BSc-MSc (Integrated)

Yam (Dioscorea spp) is a multi-species, polyploid and vegetatively propagated tuber crop that is cultivated widely in the tropics and subtropics. Yam belongs to the order Liliflorae, family Dioscoreaceae, and genus Dioscorea and are mainly depended for tubers, which are enlarged storage organs containing food reserves in the form of starch and carbohydrate. Dioscorea alata L., commonly known as water yam is one amongst the top ten most important yam species that contribute to food security of millions of people of the world. The major limitation to the sustainable production of D. alata is its susceptibility to anthracnose disease, caused by the fungus Colletotrichum gleosporioides and it can cause yield reduction upto 80%. Hence the present study was carried out with an objective to identify resistant genotypes and molecular markers linked to genes conferring resistance to anthracnose in greater yam using ISSR, RAPD and SSR primers.
Field and laboratory screening was done by whole plant area scoring method and excised leaf assay technique respectively. In both the methods, the lesions observed were rated visually on a 0-5 scale based on the percentage of lesion on leaves and vines, where 0= no infection, 1= 1-10%, 2= 10-25%, 3= 25-50%, 4= 50-75% and 5= >75%. This helped to classify the 40 genotypes used into resistant and susceptible groups, from which 17 resistant and 13 susceptible genotypes were selected for molecular marker analysis.
DNA was isolated from 30 greater yam (Dioscorea alata L.) genotypes. Based on preliminary screening, 6 ISSR, 5 RAPD and 24 SSR primers with high polymorphism were selected for the molecular characterization of greater yam genotypes.
Total number of bands per ISSR primer ranged from 13 (UBC 807 & UBC 836) to 26 (UBC 809). All the bands of UBC 825, (GA)9AT and (GA)9AC were found to be polymorphic. The polymorphism of the ISSR primers studied ranged from 84.61% to 100%. The expected heterozygosity value (He) of the ISSR primers ranged between 0.53 (UBC 836) to 0.81 ((GA)9AC). The polymorphism Information content (PIC) of the primers ranged from 0.5164 (UBC 836) to 0.7929 ((GA)9AC). The gene diversity values ranged from 0.1680 to 0.2104. Total number

of bands per RAPD primer ranged from 14 to 24. All the primers except OPE 06 showed 100% polymorphism. The expected heterozygosity values ranged from 0.5120 (OPF 06) to 0.7476 (OPE 06). All the primers showed gene diversity values <0.2. Among the sixteen SSR markers studied, the number of alleles per marker ranged from 1 to 3 while the number of polymorphic alleles ranged from 0 to 3. The expected heterozygosity values ranged from 0 to 0.6612. All the primers recorded PIC value <0.5872. The gene diversity values ranged from 0 to 0.32.
Mantel test revealed non-significant correlation of the clustering of greater yam genotypes between SSR and other markers. However significantly high correlation of 0.51385 existed between clustering of genotypes based on RAPD and ISSR markers. Among the three different types of molecular markers viz. ISSR, RAPD and SSR used in the present study, SSR markers were found to be better in elucidating variation among greater yam genotypes based on principal component analysis.
The frequency of the different alleles in resistant and susceptible genotypes with regard to ISSR, RAPD and SSR markers were recorded. The difference between the percentage of the resistance genotypes and susceptible genotypes with specific alleles and their deviation were calculated and shortlisted with respect to ISSR, RAPD and SSR markers. The results indicated the association of three ISSR markers (UBC 807, UBC 836 and (GA)9AT) with anthracnose resistance in greater yam. The presence of UBC 836 (1754bp) in the three resistant genotypes and absence in three susceptible genotypes, used as reference for final validation, suggests it as the best marker studied.
The results suggests UBC 807, UBC 836 and (GA)9AT as good markers associated with anthracnose resistance. However these markers needs further validation on mapping population preferably recombinant breeding lines that can be developed using the highly resistant genotypes identified in the present investigation. Also highly resistant genotypes viz. Da110, JAS2 and DaH9/196 identified can be further evaluated to develop a variety with high level of resistance to anthracnose disease for cultivation in Kerala.