Using Next Generation Sequencing for Tagging Stress Resistance Genes for Energycane.

In order to be viable in low input environments and avoid competition with food and fiber, biofuel crops need to be adapted to various stresses, such as drought, salt, cold and low nutrient availability. Here is where Saccharum spontaneum, a wild relative of sugarcane, is most advantageous as a source of genes: in addition to high fiber, the species is highly resistant to a number of stresses. It tolerates a range of temperatures, from tropic heat to temperate cold conditions and is highly adaptable, with genotypes found under waterlogged conditions in marshes, under drought stress in deserts and near the sea, under saline conditions. In addition, it is the germplasm most widely used for energy cane genetic breeding, given its natural ability to intercross with sugarcane, allowing the introduction of positive genes without the need for genetic modification. This gives plant breeders the ability to create genotypes adapted to temperate zones, re-engineering the crop to expand its growing area in the U.S.In order to investigate the gene(s) involved in cold stress tolerance in this species 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 involved on cold stress. 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, which activates transcription factor(s), leading to the expression of genes related to cold tolerance in S. spontaneum. Their lower expression intensity in CP72-1210 would explain its lack of cold resistance. 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.