Source: ALCORN STATE UNIVERSITY submitted to NRP
EXPLORING MELON GENETIC DIVERSITY TO BREED NOVEL MORPHOTYPES FOR DISSEMINATION INTO SMALL FARMS THROUGH A PARTICIPATORY SELECTION APPROACH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0230461
Grant No.
2012-38821-20036
Cumulative Award Amt.
$498,645.00
Proposal No.
2012-02511
Multistate No.
(N/A)
Project Start Date
Sep 1, 2012
Project End Date
Aug 31, 2016
Grant Year
2012
Program Code
[EQ]- Research Project
Recipient Organization
ALCORN STATE UNIVERSITY
(N/A)
ALCORN STATE,MS 39096
Performing Department
Agriculture
Non Technical Summary
Melon breeders routinely use morphological markers for selection such as presence of orange flesh to improve β-carotene in melon (linked to flesh). Traits like seed germination, seedling vigor, fruit yield, early maturity, fruit size and sugar content are manifested by complex inheritance represented by multiple genes. Improvement in quantitative traits using traditional strategies is time consuming. For example, selecting for higher carotenoid concentration is difficult using visual appearance as was done in the past. Further, morphological markers will have an intrinsic disadvantage if the trait is influenced by the environment. CPW and FW are good examples of the devastating diseases, where developing new cultivars with resistance has thus far proven difficult in all the cucurbit crops, and has been indentured by producers as the major problems needing identification of linked markers for resistant alleles and further marker assisted selection to pyramid genes into cultivars. Development of disease and pest-resistant, high-quality melon varieties is critical to the economic prosperity of US melon farmers. Most importantly, our project activities include comprehensive demonstrations of the fruit quality traits, taste and flavor of the new introgressed morphotypes to the growers and consumers in four states in an effort to involve them to satisfy their requirements for good flavor, fruit quality, and high market value and address any concerns related to production. The major effort in this thrust will be conducting on-farm trials for participatory breeding to identify superior melon types to local cultivars. Selections for superior types will be made from participatory breeding at the farms of underserved communities. Farmers will be educated on how to harvest, store, and market the crop for fresh consumption. Farmers will also be taught how to save their seed for future production. During the field days, surveys will be conducted to elicit consumer preferences for fruit attributes (including shape, size, skin color and pattern, flesh color and texture, sweetness, flavor and aroma). On-line leaflets will be created for all appropriate new material that is created in the project. These will support the webinars, videos, and field days. One series of leaflets will be directed to the production, post-harvest handling, and marketing of cucurbits. An undergraduate internship program will be organized with cooperating scientists on the project.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011420108030%
2021420108020%
2031420108020%
2041420108115%
2121420108115%
Goals / Objectives
Melons (Cucumis melo L.) are a diverse group of cucurbits that includes the orange flesh cantaloupes, the green flesh honeydew, and the mixed melons (Casaba, Crenshaw, Persian, Santa Claus, and Juan Canari). The primary goal of our melon program is to use genomic assisted characterization of exotic melons for fruit traits and breeding of superior melon types by pyramiding favorable allele combinations into the US melons. We made a nested association mapping population (NAM) to maximize recombination from diverse world melon collections with the aim to carry out association mapping for improvement of fruit yield and quality traits. The ultimate goal of this three year project is the development of high-yielding, disease resistant melon varieties with improved fruit quality by selective crosses with exotic melon types in conjunction with molecular-based gene-introgression tracking methodologies. To address these challenges, we have assembled a team of inter institutional researchers involving breeders, horticulturists and geneticists to function at various levels of the project. The current proposal will address the need to integrate modern technologies that can create a set of common tools for marker assisted breeding through the specific objectives are 1) Development of high-throughput DNA marker platforms for melon by application of genotyping technologies to resolve public domain melon SNPs, 2) Evaluation of fruit yield, quality and resistance traits, 3) Association mapping for location of QTLs/Markers of importance, 4) Initiating marker-assisted breeding for improved traits, 5) ECO-TILLING of new alleles for genes of importance using GenBank resources and other publicly available candidate nutraceutical genes as well as publically available resistance and quality traits, and 6) Integration into a platform that facilitates data capture, storage and mining relevant to marker assisted breeding process. The combination of SNP polymorphism data over a range of morphotypes will allow us to identify functional marker panel consisting a set of highly informative markers for marker-assisted selection experiments in melons. This resource will facilitate the selection of novel traits for the introgression of exotic germplasm into elite backgrounds. We will develop simple horticultural procedures to maintain breeding lines, seed production, taste and flavor panels and cost-effective genomic protocols such as SNP genotyping and DNA diagnostics for melon trait identification. A program of exchange visits of students, scientists and extension personnel will efficiently allow technology and material transfer between the institutions. Resources will include a series of webinars to provide interactive education about the breeding efforts; a series of YouTube videos to describe the breeding lines, breeding techniques, and production methods; on-line leaflets on production, post-harvest handling, and marketing of cucurbits; and a "match the scientist to the student" undergraduate internship program. Demonstration plots of the advanced breeding lines, field days, and taste tests will allow farmers and consumers to be directly engaged in the project.
Project Methods
Genotyping: The proposal is cost effective as a large number of SNPs is available for public use. Currently there are 10387 SNPs available for public use on the Cucurbit Genomic Resource site (http://www.icugi.org/) that are identified from the aligned sequences of unigenes/DNA/BAC end of reference melon collections (Piel de Sapo, Vedrantais, PI161375, Dulce, agrestis and cantaloupe). Many new marker technologies are currently available for genotyping using SNPs, reflecting the increasing popularity of these markers. Once association mapping is carried out using a 200 DNA panel to identify QTLs, the selected SNPs can be further assayed for marker assisted selection or any targeted region genotyping or fine-mapping with Custom Infinium DNA Analysis products. Evaluation of fruit yield, quality and resistance traits: A set of NAM lines will be selected based on the screens from the existing DNA marker panels and superior NAM lines will be used for the participatory selection program. The lines identified at on-farm location studies from the two states (MS and WV) will be further screened for disease resistance for powdery mildew (PM) and (F.o.m) Fusarium oxysporum f. sp melonis at ASU to identify resistant lines. During the summers of 2013, 2014 and 2015, field tests will be run to measure various traits in the individual advanced progeny lines and morphotypes collections. Fruit yield/quality traits to be measured are Days to first flower, Days to first fruit maturity, fruit weight, fruit shape, fruit length, fruit width, thickness of pericarp, flesh color, fruit flavor, juiciness of flesh, sugar content, fruit number per plant, average fruit weight, number of lateral branches, exocarp and endocarp pressure (transportability) and shelf life (number of days in storage after harvest). Association mapping for location of QTLs/Markers of importance: Resolution of population structure is the most important activity before we undertake association mapping. Since this proposal aims to use all the world collections for association mapping, we propose to conduct analysis for population structure using the large genotyping data sets. Association mapping analysis will be conducted using the freely useable software TASSEL 2.1 (www.maizegenetics.net). Marker assisted selection (MAS) to identify lines with QTLs and pyramiding favorable alleles using recurrent selection We have identified markers linked to various fruit yield, quality and resistance traits using association mapping. Information on more QTLs, gene specific markers and NAM progenies that are homozygous for various important loci will be available from the association mapping activity. We propose to launch MAS in the first year itself using the markers that are already available from previous research. By the end of summer 2013, we will have our first MAS selected early generation progenies ready to test in the on farm trials. Our main focus will be to eliminate progenies that are heterozygous at favorable marker locus and forward homozygotes for contributing allele of marker. MAS allows the transfer of genes from a donor into an elite recipient genotype, the recurrent parent

Progress 09/01/12 to 08/31/16

Outputs
Target Audience:Target audience include graduate and undergraduate students at ASU and WVSU, and local farmers. Around 250 local farmers visited the melon filed during 2015 and 2016 field days at ASU. Besides filed day, we also have distributed melon heirloom collections to 5 local farmers in West Virginia and Mississippi. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Six undergrad students and two graduate students participated at various levels of the project including horticultural evaluation, genomics, plant breeding and phytochemicalevaluation. How have the results been disseminated to communities of interest?Filed days, emails and personal visits. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have generated a large number of genome anchored SNPs(13,789) for melon, which will be of immense use for melon improvement programs across the world. In addition, we made a genetic map using which we can identify linked markers for various traits. This melon genomics program provided hands on training to five undergraduate and three graduate students. All these students are interested to continue further studies (MS/PhD) in plant breeding and Genetics. Exposing young scientists to a variety of hands-on training will improve their scientific skills and augment their career path choices. This will encourage the recruitment of minority undergraduate and graduate students by emphasizing molecular breeding as an interesting and rewarding career. Our NAM progenies will be of immense use for identifying unique value added melon morphotypes for use in small farms. Using 13,789 genome anchored SNPs, we estimated genome-wide fixation indices (Fst) between various melon morphotypes and genome-wide linkage disequilibrium (LD) decay The FST between accessions of cantalupensis and inodorus was 0.23. The FST between cantalupensis and various agrestis accessions was in a range of 0.19-0.53 and between inodorus and agrestis accessions was in a range of 0.21-0.59 indicating sporadic to wide ranging introgression. Melon has climacteric and non-climacteric morphotypes and wide variation for fruit firmness, a very important trait for transportation and shelf life. Various levels of QTLs were identified with high to moderate stringency and linked to fruit firmness using both genome-wide association study (GWAS) and biparental mapping. Gene annotation revealed some of the SNPs are located in β-D-xylosidase, glyoxysomal malate synthase, chloroplastic anthranilate phosphoribosyl transferase, and histidine kinase, the genes that were previously characterized for fruit ripening and softening in other crops. Additional information can be found from the following Journal article published recently. Nimmakayala P, Tomason Y, Abburi VL, Rodríguez A, Saminathan T, Vajja VG, Salazar G, Panicker G, Levi A, Wechter W, McCreight J, Korol A, Ronin Y, Garcia-Mas J, Reddy U (2016) Genome-wide differentiation of various melon horticultural groups for use in GWAS for fruit firmness and construction of a high resolution genetic map. Frontiers in Plant Science 7 (1437). doi:10.3389/fpls.2016.01437

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Nimmakayala P, Tomason Y, Abburi VL, Rodr�guez A, Saminathan T, Vajja VG, Salazar G, Panicker G, Levi A, Wechter W, McCreight J, Korol A, Ronin Y, Garcia-Mas J, Reddy U (2016) Genome-wide differentiation of various melon horticultural groups for use in GWAS for fruit firmness and construction of a high resolution genetic map. Frontiers in Plant Science 7 (1437). doi:10.3389/fpls.2016.01437


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

Outputs
Target Audience:Target audience includegraduate and undergraduate students at ASU and WVSU, and local farmers. Around 250 local farmers visited the melon filed during field day at ASU. Besides filed day, we also have distributed melon heirloom collections to 5 local farmers in West Virginia and Mississippi. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students are involved in filed work starting from sowing seeds to harvesting. They are also trained in selfing and crossing techniques. One graduate student is working on melon association mapping and genetic diversity at WVSU. How have the results been disseminated to communities of interest?Field Days, emails and conference presentations What do you plan to do during the next reporting period to accomplish the goals?We are planning tocarry out SNP validation, candidate gene identification for various traits and forward NAM progenies for participatory testing in farmers' fields.

Impacts
What was accomplished under these goals? As per the objectives, we have developed 12,000 new SNPs using Nextgen sequencing technologies and mapped them to whole genome sequence draft of melon to identify chromosomal location for all the SNPs. We made a biparental cross and using F2s, we have mapped all the SNPs into a robust genetic map. F2s and F3s are evaluated and phenotyping data pertaining to fruit traits, resistance and nutraceutical traits are recorded and QTL mapping is in progress. In addition to this work, we have field evaluated several melon morphotypes panels collected from various countries to record various traits including fruit quality, resistance and nutraceutical traits from two different locations and two seasons. Currently, we are performing genomewide association studies (GWAS). If extension is granted, we will validate QTLs identified in biparental crosses in GWAS and vice versa. Since the SNPs are already mapped to various chromosomes and since we have high density SNPs across the genome, it would be possible to identify candidate genes underlying various traits. In addition, Nested Association Mapping (NAM) population is currently in progress.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Comparison of Fruit Qualities of Melons (Cucumis melo L.) from Around the Globe Grown on a Heavy Soil Using Organic Agricultural Practices


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

Outputs
Target Audience: Target audience includes graduate and undergraduate students at ASU and WVSU, and local farmers. Around 200 local farmers visited the melon filed during field day at ASU. Besides filed day, we also have distributed melon heirloom collections to 5 local farmers in West Virginia and Mississippi. Two undergraduate students are involved in filed work starting from sowing seeds to harvesting. They are also trained in selfing and crossing techniques. One graduate student is working on melon association mapping and genetic diversity at WVSU. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two undergrads and a grad student are being trained in phenotyping melon morphotypes. Association mapping was incorporated in the crop evolution and biodiversity syllabus. Five students attended crop diversity and evolution class, in which crop genetic diversity and concept of secondary centers of origin was explained using the collection used in this project. How have the results been disseminated to communities of interest? Filed days, emails and personal visits. Local farmers in Mississippi were exposed to melon morphotypes with diversefruit shapes and sizes. Segregants of various crosses involving diverse melon morphotypes were useful for farmers to select melon types of their choice. What do you plan to do during the next reporting period to accomplish the goals? Nested association mapping population is being phenotyped for fruit quality. Currently association mapping using GWAS modules is in progress to identify genes for various fruit traits. A manuscript on SNP development and haplotyping of melon morphotypes is under review.

Impacts
What was accomplished under these goals? We have developed 25,000 SNPs (single nucleotide polymorphism) using Genotyping by Sequencing for 96 diverse group of melon accessions that includes the orange flesh cantaloupes, the green flesh honeydew, and the mixed melons (Casaba, Crenshaw, Persian, Santa Claus, Juan Canari). Using these SNPs, we identified specific haplotypes for each melon morphotype that can be used to identify passport for each kind. Linkage disequilibrium (LD) decay in melon cultivars was characterized. Since all the SNPs were mapped to various chromosomes of whole genome sequence draft, it was possible to generate pairwise FST (morphotypes differentiation index) distribution to identify the locations of importance for breeding across the genome. A high resolution genetic map was constructed using 2000 SNPs. We estimated colinearity of genetic map and whole genome sequence draft that allowed us to identify chromosomal rearrangements across the genome. Entire collection in this study was field evaluated for two seasons under two locations (ASU and WVSU) for the fruit quality data (beta-carotene, total soluble solidsand sucrose levles).

Publications

  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Association mapping and haplotyping in melon using SNP markers


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: Target audience includes graduate and undergraduate students at ASU and WVSU, and local farmers. Around 200 local farmers visited the melon filed during field day at ASU. Besides filed day, we also have distributed melon heirloom collections to 5 local farmers in West Virginia and Mississippi. Two undergraduate students are involved in filed work starting from sowing seeds to harvesting. They are also trained in selfing and crossing techniques. One graduate student is working on melon association mapping and genetic diversity at WVSU. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? All the graduate and undergraduate students working in this project are exposed to field techniques like selfing and crossing.In the lab, students are trained in DNA technologies starting from DNA isolation to SSR amplification and SNP genotyping. How have the results been disseminated to communities of interest? Filed days, emails and personal visits. What do you plan to do during the next reporting period to accomplish the goals? A genetic map will be created that will have colinearity with the whole genome sequence draft. This genetic map will be used to locate recombination hot and cold spots and precise LD blocks will be characterized. SNP dataset that is used for diversity analysis of melons will be further characterized based on chromosome location, gene/non genic location and selection pattern across the genome. Quantitative nucleotide (QTNs) for various fruit traits will be identified. Nested association mapping populations will be carried over to the future generations. Some of the superior segregants will be tested for their superiority at both Alcorn State University and West Virginia State University.

Impacts
What was accomplished under these goals? Ninety six diverse group of melons that includes the orange flesh cantaloupes, the green flesh honeydew, and the mixed melons (Casaba, Crenshaw, Persian, Santa Claus, Juan Canari) are currently grown at both Alcorn State University and West Virginia State University. The fruit data that is collected from these will be used to perform association mapping and thereby identify genes and markers that control various quality traits. A team of melon breeders at West Virginia State University are currently working to characterize the eastern European melon, adana, varieties and introgress desirable fruit traits into the US melons. We have introgressed melons with part of adana genome into the cantaloupe types. The primary goal of our melon program is to use genomic assisted characterization of exotic melons for fruit traits and breeding of superior melon types by pyramiding favorable allele combinations into the US melons. We made a nested association mapping population (NAM) to maximize recombination from diverse world melon collections. Currently, we designed a melon screening program to assess advanced lines of the different melon types and determine their adaptability to West Virginia and Mississippi growing conditions by use of a participatory selection approach involving small farms.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tomason Y, Nimmakayala P, Levi A and Reddy U.2013.Map-based molecular diversity, linkage disequilibrium and association mapping of fruit traits in melon. Molecular Breeding, 2013. 31(4): p. 829-841