Progress 05/01/20 to 04/30/24
Outputs
Target Audience:Watermelon Geneticists, plant breeders and Private seed companies Graduate and undergraduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have organized four workshops for local graduate students, undergraduate students, postdoctoral associates, and faculty in various bioinformatics pipelines, emphasizing machine learning, transcriptomics, and genomics. We integrated these pipelines into the curriculum of Biotechniques (BT 571), Molecular Genetics (BT 573), and Crop Diversity Analysis (BIol 599). We also organized virtual RNA sequence analysis for other 1890 students, postdocs and faculty from Delaware state university, Florida A&M university and Fort Valley State University. We also provided training in Nanopore sequencing. Both the graduate students and technicians were actively involved in selfing and intermating RILs that have potential for use as prebreeding lines. How have the results been disseminated to communities of interest?Through hands-on training, Emails and presentations in national and international conferences. PACE assays that can discriminate resistant and susceptible alleles are made publicly available for use in genomic selection. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Gummy Stem Blight (GSB), caused by three cryptic Stagonosporopsis species, is one of the most devastating diseases affecting watermelon in the US, impacting most of the plant's above-ground parts. Current research project aimed to identify key Quantitative Trait Variants (QTVs) that include SNPs and In/Dels associated with GSB resistance in selfed derivatives of advanced multicross interspecific derivatives population derived from intercrosses between the most resistant lines ofCitrullus amarusand highly susceptible cultivars ofCitrullus lanatus. Based on the disease scores from 288 interspecific derivatives in the field (7 days, 14 days, 21 days, and 27 days) and greenhouse, 30 lines exhibiting extreme resistance and another 30 susceptibility to gummy stem blight disease were chosen using an outlier algorithm (https://www.r-bloggers.com/2020/08/outliers-detection-in-r/). These extremes were utilized to form a Resistant bulk (R-Bulk) and Susceptible bulk (S-Bulk). Genomic DNA was extracted from young leaves using the DNeasy Plant Pro kit (Qiagen, Germany). The integrity and concentration of the extracted genomic DNA were assessed through agarose gel electrophoresis and a Qubit fluorimeter (Thermo Scientific, USA). Genomic DNA from each individual was pooled in equal quantities to construct R-Bulk and S-Bulk. This combined DNA was subsequently used for the DNA library preparation and genome sequencing.These bulks underwent whole-genome sequencing, generating over 1 billion reads per bulk to achieve comprehensive genome coverage. The mapping percentage of the bulks to the parental genomes ranged from 92% to 99%. More than 6 million SNPs and 1 million indels were identified from the resistant parental genome. In comparison, fewer than 2 million SNPs and 0.4 million indels were found using the susceptible parental genome. QTNs associated with GSB resistance were identified using single-nucleotide polymorphism-index and Gprime methods. Significant quantitative trait loci (QTLs) associated with Gummy Stem Blight (GSB) resistance were identified on chromosomes 1, 2, 3, 5, 7, 10, and 11. Among the key genes identified were the Lipase Class 3 Family Protein on chromosome 1, which is involved in lipid-based signaling pathways essential for plant stress responses, and the Amine Oxidase gene, which produces hydrogen peroxide, a crucial molecule in plant defense. Myosin-1 on chromosome 1 plays a significant role in maintaining cell wall integrity, while the 2-oxoglutarate (2OG) and Fe(II)-dependent Oxygenase Superfamily Protein enhances plant defense mechanisms. On chromosome 2, the Ribosome Hibernation Promotion Factor influences the plant's ability to respond to stress and pathogen attacks. Isoflavone Reductase Like is involved in the biosynthesis of antifungal compounds, and the Exostosin Family Protein is associated with the synthesis of cell wall polysaccharides. The Ubiquitin-like-specific Protease 1D and Zinc Finger CCCH Domain-Containing 15 genes on chromosome 3 are critical for regulating defense responses and RNA stability, respectively. Chromosome 5 hosts the Dihydroxy-acid Dehydratase Putative gene, which participates in the synthesis of defensive compounds. Notably, the Avr9/Cf-9 Rapidly Elicited Protein on chromosome 7 recognizes pathogen effectors and activates defense mechanisms. On chromosome 10, the Transmembrane Protein Putative is likely involved in signal transduction processes, and the Leucine-Rich Repeat Receptor-Like Protein Kinase Family Protein is crucial for detecting pathogenic attacks. Finally, chromosome 11 includes the Zinc Finger Protein 1, which is involved in DNA binding and transcription regulation, and Xyloglucan Endotransglucosylase/Hydrolase, which remodels the cell wall to enhance physical barriers against pathogens. Significant loci associated with GSB resistance were used to develop PACE assays. These findings will aid in the development of GSB-resistantwatermelon cultivars.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Natarajan P Rathnagiri A, Rivera-Burgos LA, Lopez-Ortiz C, Tomason Y, Nimmakayala P, Sari N, Wehner TC, Levi A, Reddy UK (2024). Exploring the Genomic Landscape of Gummy Stem Blight Resistance in Watermelon Through QTL-Seq. Theoretical and Applied Genetics (in review).
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:Watermelon Geneticists, plant breeders and Private seed companies Graduate and undergraduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have organized six workshops for local graduate students, undergraduate students, postdoctoral associates, and faculty in various bioinformatics pipelines with an emphasis on machine learning, transcriptomics, and genomics. We integrated these pipelines into the curriculum of Biotechniques (BT 571), Molecular Genetics (BT 573), and Crop Diversity Analysis (BIol 599). We also organized virtual RNA sequence analysis for other 1890 students, postdocs and faculty from Delaware state university, Florida A&M university and Fort Valley State University. We also provided training in Nanopore sequencing. Both the graduate students and technicians were actively involved in selfing and intermating RILs that have potential for use as pre-breeding lines. How have the results been disseminated to communities of interest?Through classroom teaching and training, Emails and zoom presentations. National and International Conference presentations. What do you plan to do during the next reporting period to accomplish the goals?Currently, we are in the process of resequencing the 200 RILs and 180 lines of GWAS panel. We will be developing PACE assays targeting candidate genes to perform high throughput screening of lines from RILS, GWAS panel and PI collection to characterize gene pools that are resistant to gummy stem blight and advance ongoing genomic selection. We will conduct two more workshops for undergraduate students in the field of genomics and machine learning tools.
Impacts
What was accomplished under these goals?
We built a 288 MAGIC RIL population and phenotyped for gummy stem blight for three years. This RIL population involves intermating of Citrullus amarus and resulting 4th generation unselected segregants were mated with high quality type cultivars of Citrullus lanatus and forwarded to the F8 generation. We performed Genotyping by Sequencing for the entire population to identify more than 30,000 segregating SNPs mapped to both amarus (USVL246) and lanatus (Charleston Grey) reference genomes. GWAS was performed using the entire population as well as on the individuals of 30 extreme resistant and susceptible segregants for use in XP-GWAS. This analysis revealed strong associations between the SNPs located in known disease-resistant genes and their regulators. In addition, we performed 500X coverage whole-genome sequencing of bulked DNA of the 30 extreme resistant and susceptible individuals. This data generated more than six million polymorphic SNPs between the bulks. We used QTL-Seq analysis and identified a set of QTNs and causative genes. PCR Allele Competitive Extension (PACE) genotyping assays for highly associated SNPs were developed for use in Marker Assisted Selection and used to screen larger cultivar collections. These PACE markers can be highthroughput, hence can be useful to readily incorporate GSB resistance in watermelon and melon.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
3. Natarajan P, Nimmakayala P, Lopez-Ortiz C, C Rathnagiri A, Luis A. Rivera-Burgos, Sari N, Wehner TC, Levi A, Tomason Y, Reddy UK (2023) Whole-genome scanning using QTL-Seq and GWAS for gummy stem blight resistance in watermelon. The International Plant and Animal Genome Conference 2023 (PAG 30), USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
2. Natarajan P, Nimmakayala P, Lopez-Ortiz C, C Rathnagiri A, Luis A. Rivera-Burgos, Sari N, Wehner TC, Levi A, Tomason Y, Reddy UK (2022) Whole-genome scanning using QTL-Seq and GWAS for gummy stem blight resistance in watermelon. Cucurbitaceae 2022, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Natarajan P, Nimmakayala P, Lopez-Ortiz C, Tomason Y and Umesh K. Reddy* (2023) Mapping genomic regions for Gummy Stem Blight Resistance Using a Watermelon MAGIC population. ISHS International Symposium on Cucurbits June 11-15, 2023, China.
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:Watermelon Geneticists,plant breeders and Private seed comapnies Graduate and undergraduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have conducted in-person hands on workshops providing easy to learn training modules for NextSeq based QTL-seq and RNA seq analyses pipelines in Biotechniques (BT571) and Genetics (Biol270) courses. 20 undergraduate and 9 graduate students have attended these training classes and became proficient in performing genomics and transcriptomics. Both the graduate student and technician were actively involved in selfing the RIL population under greenhouse conditions. How have the results been disseminated to communities of interest?Through classroomteaching and training, Emails and zoom presentations. What do you plan to do during the next reporting period to accomplish the goals?We will be developing the PCR Allele Competitive Extension (PACE) genotyping assays for highly associated SNPs so that large populations can be assayed using these PACE in watermelon as well as in melon. These PACE markers are highthroughput hence can be useful to readily incorporate GSB resistance in watermelon and melon. Training and teaching would continue to the students on GBS analysis and genotyping techniques. We have currently GBS data for 1165 accessions in GRIN and have obtained gummy stem blight resistance scores. We plan to perform GWAS to identify any hidden allelic effects that are not resolved yet in our MAGIC population.
Impacts
What was accomplished under these goals?
Gummy stem blight (GSB), a major disease caused by a combination of species involving Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citruli and Stagonosporopsis caricae and is the serious devastating disease causing devastation for watermelon cultivation in the United States. Three hundred MAGIC derivatives of F8 generation have been created using intercrosses involving resistant accessions of Citrullus amarus (PI 482342, PI 189225, PI 526233, PI 482283, PI 482374) and susceptible accessions of Citrullus lanatus (Charleston Grey, Calhoun Gray, Mickylee, Minilee, Allsweet, Crimson Sweet, Petite Sweet) in which GSB (inoculated with Stagonosporosis cucurbitacearum) and we noted segregation for resistance as well as fruit quality. MAGIC lines were evaluated for disease severity and fruit morphological and quality traits under greenhouse and field conditions in a randomized complete block design with 10 replications for 3 years in collaboration with Dr. Todd Wehner's lab at NCSU. Genomic DNA was isolated from 30 GSB susceptible individuals and 30 GSB resistant individuals using the E.Z.N.A. Plant DNA DS Kit (Omega Bio-Tek, USA). The genomic DNA was quantified using Qubit fluorimeter (Thermo Scientific, USA) and the quality was assessed using agarose gel electrophoresis. The resistant and susceptible bulks were constructed by combining equimolar concentrations of DNA from 30 extreme resistant RILs and 30 extreme susceptible RILs. The quality of the sequencing library was assessed using Bioanalyzer (Agilent, USA) and the quantity was measured using Qubit fluorimeter (Thermo Scientific, USA). The two bulks were subjected to whole-genome sequencing using Illumina's NextSeq500 with paired-end sequencing chemistry (2x150 bp) to generate more than 1 billion reads per bulk and achieve the genome's in-depth coverage. The mapping percentage of bulks to the parental genomes ranged from 92 to 99. More than 6,001,242 SNPs were identified from the bulks based on the resistant parental genome, while 1,821,568 SNPs were identified using the susceptible parental genome. The QTLs associated with the GSB resistance were identified using single-nucleotide polymorphism-index and Gprime methods. We have identified statistically significant variants/loci associated with the GSB resistance on chromosomes 1, 2, 3, 5, 10 and 11. Several important candidate genes were assigned to GSB resistance based on the physical location of the genes in the important QTL regions on these chromosomes. The significant loci associated with GSB resistance will be used to develop PACE genotype markers.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Natarajan P, Nimmakayala P, Abburi VL, Ortiz C, Levi A, Wehner T, Reddy UK.(2022) QTL Seq for Gummy Stem Blight Resistance in Watermelon. ARD Research Symposium 2022.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Natarajan P, Nimmakayala P, Abburi VL, Ortiz C, Levi A, Wehner T, Reddy UK (2022) Whole genome scanning using QTL-Seq and GWAS for gummy stem blight resistance in watermelon. Frontiers in Plant Science.
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:Watermelon Geneticists and plant breeders Graduate and undergraduate students Changes/Problems:We could not conduct field evaluations due to the pandemic conditions. Student participation is also restricted. What opportunities for training and professional development has the project provided?We have conducted online training modules onNextSeq and RNA seq analysis pipeline in Crop Evolution and Plant genetics courses. 32 undergraduate and 6 graduate students attended these training classes. We could not conduct in-person activities due to the Pandemic restrictions. Both the graduate student and technician were actively involved in selfing the RIL population under greenhouse conditions. How have the results been disseminated to communities of interest?Through Emails and Zoom presentations What do you plan to do during the next reporting period to accomplish the goals?We will be analyzing raw sequencing data from deep sequencing for candidate genes or QTLs for GSB resistance. Genotyping 288 RILs using genotyping by sequencing for QTL mapping. We have already collected the leaf tissue and DNA extraction of 288 lines is in progress.
Impacts
What was accomplished under these goals?
Gummy stem blight (GSB), a major disease caused by a combination of species involving Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citruli and Stagonosporopsis caricae and is the serious devastating disease causing devastation for watermelon cultivation in the United States. Three hundred recombinant inbred line (RILs) have been created using intercrosses using MAGIC methods involving resistant accessions of Citrullus amarus (PI 482342, PI 189225, PI 526233, PI 482283, PI 482374) and susceptible accessions of Citrullus lanatus (Charleston Grey, Calhoun Gray, Mickylee, Minilee, Allsweet, Crimson Sweet, Petite Sweet) in which GSB (inoculated with Stagonosporosis cucurbitacearum) and fruit quality segregating. 300 RILs were evaluated for disease severity and fruit morphological and quality traits under greenhouse and field conditions in a randomized complete block design with 10 replications and 3 years in collaboration with Dr. Todd Wehner's lab. From the disease severity scores, we have constituted bulks of resistance and susceptible with 10 extreme RILs and equimolor concentrations of DNAs are pooled. Deep sequencing of these bulks and parents is in process. Genomic DNA was isolated from 10 GSB susceptible individuals and 10 GSB resistant individuals using the E.Z.N.A. Plant DNA DS Kit (Omega Bio-Tek, USA). The genomic DNA was quantified using Qubit fluorimeter (Thermo Scientific, USA) and the quality was assessed using agarose gel electrophoresis. The GSB resistant and GSB susceptible DNA bulks were prepared by pooling equal amounts of DNA from 10 resistant plants and 10 susceptible plants. About 1 microgram of genomic DNA from each of the two bulks were used to construct two seperate (one for each bulk)DNA sequencinglibraries using NEBNext Ultra II DNA Library Prep Kit, according to the manufacturer's protocol (NEB, USA). The quality of the sequencing library was assessed using Bioanalyzer (Agilent, USA) and the quantity was measured using Qubit fluorimeter (Thermo Scientific, USA). The libraries were sequenced using Illumina's NextSeq500 with paired-end sequencing chemistry (2x150 bp). The sequence analysis is in progress. Organized two NextGen sequncing and Transcriptome analysis trainng classes via Zoom to 40 undergarduate and graduate students.
Publications
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