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INVESTIGATOR: Panicker, G.

PERFORMING INSTITUTION:
ALCORN STATE UNIVERSITY
ALCORN STATE, MISSISSIPPI 39096

PHENOTYPING A DIVERSITY PANEL FOR EXPLORING NATURAL VARIATION WITHIN NUTRACEUTICALLY IMPORTANT METABOLITES FOR USE IN WATERMELON BREEDING

NON-TECHNICAL SUMMARY: This CBG project will expose students to the nation's strengths in crop improvement and genetic resources and will enhance their abilities to pursue high-tech jobs in the future and contribute to US food security. We propose to accomplish this important goal through the establishment of multi-institutional networking with a similar student population as Alcorn State University (ASU) that desperately needs modern technologies and training. We will create 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. In so doing, we will bring our students up to speed so that they can be part of the nation's strength in crop genome analysis. The results of this research will provide researchers the tools needed to increase fruit size, quality and nutraceutically important genes, which will facilitate increased watermelon production, and provide basic knowledge that can also be applied to other crops to enhance food security. Breeding and genomic technologies to improve watermelons at WVSU and ASU will bring national and international recognition to our land-grant programs. The current proposal is based on sound research results in genome analysis, and the generation of SNP markers and a GWAS panel in watermelons. This proposal has a strong educational component that will improve the knowledge and skill levels of our students.

OBJECTIVES: Evaluation of 192 world watermelon collections for important yield traits and fruit quality traitsAssemble a GWAS panelMetabolomic profiling using LC-MS Q-TOF Analysis (High performance Liquid Chromatography couples with Quadruple Time of Flight mass spectrometer)Resequencing of GWAS panel and identification of SNPsAssociation mapping for location of QTLs/Markers of importance using genome-wide polymorphisms (GWAS)Combined horticultural evaluation of value added watermelon accessionsParticipatory selection approach

APPROACH: During the summers of 2018 and 2019, 192 watermelon genotypes from world collection will be grown at the Agricultural Experiment Stations of ASU and WVSU for evaluation of fruit-related traits. The experimental design will be a randomized block design (RBD) 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 following fruit yield and quality traits: a) days to first flower, b) days to first fruit maturity, c) fruit length and d) diameter (cm), e) fruit length-diameter ratio, f) number of fruits per plant, g) fruit yield per plant (kg/plant), h) rind thickness (cm), i) rind hardness (kg/mm), j) mesocarp pressure (kg/mm), k) endocarp pressure (kg/mm), and l) soluble solids (Brix%). Rind hardness, mesocarp and endocarp pressure will be measured by a penetrometer (Fruit pressure tester, FT 011) as puncture force (kg/mm) using 2, 3, and 8 mm tips. The Brix% of flesh juice will be measured using a hand refractometer. The phenotypic data will be subjected to ANOVA using JMP 7.0 software (SAS Institute Incorporation, Cary, NC).Metabolomic profiles for flesh of ripe watermelons of the genetic populations will be carried out. 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. 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®.Re-sequencing of watermelon GWAS panel to develop millions of SNPs is the most important strategy in this proposal. The identification of watermelon SNPs, as in this and previous studies, will allow for genome-wide association mapping and marker-assisted selection to support breeding programs in development of new, superior varieties with improved quality, production efficiency, and abiotic stress tolerance. The watermelon reference genome and its annotation are freely available on the internet from the International Cucurbit Genomics Initiative (ICuGI). Scaffolds anchored to various chromosomes will be further characterized for presence of SNPs. We will use SOAP2 to map all reads from each line against the reference genome, with mismatches of 3 bp allowed in a single read. The mapping results will then be split into chromosomes and sorted by mapping coordinates. The likelihood of genotypes of each line at every genomic site will be calculated by use of SOAPsnp39 with parameters −L 50 −u −F 1. Then, SNPs will be filtered using the criteria of the SNP quality value given by SOAPsnp >20 and the base quality >15 as described. The likelihood of each individual's genotype in "glf" format data will be generated for each chromosome of each line. We will also reveal SNPs in the core set by use of GLFmulti, software based on the maximum-likelihood estimation of site frequency at each site. This software integrates the likelihoods of genotypes of each line at each site generated by SOAPsnp. The core set of SNPs will be obtained by filtering according to the site frequency and quality score given by GLFmulti, which considers sequencing quality and mapping quality.The core set of SNPs will be further filtered by the following criteria: (i) 2 alleles existing in the population, which confirms the respective position as polymorphic in the population; (ii) the total sequencing depth >15× and <2,400× (excluding possible errors resulting from low-quality mapping and repetitive sequences); (iii) the average mapping rate of reads mapped to the position <1.5, which rules out effects caused by duplications; and (iv) the nearest SNPs >1 bp away. In addition, positions with depth <5× in each line will be treated as "genotype missing" instead of using the reference genotype. These SNPs will be used as the putative SNPs in the core set.Using genome-wide SNPs, we can evaluate the genome-wide LD pattern for a diverse collection of sweet watermelon. Because genomes with high LD will require low marker density for GWASs or GS, our SNPs will be of immense use for GWASs of watermelon breeding. GWASs based on LD provide a promising tool for detecting and fine-mapping quantitative trait loci underlying complex traits.An examination of population structure, genetic diversity at the molecular level, and LD distribution across various chromosomes will allow for the identification of QTLs through the model-based association mapping of various traits.

PROGRESS: 2019/04 TO 2020/03
Target Audience:Graduate and undergraduate students Watermelon breeders, geneticists and seed companies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergradaute students working in this project are getting exposed to watermelon production and breeding technologies. Two graduate students worked for their thesis research and 2 undergraduate students did their research projects. How have the results been disseminated to communities of interest?Field day at Alcorn State University Through emails,conference presentations and peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals?We plan to perform haplotype networking to understand the role of various metabolites in domestication. Functional validation of genes identified using GWAS for various metabolites is in progress. Graduate students and undergraduates will be given intensive training to handle big datasets. During the next reporting period, we will conduct a hands on big data workshop for undergraduates.

IMPACT: 2019/04 TO 2020/03
What was accomplished under these goals? Using this research capacity building grant, we developed 25,000 SNPs for 192 world watermelon collections and grown this panel to estimate various fruit specific compounds including Nonanal, 2,6-Nonadienal, E,Z, 3-Nonen-1-ol, Z, 3,6-Nonadien-1-ol, E,Z, 2-Nonenal, E and 2-Decenal. These compounds produce flavor and aroma to watermelons and important for insect resistance. In addition, we have profiles for flavonoids, carotenoids, phenols, citrulline, histidine, arginine, asparagine, glutamine, serine, glutamic acid, aspartic acid, threonine, glycine, glycine, alanine, GABA, proline, L-ornithine, tyrosine, valine, isoleucine, leucine, phenylalanine, ethanolamine, hydroxylysine, Alpha Aminoadipic Acid, Kynurenine and Tryptophan. Subsequently we performed GWAS to identify candidate genes responsible for natural variation for various metabolites.

PUBLICATIONS (not previously reported): 2019/04 TO 2020/03
1. Type: Journal Articles Status: Published Year Published: 2019 Citation: Joshi, V.; Shinde, S.; Nimmakayala, P.; Abburi, V.L.; Alaparthi, S.B.; Lopez-Ortiz, C.; Levi, A.; Panicker, G.; Reddy, U.K. Haplotype Networking of GWAS Hits for Citrulline Variation Associated with the Domestication of Watermelon. Int. J. Mol. Sci. 2019, 20, 5392.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Umesh K. Reddy, Shinde, S., Nimmakayala, P., Abburi, V. L., Girish Panicker, Alaparthi, S. B., Lopez-Ortiz, C.. Haplotype Networking of GWAS Hits for Citrulline Variation Associated with the Domestication of Watermelon. International Plant & Animal Genome XXVIII Conference, San Diego California, 2020.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dana Gaines, Kelsey Henry, Kibet Leonard, Willie Mims, Nimmakayala P, Tomason Y, Reddy UK,Girish Panicker. 2020.Evaluation of grafted watermelons (citrulus lanatus) grown on a heavy soil with organic treatments for their quality, yield, biomass, and adaptability in Mississippi. Mississippi Academy of Sciences, eighty fourth annual meeting, February 20, 2020.
4. Type: Journal Articles Status: Published Year Published: 2019 Citation: Garcia-Lozano, M., Dutta, S.K., Natarajan, P. Tomason YR, Lopez C, Katam R, Levi A, Nimmakayala P, Reddy UK.2019. Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids. Plant Mol Biol 102, 213â223 (2020). https://doi.org/10.1007/s11103-019-00942-7