GENETIC AND BIOCHEMICAL STUDIES ON GALACTOMANNAN BIOSYNTHESIS IN CLUSTER BEAN

Abstract

Guar pods were collected from different developmental stages by tagging the flowers on the day they open as first day after flowering. Pods from 5th day after flowering to 35th day after flowering were collected in this way. RNA from their seeds was used to study the pattern of expression of four enzymes of the galactomannan biosynthesis pathway i.e. mannan synthase which makes -1, 4-linked mannan backbone, galactomannan galactosyltransferase which adds galactosyl residues to the mannan backbone, alpha-galactosidase which is known to remove some galactose residues in the end and phoshomannoisomerase which is involved in the conversion of fructose-6-phosphate to mannose-6-phosphate. The expression of mannan synthase and galactosyltransferase was found to increase gradually with increasing developmental stages. Alpha-galactosidase was not found to express in any of the stages suggesting that in cluster bean it may not have any role in regulating the man/gal ratio in the final stages by post-depositional modification. Expression analysis for these enzymes was also done in other tissues like leaf, stem, root and flower bud. None of the enzymes were found to express in these tissues suggesting that they are endosperm specific enzymes involved in galactomannan biosynthesis which occurs only in the walls of the endosperm. Two marker techniques i.e. EST-SSR and AFLP were taken up to study 12 varieties of cluster bean. Among these 12 selected varieties, five were landraces, five commercial varieties and two wild varieties. Moreover these varieties have been chosen from different clusters formed on the basis of RAPD analysis (Nagesh K. A.; unpublished results). Twelve varieties were analyzed using 26 SSR primers. Out of these amplification was obtained with 20 primers. Six were found to be polymorphic and the polymorphic bands obtained were analyzed. The polymorphic bands were obtained with the wild variety, Cyamopsis senegalensis, in the 180-220 bp range. For AFLP, standardization of the technique was done with one variety i.e. M-83. Eight MseI and eight EcoRI selective primers were chosen and all 64 combinations were used in selective amplification. Out of these 64 combinations, 8 combinations i.e. M-CAA/E-AAG, M-CAA/E-ACC, M-CAC/E-ACT, M-CAC/E-ACA, M-CAG/E-ACT, M-CAT/E-ACT, M-CAT/E-ACA, M-CTA/E-ACT generating more fragments than others, were chosen after analyzing the explorer gel for further analysis in 12 varieties. All the accessions were then subjected to selective amplification with these primer combinations. Proper bins were obtained and the results were arranged in (1/0) format. Similarity matrix and phylogenetic tree were generated with the help of this data. The binary data obtained by gene mapper software was converted to a similarity matrix using Jaccard's coefficient. A dendrogram was generated by Unweighted Pair-Group Average (UPGMA) method using the similarity matrix by Jaccard's similarity coefficient [51] to determine the relatedness of 13 genotypes analyzed. Using 30% similarity, three clusters were formed. Cyamopsis senegalensis clearly out-grouped in both the marker analysis studies used.