Assessment of genetic variation in lucerne (Medicago sativa L.) using protease inhibitor activities and RAPD markers.

ABSTRACT

The aim of the present study is to identify and characterize lucerne lines resistance to weevil infestation. After three years of field screening for resistance to weevil infestation, 13 lines of lucerne were selected to assess the genotypic variations for lucerne weevil (Hypera postica Gyll.) at biochemical and molecular levels. Total phenols varied from 0.15 to 0.91 mg g-1 (DM) in these genotypes. The highest trypsin (11.11 unit mg-1 protein) and chymotrypsin (93.0 unit mg-1 protein) inhibitors activities were recorded in G-1-02 and B-4-03 lines respectively, whereas highest a-amylases inhibitor activity (14.2 unit mg-1 protein) in C-6-01. Zymogram patterns for trypsin inhibitor activity showed quantitative variations among the lines. In total 262 DNA fragments were generated when 45 deca-mer random primers were employed. Genetic variation in terms of genetic distance ranged from 0.65 to 0.85. Sequential Agglomerative Hierarchical and Nested (SAHN) clustering using the Un-weighted Pair Group Method with Arithmetic mean (UPGMA) algorithm yielded two clusters (cluster I and II) which converged at 72% similarity level. Cluster I contained most of the lines having low level of weevil infestation. High bootstrap values (>40) indicated the significance of nodes embodied in these two clusters. However, SDSPAGE analysis of the leaf proteins of these 13 lines showed no major variations except minor difference in the protein bands of molecular weights between 14 to 20 kD.