Browsing by Author "Chopra, Ratan"
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Item Identification of Single Nucleotide Polymorphisms (SNPs) in Arachis (Peanut) Wild Species and Cultivated Accessions by Solexa Transcriptome Sequencing and Utilization of SNPs for Linkage Mapping in an Arachis A Genome Diploid F2 Population(2014-12-09) Chopra, Ratan; Burow, Mark D.; Sharma, Jyotsna; Payton, Paxton; Simpson, Charles E.; Mudge, JoannWe report the utilization of de novo assembly for tetraploid and diploid peanut to achieve optimal quality transcriptome references using three different assemblers – Trinity, TransAByss and SOAPdenovo-trans. Results of de novo assembly were used to carry out SNP identification in a panel of twenty two accessions of the Arachis genus, including cultivars representing Arachis hypogaea subsp. hypogaea - var. hypogaea, var. hirsuta, and Arachis hypogaea subsp. fastigiata - var. fastigiata, var. vulgaris, var. peruviana, and var. aequtoriana, and wild species representing the A-, B-, and K- genomes, a wild tetraploid – Arachis monticola, and a synthetic amphidiploid - TxAG-6. SNPs identified in the panel provided an insight into the extent of transcript polymorphism present in the genus. Expression analysis performed on three allopolyploids with their ancestral diploid genome donors indicated that B-genome transcripts of the tetraploids were expressed in a pattern more closely related to the B- genome diploids. Validation of SNP calls was performed using allele-specific chemistry on a LightCycler 480. Polymorphic SNPs between A. duranensis x A. cardenasii from the above dataset were used to develop a genetic map on LightCycler 480 and on a high-throughput genotyping platform the Fluidigm Biomark HD. A total of 31 QTLs using composite interval mapping were identified for ten plant architectural traits such as leaf measurements, main stem height, presence of main stem flowers, and seed weight. In summary, this study generated a transcriptome resource for twenty-two peanut accessions and provided an insight into transcript variability in peanut accessions. Also, the genetic map and QTL studies provide a base to assist future utilization of the transcript-based SNPs in peanut.Item Phenotypic Evaluation for Water Deficit Stress Tolerance and the Study of Targeted Re-Sequencing Approach in Peanut(2017-05) Kulkarni, Roshan; Burow, Mark D.; Ritchie, Glen; Chopra, Ratan; Baring, MichaelThe segregating F2:4 and F2:5 generations from the cross between COC270 and TX071304 were screened for physiological traits SPAD reading, flower count, canopy temperature and leaf closure over two years 2015 and 2016 respectively, under water deficit conditions over different time periods. Significant differences were observed among the genotypes for SPAD, canopy temperature and leaf closure across years. There were significant differences among the genotypes for SPAD and leaf closure readings within years. The use of a stepwise regression model gave a significant correlation (r = 0.38) between the predicted yield and observed yield. The predicted yield based on this model was consistent with actual yield for most of the top ranks. An integrated approach of improved bioinformatics and targeted resequencing was used to identify and select homeologous SNPs in tetraploid peanuts for mapping studies. A SNP identification pipeline was developed using GATK software to identify SNPs. SNPs were identified using four different approaches, GATK pipeline, SWEEP filter, alignment against OLin transcriptome and few SNPs were selected from the peanut SNP chip. Forty-eight targets of around 400 bp length were selected for validation on a Fluidigm Access Array as a proof of concept, followed by mass sequencing on an Illumina MiSeq. Eighty –one percent of the SNP calls derived from the Fluidigm – MiSeq were validated for diploids, and 72% of the SNP calls were validated for tetraploids. This approach will benefit tetraploid breeding programs by reducing the cost of genotyping of QTL mapping populations and used in QTL studies on water deficit stress tolerance populations.Item Use of targeted amplicon sequencing in peanut to generate allele information on allotetraploid sub-genomes(2020) Kulkarni, Roshan (TTU); Chopra, Ratan; Chagoya, Jennifer; Simpson, Charles E.; Baring, Michael R.; Hillhouse, Andrew; Puppala, Naveen; Chamberlin, Kelly; Burow, Mark D. (TTU)The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost.