Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: We have developed mapping populations for watermelon, melon and pumpkin. These populations are characterized for various fruit related traits and developed advanced segregating mapping populations. Molecular markers (SNPs, SSRs and AFLPs) are developed for these populations. Several syntenic fruit related QTLs are identified. 5000 BAC end sequences are generated to identify 234 BAC-end-SSRs. These markers linked watermelon genetic map with the physical BAC resources. SSR markers mapped on to watermelon were also used to map the genomes of cucurbit species to define syntenic relationships and common QTLs across the species are identified for various fruit related traits. Methylation profiles are generated for various tissues of diploids and tetraploids and epigenetic marks are characterized across the ploidy differences. Methylated markers are identified in watermelon genome and tracked them to be present across the genealogies. DNA profiles were generated using Methylation-sensitive AFLP assay for 47 watermelon heirlooms. Results indicated that methylation specific diversity (43%) in US watermelon heirlooms is higher than the diversity (19.8%) estimated by several investigators using conventional DNA markers. RNA sequencing using next gen methods (Hy-Seq Illumina) of collected fruit tissues at various developmental stages from the grafted cucurbit species in various combinations of scion and graft has revealed valuable information related to long distance signaling and role in disease resistance. Fruit specific unigenes were assembled for watermelon, Lagenaria and Cucurbita species. RNA sequencing using next gen methods (Hy-Seq Illumina) of collected fruit tissues at various developmental stages from the diploid and tetraploid watermelon has revealed genic information pertaining to neofunctionalization, subfunctionalization and gene silencing. We have developed FISH (Fluorescent in situ hybridization) techniques using BAC/rDNA probes across various cucurbit species to understand syntenic and phylogenetic relationships. FISH techniques developed for various cucurbit species helped to integrate genetic maps with cyto-molecular maps. Association mapping of various fruit related traits have been carried out for various cucurbit species and breeding values are predicted for various accessions in watermelon, melon and pumpkin collections. Cucurbit family includes several economically important crop species and miRNA component in any of the Cucurbitaceae members has not been investigated. Also, identification of miRNAs in these plant species is not possible due to the lack of genomic information. Four small RNA libraries from the pooled RNA isolated from different tissues and sequenced to identify not only conserved miRNAs but also novel miRNAs expressed in Cucurbita pepo, Cucurbita moschata Citrullus lanatus, and Lagenaria siceraria. Results suggest that Cucurbitaceae members possess conserved and novel miRNA-guided post-transcriptional gene regulations. Two small RNA libraries from tetraploid and diploid watermelon tissues are constructed and deep sequencing revealed miRNA regulation across the tissues of diploids and tetraploid watermelons. PARTICIPANTS: This is a multi-institutional project and all the Co-Principal Investigators are actively involved in the proposed research work. Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with Dr. Hongbin Zhang, Director of the Laboratory for Plant Genomics and Molecular Genetics and the TAMU GENE-finder Genomic Resources Center, Texas A&M University, College station, Texas. The ARS Research Geneticist Dr. Amnon Levi has developed next generation sequencing data. At WVSU total of ten students were involved, out of which seven undergraduates and three MS students. At Oklahoma State University, Drs. Sunkar and Jagadeeswaran generated small RNA libraries. Undergraduate students carried out independent experiments using microsatellite markers and also recorded observations on plant morphology including fruit traits for genetic mapping studies. Graduates students were involved in northern blotting and RT PCR of miRNAs across various species. TARGET AUDIENCES: Graduate students, Geneticists, Melon and watermelon Breeders, melon growers and seed companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
DNA markers, maps, genome sequences and SNP platforms developed in this study using 454 and Solexa sequencing technologies will have immense impact to speed up various breeding goals. Genetic map with the whole genome coverage will facilitate the identification and genetic mapping of putative resistance gene sequences that will be useful in future studies to enhance disease and pest resistance in cucurbit cultivars. Cucurbits are the important vegetable crops, grown in 44 states in the United States. We found that although cucurbit crop cultivars are widely diverse in their fruit shape, size, color, and quality, they share a narrow genetic base. This narrow genetic base is a result of many years of cultivation and selection for varieties with desirable fruit qualities. As a result, the American cultivars are susceptible to a large number of diseases and pests, and there is a continuous need to improve them and make them more resistant to diseases and pests. In this study, we have collaborated with the scientists at USDA-ARS and Oklahoma State University on conducting experiments aiming to elucidate the molecular events that control the genes affecting the quality of cucurbits. The knowledge gained in this study should be useful for researchers and plant breeders interested in understanding and the genetic and biological mechanisms that produce the differences among cucurbit crop cultivars, and in utilizing these mechanisms to improve quality suitable to consumer needs. Six workshops are conducted to impart training in marker assisted selection. Three graduate students submitted thesis in cucurbit research. In addition, five undergrad students conducted independent research experiments in cucurbit research and presented the data in national and international conferences. Five papers are published in international standard peer reviewed journals and three more manuscripts are currently in preparation. Through this project support, we have created a million dollar worth molecular markers and micro RNAs spanning across the cucurbit genomes. These resources led to identification of disease resistant genes and genes for fruit quality and yield. Resistant varieties for diseases esp. powdery mildew and seedless watermelon technology collectively can impact up to $1 million dollar worth income for the cucurbit producers per year.
Publications
- Nimmakayala P, Faridi NI, Tomason YR, Ponniah SK, Reddy UK. 2010. Molecular and Cytogenetic Characterization of Watermelon using DNA Markers and FISH. International Plant & Animal Genome XVIII Conference, January 9-13, 2010.W188.
- Tomason YR, Nimmakayala P, Vajja VG, Tomason MI, Reddy UK. 2010. LD mapping for melon fruit related traits. International Plant & Animal Genome XVIII Conference, January 9-13, 2010.P677.
- Rahman M, Reddy UK , Tomason YR , Ponniah SK , Vajja VG, Chinta RR, Nimmakayala P.2010 Mapping QTL in Synthetic Tetraploid Population Of Watermelon. International Plant & Animal Genome XVIII Conference, January 9-13, 2010.P680.
- Nimmakayala P, Tomason YR, Vajja VG and Reddy UK. 2010. Population Structure and Association Mapping in Watermelon. 108th Annual conference of the American Society for Horticultural Science, August 2-5, 2010. S155.
- Tomason YR, Nimmakayala P, Vajja VG, Levi A, Gibson P and Reddy UK. 2010. LD Mapping for Melon fruit related traits. 108th Annual conference of the American Society for Horticultural Science, August 2-5, 2010. S123.
- Jagadeeswaran Guru, Padma Nimmakayala, Yun Zheng, Kanchana Gowdu, Umesh Reddy and Ramanjulu Sunkar. 2011. Characterization of the small RNA component of the transcriptome in leaves and fruits of four cucurbits revealed dynamic regulation of conserved and novel miRNAs. BMC Genomics (in review)
- Dawei Li, Jordi Garcia-Mas, Juan Zalapa, Jack E Staub, Umesh K. Reddy, Xiaoming He, Zhenhui Gong, Yiqun Weng 2011. Syntenic Relationships between Cucumber (Cucumis sativus L.) and Melon (C. melo L.) chromosomes as revealed by comparative genetic mapping. BMC Genomics 2011, 12:396
- Nimmakayala P, Faridi NI, Tomason YR, Lutz F, Levi A and U K. Reddy. 2011. Citrullus. (Book chapter). Wild Crop Relatives: Genomic and Breeding Resources, Vegetables. Springer-Verlag, Berlin Heidelberg. Pages 59-66. (Book Chapter)
- Nimmakayala P, Vajja, G., Gist, R., Tomason, Y.R., Levi, A., Reddy. U.K. 2011. Effect of DNA methylation on molecular diversity in watermelon heirlooms, and stability of methylation specific polymorphisms across the genealogies. Euphytica. 177: 79-89.
- Aryal N, Nimmakayala P, Tomason YR, Jeong J, Vajja VG, Levi A and Reddy UK. Molecular and cytogenetic characterization of watermelon using DNA Markers and FISH. 108th Annual conference of the American Society for Horticultural Science, August 2-5, 2010.S303.
- Lutz FG, Ponniah SK, Nimmakayala P, Tomason YR, Levi A and Reddy UK. 2010. Syntenic Analysis of Cucurbit Cultivar Complex Using Microsatellite Markers. 108th Annual conference of the American Society for Horticultural Science, August 2-5, 2010. S90.
- Nimmakayala P, Tomason Y, Jeong J, Vajja G, Levi A, Gibson P,Reddy UK. 2009. Molecular diversity in the Ukrainian melon collection as revealed by AFLPs and microsatellites. Plant Genetic Resources 7 (2): 127-134.
- Nimmakayala P, Jeong J, Tomason Y, Levi A, Ramasamy P, UK. Reddy. 2010. Genetic reticulation and interrelationships among Citrullus species as revealed by joint analysis of shared AFLPs and species-specific SSR alleles. Plant Genetic Resources 8 (1): 16-25.
- conference presentations 2009:
- Nimmakayala P, Tomason YR, Sokolova VK, Vajja VG, Ponniah SK, Reddy UK. 2010. Population Structure and Association Mapping in Watermelon Heirloom Collections. International Plant & Animal Genome XVIII Conference, January 9-13, 2010.P679.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Mapping data, populations and DNA markers (SNPs, SSRs and AFLPs) are developed. Several syntenic fruit related QTLs are identified. 5000 BAC end sequences are generated to identify 234 BAC-end-SSRs. This resource will link watermelon genetic map with the physical BAC resources. Currently, we are genotyping common SSR markers (heterologous)on to watermelon and cucumber maps to define syntenic relationships and common QTLs across the species that would illustrate similarities within the cucurbitaceous family for various fruit related traits. We grafted cucurbit species in various combinations of scion and graft and collected fruit tissues at various developmental stages. In addition to identified cDNA AFLP generated transcript fragments across the species and typical to grafting, we are making small RNA libraries on various tissues. Apart from DNA markers and functional genomics tools, we developed FISH (Fluorescent in situ hybridization) techniques using BAC/rDNA probes across various cucurbit species to understand syntenic relationships. Cucurbit family includes several economically important crop species and miRNA component in any of the Cucurbitaceae members has not been investigated. Also, bioinformatic identification of miRNAs in these plant species is not possible due to the lack of genomic information. We generated four small RNA libraries from the pooled RNA isolated from different tissues (leaf, stem, as well as flesh, rind and placenta from the fruits) and sequenced to identify not only conserved miRNAs but also novel miRNAs expressed in Cucurbita pepo, Cucurbita moschata Citrullus lanatus, and Lagenaria siceraria. Approximately 5 million reads were obtained from each of these libraries and initial sequence analysis indicated the identification of 130 miRNA homologs belonging to 23 conserved miRNA (miR156/157, miR159, miR160, miR162, miR164, miR165/166. miR167, miR168, miR169, miR170/171, miR172, miR319, miR390, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR408, miR528, miR894 and miR2111) families in these four species. Of these conserved miRNAs, miR165/166 is the most abundantly expressed in all three plant species investigated. However the abundance of other conserved miRNAs (for instance miR159 family) is not uniform but greatly varied among the species. Most importantly, our analysis also has found more than two dozens of novel miRNAs that are conserved between these four cucurbit species but are absent in sequenced genomes such as Arabidopsis and rice. These initial results suggest that Cucurbitaceae members possess conserved and novel miRNA-guided post-transcriptional gene regulations. PARTICIPANTS: This is a multi-institutional project and all the Co-Principal Investigators are actively involved in the proposed research work. Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with Dr. Hongbin Zhang, Director of the Laboratory for Plant Genomics and Molecular Genetics and the TAMU GENE-finder Genomic Resources Center, Texas A&M University, College station, Texas. The ARS Research Geneticist Dr. Amnon Levi has developed next generation sequencing data. At WVSU total of ten students were involved, out of which seven undergraduates and three MS students. At Oklahoma State University, Drs. Sunkar and Jagadeeswaran generated small RNA libraries. Undergraduate students carried out independent experiments using microsatellite markers and also recorded observations on plant morphology including fruit traits for genetic mapping studies. Graduates students were involved in northern blotting of miRNAs across various species. TARGET AUDIENCES: Graduate students, Geneticists, Melon Breeders, melon growers and seed companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Fruit yield and quality related genes will be identified and their expression across cucurbit species is an important genomic resource for developing high quality cucurbits. Four graduate students, an international researcher from Ukraine and seven undergraduate students are trained in cucurbit breeding and functional genomics.
Publications
- Padmavathi Nimmakayala, Yan R. Tomason, Jooha Jeong, Gopinath Vajja, Amnon Levi Paul Gibson, Umesh K. Reddy 2010. Genomic Resources for wild Citrullus species. (A Book chapter submitted to Springer Verlog publication to be published in the book entitled as Genomic Resources for Wild Vegetable Species.
- Padmavathi Nimmakayala, Yan R. Tomason, Jooha Jeong, Gopinath Vajja, Amnon Levi Paul Gibson, Umesh K. Reddy 2010. Effect of DNA methylation on molecular diversity of watermelon heirlooms and stability of methylation specific polymorphisms across the genealogies. Euphytica. DOI 10.1007/s10681-010-0259
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Identification of fruit related QTLs across various cucurbit species is in progress. Various segregating (F2 and F3) mapping populations of watermelon (diploid and tetraploid), melon and also an interspecific pumpkin population involving the species Cucurbita pepo and C. moschata were evaluated. Currently, we are developing and genotyping common SSR markers (heterologous) to define syntenic relationships and common QTLs across the species that would illustrate similarities within the cucurbitaceous family for various fruit related traits. We grafted cucurbit species in various combinations of scion and graft and collected fruit tissues at various developmental stages. We did cDNA AFLP on various tissues and identified several similar transcript fragments across the species and typical to grafting. 2500 of these TFs are sequenced and currently annotation is in progress. We also characterized diploid and tetraploid watermelon lines for methylation differences and gene expression using mAFLP, cDNA AFLP and microarrays. Apart from DNA markers and functional genomics tools, we are currently exploring FISH (Fluorescent in situ hybridization) using BAC/rDNA probes across various cucurbit species to understand syntenic relationships. PARTICIPANTS: This is a multi-institutional project and all the Co-Principal Investigators are actively involved in the proposed research work. Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with Dr. Hongbin Zhang, Director of the Laboratory for Plant Genomics and Molecular Genetics and the TAMU GENE-finder Genomic Resources Center, Texas A&M University, College station, Texas. The ARS Research Geneticist Dr. Amnon Levi has developed EST resources and made a microarray chip for 4000 fruit related genes. At WVSU total of six students were involved, out of which four were undergraduates and two MS students. Undergraduate students carried out independent experiments using microsatellite markers and also recorded observations on plant morphology including fruit traits for genetic mapping studies. Graduates students were involved in DNA, RNA extractions, cDNA AFLP analysis and sequencing. TARGET AUDIENCES: Graduate students, Geneticists, Melon Breeders, melon growers and seed companies. PROJECT MODIFICATIONS: There are no major modifications.
Impacts
Fruit yield and quality related genes will be identified and their expression across cucurbit species is an important genomic resource for developing high quality cucurbits. A graduate student, an international researcher from Ukraine and three undergraduate students are trained in cucurbit breeding and functional genomics.
Publications
- 1.Padmavathi Nimmakayala, Yan R. Tomason, Jooha Jeong, Gopinath Vajja, Amnon Levi Paul Gibson, Umesh K. Reddy 2009. Molecular Diversity in the Ukrainian Melon Collection as Revealed by AFLPs and Microsatellites. Plant Genetic Resources. 7, 2, 127-134.
- 2.Padmavathi Nimmakayala, Yan R. Tomason, Jooha Jeong, Gopinath Vajja, Amnon Levi, Paul Gibson, Umesh K. Reddy 2009. Genetic reticulation and interrelationships among Citrullus species as revealed by joint analysis of shared AFLPs and species specific SSR alleles. Plant Genetic Resources. 1-10 (currently online)
- 3.Umesh K. Reddy, Rene Gist, Yan R. Tomason, Jooha Jeong, Gopinath Vajja, Amnon Levi, Padmavathi Nimmakayala. 2009. Hitchhiking effects on genetic diversity induced by DNA methylation in US watermelon heirlooms. Plant Breeding (Submitted).
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We developed mapping populations for watermelon, melon and pumpkin. These mapping populations are characterized for various fruit related traits. Currently, we are developing and genotyping common markers to define syntenic relationships and common QTLs across the species that would illustrate similarities within the cucurbit family for various fruit related traits. We grafted cucurbit species in various combinations of scion and graft and collected fruit tissues at various developmental stages. We did cDNA AFLP on various tissues and identified several similar transcript fragments across the species and typical to grafting. We are in process of sequencing these TFs for use in understanding their role in long distance signaling from scion to graft across the species and conserved similarity across the genomes. PARTICIPANTS: This is a multi-institutional project and all the Co-Principal Investigators are actively involved in the proposed research work. Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with Dr. Hongbin Zhang, Director of the Laboratory for Plant Genomics and Molecular Genetics and the TAMU GENE-finder Genomic Resources Center, Texas A&M University, College station, Texas. At Florida A&M University, Dr. Mehboob Sheik and Dr. Ramesh Katam are evaluating the watermelon segregating population in experimental fields. The ARS Research Geneticist Dr. Amnon Levi has developed EST resources and made a microarray chip for 4000 fruit related genes. At WVSU total of six students were involved, out of which four were undergraduates and two MS students. Undergraduate students carried out independent experiments using microsatellite markers and also recorded observations on plant morphology including fruit traits for genetic mapping studies. Graduates students were involved in DNA, RNA extractions, cDNA AFLP analysis and sequencing. TARGET AUDIENCES: Graduate students, Geneticists, Melon Breeders, melon growers and seed companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Fruit yield and quality related genes will be identified and their expression across cucurbit species is an important genomic resource for developing high quality cucurbits. A graduate student, an international researcher from Ukraine and three undergraduate students are trained in cucurbit breeding and functional genomics.
Publications
- Nimmakayala, Padma; Tomason,Yan R.; Jeong Jooha; Vajja, Gopinath; Levi, Amnon;Gibson Paul; Reddy, Umesh K, 2008. Molecular Diversity in ,the Ukrainian Melon Collection as Revealed by AFLPs and Microsatellites. Plant Genetic Resources (In Press).
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