Working group session:
Functional Genomics
Presentation type:
poster
Authors:
Hu, Hongtao ; Pant, Shankar; Singh, Narendra; David, Weaver; Locy, Robert
Presenter:
Hu, Hongtao
Correspondent:
Locy, Robert
Abstract:
Previously we have screened the Gossypium hirsutum L. USDA germplasm collection for accessions that were more tolerant of heat stress using a chlorophyll fluorescence assay. Seven accessions were identified that appeared to be significantly more thermotolerant than the DeltaPine 90 (DP90) variety used as the thermosensitive control. Of these accessions, five accessions TX337, TX1427, TX2287, TX2288, and TX2454 have been used to produce segregating F2 populations. Leaves from greenhouse grown plants of these five accessions along with DP90 and a second commercial variety SureGrow 747 (SG747) were used for preparation of small RNA libraries either from control (0 hr heat stress), 4 hr heat stress, or 24 hr heat stress at 42 C (growth chamber). RNA-seq was conducted on these small RNA libraries derived from total RNA from these samples (note that the 4 and 24 hr HS samples were combined for library sequencing generating 14 leaf libraries) using the ABI Solid platform with single-end sequencing, and the resulting sequence libraries were submitted to a small RNA library pipeline (based on MIREAP) that we have developed. Normalized abundance of each small RNA found in each library was determined. The CLC Genomics Workbench platform was utilized to statistically determine the heat stress up- and down-regulated miRNAs in each of the 7 genotypes. The subset of miRNAs that are differentially expressed differently in control versus 1 or more accessions were selected for further analysis. Where possible these were mapped to D-genome chromosome scaffolds and A-genome larger contigs. By then assembling transcriptome libraries for each genotype to the defined miRNA precursor sequences a set of SNPs has been defined which we will subsequently utilize to follow these miRNA precursor sequences in segregating F2 populations. Heat stress tolerance of individual plants the F2 populations will be correlated with the SNPs to validate a role for specific miRNA genes in heat stress tolerance and hopefully generate a set of useful markers that can be utilized in subsequent breeding work.