1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
The goal of this project is to use the next-generation sequencing (NGS) technology to generate genomic information from genes expressed and/or involved in plant response to water deficit stress, thermal stress, and biotic stress such as disease resistance. Molecular and regulatory responses between contrasting stress-tolerant and disease-resistant phenotypes will generate profiles that will help us to determine genetic mechanisms controlling biochemical and physiological processes; identify and characterize the diversity of expressed genes on diverse genotypes; and identify new biomarkers for marker-assisted breeding methods to develop stress- and disease-tolerant peanut and cotton genotypes. A NGS sample preparation (RNA isolation and library assembly) space-laboratory has been set up at the Center for Biotechnology and Genomics (CBG) at Texas Tech University (TTU) and at the USDA-ARS Plant Stress and Germplasm Development Unit. In addition, USDA-ARS and TTU computers and softwares were set up and made available to our project for DNA and transcriptome (a set of expressed genes from an entry or specific tissue) sequence assembly, gene expression, and biomarker discovery. For cotton, RNA-sequencing was done on an Illumina MiSeq instrument. Overall the sequence quality was very high, with average QScore of 35, meaning 1 error in 5000 bases. We have obtained from 26,000 to 31,000 assembled expressed genes with an average gene-sequence length of 1 kb or 1000 bp. We have generated a reference transcriptome for cotton genotype Pima-S6 from leaf and root tissue. For peanut, an experiment was designed to study the effects of water-deficit stress on developing peanut pods. During the year, a transcriptome data set was generated by NGS from well-watered control and water-deficit-stressed pod tissues. A reference pod transcriptome was created with six-frame-translation to generate the peanut pod proteome (a set of proteins) database. We have used this proteome database to search and quantify differential proteins altered during stress response. Activities during the project were documented through peer-review publications, presentations to cotton and peanuts producers, commodity groups, and professional societies, and through telephone contacts and site visits with collaborator.