Characterization of Allelic Variants in Sugarcane Genes Related to Drought Stress

Drought is the most important abiotic stress that affects sugarcane productivity. Analyses of microarray and RNAseq conduced with two drought contrasting genotypes (IACSP94-2094/tolerant and IACSP97-7065/sensitive)  on field and at greenhouse have identified several genes that are up/down-regulated on drought condition. From the set of genes validated by quantitative PCR, Dehydrin, Dirigent-jacalin and Lipoxygenase (ScLOX) were chosen as target for functional analyses using rice and sugarcane transgenic plants. The full-length coding sequences of these genes were accessed by SMARTer RACE cDNA Amplification Kit (Clontech) using the tolerant genotype IACSP94-2094 mRNA. The cloning was performed using the vector pGEM-T Easy (Promega) and E. coli DH10B lineage, and sequences subcloned into pDONR 211 gateway vector.  The full length coding sequence of Dehydrin and Dirigent-jacalin genes were successful isolated and cloned. From the 10 Dehydrin clones sequenced, two allelic variant were identified with a frameshift mutation and one sequence showed identical to the Dehydrin sequences from the nucleotide databases queried. From 16 Dirigent-jacalin clones sequenced, it was found one allelic variant with frameshift mutation and only one sequence matching the sequences from the database queried. On the other hand, the coding sequence of ScLOX is incomplete at SUCEST (Sugarcane Functional Genomics Database). After many attempts of full-length transcripts ScLOX amplifications, only 3'RACE fragment from five clones were identified as the target gene. However, the sequences from the 5'RACE amplification clones matched with others members from Lipoxygenase gene family, but not with the queried sequence. The allelic variants representing apparently nonfunctional alleles, attributed to the polyploidy and complexity nature of sugarcane genome represent one of major difficulties in cloning desired genes from coding regions.