Analysis of Genetic Diversity of Indian Tea Accessions Using Two Modified Amplified Fragment Length Polymorphism Methods

Abstract

The authors compared the utility of two variants of Amplified Fragment Length Polymorphism (AFLP) technique, viz. 4-selective AFLP and three Endonuclease Amplified Fragment Length Polymorphism (TE-AFLP), for accurate assessment of genetic diversity in tea. These assays were optimized to resolve the reported discrepancies in detected levels of genetic variation in tea through conventional AFLP assay. The 4-selective AFLP assay involved the use of four selective nucleotides in MseI primer instead of three, in combination with EcoRI primers with three selective nucleotides. This led to a significant reduction in the total number of bands and improved the overall band readability as compared to conventional AFLP. However, presence of a large number of “shadow bands” of varying intensities across lanes, still disallowed an error free scoring. This was effectively resolved by TE-AFLP assay where selective adapter ligation, in addition to selective amplification, contributes to reduction in the complexity of fingerprint pattern. The major advantage of TE-AFLP over 4-select AFLP was its clear and unambiguous banding profiles that simplified the scoring of bands. Both methodologies yielded comparable results with China types clearly separating from Assam types. In contrast to previous reports, the dendrogram revealed an overall tendency of the accessions to cluster based on their morphotypes and their geographic origin. Although, 4-select AFLP detected higher polymorphism information content values, the overall reliability of TE-AFLP bands was higher due to their unambiguous nature and easy scorability. A major fraction of variation (75 %) was found to be partitioned within the morphotypes indicating predominant out-crossing in this species.