Using Next Generation Sequencing for Tagging Stress Resistance Genes from S. spontaneum

Abstract

S. spontaneum provides sugarcane researchers with a valuable genetic resource to improve the genetic traits of commercial sugarcane hybrids. Abiotic stress tolerance (e.g. cold, drought and salt) is of great interest among sugarcane breeders. In order to investigate the gene(s) involved in cold stress tolerance in S. spontaneum, our lab compared the gene expression profiles, by RNA-seq, of a commercial sugarcane hybrid (CP72-1210, cold susceptible) and a cold tolerant S. spontaneum (TUS05-05), before and after cold stress. RNA-seq data showed that the major differences in the gene expression profiles between the cold susceptible and tolerant genotypes are in those genes with catalytic and binding activities. Based on the RNA-seq data, we have selected seven genes for further study, from a list of genes that are induced after cold stress. Real-time quantitative PCR (RT-qPCR) data confirmed that four, out of seven genes further studied, are consistently induced after cold stress.  A Bacterial Artificial Chromosome (BAC) library from TUS05-5, composed of ~73,000 BAC clones (~150Kb fragment genomic DNA) has been created to allow the identification of the promoter sequences involved. DNA sequencing confirmed that we have cloned two derivatives of promoters of two genes. Preliminary data confirmed promoter activity on sugarcane callus and onion epidermal layer. Our working hypothesis is that the specific proteins, encoded by the contigs detected, are induced in TUS05-05 by cold stress, but in lower intensity in CP72-1210, which activates transcription factor(s), leading to the expression of genes related to cold tolerance in S. spontaneum.  TUS05-05 is part of our S. spontaneum collection and is planned to be used in hybridization crosses with sugarcane.  Having the DNA sequence information of these genes, we plan to perform marker-assisted selection in order to speed up the introgression of cold resistance in sugarcane and energycane.