Genetic Diversity Estimates in Some Walnut (Juglans regia L.) Accessions Using Molecular Markers and Phenotypic Data

The present study was conducted on twenty seven walnut genotypes/selections to estimate the extent of genetic relationship by morphological and molecular characterization using UPOV descriptors (International union for protection of new varieties of plants) (UPOV, 1999) and molecular marker analysis during the 2009-2010. In this study, a total of 34 traits, 16 RAPD (Randomly Amplified Polymorphic DNA) primers, and 13 simple sequence repeat (SSR) loci were used. Multivariate analyses which include Principal Component analysis and Cluster Analyses, was employed to examine the extent of phenotypic variation in the genotypes/selections of walnut. Based on morphological data, the genotypes were grouped into nine clusters. The cluster III and IV consisted of highest number of genotypes (7), the cluster I, cluster V and cluster IX contained the lowest number of genotypes (1). Principal component analysis was performed to assess relationship among different components. First two principal components accounted for 98% of total variation. Sixteen RAPD primers were initially used out of which only 14 could be amplified which resulted in the generation of DNA fragments that ranged from 500-1500 base pairs. RAPD data was used to calculate similarity matrix using Jaccards coefficient and similarity coefficient ranged from 0.05 (SKAUW-15 & SKAUW-7) to 0.86(SKAUW-008 & SKAUW-040). Dendrograms revealed that all the walnut genotypes/selections were grouped into seven major clusters. Maximum numbers of genotypes were observed in cluster III (eleven genotypes) and minimum in cluster V, VI and VII (one genotype). Polymorphic Information Content (PIC) value ranged from 0.11 (A-15)-0.39 (A-11) and resolving power ranged from 0.12-1.29. DNA fragments generated from simple sequence repeat (SSR) data ranged from 100-1100 base pairs. Similarity matrix using SSR data revealed that similarity coefficients ranged from 0.10 (SKAUW-008 & SKAUW-006) to 0.95 (SKAUW-024 & SKAUW-027). All the genotypes got grouped into nine clusters with maximum number of genotypes in cluster(13) and minimum number in cluster I,IV,VIII and cluster IX (only one). PIC value and resolving power ranged from 0.11 (WGA5) to 0.36 (WGA1) and 0.24 to1.32. The Mantel matrix correspondence test was used to compare the molecular and morphological similarity matrices.Correlation between genetic distances obtained through RAPD and SSR markers was relatively high (r=0.48, P=0.99), indicating that SSR technique was more efficient for evaluating genetic diversity in the genotypes of walnut that we evaluated. However results of cluster analysis based on molecular data did not show any correlation with morphological characters based on Mantels test (r=0.09, P=0.87). Present studies revealed that considerable number of SSR and ISSR markers must be used to depict the genetic relationships among walnut genotypes /selections before reaching to a comprehensive conclusion