Project Number: 3096-21000-019-06-A
Project Type: Cooperative Agreement
Start Date: Nov 1, 2012
End Date: Dec 31, 2014
Objective 1: Identify and characterize genetic diversity in cotton and peanut for tolerance and susceptibility to biotic (fusarium) and abiotic (water deficit and heat extremes) stress. Objective 2: Determine genetic mechanisms controlling biochemical and physiological processes that contribute to biotic and abiotic stress avoidance and/or tolerance in cotton and peanut using next-generation sequencing, transcriptome profiling, and proteome profiling. Objective 3: Identify new molecular markers (from next-generation sequencing) and use integrated marker-assisted breeding methods to develop stress-tolerant (abiotic and biotic) peanut and cotton genotypes.
Research will be carried out at the USDA-ARS Cropping Systems Research Laboratory (CSRL) and Texas Tech University Center for Biotechnology and Genomics (CBG). Briefly, we will use a combination of field and glasshouse physiological techniques to evaluate whole-plant response to water deficit stress, thermal stress, and biotic stress. Using the next-generation sequencing (NGS) technology, we will generate genomic information from expressed genes. Transcript profiling: For all samples, total RNA will be extracted using standard techniques developed at CSRL. cDNA libraries will be generated from unstressed and stressed peanut and cotton tissues. Transcriptome sequencing will be conducted on double stranded cDNA by paired-end sequencing on an Illumina MiSeq sequencer. Comparative gene expression profiling of sequencing data will be done to identify molecular and regulatory responses between contrasting stress tolerant and disease resistant phenotypes. Differential cDNA sequences obtained from either QSeq or Cufflinks software will be assigned bin identifiers for mapping into pathways and expression validation will be carried out using semi-quantitative PCR. Protein profiling: Total protein will be isolated from different tissues using a standard protein extraction protocol developed at the CBG. After Gel or Off-Gel fractionation of digested proteins, the peptides from each fraction will be separated by liquid chromatography (Dionex RS 3000 nanoLC) and detected using Thermo LTQ XL mass spectrometer. Differential protein expression will be measured by label-free quantitation using normalized spectral abundance factors. Significantly differentially expressed proteins will be assigned bin identifiers for MapMan analysis. Mapping of transcripts, proteins and metabolites: MapMan software (ver. 3.50) will be used for annotation and display of differentially expressed transcripts and proteins. Genotyping by sequencing approach or GBS: GBS will be used to identify single nucleotide polymorphism (SNP) genetic bio-markers from selected cotton genotypes and progeny with diverse genetic backgrounds. Preparation of libraries for NGS and filtering raw sequenced data will follow procedures and protocols for sequencing on an Illumina MiSeq sequencer. The Lasergene Genomic Suite and Work Bench software packages will use for sequence alignments.