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INVESTIGATOR: REDDY, U.

PERFORMING INSTITUTION:
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE, WEST VIRGINIA 25112

DIVERSIFYING THE WATERMELON CULTIVAR GENETIC BASE USING GENOMIC SELECTION TO IMPROVE NUTRACEUTICAL TRAITS AND USE THEM AS PARENTAL LINES

NON-TECHNICAL SUMMARY: Seedless watermelons currently account for 98% of total watermelon production with a farm value of $492 million in the U.S. and an average annual consumption of 15.4 pounds per capita. Despite their importance, the entire production technology is in the hands of seed companies, who have inflated seed cost to extremely high levels. Other seed production technologies are almost out of reach of small farmers, whose primary livelihood is based on selling produce of truck crops such as watermelons. Integrating advanced genomic methods with genome-wide association mapping, genomic selection and RNAseq-based transcriptomic analysis should provide watermelon researchers the tools needed to increase fruit size, quality and nutritional value, which will facilitate increased production and consumption of seedless types. Breeding and genomic technologies to improve seedless watermelons at WVSU will bring national and international recognition to our land-grant program. The current proposal is based on sound research results that already have been generated in genome analysis and generation of tetraploids, the progenitor lines of seedless types in watermelons. This proposal has a strong educational component that will improve the knowledge and skill levels of our students.

OBJECTIVES: Identification of DNA markers linked to positive alleles involved in carotenoid biosynthesis and the other pathways that are important for cardiovascular health-promoting amino acids to conduct marker-assisted selection of value-added watermelon cultivars can become a model for understanding the molecular basis of value-added properties. Toward this goal we propose the objectives as below. Objective 1: Generation of tetraploids from the diverse watermelon cultivar collections for use in breeding high quality triploids Objective 2: Evaluation of diploids, tetraploids and their triploid derivatives for important yield traits, fruit quality traits and QTL analysis Objective 3: Association mapping for location of QTLs/Markers of importance Objective 4: Combined horticultural evaluation of diploids, tetraploids and their triploid derivatives and marker assisted breeding for improved tetraploid lines. Objective 5: Assays for antioxidant properties and for biological activity Objective 6: Participatory selection approach

APPROACH: Lack of knowledge concerning gene regulation, inheritance of various traits, QTL information and breeding behavior of tetraploid forms of watermelon represents a major bottleneck for improvement of seedless watermelons. We generated 23000 Single Nucleotide Polymorphisms (SNPs) using the most modern approach which is known as "Genotyping by Sequencing" or GBS. GBS utilizes a genome complexity reduction technique in a library making combined with next gen sequencing (Elshire et al., 2011; Poland et al., 2012). We assembled a set of software to process such a robust dataset and used it for imputation (predicting missing nucleotides), call rates and purging minor allele frequencies. After limiting the dataset to for retaining a minimum call rate of 90% with a maximum of two alleles and removing minor allele frequency below 0.05%, we were able to retain a dataset of 14,247 SNPs among 181 genotypes of cultivated watermelon forms. We characterized genome-wide linkage disequilibrium and population stratification, which are the foundations for performing association mapping. To start with, association mapping will be carried out for the characters soluble solids and the other fruit traits while keeping the population stratification based on ten eigen vectors of principal component analysis as the cofactor. Field Evaluation: During the summer of 2014, all the identified tetraploids along with their corresponding diploids will be grown in replications at the Agricultural Experiment Stations of WVSU and AAMU for evaluation of different fruit-related traits. The experimental design will be randomized block design (CRD) with three replications. Plant-to-plant and row-to-row distances will be maintained at 200 cm and 100 cm, respectively. Standard management practices will be followed throughout the growing season. Data will be recorded on all of the fruit yield and quality traits for tetraploid and diploid progeny: days to first flower, days to first fruit maturity, fruit length and diameter (cm), fruit length-diameter ratio, number of fruits per plant, fruit yield per plant (kg/plant), rind thickness (cm), rind hardness (kg/mm), mesocarp pressure (kg/mm), endocarp pressure (kg/mm), and soluble solids (Brix%). Rind hardness, mesocarp and endocarp pressure will be measured by the penetrometer (Fruit pressure tester, FT 011, Facchini, Italy) as puncture force (kg/mm) using a 2, 3, and 8 mm tip, respectively. The Brix% of flesh juice will be measured using a hand refractometer (ATAGO, Japan). The phenotypic data will be subjected to ANOVA using JMP 7.0 software (SAS Institute Incorporation, Cary, NC). Metabolomic Studies: profiles for flesh of ripe watermelons of the genetic populations will be carried out by the service center of University of Illinois (please see the letter of commitment). Polar compounds (such as the phenolic acids), and less polar compounds (such as anthocyanins) will be extracted from freeze dried watermelon flesh samples of the genetic populations using our established methods. Carotenoids and fat soluble vitamin, the least polar compounds, will be extracted using non polar solvents, such as ethanol, under protective lighting followed by hexane extraction (Davis et al., 2003). Compound identification will be performed using an LC-MS Q-TOF (High performance Liquid Chromatography couples with Quadruple Time of Flight mass spectrometer with MS/MS capability). Q-TOF is a high resolution electrospray ionization system (HR ESI) with high sensitivity, resolution, and mass accuracy. The high mass accuracy allows exact mass measurements of small molecules such as phenolic acids, and of larger molecules such as polymeric proanthocyanidins. This system accurately identifies compounds based on retention time, absorbance values, and molecular weight. Many compounds can be identified by comparison with existing metabolomic databases such as MetAlign®. Association mapping: Resolution of population structure is a necessary pre-requisite for association mapping. Since this proposal aims to carry out association mapping using the large genotyping data sets as collected from the previous study. If structure matrix (Q matrix) is not included when conducting association analysis, there is a strong possibility of including spurious associations between candidate markers and phenotypes that can result from the presence of population structure (Prichard etal., 2000). Association mapping analysis will be conducted using the freely available software TASSEL 4.0 (www.maizegenetics.net). Mixed Linear Model (MLM) and Best Linear Unbiased Prediction (BLUP) will be carried out, while taking population structure and kinship into consideration. This analysis identifies QTLs linked to various traits separately and jointly along with the breeding values based on the presence of favorable QTLs in the diploids and tetraploids. We expect that the comparative association mapping of diploids and tetraploids will provide interesting findings pertaining to ploidy level changes and genome perturbations that occurred because of genome duplications in tetraploids. Cell culture: Vascular smooth muscle cells will be used for the bioassays. We will culture cells as we have previously described (Hankins et al., 2008). Stock solutions of watermelon flesh extracts will be made by diluting the extracts in DMEM. The stock solutions will be filter sterilized and stored at 4°C until use in the bioassays. Determination of oxidative damage to proteins: Oxidation of amino acid residues on proteins results in formation of carbonyls that are introduced into protein side chains. In as much as protein carbonyl derivatives are chemically stable, they are convenient markers of oxidative stress (Nakamura and Sataro, 1996). These will be measured using the OxiSelect™ Protein Carbonyl Spectrophotometric Assay (Cosmo-Bio, Tokyo, Japan). Protein samples will be isolated, after which derivitization of the carbonyl group with 2,4- dinitrophenylhydrazine (DNPH) is followed by detection with an anti-DNP antibody. With transfer to a minicuvette, absorbance of protein-hydrozone is measured at 375 nm, and the concentration of protein carbonyl is calculated from comparison to a standard curve.

PROGRESS: 2013/09 TO 2017/08
Target Audience:Watermelon breeders, geneticists and seed companies got immensely benefitted with our research results. Undergraduate and graduate students successfully completed. Four thesis were submitted by students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students submitted their thesis research. Two technicians and one postdoc were trained in watermelon breeding and genomics. Developed GWAS panels that are useful for teaching crop improvement in duallevel classes. How have the results been disseminated to communities of interest?Results are currently archived in cucurbit genome database maintained at www.icgui.org Six peer reviwed publications and ten conference presentations were accomplished so far. Seeds of value added and diverse germplasm were shared to USDA ARS and other public as well as private breeders. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2013/09 TO 2017/08
What was accomplished under these goals? We expanded SNP panel with additional 10,000 SNPs that have minor allele frequency above 0.05. We successfully developed GWAS models useful to identify genes forvarious nutraceutical traits such as flavor,Citrulline and lycopenein watermelon Most significantly, we identified eight major QTLs for citrullineand 8 QTLs of varying degree for flavor compounds. Evaluated several important watermelon lines for use in commercial breeding We made crosses involving diverse lines and segregants were subjected to marker assisted selection for various fruitquality traits and currently being evaluated for their suitability to use them as prebreeding lines for seedless watermelons. Published six important papers in watermelon genomics that can impact world watermelon breeding programs.

PUBLICATIONS (not previously reported): 2013/09 TO 2017/08
1. Type: Journal Articles Status: Published Year Published: 2017 Citation: 3. Levi, A.; Simmons, A. M.; Massey, L.; Coffey, J.; Wechter, W. P.; Jarret, R. L.; Tadmor, Y.; Nimmakayala, P.; Reddy, U. K. Genetic Diversity in the Desert Watermelon Citrullus colocynthis and its Relationship with Citrullus Species as Determined by High-frequency Oligonucleotides-targeting Active Gene Markers. Journal of the American Society for Horticultural Science 2017, 142 (1), 47-56.
2. Type: Book Chapters Status: Published Year Published: 2017 Citation: 1. Nimmakayala, P.; Saminathan T.; Abburi, V.L.; Yadav, L.K.; Levi, A.; Weng, Y.; Reddy, U. K. Comparative Genomics of the Cucurbitaceae. (Book chapter). R. Grumet et al. (eds.), Genetics and Genomics of Cucurbitaceae, Plant Genetics and Genomics: Crops and Models, DOI 10.1007/ 7397 2016 34. Springer International Publishing AG 2017
3. Type: Book Chapters Status: Published Year Published: 2017 Citation: 2. Levi, A.; Jarret, R. L.; Kousik, S.; Wechter, W. P.; Nimmakayala, P.; Reddy, U. K. Genetic Resources of Watermelon. (Book chapter). R. Grumet et al. (eds.), Genetics and Genomics of Cucurbitaceae, Plant Genetics and Genomics: Crops and Models, DOI 10.1007/ 7397 2016 34. Springer International Publishing AG 2017
4. Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Nimmakayala, P., Levi, A., Abburi, L., Abburi, V. L., Tomason, Y. R., Saminathan, T., Vajja VG, Malkaram S, Reddy R, Wehner T, and Reddy, U. K. (2014). Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. BMC genomics, 15(1), 1-15. doi: 10.1186/1471-2164-15-767
5. Type: Journal Articles Status: Published Year Published: 2014 Citation: 2. Reddy, U. K., Nimmakayala, P., Levi, A., Abburi, V. L., Saminathan, T., Tomason, Y. R., Vajja G, Reddy R, Abburi L, Wehner TC, Ronin Y Karol, A. (2014). High-Resolution Genetic Map for Understanding the Effect of Genome-Wide Recombination Rate on Nucleotide Diversity in Watermelon. G3: Genes|Genomes|Genetics, 4(11), 2219-2230. doi: 10.1534/g3.114.012815

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience:Graduate and undergraduate students, watermelon growers, Watermelon Geneticists and Plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students submitted their thesis research. Nine undergraduate students worked in the project. We have trained two postdoctoral associates and a technician to work at various levels of the project. How have the results been disseminated to communities of interest?Farmers in Alabama were given two on farm visits to see diversity of watermelon cultivars. Four important papers were published in BMC Genomics, Molecular Breeding, G3 (Genes, Genomes and Genetics) and Journal of Heredity. We presented entire study in Plant and Animal Genome Conference. What do you plan to do during the next reporting period to accomplish the goals?We are currently performing metabolite profiling on various watermelons. We plan to estimate various important metabolites like lycopene, citrulline and vitamin C across the diversity panel and perform GWAS for identifying respective genes that are important for these.

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? A diversity panel consisting 180 watermelon accessions was used for genome wide association study for various fruit traits. Most significantly, we identified three major QTLs for soluble solids and 5 QTLs of varying degree for trichome density and length. We identified QTLs that explain fruit weight and length. We subjected entire panel for SNP genotyping and generated 5000 SNPs that have minor allele frequency above 0.05. Diversity analysis indicated that cultivated watermelons originated from West Africa. Accessions of Africa were grouped into three major clusters, out of which one of the cluster relates entire world watermelon cultivars. Population structure analysis resolved the genetic differentiation of Asian and American ecotypes. We used 201 genome-wide microsatellites to resolve selection sweeps across the genome. In addition, 5000 SNPs were used to estimate pairwise FST among the cultivar groups to identity two strong selection sweeps across watermelon genome. We located genes that are important for watermelon domestication. A high resolution genetic map was constructed consisting 10,480 SNPs. We characterized genome wide LD decay as well as individual genes.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
1. Type: Journal Articles Status: Published Year Published: 2015 Citation: Reddy UK, Abburi L, Abburi VL, Saminathan T, Cantrell R, Vajja VG, Reddy R, Tomason YR, Levi A, Wehner TC, Nimmakayala P (2015) A Genome-Wide Scan of Selective Sweeps and Association Mapping of Fruit Traits Using Microsatellite Markers in Watermelon. Journal of Heredity. doi:10.1093/jhered/esu077
2. Type: Journal Articles Status: Published Year Published: 2015 Citation: Saminathan T, Nimmakayala P, Manohar S, Malkaram S, Almeida A, Cantrell R, Tomason Y, Abburi L, Rahman MA, Vajja VG, Khachane A, Kumar B, Rajasimha HK, Levi A, Wehner T, Reddy UK (2015) Differential gene expression and alternative splicing between diploid and tetraploid watermelon. J Exp Bot. 66(5):1369-85. doi: 10.1093/jxb/eru486.
3. Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Levi A, Simmons AM, Massey L, Coffey J, Wechter P, Tadmor Y, Reddy UK, Nimmakayala P , 2016. Genetic Relationships in the Desert Watermelon Citrullus colocynthis As Viewed with High Frequency Oligonucleotide?Targeting Active Gene (HFO?TAG) Markers. International Plant & Animal Genome XXI Conference, January 8-13, 2016- San Diego, CA

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:Until recently, WVSU students had little contact with the latest developments in genomics and crop improvement. As a result, our students have been unable to compete for jobs and have been ill prepared to pursue advanced degrees or additional education in one of the fastest growing areas of research and industry. This CBG project exposed ourstudents to the nation's strengths in crop improvement and genetic resources and enhanced their abilities to pursue high-tech jobs in the future and contribute to US food security. We accomplished this important goal through the establishment of multi-institutional networking with a similar student population as Alabama A&M University (AAMU) that desperately needs modern technologies and training. We created a plethora of common opportunities for our students to pursue research in the areas of genome analysis and modern methods in plant breeding, horticulture, genomics, and molecular biology through a multi-disciplinary approach. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three undergraduate students and two graduate students worked in this project. Graduate students submitted their thesis in topics of identifying fruit specific DNA markers and GWAS for trichome features. A postdoc has published a diploid and tetraploid transcriptome map in Journal of Experimental Botany. In total there are five papers published in peer reviewed journals. How have the results been disseminated to communities of interest?We developed a database system that will contain SNP marker data, genotyping and the quantitative data related to value added traits and mapping information and make this information available to the public through Cucurbit Genomic Resource site (http://www.icugi.org/). All the sequence information was already supplied to this website for easy access by national and international researchers. We also presented our accomplishments at the annual meetings of the American Society of Horticultural Sciences, American Society of Agronomy, and the Plant & Animal Genome Conference. These abstracts were developed into full length peer-reviewed manuscripts. What do you plan to do during the next reporting period to accomplish the goals?Using molecular markers in breeding will allow efficient and accurate selection, saving the time and expense of phenotyping. Therefore, we propose to assemble a genome-wide association study (GWAS) panel, resequence selected cultivars, identify single nucleotide polymorphisms (SNPs), and examine variation in L-citrulline in this watermelon germplasm collection across genotypes and environments. We will then use association mapping to identify candidate genes/SNPs associated with citrulline contents. Examination of agronomic traits, including fruit quality will allow us to monitor potential linkage of candidate genes to deleterious traits. These markers will enable breeders to use marker-assisted selection to generate watermelon varieties with increased L-citrulline concentrations, and to enhance the watermelon germplasm base for U.S. growers, producers and consumers. Moreover, development of the GWAS panel, fully genotyped for a genome-wide set of SNPs, will also provide a genomics resource that enables enhancement of important agronomic traits of interest, as GWAS for other traits will require only phenotypic data for the lines in the GWAS panel.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? Recently, we resolved population structure using a robust set of 8,125 SNPs among 181 genotypes of cultivated watermelon belonging to Africa, Asia, Europe and America and analyzed genetic diversity as well as Identity by Descent (IBD), which resolves the common ancestry of various collections. Furthermore, we resolved linkage disequilibrium (LD) and identified the chromosomes that contain high and low LD as well as LD in the genic portion of watermelon genome. We successfully used the LD for association mapping of various traits in watermelon. We performed GWAS of the characters soluble solids and other fruit traits while keeping the population stratification as the cofactor. We also identified several candidate genes to improve fruit quality in watermelon tetraploids that are crucial for seedless type breeding. We have currently several imporatnt SNP markers linked various important traits of watermelon.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Reddy UK, Abburi L, Abburi VL, Saminathan T, Cantrell R, Vajja VG, Reddy R, Tomason YR, Levi A, Wehner TC, Nimmakayala P (2014) A Genome-Wide Scan of Selective Sweeps and Association Mapping of Fruit Traits Using Microsatellite Markers in Watermelon. Journal of Heredity. doi:10.1093/jhered/esu077 2. Saminathan T, Nimmakayala P, Manohar S, Malkaram S, Almeida A, Cantrell R, Tomason Y, Abburi L, Rahman MA, Vajja VG, Khachane A, Kumar B, Rajasimha HK, Levi A, Wehner T, Reddy UK (2014) Differential gene expression and alternative splicing between diploid and tetraploid watermelon. Journal of Experimental Botany. doi:10.1093/jxb/eru486 3. Padma Nimmakayala, Amnon Levi, Lavanya Abburi, Venkata Lakshmi Abburi, Yan R. Tomason, Thangasamy Saminathan, Venkata Gopinath Vajja, Sridhar Malkaram, Rishi Reddy, Todd C. Wehner, and Umesh K. Reddy 2014. Single nucleotide polymorphisms generated by genotyping by sequencing used to character

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: Graduate and undergraduate students, watermelon growers, Watermelon Geneticists and Plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students submitted their thesis research. We have currently a PhD student who is working at both Alabama A&M and West Virginia State University. Four undergraduate students worked in the project. We have trained two postdoctoral associates and a technician to work at various levels of the project. How have the results been disseminated to communities of interest? Farmers in Alabama were given an on farm visits to see diversity of watermelon cultivars. . Four important papers were published in BMC Genomics, Molecular Breeding, G3 (Genes, Genomes and Genetics) and Journal of Heredity. We presented entire study in Plant and Animal Genome Conference(2014). What do you plan to do during the next reporting period to accomplish the goals? We are currently performing metabolite profiling on various watermelons. We plan to estimate various important metabolites like lycopene, citrulline and vitamin C across the diversity panel and perform GWAS for identifying respective genes that are important for these.We are also planning to evaluate the exisiting parental lines for seedless watermelon production and devlop diverse parental lines.

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? A diversity panel consisting 180 watermelon accessions was used for genome wide association study for various fruit traits. Most significantly, we identified three major QTLs for soluble solids and 5 QTLs of varying degree for trichome density and length. We identified QTLs that explain fruit weight and length. We subjected entire panel for SNP genotyping and generated 5000 SNPs that have minor allele frequency above 0.05. Diversity analysis indicated that cultivated watermelons originated from West Africa. Accessions of Africa were grouped into three major clusters, out of which one of the cluster relates entire world watermelon cultivars. Population structure analysis resolved the genetic differentiation of Asian and American ecotypes. We used 250 genome-wide microsatellites and used them to resolve selection sweeps across the genome. In addition, 5000 SNPs were used to estimate pairwise FST among the cultivar groups to identity two strong selection sweeps across watermelon genome. We located genes that are important for watermelon domestication. A high resolution genetic map was constructed consisting 10,480 SNPs. We characterized genome wide LD decay as well as individual genes. The details are in the publications listed.

PUBLICATIONS: 2013/09/01 TO 2014/08/31
1. Type: Journal Articles Status: Published Year Published: 2014 Citation: Padma Nimmakayala, Amnon Levi, Lavanya Abburi, Venkata Lakshmi Abburi, Yan R. Tomason, Thangasamy Saminathan, Venkata Gopinath Vajja, Sridhar Malkaram, Rishi Reddy, Todd C. Wehner, and Umesh K. Reddy 2014. Single nucleotide polymorphisms generated by genotyping by sequencing used to characterize genome-wide diversity, linkage disequilibrium and selection sweep for worldwide cultivated watermelon. BMC Genomics 2014, 15:767 (Highly accessed)
2. Type: Journal Articles Status: Published Year Published: 2014 Citation: Reddy UK, Nimmakayala P, Levi A, Abburi VL, Saminathan T, Tomason YR, Vajja G, Reddy R, Abburi L, Wehner TC, Ronin Y, Karol A. 2014 High-Resolution Genetic Map for Understanding the Effect of Genome-Wide Recombination Rate on Nucleotide Diversity in Watermelon. G3: Genes|Genomes|Genetics, 4:2219-2230.
3. Type: Journal Articles Status: Published Year Published: 2014 Citation: 3. Nimmakayala, P., Abburi VL, Bhandari A, Abburi L, Vajja VG, Reddy R, Malkaram S, Venkatramana P, Wijeratne A, Tomason YR, Levi A, Wehner T and Reddy UK. 2014. Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats. Molecular Breeding, 34(2): 537-548
4. Type: Journal Articles Status: Accepted Year Published: 2015 Citation: 4. Reddy UK, Nimmakayala P, Levi A, Abburi VL, Saminathan T, Tomason YR, Vajja G, Reddy R, Abburi L, Wehner TC, Ronin Y, Karol A. 2014 A genome-wide scan of selective sweeps and association mapping of fruit traits using microsatellite markers in watermelon. Journal of Heredity. JOH-2014-163.R1