1a. Objectives (from AD-416):
To discover biomarker-genes associated with physiological and morphological traits that improve drought stress tolerance and Fusarium wilt resistance, and to validate biomarker associations with stress resistance traits.
1b. Approach (from AD-416):
Recent advances in genomics have provided considerable information regarding the discovery and expression of genes controlling important crop traits. Despite the significant data on gene expression responses, there is still limited information on biomarker-associations with physiological and morphological traits related to drought tolerance and disease resistance. In this research project, we are proposing to discover molecular markers or biomarker-genes associated with drought stress and Fusarium wilt (FOV race 4) resistance and to validate biomarker associations on diverse cotton populations with different genetic backgrounds. Using conventional and next-generation sequencing (NGS) technologies, we will generate DNA sequences from genes expressed in known FOV-resistant and -susceptible cotton genotypes. We will utilize the most recently assembled cotton genome data to identify single nucleotide polymorphisms (SNP) in the resistant and susceptible genotypes. Evaluation phenotypic-data on developed mapping populations will be used in biomarker-association analyses to validate biomarkers associated with disease and stress resistance traits. Data generated by these molecular systems will then be analyzed using SAS, genetic and QTL, and association mapping programs. We will employ these SNP-biomarkers for marker assisted selection (MAS) studies. These biomarkers will have the potential to increase the efficiency of breeding by facilitating MAS resulting in significant decreases in cost, time, and the risk associated with subjective phenotypic assays.
3. Progress Report:
The goal of this project is to discover biomarker associations with plant stress responses for improving drought tolerance and disease resistance through marker-assisted selection. Selected genotypes were identified as the best candidates for this project based on our previous Fusarium wilt (FOV) research. Pima-S6 is the source of resistance genes for FOV races 1 and 4. Pima-S7 and Pima 3-79 are susceptible to FOV race 4 and are tolerant to FOV race 1. Upland Shorty and Upland TM-1 are very susceptible to FOV race 1. Shorty also is susceptible to FOV race 4, while TM-1 carries some resistance to FOV race 4. Acala FBCX-2 is tolerant to FOV race 1, but mildly tolerant to FOV race 4. Selected cotton genotypes were planted in the greenhouse, and leaves and roots were harvested for future RNA isolation. In addition, root tissue used for the RNA isolation was obtained from greenhouse FOV evaluations with the support of the University of California (Davis and Riverside) collaborators. RNA was isolated from root tissue of control (non-infected roots) and roots infected with Fusarium wilt (FOV) race 4 of Pima-S6. Control-roots (non-infected roots), FOV race 4 infected-roots, and leaves of Pima-S6 were subjected to RNA isolation, cDNA library preparation, and subsequent sequencing. We have been able to capture more than 85% of all expressed genes (transcriptome set) in leaf and root tissue. After our transcriptome assembly of around 1.0 Gb (billion bases) sequenced reads (read = to around 150 bp of DN sequence) from each tissue sample, we obtained from 26,000 to 31,000 assembled contigs or expressed genes with an average gene-sequence length of 1 kb or 1000 bp. Differential gene expression analysis between the control-roots and infected-root samples will be done to discover the genes involved in the key pathways of FOV disease infection. In addition, possible biomarkers discovered through this project will increase the efficiency of breeding by facilitating MAS, resulting in significant decreases in cost, time, and the risk associated with subjective phenotypic assays. Activities during the project were documented through presentations to cotton and peanuts producers, commodity groups, and professional societies, and through telephone contacts and site visits with collaborator.