x Vignoles Grapes Using SSR Markers
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Surya Sapkota, Li-Ling Chen, Shanshan Yang, Katie E. Hyma, Lance Cadle-Davidson and Chin-Feng Hwang. Construction of a High-density Linkage Map and QTL Detection of Downy Mildew Resistance in Vitis aestivalis-derived Norton.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Elizabeth A. Burzynski, Imelda Ryona, Bruce I. Reisch, Chin-Feng Hwang and Gavin L. Sacks. Quantification of Compound x Sample Dependent Effects Introduced by Unmatched Internal Standards Using SPME-GC-TOF-MS for Analysis of Grape and Wine Volatiles

Progress 09/01/13 to 08/31/18

Outputs
Target Audience:Using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-released, Norton-based cultivars.This research has led to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects.Research results have been presented at the bi-annual North America Grape Breeders' Conference in August, Fayetteville, AR 2013, Geneva, NY, 2015 and Davis, CA 2017, respectively, XI International Conference on Grapevine Breeding and Genetics in July 2014, Beijing, China, annual meetings of American Society for Enology and Viticulture (ASEV) in July, Portland, OR 2015 and ASEV-East Section in July, St. Louis 2016 and Charlottesville, VA 2017, VitisGen Annual Advisory meeting, January, 2014-2018, Craft Beverages Unlimited Midwest, St. Charles, MO 2016, XII International Conference on Grapevine Breeding and Genetics, in July 2018, Bordeaux, France, Show Me State Grape and Wine Conference and Missouri Wine and Grape Research Board (MWGRB), March, 2014-2018.In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. Changes/Problems:None What opportunities for training and professional development has the project provided?Linking theVitisGenI&II educational and research resources with theV. aestivalis-derived 'Norton' grape breeding program, this proposed work includes interdisciplinary training opportunities for students in plant breeding, genetics, genomics and plant pathology with a specific focus on viticulture.The following are the core competencies that have been provided: Build Research Capacity 1. Flower emasculation and pollination as well as mapping population generation 2. Vine pruning, leaf removal, cordon training and vineyard management 3. Linkage map construction using both SSR and SNP markers 4. Integration of a high-resolution linkage map for future Norton grape breeding program 5. Utilization of molecular markers to be applied for marker-assisted selection Build Educational Capacity 1. Comprehension of the basics and application of various molecular marker techniques 2. Familiarity with GBS protocols and their connection with statistical software 3. Implement GBS technology into teaching materials for students to understand the plant molecular breeding concept 4. Ability to make public presentations and write reports and manuscripts for publication Undergraduates: (Summer Intern; June-August 2014-2018) Mia Mann, Jesse Carroll, Joshua McCormick, Patrick Mahoney, Rayanna Bailey, Bryce Bentley, Samuel Sergent, Makayla Vandiver and Karlene Negus Key skills learned include emasculation of grapevine flowers, pollen preparation, pollination, seed germination, propagation of cuttings, assessment of disease index and berry quality as well as vineyard management; laboratory skills include DNA extraction, PCR, gel electrophoresis, analysis of SSRs via capillary electrophoresis and linkage map construction using JoinMap 4 software. Graduate Students: Rayanna Bailey (January 2018 - Present): Segregation analysis berry quality related traits Sadie Land (January 2018 - Present): Marker-assisted selection on downy mildew resistance locusRpv3in Chambourcin x Cabernet Sauvignon population Bryce Bentley (June 2018 - Present): Study of Chambourcin and Cabernet Sauvignon to Dicamba simulated drift Postgraduate Researcher: Li-Ling Chen (December 2013 - Present): TASSEL software and the integration of both Norton SSR and SNP maps Graduate Students Receiving degrees Pragya Adhikari, MS degree, December 2014 Logan Duncan, MS degree, May 2016 Mia Mann, MS degree, May 2016 Brigette Williams, MS degree, May 2016 Daniel Adams, MS degree, May 2017 Surya Sapkota, Ph.D. degree, December 2017 Jacob Schneider, MS degree, May 2018 Will Knuckles, MS degree, August 2018 How have the results been disseminated to communities of interest?Oral/Poster Presentations Dr. Hwang and Li-Ling Chen (Research Specialist)wereinvited to give a series of 4seminars entitled "Optimizing Vitis aestivalis-derived 'Norton' Grape Breeding withMarker-Assisted Selection",Ningxia University, Yinchuan, Ningxia, China, September10-14, 2018. Dr. Hwang was invited to give a seminar entitled "QTL Mapping of Downy Mildew and Botrytis Bunch Rot Resistance in aVitis aestivalis-derived 'Norton'-based Population",XII International Conference on Grapevine Breeding and Genetics,Bordeaux, France, July 18, 2018. Dr. Hwang was invited to attend the Research Committee meeting of the Missouri Wine and Grape Board and reported the progress of the program to the Committee on October 30, 2014, June 02, 2015, October 28, 2015, May 18, 2016, October 26, 2016, June 06, 2017,October 17, 2017, June 13, 2018 and October 23, 2018. Dr. Hwang, Li-Ling Chen (Research Specialist) and two students (Will Knuckles and Jacob Schneider) attended the 25thMissouri State University Interdisciplinary Forum, Springfield, M,May 05, 2018.Schneider presented a talk entitled "Genetic study of rooting potential of grape hardwood cuttings".Knuckles presented a poster entitled "The Identification of Interspecific Hybrids, Jaeger 70 x Vignoles Grapes Using SSR Markers". Dr. Hwang gave a seminar entitled "Expanding Research on Berry and Juice Chromatographic Analysis to Expedite Grape Cultivar Improvement and Build Education Capacity", 2017 USDA-NLGCA Project Directors' Meeting, Morro Bay, California, October 3, 2017. Dr. Hwang gave a seminar entitled"Vitis aestivalis-derived 'Norton' Grape Breeding Progress Report from Missouri State University", 2017 North America Grape Breeding Conference, Davis, California, August 25, 2017. Dr. Hwang, Li-Ling Chen (Research Specialist) and three students (Surya Sapkota, Will Knuckles and Jacob Schneider) attended the 42ndAmerican Society for Enology and Viticulture (ASEV) Eastern Section Conference on July 10-12, 2017 in Charlottesville, Virginia.Surya Sapkota was invited to give a seminar entitled "Fine Mapping and Detection of Downy Mildew Resistance Locus in Norton-based Population".Will Knuckles and Jacob Schneider, presented posters on their research and participated in the 'Flash Talk' where students gave timed, three-minute presentations on their research.Knuckles presented a poster entitled "The Identification of Interspecific Hybrids, Jaeger 70 x Vignoles Grapes Using SSR Markers".Schneider presented a poster entitled "Genetic Study of Dormant Rooting Ability in Grapes". Dr. Hwang gave a seminar entitled "Optimizing Marker-Assisted Selection for Norton Grape Breeding", 2017 Show Me State Grape & Wine Conference, Columbia, Missouri, March 08, 2017.Graduate students, Surya Sapkota, presented a talk entitled "Construction of a high-density linkage map ofVitis aestivalis-derived 'Norton' using both SSR and SNP markers and identification of quantitative trait loci (QTLs) associated with downy mildew resistance" in the 6thMissouri Grape and Wine Symposium on March 09, 2017. Dr. Hwang gave a seminar entitled "Norton Grape Improvement Using Marker-Assisted Selection", Agricultural Biotechnology Research Center, Sinica Academia, Taipei, Taiwan, December 16, 2016. Dr. Hwang gave a seminar entitled "OptimizingVitis aestivalis-derived 'Norton' Grape Breeding using Molecular Genetic and Genomic Approaches", Department of Viticulture and Enology, University of California, Davis, California,September 20, 2016. Dr. Hwang and three students attended the 41stAmerican Society for Enology and Viticulture (ASEV) Eastern Section Conference on July 19-20, 2016 in St. Louis, Missouri.He was invited to give a seminar entitled "Molecular Genetic Approaches to Norton Grape Improvement".Two graduate students, Surya Sapkota, and Daniel Adams, presented posters on their research and participated in the 'Flash Talk' where students gave timed, three-minute presentations on their research.As a result, Daniel Adams won the "Best Poster" award in the Viticulture category.Sapkota presented a poster entitled "Construction of High-Density Linkage Maps and Detection of Downy Mildew Resistance Locus inVitis aestivalis-derived 'Norton' Population". Adams presented a poster entitled "Genetic Analysis of Cold Hardiness inVitis aestivalis-derived 'Norton' Grape". Dr. Hwang gave a seminar entitled "OptimizingVitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" in the 2016 Craft Beverages Unlimited Midwest, St. Charles Convention Center, St. Charles, Missouri, February 5, 2016. Two graduate students, Logan Duncan and Brigette Williams, presented two talks entitled "QTL analysis of problematic traits associated with North American grape species" and "Investigation into the Genetic Basis of Leaf Shape in Grapes" in the 5thMissouri Grape and Wine Symposium, Columbia, Missouri,March 18, 2016. Dr. Hwang gave a seminar entitled"Molecular Genetic Approaches to Norton Grape Improvement" in the 2015 USDA Non-Land Grant College of Agriculture (NLGCA) Project Director Meeting, Kansas City, Missouri, October 06, 2015. Dr. Hwang gave a seminar entitled "Progress in the Norton Grape Breeding Program at the Missouri State University" in the North American Grape Breeders Conference,NYS Agricultural Experiment Station, Geneva, New York, August 27, 2015. Dr. Hwang and four students attended the 66thAmerican Society for Enology and Viticulture (ASEV) National Conference on June 17-18, 2015 in Portland, Oregon.He was invited served as a moderator for a "Viticulture-Pests and Diseases" session.All four graduate students, Surya Sapkota, Logan Duncan, Mia Mann and Brigette Williams, presented posters on their research.Mann was also selected to participate in the flash talk where students gave three-minute presentations on their research.Sapkota presented a poster entitled "Genetic Study of Downy Mildew Resistance inVitis aestivalis-derived 'Norton' Based Population". Duncan presented a poster entitled "Genetic Analysis of Dormant Rooting Potential inVitis aestivalis-derived 'Norton' Grape". Mann presented a poster entitled "Interspecific Hybrid Identification and Linkage MapConstruction of a Chambourcin x Cabernet Sauvignon Population". Williams presented a poster entitled "Investigation into the Genetic Basis of Leaf Shape in Grapes". Dr. Hwang, a research specialist, 5 graduate students and 2 summer interns were invited to MissouriDepartment of Agriculture in Jefferson City, Missouri to meet with Mr. Jim Anderson,MO Wine Director,and reported the progressofVitis aestivalis-derived 'Norton' breeding program on June 04, 2015. Dr. Hwang gave a seminar entitled "OptimizingVitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" in the 4thMissouri Grape and Wine Symposium, University of Missouri, Columbia, Missouri, May 08, 2015. Dr. Hwang was invited to give a seminar entitled "Optimization ofVitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" at the USDA-Crop Diseases, Pests & Genetics Unit, Parlier, California, March 24, 2015. Dr. Hwang was invited to give a seminar entitled "Mapping Genetic Loci for Resistance to Botrytis Bunch Rot in Grapevine", The 11th International Conference on Grapevine Breeding and Genetics, Beijing, China, August 1, 2014. Dr. Hwang was invited to give a seminar entitled "Optimization of Vitis aestivalis-derived 'Norton' Grape Breeding Using Molecular Genetic and Genomic Approaches", The 65th American Society for Enology and Viticulure National Conference, Austin, Texas, June 26, 2014 Dr. Hwang was invited to give a seminar entitled "True Hybrid Progenies - Identification and Applications of Vitis aestivalis 'Norton'-based Populations", 3rdAnnual MO Grape and Wine Research Symposium, Columbia, Missouri, December 06, 2013. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Develop a high-density Norton linkage map using both SSR and SNP markers A mapping population with 182 genotypes was developed from a cross between 'Norton' andV. vinifera'Cabernet Sauvignon' from which a consensus map was constructed via 411 SSR markers on 19 linkage groups with a total distance of 1,678.6 cM.Genome-wide SNPs were identified using the TASSEL-GBS pipeline with the sequence data from both parents and 182 F1hybrids relative to the 12X.v2 ofV. viniferareference genome PN40024.A total of 43,971 polymorphic SNPs were identified and saved as a Variant Call Format (VCF) file. This file was uploaded into TASSEL 5.0 and filtered to 40,724 SNPs to create an hapmap file.Markers with >10% missing data across genotypes and those with >96% similarity were excluded, rendering 3,825 SNP markers.Combining these 3,825 SNP markers with 411 previously genotyped SSR markers yielded a total of 4,236 markers.A linkage map was constructed with 2,072 markers (407 SSRs and 1,665 SNPs) clustering into 19 linkage groups and spanning a genetic distance of 2,203.5 cM. 2. Botrytis bunch rot disease evaluation under a laboratory condition In years 2016 and 2017, the response toP. viticolain parents and 158 F1progenies was evaluated under the laboratory condition.Berry infection was evaluated based on the appearance of mycelium and conidia on berry surfaces.Berries were monitored for 10 days. Disease severity (proportion of each berry infected byB. cinerea) was assessed and evaluated as a visual semiquantitative scale of notation ranging from 1 to 5.1- no disease with no fungus establishment (highly resistant; Norton), 2- fungus establishment but growth restricted, 3- fungus growth with less than 1/3rd of the berries, 4- sufficient fungus growth with more than 50 % of a berry covered, and 5- full growth of fungus and berries completely covered by fungal hyphae (highly susceptible; Cabernet Sauvignon).The Botrytis bunch rot phenotype segregated quantitatively in the mapping population with a continuous variation irrespective of the data from these two years. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot A significant QTL was identified on LG 2 flanked by SSR markers VMC6F1 and VMC3B10 at 4.2 cM apart.Using the general linear model in TASSEL, seven significant SNP-trait associations were detected between the two flankingSSR markers.However, only four SNPs were genetically positioned in the map constructed by JoinMap 4.1, further reducing the confidence interval to a genomic distance of 0.3-1.3 cM.It explained 18.4% of total phenotypic variation observed (R2) for Botrytis bunch rot with a maximum LOD score of 7.1, surpassing the permutation test significance threshold of 4.0.In addition, composite interval mapping in R/QTL confirmed these maker-trait associations.Flanking markers closely linked with the trait have been used for marker-assisted selection in the development of new cultivars with resistance to Botrytis bunch rot. 4. Evaluate the viticultural and enological traits and determine the commercial potential of Norton /V. viniferaF1hybrids with Botrytis bunch rot resistance In 2012, we made crosses between Norton and Vignoles (NxV), Norton and Point Noir (NxPN), Norton and Chardonnay (NxC) as well as Norton and Syrah (NxS).The initial harvest was obtained in 2016.Several of the F1hybrids from these crosses demonstrated strong resistance toB. cinereausing both marker and laboratory assays.We will continue to perform assays including the evaluation of cluster architecture, downy mildew resistance, dormant hardwood rooting ability, malic acid content and sulfur sensitivity to evaluate commercial potential. 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment and education Dr. Anson Elliott and Dr. Hwang offered the Plant Breeding and Genetics course (AGP 575/675), which serves as a general introduction to the principles of classical breeding and biotechnology.The main purpose of this course is to examine concepts relevant to students in plant breeding, genetics, biotechnology, crop sciences and allied fields of plant pathology.The course content presented a broad range of applicable information pertaining to the modification and improvement of a wide range of crops, including both agronomic and horticultural crop species.A total of one hundred and sixty-four students registered for this course from Spring 2014-2018 and lectures on the GBS technology for SNP marker development, TASSEL (Trait Analysis by aSSociation Evolution and Linkage) pipeline and association mapping have been emphasized.Dr. Hwang and Li-Ling Chen, research specialist, also offered the laboratory hands-on course (Plant Breeding and Genetics Lab, AGP 576/AGP 676) from Spring 2014-2018 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) perform polymerase-chain reaction (PCR), 4)visualize DNA via electrophoresis,and 5) run DNA fragment analysis via capillary electrophoresis.The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers.One hundred and sixty-four (125 undergraduate and 39 graduate students) completed both courses.Fifty-six out of the 125 undergraduate students are from Ningxia University, China.They are part of one-year study away/exchange between Missouri State University and Ningxia University.Graduate students, Surya Sapkota, Pragya Adhikari, Brigette Williams, Logan Duncan, Mia Mann, Daniel Adams, Rayanna Bailey, Will Knuckles, Sadie Land and Jacob Schneider served as teaching assistants for the class in the past five years.The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Pragya Adhikari, Li-Ling Chen, Xu Chen, Surya Sapkota and Chin-Feng Hwang (2014). Interspecific Hybrid Identification of Vitis aestivalis-derived Norton-Based Populations Using Microsatellite Markers. Scientia Horticulturae 179: 363-366.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Surya Sapkota, Li-Ling Chen and Chin-Feng Hwang (2015). Genetic study of Botrytis Bunch Rot Resistance in Grapevine. Acta Horticulturae (ISHS) 1082: 149-152.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Jacob Schneiders dissertation Genetic Study of Rooting Ability in Vitis aestivalis-derived Norton Grape
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Will Knuckless dissertation The Identification of Intraspecific Hybrids Between Jaeger 70 x Vignoles Grapes Using SSR Markers
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Surya Sapkotas dissertation High Density Mapping and Quantitative Trait Loci Analysis for Fungal Diseases in Vitis aestivalis-derived Norton
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Daniel Adams dissertation Genetic Analysis of Cold Hardiness in a Population of Norton (Vitis aestivalis) and Cabernet Sauvignon (Vitis vinifera) Hybrids
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Brigette Williamss dissertation Investigation into the Genetic Basis of Leaf Shape in Grapes
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Surya Sapkota, Li-Ling Chen, Shanshan Yang, Katie E. Hyma, Lance Cadle-Davidson and Chin-Feng Hwang (2018). Construction of a High-density Linkage Map and QTL Detection of Downy Mildew Resistance in Vitis aestivalis-derived Norton. Theoretical and Applied Genetics (https://doi.org/10.1007/s00122-018-3203-6)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mia Hammers, Surya Sapkota, Li-Ling Chen and Chin-Feng Hwang (2017). Construction a Genetic Linkage Map of Vitis aestivalis-derived Norton and Its Use in Comparing Norton and Cynthiana. Molecular Breeding 37: 64.
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Mia Manns dissertation Utilization of Microsatellite Markers for a Comparative Assessment of Norton and Cynthiana and the Linkage Map Construction of a Chambourcin x Cabernet Sauvignon Population
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Logan Duncans dissertation Quantitative Trait Loci (QTLs) Analysis of Sulfur Sensitivity in Vitis aestivalis-derived Norton grapes
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Adhikaris dissertation Molecular Genetic Study of Rooting Ability and Sulfur Sensitivity in Vitis aestivalis-derived Norton grapes
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Jonathan Fresnedo-Ram�rez, Qi Sun, Chin-Feng Hwang, Craig A. Ledbetter, David W. Ramming, Anne Y. Fennell, M. Andrew Walker, James J. Luby, Matthew D. Clark, Jason P. Londo, Lance Cadle-Davidson, Gan-Yuan Zhong, Bruce I. Reisch (2016). Towards the elucidation of cytoplasmic diversity in North American Grape Breeding Programs. Molecular Breeding. Molecular Breeding 36: 116.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Surya Sapkota, Li-Ling Chen, Kathy Schreiner, Hei Ge, and Chin-Feng Hwang (2015). A Phenotypic Study of Botrytis Bunch Rot Resistance in Vitis aestivalis-derived Norton Grape. Tropical Plant Pathology 40: 279-282.

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:In this proposed project, using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-released, Norton-based cultivars. This research will lead to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects. Research results have been presented at the annual meetings of 42nd annual American Society for Enology and Viticulture (ASEV)-Eastern Section, 2017 Shoe Me State Grape and Wine Conference and Missouri Wine and Grape Research Board (MWGRB). In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. Changes/Problems:We would like to request a one year no-cost extension for the above referenced award. If approved, the new ending date would be 08/31/2018. The request is necessary as we were unable to produce enough healthy berries in the past two years due to various diseases that accompany humid summers in Missouri. In order to obtain healthy berries for the evaluation of berry-B. cinerea interactions, the vineyard was sprayed in the growing season of 2015. We are currently adding appropriate vineyard management techniques as well as pesticide applications to increase the number of sound berries produced. The student, Jacob Schneider, will continue to construct a Norton linkage map using single nucleotide polymorphism (SNP) markers provided by the Cornell University-based VitisGen project. He will be using several layers of filter to identify the polymorphic markers and exclude the repeat and redundant markers from our previously identified 43,000 SNPs pool. A collaborative effort between Cornell University and Missouri State University is in process, and we are currently almost 75% finished with this SNP-based Norton map. We would need time for Jacob to learn the required software and technology to complete the construction of the Norton linkage map via SNP markers for Botrytis bunch rot QTL mapping. What opportunities for training and professional development has the project provided?1. Flower emasculation and pollination as well as mapping population generation 2. Vine pruning, leaf removal, cordon training and vineyard management 3. Linkage map construction using both SSR and SNP markers 4. Integration of a high-resolution linkage map for future Norton grape breeding program 5. Utilization of molecular markers to be applied for marker-assisted selection 6. Comprehension of the basics and application of various molecular marker techniques 7. Familiarity with GBS protocols and their connection with statistical software 8. Ability to make public presentations and write reports and manuscripts for publication Undergraduates: Rayanna Bailey, Bryce Bentley and Samuel Sergent (Summer Intern; June-August 2017) Key skills learned include emasculation of grapevine flowers, pollen preparation, pollination, seed germination, propagation of cuttings, assessment of disease index and berry quality as well as vineyard management; laboratory skills include DNA extraction, PCR, gel electrophoresis, analysis of SSRs via capillary electrophoresis and linkage map construction using JoinMap 4 software. Graduate Students: Surya Sapkota (August 2013 - Present): Phenotyping of grape berry-B. cinerea interactions and integration of a high-resolution linkage map using both SSR and SNP markers Jacob Schneider (June 2016 - Present): Genetic study of rooting potential of hardwood cuttings Will Knuckles (January 2017 - Present): Constructing a mapping population of Jaeger 70 x Vignoles grapes via DNA markers Daniel Adams (June 2015 - May 2017): Increase the resolution of Norton linkage map using SSR markers Postgraduate Researcher: Li-Ling Chen (December 2013 - Present): TASSEL software and the integration of both Norton SSR and SNP maps Graduate Students Receiving degrees Daniel Adams, MS degree, May 2017 How have the results been disseminated to communities of interest?Oral/ Poster Presentations Dr. Hwang gave a seminar entitled "Optimizing Vitis aestivalis-derived 'Norton' Grape Breeding using Molecular Genetic and Genomic Approaches", Department of Viticulture and Enology, University of California, Davis, September 20, 2016. Dr. Hwang gave a seminar entitled "Norton Grape Improvement Using Marker-Assisted Selection", Agricultural Biotechnology Research Center, Sinica Academia, Taipei, Taiwan, December 16, 2016. Dr. Hwang gave a seminar entitled "Optimizing Marker-Assisted Selection for Norton Grape Breeding", 2017 Show Me State Grape & Wine Conference, Columbia, Missouri, March 08, 2017. Graduate students, Surya Sapkota, presented a talk entitled "Construction of a high-density linkage map of Vitis aestivalis-derived 'Norton' using both SSR and SNP markers and identification of quantitative trait loci (QTLs) associated with downy mildew resistance" in the 6th Missouri Grape and Wine Symposium on March 09, 2017. Dr. Hwang was invited to attend the Research Committee meeting of the Missouri Wine and Grape Board and reported the progress of the program to the Committee on October 26, 2016 and June 06, 2017. Dr. Hwang, Li-Ling Chen (Research Specialist) and three students (Surya Sapkota, Will Knuckles and Jacob Schneider) attended the 42nd American Society for Enology and Viticulture (ASEV) Eastern Section Conference on July 10-12 in Charlottesville, Virginia. Surya Sapkota was invited to give a seminar entitled "Fine Mapping and Detection of Downy Mildew Resistance Locus in Norton-based Population". Will Knuckles and Jacob Schneider, presented posters on their research and participated in the 'Flash Talk' where students gave timed, three-minute presentations on their research. Knuckles presented a poster entitled "The Identification of Interspecific Hybrids, Jaeger 70 x Vignoles Grapes Using SSR Markers" Schneider presented a poster entitled "Genetic Study of Dormant Rooting Ability in Grapes"? What do you plan to do during the next reporting period to accomplish the goals?1. Preserve the existing mapping population of Norton x Cabernet Sauvignon (286 genotypes) at the Missouri State Fruit Experiment Station (MSFES) 2. Integrate a Norton linkage map with both SSR and SNP markers 3. Continue to phenotype segregation testing on Botrytis bunch rot 4. Continue to develop protocols for conducting segregation analysis of the mapping population for hardwood cutting rooting ability, sulfur sensitivity and cold hardiness 5. Combine the existing phenotyping and genotyping data and Identify markers tightly linked to the major quantitative trait locus (QTL) involved in the defense against Botrytis bunch rot 7. Continue to teach courses in "Plant Breeding and Genetics" and " Plant Breeding and Genetics Lab" with input from students 8. Implement GBS technology into the NSF-Funded Viticulture and Enology Science and Technology Alliance (VESTA) online courses 9. Attend the project director meeting in August 2017 and submit annual progress and final report

Impacts
What was accomplished under these goals? 1. Develop a Norton linkage map using genetic markers A mapping population consisting of 182 F1 genotypes between V. aestivalis-derived 'Norton' and V. vinifera 'Cabernet Sauvignon' has been expanded and developed. We tested 1,157 SSR markers with the parents and six F1 progenies, 859 were amplified via PCR and 413 were polymorphic for at least one parent. The remaining 446 SSR markers were unusable because they were either monomorphic or displayed an unclear banding pattern. Of the 413 polymorphic SSR markers, 376 anchored on the Norton linkage map and covered 1,496.6 centimorgan (cM) on 19 chromosomes. The consensus map consisted of 411 markers on 19 linkage groups with a total distance of 1,678.6 cM. We also identified 290 polymorphic markers and mapped 279 markers on the Cabernet Sauvignon linkage map. GBS has been completed for the parents and 182 progeny of the population. Currently, 43,320 SNP markers have been identified in this population. A consensus linkage map with 4,486 SNP markers also has been constructed. 2. Identify genetic markers that are associated with Botrytis bunch rot resistance to use in future marker-assisted selection The 2014 season had fewer healthy berries produced due to various diseases that accompany a humid summer in Missouri. Therefore, the phenotyping data on the evaluation of 92 F1 progeny from 2014 was not representative. In order to obtain healthy berries for the evaluation of berry-B. cinerea interactions, the vineyard was sprayed in the growing season of 2015. Additional vineyard management has been imposed in 2016 including collection of dormant season cane weight data followed by bud adjustment to balance vine size with the future crop; early season pesticide applications; and shoot thinning and shoot positioning. We will continue with leaf removal as needed from fruit set to veraison in 2017. This protocol is designed to increase the number of sound berries for phenotyping analysis. The data will then be incorporated with the genotyping platform to identify the tightly linked markers associated with the resistance. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot A high-resolution linkage map of Norton was integrated with 1,659 SNP and 406 SSR markers and clustered into 19 linkage groups for a total of 2,065 markers spanning a genetic distance of 2,203.5 cM. Disease progression and resistance reaction in response to B. cinerea was studied 10 days' post-inoculation in this population for two consecutive years under controlled conditions. A quantitative trait loci (QTL) analysis indicated a putative resistance locus on chromosome 2 that explained 18% of the total phenotypic variation. Flanking markers closely linked with the trait can be used for marker-assisted selection in the development of new cultivars with resistance to Botrytis bunch rot. 4. Evaluate the viticultural and enological traits and determine the commercial potential of Norton/V. vinifera F1 hybrids with Botrytis bunch rot resistance Vignoles, a mixed hybrid, is a popular white wine grape in Missouri and the eastern United States. Despite consumer demand for Vignoles, its production is limited by bunch rot susceptibility associated with compact cluster architecture. In 2012, we made crosses between Norton and Vignoles (NxV) and initial harvest was obtained in 2016. Two of the F1 hybrids (NxV 10 and NxV 13) demonstrated strong resistance to B. cinerea using both marker and laboratory assays. We will perform additional assays in 2017 including the evaluation of cluster architecture, downy mildew resistance, dormant hardwood rooting ability, malic acid content and sulfur sensitivity to evaluate commercial potential. 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment and education Dr. Anson Elliott and Dr. Hwang continued to offer the Plant Breeding and Genetics course, which serves as a general introduction to the principles of classical breeding and biotechnology. The main purpose of this course is to examine concepts relevant to students in plant breeding, genetics, biotechnology, crop sciences and allied fields of plant pathology. The course content presented a broad range of applicable information pertaining to the modification and improvement of a wide range of crops, including both agronomic and horticultural crop species. Forty-two students registered for this course in Spring 2017 and lectures on the GBS technology for SNP marker development, TASSEL (Trait Analysis by aSSociation Evolution and Linkage) pipeline and association mapping have been emphasized. Dr. Hwang also continued to offer the laboratory hands-on course (Plant Breeding and Genetics Lab, AGP 576/AGP 676) in Spring 2017 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) visualize DNA via electrophoresis, 4) perform polymerase-chain reaction (PCR) and 5) run DNA fragment analysis via capillary electrophoresis. The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers. Forty-two (34 undergraduate and 8 graduate students) completed the course. Eighteen out of the 34 undergraduate students are from Ningxia University, China. They are part of one-year study away/exchange between Missouri State University and Ningxia University. Research Specialist, Li-Ling Chen, and graduate students, Daniel Adams, Will Knuckles, Surya Sapkota and Jacob Schneider served as teaching assistants for the class. The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mia Hammers, Surya Sapkota, Li-Ling Chen and Chin-Feng Hwang (2017). Construction a Genetic Linkage Map of Vitis aestivalis-derived Norton and Its Use in Comparing Norton and Cynthiana. Molecular Breeding 37: 64.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Jonathan Fresnedo-Ram�rez, Qi Sun, Chin-Feng Hwang, Craig A. Ledbetter, David W. Ramming, Anne Y. Fennell, M. Andrew Walker, James J. Luby, Matthew D. Clark, Jason P. Londo, Lance Cadle-Davidson, Gan-Yuan Zhong, Bruce I. Reisch (2016). Towards the elucidation of cytoplasmic diversity in North American Grape Breeding Programs. Molecular Breeding 36: 116.
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2017 Citation: Daniel Adams dissertation Genetic Analysis of Cold Hardiness in a Population of Norton (Vitis aestivalis) and Cabernet Sauvignon (Vitis vinifera) Hybrids

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:In this proposed project, using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-released, Norton-based cultivars. This research will lead to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects. Research results have been presented at the annual meetings of 41st annual American Society for Enology and Viticulture (ASEV)-Eastern Section, Midwest Grape and Wine Conference (MGWC), 2016 Craft Beverages Unlimited Midwest, Missouri Wine and Grape Research Board (MWGRB), bi-annual North America Grape Breeders' Conference and VitisGen Annual Advisory meeting. In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. There are approximately 4,000 people worldwide may be affected by the distribution of this new knowledge. Changes/Problems:None What opportunities for training and professional development has the project provided?1. Flower emasculation and pollination as well as mapping population generation 2. Linkage map construction using both SSR and SNP markers 3. Integration of a high-resolution linkage map for future Norton grape breeding program 4. Utilization of molecular markers to be applied for marker-assisted selection 5. Comprehension of the basics and application of various molecular marker techniques 6. Attend GBS workshop and VitisGen Annual Conference at Cornell University 7. Familiarity with GBS protocols and their connection with statistical software 8. Ability to make public presentations and write reports and manuscripts for publication Undergraduates Patrick Mahoney (Summer Intern; June-August 2016) Key skills learned include emasculation of grapevine flowers, pollen preparation, pollination, seed germination, propagation of cuttings, assessment of disease index and berry quality as well as vineyard management; laboratory skills include DNA extraction, PCR, gel electrophoresis, analysis of SSRs via capillary electrophoresis and linkage map construction using JoinMap 4 software. Graduate Students: Surya Sapkota (August 2013 - Present): Phenotyping of grape berry-B. cinereainteractions and integration of a high-resolution linkage map using both SSR and SNP markers Daniel Adams (June 2015 - Present): Increase the resolution of Norton linkage map using SSR markers Jacob Schneider (June 2016 - Present): Genetic study of rooting potential of hardwood cuttings Mia Mann (January 2015 - May 2016): Constructing a Norton linkage map using SSR markers Logan Duncan (August 2014 - May 2016): Genetic study of Sulfur Sensitivity inVitis aestivalis-derived 'Norton' grapes Brigette Williams (June 2014 - May 2016): Genetic Basis of Leaf Shape in Grapes Postgraduate Researcher: Li-Ling Chen (December 2013 - Present): TASSEL software and the integration of both Norton SSR and SNP maps Minority Students: Mia Mann was recruited into the Plant Science graduate program in January 2015 and graduated with a MS degree in May 2016. She was an undergraduate student worker under the support of this project. Graduate Students Receiving degrees Logan Duncan, MS degree, May 2016 Mia Mann, MS degree, May 2016 Brigette Williams, MS degree, May 2016 How have the results been disseminated to communities of interest?Oral/ Poster Presentations Dr. Hwang gave a seminar entitled "Progress in the Norton Grape Breeding Program at the Missouri State University" in the North American Grape Breeders Conference, NYS Agricultural Experiment Station, Geneva, New York, August 27, 2015. Dr. Hwang gave a seminar entitled "Molecular Genetic Approaches to Norton Grape Improvement" in the 2015 USDA Non-Land Grant College of Agriculture (NLGCA) Project Director Meeting, Kansas City, Missouri, October 06, 2015. Dr. Hwang attended the fourth annual meeting of the Cornell University-based VitisGen program which included a GBS Workshop on January 7-8, 2016. He gave a progress report on the V. aestivalis-derived 'Norton' grape breeding program. Dr. Hwang gave a seminar entitled "Optimizing Vitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" in the 2016 Craft Beverages Unlimited Midwest, St. Charles Convention Center, St. Charles, MO, February 5, 2016. Two graduate students, Logan Duncan and Brigette Williams, presented two talks entitled "QTL analysis of problematic traits associated with North American grape species" and "Investigation into the Genetic Basis of Leaf Shape in Grapes" in the 5th Missouri Grape and Wine Symposium on March 18, 2016. Dr. Hwang was invited to attend the Research Committee meeting of the Missouri Wine and Grape Board and reported the progress of the program to the Committee on October 28, 2015 and May 18, 2016. Dr. Hwang and three students attended the 41st American Society for Enology and Viticulture (ASEV) Eastern Section Conference on July 19-20 in St. Louis, Missouri. He was invited to give a seminar entitled "Molecular Genetic Approaches to Norton Grape Improvement". Two graduate students, Surya Sapkota, and Daniel Adams, presented posters on their research and participated in the 'Flash Talk' where students gave timed, three-minute presentations on their research. As a result, Daniel Adams won the "Best Poster" award in the Viticulture category. Sapkota presented a poster entitled "Construction of High Density Linkage Maps and Detection of Downy Mildew Resistance Locus in Vitis aestivalis-derived 'Norton' Population" Adams presented a poster entitled "Genetic Analysis of Cold Hardiness in Vitis aestivalis-derived 'Norton' Grape" What do you plan to do during the next reporting period to accomplish the goals?1. Preserve the existing mapping population of Norton x Cabernet Sauvignon (256 genotypes) at the Missouri State Fruit Experiment Station (MSFES) 2. Integrate a Norton linkage map with both SSR and SNP markers 3. Continue to phenotype segregation testing on Botrytis bunch rot 4. Continue to develop protocols for conducting segregation analysis of the mapping population for resistance against black rot (Guignardia bidwellii) and bitter rot (Greeneria uvicola) 5. Combine the existing phenotyping and genotyping data and Identify markers tightly linked to the major quantitative trait locus (QTL) involved in the defense against Botrytis bunch rot 6. Begin to screen the developed Norton bacterial artificial chromosome (BAC) library for the identification of gene(s) associated with Botrytis bunch rot resistance 7. Continue to teach courses in "Plant Breeding" and "Advanced Plant Breeding Techniques" with input from students 8. Implement GBS technology into the NSF-Funded Viticulture and Enology Science and Technology Alliance (VESTA) online courses 9. Attend the project director meeting in January 2017 and submit annual progress report

Impacts
What was accomplished under these goals? 1. Develop a Norton linkage map using genetic markers A mapping population consisting of 182 F1 genotypes between V. aestivalis-derived 'Norton' and V. vinifera 'Cabernet Sauvignon' has been expanded and developed. We tested 1,157 SSR markers with the parents and six F1 progenies, 859 were amplified via PCR and 413 were polymorphic for at least one parent. The map for Norton consisted of 376 markers and covered 1,496.6 centimorgan (cM) on 19 chromosomes. The consensus map consisted of 411 markers on 19 linkage groups with a total distance of 1,678.6 cM. We also assembled 279 polymorphic markers on the Cabernet Sauvignon linkage map. GBS has been completed for the parents and 182 progeny of the population. Currently, 43,320 SNP markers have been identified in this population. A consensus linkage map with 4,486 SNP markers also has been constructed. The integration of a high-resolution Norton linkage map using JoinMap 4 and R-QTL are current under construction. 2. Identify genetic markers that are associated with Botrytis bunch rot resistance to use in future marker-assisted selection As in 2013, the 2014 season had fewer healthy berries produced due to various diseases that accompanied a humid summer in Missouri. Therefore, the phenotyping data on the evaluation of 92 F1 progeny from 2014 was not representative. In order to obtain healthy berries for the evaluation of berry-B. cinerea interactions, the vineyard was sprayed in the 2015 and 2016 growing seasons. We will be performing the phenotyping analysis on Botrytis bunch rot resistance in September 2016. The data will then be incorporated with the genotyping platform to identify the tightly linked markers associated with the resistance. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot The construction of a Norton bacterial artificial clone (BAC) library is completed through Amplicon Express (Pullman, WA). The library has coverage of more than a 7X genome equivalent with an average insert size of more than 100 kb. Three sets of the filters containing the whole BAC library were also constructed for the purpose of identification of potential Botrytis bunch rot resistance genes. 4. Evaluate the viticultural and enological traits and determine the commercial potential of Norton/V. vinifera F1 hybrids with Botrytis bunch rot resistance The propagation of Norton through traditional hardwood cutting has been a major hindrance in providing enough plants to meet commercial demand. Thus, it is interesting to study the vegetative propagation capacity of Norton grape and to determine whether it can be improved in the hybrid population. In viewing of this, a laboratory assay using dormant hardwood cutting for rooting potential was developed and applied to the mapping population from Norton x Cabernet Sauvignon. Assessments were conducted using the parents and 133 hybrid progenies. Rooting potential was evaluated as total number of primary roots ranging from 1 to 4. 1 = less than 2 roots/cutting; 2 = 3-4 roots/cutting; 3 = 5-7 roots/cutting; 4 = more than 7 roots/cutting. The F1 hybrid progeny showed segregation in dormant tooting ability. The preliminary data indicated a quantitative trait locus (QTL) for dormant rooting potential was detected on Cabernet Sauvignon linkage group 5 using multiple linear regression (MLM) in TASSEL and R-QTL analysis. Additional trials are currently conducted to confirm the results. There is always a risk of vine loss due to cold injury during winter. To manage this risk, a total of 136 genotypes with at least six 18-inch cuttings per seedling (plus parents) were again shipped to E.J. Gallo Winery, Modesto, CA in March 2016 to establish a backup population. This backup planting is an important resource for us to reduce the possibility of loss of important genotypes. It could also provide additional phenotyping material. 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment and education Dr. Anson Elliott and Dr. Hwang continued to offer the Plant Breeding course, which serves as a general introduction to the principles of classical breeding and biotechnology in Spring 2016. The main purpose of this course is to examine concepts relevant to students in plant breeding, genetics, biotechnology, crop sciences and allied fields of plant pathology. The course content presented a broad range of applicable information pertaining to the modification and improvement of a wide range of crops, including both agronomic and horticultural crop species. Fifty-three students registered for this course and lectures on the GBS technology for SNP marker development, TASSEL (Trait Analysis by aSSociation Evolution and Linkage) pipeline and association mapping have been emphasized. Dr. Hwang continued to offer the laboratory hands-on course (Techniques in Plant Breeding, AGN 495/AGP 730) in Spring 2016 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) visualize DNA via electrophoresis, 4) perform polymerase-chain reaction (PCR) and 5) run DNA fragment analysis via capillary electrophoresis. The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers. Forty-three (35 undergraduate and 8 graduate students) completed the course. Twenty-eight out of the 35 undergraduate students are from Ningxia University, China. They are part of one-year study away/exchange between Missouri State University and Ningxia University. Research Specialist, Li-Ling Chen, and graduate students, Brigette Williamson, Mia Mann, Logan Duncan, Daniel Adams and Surya Sapkota served as teaching assistants for the class. The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Jonathan Fresnedo-Ram�rez, Qi Sun, Chin-Feng Hwang, Craig A. Ledbetter, David W. Ramming, Anne Y. Fennell, M. Andrew Walker, James J. Luby, Matthew D. Clark, Jason P. Londo, Lance Cadle-Davidson, Gan-Yuan Zhong, Bruce I. Reisch (2016). Towards the elucidation of cytoplasmic diversity in North American Grape Breeding Programs. Molecular Breeding (In Press).
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Surya Sapkota, Li-Ling Chen, Kathy Schreiner, Hei Ge, and Chin-Feng Hwang (2015). A Phenotypic Study of Botrytis Bunch Rot Resistance in Vitis aestivalis-derived Norton Grape. Tropical Plant Pathology 40: 279-282.
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2016 Citation: Logan Duncans dissertation Quantitative Trait Loci (QTLs) Analysis of Sulfur Sensitivity in Vitis aestivalis-derived Norton grapes
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2016 Citation: Mia Manns dissertation Utilization of Microsatellite Markers for a Comparative Assessment of Norton and Cynthiana and the Linkage Map Construction of a Chambourcin x Cabernet Sauvignon Population
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2016 Citation: Brigette Williamss dissertation Investigation into the Genetic Basis of Leaf Shape in Grapes

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:In this proposed project, using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-released, Norton-based cultivars. This research will lead to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects. Research results have been presented at the annual meetings of American Society for Enology and Viticulture (ASEV), Midwest Grape and Wine Conference (MGWC), Missouri Wine and Grape Research Board (MWGRB), VitisGen Annual Advisory Meeting and the 11th International Conference on Grapevine Breeding and Genetics (ICGBG) in July 2014, Beijing, China. The data will also be presented at the bi-annual North America Grape Breeders' Conference at Cornell University on August 27-28, 2015. In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. There are approximately 4,000 people worldwide may be affected by the distribution of this new knowledge. Changes/Problems:Presently the project is on time and on track. What opportunities for training and professional development has the project provided? 1. Flower emasculation and pollination as well as mapping population generation 2. Linkage map construction using both SSR and SNP markers 3. Integration of a high-resolution linkage map for future Norton grape breeding program 4. Utilization of molecular markers to be applied for marker-assisted selection 5. Comprehension of the basics and application of various molecular marker techniques 6. Attend GBS workshop and VitisGen Annual Conference at Cornell University 7. Familiarity with GBS protocols and their connection with statistical software 8. Ability to make public presentations and write reports and manuscripts for publication Undergraduates: Jesse Carroll and Joshua McCormick (Summer Interns; June-August 2015) Key skills learned include emasculation of grapevine flowers, pollen preparation, pollination, seed germination, propagation of cuttings, assessment of disease index and berry quality as well as vineyard management; laboratory skills include DNA extraction, PCR, gel electrophoresis, analysis of SSRs via capillary electrophoresis and linkage map construction using JoinMap 4 software. Graduate Students: Daniel Adams: Increase the resolution of Norton linkage map using SSR markers(May 2015 - Present) Surya Sapkota: Phenotyping of grape berry-B. cinerea interactions and integration of a high-resolution linkage map using both SSR and SNP markers(September 2014 - August 2015) Pragya Adhikari: Constructing a Norton linkage map using SSR markers(September 2013 - December 2014) Postgraduate Researcher: Li-Ling Chen: TASSEL software and the integration of both Norton SSR and SNP maps (December 2013 - Present) Minority Students: Mia Mann was recruited into the Plant Science graduate program in January 2015. Her anticipated graduation date is in August 2016. She was an undergraduate student worker under the support of this project. Graduate Students Receiving degrees Pragya Adhikari, MS degree, December 2014 How have the results been disseminated to communities of interest?Oral/ Poster Presentations Dr. Hwang and Li-Ling Chen (Research Specialist) were invited to attend the third annual meeting of the Cornell University-based VitisGen program and a GBS Workshop in San Diego on January 8-9, 2015. Dr. Hwang also updated the progress on V. aestivalis-derived 'Norton' grape breeding program. Dr. Hwang was invited to give a seminar entitled "Optimization of Vitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" at the USDA-Crop Diseases, Pests & Genetics Unit, Parlier, CA, on March 24, 2015. Dr. Hwang gave a seminar entitled "Optimizing Vitis aestivalis-derived 'Norton' Grape Breeding with Marker-Assisted Selection" in the 4th Missouri Grape and Wine Symposium, University of Missouri, Columbia, MO, on May 08, 2015. Dr. Hwang was invited to attend the Research Committee meeting of the Missouri Wine and Grape Board and reported the progress of the program to the Committee on October 30, 2014 and June 02, 2015. Dr. Hwang, a research specialist, 5 graduate students and 2 summer interns were invited to MO Department of Agriculture to meet with Mr. Jim Anderson, MO Wine Director, and reported the progress of Vitis aestivalis-derived 'Norton' breeding program on June 04, 2015. Dr. Hwang and four graduate students attended the 66th American Society for Enology and Viticulture (ASEV) National Conference on June 17-18 in Portland, Oregon. He was invited served as a moderator for a "Viticulture-Pests and Diseases" session. All four graduate students, Surya Sapkota, Logan Duncan, Mia Mann and Brigette Williams, presented posters on their research. Mann was also selected to participate in the flash talk where students gave three-minute presentations on their research. Sapkota presented a poster entitled "Genetic Study of Downy Mildew Resistance in Vitis aestivalis-derived 'Norton' Based Population" Duncan presented a poster entitled "Genetic Analysis of Dormant Rooting Potential in Vitis aestivalis-derived 'Norton' Grape"? Mann presented a poster entitled "Interspecific Hybrid Identification and Linkage Map Construction of a Chambourcin x Cabernet Sauvignon Population" Williams presented a poster entitled "Investigation into the Genetic Basis of Leaf Shape in Grapes" What do you plan to do during the next reporting period to accomplish the goals? 1. Preserve the existing mapping population of Norton x Cabernet Sauvignon (256 genotypes) at the Missouri State Fruit Experiment Station (MSFES) 2. Integrate a Norton linkage map with both SSR and SNP markers using JoinMap 4 software 3. Continue to phenotype segregation testing on Botrytis bunch rot 4. Continue to develop protocols for conducting segregation analysis of the mapping population for resistance against black rot (Guignardia bidwellii) and bitter rot (Greeneria uvicola) 5. Combine the existing phenotyping and genotyping data and Identify markers tightly linked to the major quantitative trait locus (QTL) involved in the defense against Botrytis bunch rot 6. Begin to screen the developed Norton bacterial artificial chromosome (BAC) library for the identification of gene(s) associated with Botrytis bunch rot resistance 7. Continue to teach courses in "Plant Breeding" and "Advanced Plant Breeding Techniques" with input from students 8. Provide opportunities for students and researchers to attend the 2016 VitisGen Annual Conference and GBS workshops 9. Implement GBS technology into the NSF-Funded Viticulture and Enology Science and Technology Alliance (VESTA) online courses 10. Attend the project director meeting in January 2016 and submit annual progress report

Impacts
What was accomplished under these goals? 1. Develop a Norton linkage map using genetic markers A mapping population consisting of 182 F1 genotypes between V. aestivalis-derived 'Norton' and V. vinifera 'Cabernet Sauvignon' has been expanded and developed. There are more than 800 SSR markers that have been isolated in grapevine to date. We tested 697 SSR markers with the parents and four F1 progenies and identified 362 polymorphic markers. A haploid Norton linkage map has been constructed with 359 markers clustered in 19 chromosomes. We also assembled 262 polymorphic markers from Cabernet Sauvignon. The construction of a consensus map is in process. GBS has been completed for the parents and 182 progeny of the population. Currently, 43,320 SNP markers have been identified in this population. An additional 71 Norton x Cabernet Sauvignon F1 progenies were generated and DNA from leaf samples was used for the generation of additional SNP markers by using GBS. A consensus linkage map with 2,323 SNP markers also has been constructed. The integration of a high-resolution Norton linkage map using JoinMap 4 software is current under construction. 2. Identify genetic markers that are associated with Botrytis bunch rot resistance to use in future marker-assisted selection As in 2013, the 2014 season had fewer healthy berries produced due to various diseases that accompanied a humid summer in Missouri. Therefore, the phenotyping data on the evaluation of 92 F1 progeny from 2014 was not representative. In order to obtain healthy berries for the evaluation of berry-B. cinerea interactions, the vineyard will be sprayed in the growing seasons throughout the project. The data will then be incorporated with the genotyping platform to identify the tightly linked markers associated with the resistance. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot As planned, the construction of a Norton bacterial artificial clone (BAC) library is currently in process through Amplicon Express (Pullman, WA). The library is expected to have coverage of more than a 7X genome equivalent with an average insert size of more than 100 kb. We anticipate obtaining QTL mapping data between 2015 and 2016. 4. Evaluate the viticultural and enological traits and determine the commercial potential of Norton/V. vinifera F1 hybrids with Botrytis bunch rot resistance Leaf samples from 163 genotypes of Norton x Cabernet Sauvignon population have been submitted to the Cornell University-based VitisGen program for powdery mildew assay in June 2015. This is due to the fact that scientists at Cornell University are very skillful in the disease evaluation caused by powdery mildew. They will perform the experiment and provide us the phenotyping data to be used for QTL mapping using both SSR and SNP markers. In additional to the vineyard evaluation data collected last year, laboratory assays using detached leaves for downy mildew disease were developed and applied to the mapping population from Norton x Cabernet Sauvignon. Assessments were conducted using the parents and 182 hybrid progenies. Disease severity was evaluated as a visual semi-quantitative scale of notation ranging from 1 to 5. 1 = no visible disease system; 2 = 10% of the leaf area covered by the spores; 3 = 40% of the leaf area covered by the spores; 4 = 60% of the leaf area covered by the spores; and 5 = > 80% of the leaf area covered by the spores. A segregation analysis for downy mildew resistance was further performed, resulting in 88 resistant and 94 susceptible hybrid genotypes. The data suggests that there is a heterozygous resistance QTL with a dominant resistant allele for downy mildew disease in Norton grape. There is always a risk of vine loss due to cold injury during winter. To manage this risk, a total of 92 genotypes with at least six 18-inch cuttings per seedling (plus parents) were shipped to E.J. Gallo Winery, Modesto, CA in February 2015 to establish a backup population. This backup planting is an important resource for us to reduce the possibility of loss of important genotypes. It could also provide additional phenotyping material. 5. Initiate a molecular grape breeding program focused on undergraduate student recruitment and education Dr. Anson Elliott and Dr. Hwang continued to offer the Plant Breeding course which covers classical breeding and molecular breeding in Spring 2015. The main purpose of this course is to apply genetic principles to the improvement of crop plants including self-pollinated, cross-pollinated and asexually-propagated crops. Twenty-one students registered for this course in Spring 2015 and lectures on the GBS technology for SNP marker development, TASSEL (Trait Analysis by aSSociation Evolution and Linkage) pipeline and association mapping have been emphasized. Dr. Hwang also continued to offer the laboratory hands-on course (Techniques in Plant Breeding, AGN 495/AGP 730) in Spring 2015 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) visualize DNA via electrophoresis, 4) perform polymerase-chain reaction (PCR) and 5) run DNA fragment analysis via capillary electrophoresis. The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers. Fourteen students (6 undergraduate and 8 graduate students) completed the course. The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Surya Sapkota, Li-Ling Chen and Chin-Feng Hwang (2015). Genetic study of Botrytis Bunch Rot Resistance in Grapevine. Acta Horticulturae (ISHS) 1082: 149-152.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Pragya Adhikaris dissertation Molecular Genetic Study of Rooting Ability and Sulfur Sensitivity in Vitis aestivalis-derived Norton grapes
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Pragya Adhikari, Li-Ling Chen, Xu Chen, Surya Sapkota and Chin-Feng Hwang (2014). Interspecific Hybrid Identification of Vitis aestivalis-derived Norton-Based Populations Using Microsatellite Markers. Scientia Horticulturae 179: 363-366.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: In this proposed project, using Norton grape as a perennial woody model plant, we seek to employ advanced genetic and genomic tools to analyze Botrytis bunch rot resistance with an ultimate goal of direct-release, Norton-based cultivars. This research will lead to the construction of the first genetic linkage map for the Norton grape with both SSR and SNP markers and will serve as a foundation for future breeding projects. Research results have been presented at the annual meetings of American Society for Enology and Viticulture (ASEV), Midwest Grape and Wine Conference (MGWC), Missouri Wine and Grape Research Board (MWGRB) and the 11th International Conference on Grapevine Breeding and Genetics (ICGBG) in July 2014, Beijing, China. In additional to the professional conferences, the results have been presented at grower meetings in conjunction with viticulture/enology advisors to better educate growers on the value of molecular breeding and the benefits of improving Norton. The new knowledge produced from this proposed project also has been disseminated to the adult learner through VESTA program. There are approximately 4,000 people worldwide may be affected by the distribution of this new knowledge. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Undergraduates: Mia Mann, True hybrid identification using SSR markers (September 2013 – Present) Graduate Students: Surya Sapkota: Phenotyping of mature grape berry-B. cinerea interactions (September 2013 – November 2013) Pragya Adhikari: Constructing a Norton linkage map using SSR markers (September 2013 – Present) Postgraduate Researcher: Li-Ling Chen: TASSEL software and the integration of both Norton SSR and SNP maps (December 2013 – Present) Minority Students: Mia Mann Graduate Students Receiving degrees: Pragya Adhikari, MS degree, December 2014 How have the results been disseminated to communities of interest? Oral/ Poster Presentations: Chin-Feng Hwang. “Grape Breeding Program at Missouri State University”, The North America Grape Breeder’s Meeting, University of Arkansas, Fayetteville, AR. (Oral, August 16, 2013) Chin-Feng Hwang: “True Hybrid Progenies - Identification and Applications of Vitis aestivalis ‘Norton’-based Populations”, 3rd Annual MO Grape and Wine Research Symposium, Columbia, MO. (Oral, December 06, 2013) Li-Ling Chen, Pragya Adhikari, Mia Mann, Surya Sapkota and Chin-Feng Hwang. True Hybrid Identification of Vitis aestivalis-derived ‘Norton’-based Populations Using DNA Markers, 3rd VitisGen Annual Conference, Geneva, NY. (Poster, February 2014) Chin-Feng Hwang: “Optimization of Vitis aestivalis-derived ‘Norton’ Grape Breeding Using Molecular Genetic and Genomic Approaches”, The 65th American Society for Enology and Viticulure National Conference, Austin, Texas. (Oral, June 26, 2014) Chin-Feng Hwang: “Mapping Genetic Loci for Resistance to Botrytis Bunch Rot in Grapevine”, The 11th International Conference on Grapevine Breeding and Genetics, Beijing, China. (Oral, August 1, 2014) What do you plan to do during the next reporting period to accomplish the goals? 1. Preserve the existing mapping population of Norton x Cabernet Sauvignon (182 genotypes) at the Missouri State Fruit Experiment Station (MSFES) 2. Ship additional 71 Norton x Cabernet Sauvignon progenies to Cornell University for the construction of a linkage map with single nucleotide polymorphism (SNP) markers by using genotyping-by-sequencing (GBS) technology 3. Continue to construct the first Norton linkage map based on simple sequence repeats (SSR) and SNP markers 4. Complete the phenotype segregation testing on Botrytis bunch rot 5. Begin identifying markers tightly linked to the major quantitative trait locus (QTL) involved in the defense against Botrytis bunch rot 6. Start to develop a Norton bacterial artificial chromosome (BAC) library for the identification of gene(s) associated with Botrytis bunch rot resistance 7. Continue to teach courses in “Plant Breeding” and “Advanced Plant Breeding Techniques” with input from students and advisory committee 8. Provide opportunities for students and researchers to attend the 2015 VitisGen Annual Conference and GBS workshops 9. Implement GBS technology into the NSF-Funded Viticulture and Enology Science and Technology Alliance (VESTA) online courses 10. Attend the project director meeting and submit annual progress report

Impacts
What was accomplished under these goals? 1. Develop a Norton linkage map using genetic markers A mapping population consisting of 182 F1 genotypes between V. aestivalis-derived Norton and V. vinifera ‘Cabernet Sauvignon’ have been expanded and developed. There are more than 800 SSR markers that have been isolated in grapevine to date. We have tested 638 SSR markers with the parents and four F1 progenies and identified 362 polymorphic markers. A haploid Norton linkage map has been constructed with 359 markers clustered in 19 chromosomes. GBS has been completed for the parents and 182 progeny of the population. Currently, 43,320 SNP markers have been identified in this population. 2. Identify genetic markers that are associated with Botrytis bunch rot resistance to use in future marker-assisted selection. The 2013 season had fewer healthy berries produced due to various diseases that accompanied a humid summer in Missouri. Thereby, the phenotyping data on the evaluation of 92 F1 progeny from 2013 was not representative. We plan to evaluate mature grape berry-B. cinerea interactions in the growing seasons throughout the project. The data will then be incorporated with the genotyping platform to identify the tightly linked markers associated with the resistance. 3. Localize the major quantitative trait locus (QTL) involved in grapevine defense against Botrytis bunch rot. Expect to obtain data between 2015 and 2016 4. Evaluate the viticultural and enological traits and determine the commercial potential ofNorton/V. vinifera F1 hybrids with Botrytis bunch rot resistance. Although we had not experienced high disease pressure for downy mildew in the past 10 years, the humid summer of 2014 was conducive to disease development. One hundred eighty one F1 progeny of Norton x Cabernet Sauvignon were evaluated for downy mildew resistance on leaf samples in the vineyard. A segregation analysis for downy mildew resistance was performed, resulting in 102 resistant and 79 susceptible hybrid genotypes. 5. Initiate a molecular grape breeding program focused on undertraduate student recruitment and education. New lectures on the concepts of GBS technology and association mapping have been incorporated into an existing Plant Breeding course (AGP 575/AGP 675), Fall 2014. We also offered a new laboratory hands-on course (AGN 495/AGP 730) in Fall 2014 to cover the general technique required for molecular breeding work including 1) isolate DNA from grape leaves, 2) determine the concentration of isolated DNA using a spectrophotometer (NanoDrop 2000, Thermo Scientific), 3) visualize DNA via electrophoresis, 4) perform polymerase-chain reaction (PCR) and 5) run DNA fragment analysis via capillary electrophoresis. The purpose of these procedures is to verify the true hybrids at the seedling stage by using SSR markers. One graduate student and two seniors in the class, Brigette Williams, Mia Mann and Charles Bucher, expressed their interests in this field and have been accepted into the Plant Science graduate program with emphasis on breeding starting Summer 2014 and Spring 2015 at Missouri State University. The feedback from students indicated that the incorporation of the up-to-date molecular breeding technology is absolutely required for their competitiveness in the job market.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Genetic study of Botrytis Bunch Rot Resistance in Grapevine Surya D. Sapkota, Li-Ling Chen and Chin-Feng Hwang* State Fruit Experiment Station at Mountain Grove Campus, Darr School of Agriculture, Missouri State University, Springfield, MO 65897 *Presenting author Keywords: Vitis, interspecific hybrids, molecular marker, Botrytis bunch rot Abstract Grape bunch rot, caused by the necrotrophic fungal pathogen Botrytis cinerea, is one of the most destructive diseases in grapevines worldwide. At this point, control of Botrytis bunch rot is achieved through a combination of canopy management and fungicide usage. The identification of resistant germplasm could result in improved disease management. The Vitis aestivalis-derived Norton is one of the very few commercial red grape cultivars that can be grown under elevated B. cinerea pressure where highly susceptible V. vinifera is difficult to grow. Tests on the F1 progeny from a cross between V. aestivalis-derived Norton and V. vinifera Cabernet Sauvignon may elucidate the underlying genetic and molecular mechanisms of berry disease resistance and berry quality. To this end, we have developed a population of 92 F1 genotypes from this cross that has yielded fruit for the past three years. Also, a linkage map employing simple sequence repeat (SSR) markers clustered in 19 chromosomes has been under construction. By using genotyping-by-sequencing (GBS) technology as part of the VitisGen (www.vitisgen.org) project, we aim to improve marker-density and identify the tightly linked markers associated with Botrytis bunch rot resistance. The overall goals of this study are to use genetic markers to rapidly implement favorable alleles and accelerate breeding cycles for new improved cultivar releases that warrant decreased pesticide use in the vineyard, resulting in greater environmental and economic sustainability.