TRANSCRIPTOMIC ANALYSIS AND GENE SILENCING STUDIES IN CLUSTERBEAN

Abstract

Clusterbean or guar [Cyamopsis tetragonoloba, L. Taub.] is an important industrial crop because of the commercial applications of the galactomannan gum contained in its seeds. Plant breeding programs based on marker-assisted selection require a rich resource of molecular markers. As limited numbers of such markers are available for guar, molecular breeding programs have not been undertaken for the genetic improvement of this important crop. Hence, the present work was done to enrich the molecular markers resource of guar by identifying high quality SSR, SNP and InDel markers from the RNA-Seq data of the roots of two guar varieties. RNA-Seq analysis of the roots of two guar varieties, namely, RGC-1066 and M-83 was carried out. A total of 102,479 unigenes with an average length of 1016 bp were assembled from about 30 million high quality pair-end reads generated by an Illumina HiSeq 2500 platform. The assembled unigenes had 86.55 % complete and 97.71% partially conserved eukaryotic genes (CEGs). The functional annotation of assembled unigenes using BLASTX against six databases showed that the guar unigenes were most similar to Glycine max. GO terms were assigned to 45,200 unigenes using the UniProt database. The screening of 102,479 unigenes with MISA and SAMtools version 1.4 softwares resulted in the identification of 25,040 high-confidence molecular markers which consisted of 18,792 SSRs, 5999 SNPs and 249 InDels. These markers tagged most of the genes involved in root development, stress tolerance and other general metabolic activities. Each of the 25,040 molecular markers was characterized, particularly with respect to its position in the unigene. For 71% of the molecular markers, the names, products and functions of the unigenes were determined. About 80% of the markers, from a random sample of molecular markers, showed PCR amplification. It is expected that these markers will be useful in molecular breeding programmes and will also be helpful in studying molecular mechanisms of root development, stress tolerance and gum synthesis in guar. Guar seeds are of particular economical importance because of the presence of galactomannan in their endosperm. The BIG SEEDS (BS) gene family is known to increase the growth of lateral organs in model Arabidopsis and few other legume plants. In this study, we are reporting the role of another member of BS family, BIG SEEDS LIKE CtBSL gene in guar through RNAi mediated silencing approach. Seven non-segregating RNAi lines of guar (S3-O-2-1, S7-O-1-3, S7-O-1-5, S3-O-2-3, S3-O-2-6, S7-O-3-2 and S7-O-3-7) were selected for detailed study. Homozygous transgenic RNAi lines at T3 seeds with decreased transcript level of CtBSL showed 20-40 % increased pod and grain size. The observable phenotypic changes in transgenic lines were induced by significant increase in seed mass (12.9 folds), amino acids (14 folds) and galactose and mannose content (1 fold) due to the upregulation of growth regulatory factors and growth regulatory interacting factors. Through yeast two hybrid we observed a positive interaction between CtBSL and CtNIN protein that suppressed the downstream growth regulatory genes and cell proliferation in seeds of wild HG 2-20 variety of guar. Thus, the current research work suggests that CtBSL is a promising candidate to improve the seed size in guar which determines the overall galactomannan or gum yield in guar.