Molecular and Phytochemical Elaboration of Tea Genotypes Grown in Pakistan

Abstract

Tea is the most popular non-alcoholic beverage obtained as an infusion of the leaves of Camellia sinensis (the tea shrub). Pakistan is the second largest importer of tea and its consumption in the country is increasing day by day. Pakistan imports almost all of its tea; spending a large amount of foreign exchange annually. Tea has recently been introduced in Pakistan as a crop and needs improved genotypes suitable to the local agro climatic conditions for fulfilling the local need. This will only be possible through discovering genotypes of higher potential and adopting of appropriate production strategies in the tea growing areas of the country. Keeping in view the dire need of understanding the genetic potential of tea genotypes this project was undertaken for characterizing all the tea cultivars currently grown in Pakistan. The cultivars were analyzed through quantitative trait analysis, DNA fingerprinting and phytochemical investigations. For quantitative trait analysis 12 genotypes, including 4 commercial varieties; 4 germplasm collections and 4 land races, were grown at two locations in District Mansehra, Pakistan. For DNA analysis 130 plants, 10 plants per genotype were screened. Quantitative trait analysis of the genotypes was carried-out for 18 quantitative and seven qualitative traits. Based on statistical analysis of leaf, shoot and seed parameters all the genotypes could be placed into two groups’ i.e. broad and narrow leaved genotypes. These groups corresponded to the Chinese and Assam types of tea. The grouping was corroborated by the fact that the narrow leaved Chinese, T-05-10 and JP-01-05 genotypes exhibited similar clustering pattern both for quantitative and qualitative parameters irrespective of the locations where the genotypes were grown. Phytochemical analysis of total polyphenols, amino acids, caffeine contents and water extracted substances from fresh tea leaves of the genotypes showed significant differences among genotypes having CV-percentage values of 18.13, 14.96, 11.0 and 5.0 respectively. Cluster analysis based on Euclidean Distance Coefficient, revealed four groups and separated the broad leaved genotypes from others. Similar results were observed using Principle Co-ordinate Analysis (PCoA). The plot of the first two components based on all characters showed a clear separation of broad leaved genotypes (C. assamica), namely IN-01-07 and SL-01-05 genotypes from the narrow leaved (C. sinensis) genotypes. For DNA analysis 75 genotypes were screened with 20 RAPD primers and 103 genotypes were screened with 12 SSR primers. Genetic diversity among the genotypes was calculated according to Nei and Li’s (1979) UPGMA and bivariate data matrix which were used for making dendrogram and deciding genetic affinity or diversity among the genotypes. RAPD primers detected 115 different alleles whereas the number of alleles analyzed through SSR primers was 87. Genetic distance estimates ranged from 0-100% showing high level of diversity among screened genotypes. UPGMA cluster analysis of RAPD data showed mixed grouping profile of the samples, however, samples from different plants of particular genotypes segregated into independent sub-clusters. Average number of alleles amplified per SSR marker varied from 4 to 10. Pair wise genetic diversity among all samples ranged from 0% to 100%. Dendrogram based cluster analysis revealed five main clusters showing mixed pattern but most of the genotypes were separated in sub groups according to the genetic affinity. The findings of molecular analyses were generally cocurred with the results obtained from quantitative trait analysis which revealed a close genetic affinity among the tea genotypes based on the leaf size. The variations between and within genotypes; collected from different sites; were very high; confirming differences in origin of the tested samples. Failure of placing some broad leaf genotypes into clearly defined separate clusters may be due to introgressions as a result of cross pollination in tea cultivars. Results obtained from quantitative and qualitative trait analysis superimposed on phytochemical data clearly differentiated all the tea genotypes into two discrete groups viz. broad and narrow leaved genotypes. Molecular results also coincided with that of morphological and phytochemical analysis except that molecular analyses separated the two taxa in individual sub-clusters instead of main clusters. Ich-08 genotype was found to be best in flush production, however it has poor adoptability. On the other hand Ich-05 was found best in adaptability at the same time having flush production comparable to Ich-08. Hence Ich-08 is recommended for further evaluation and for registration as a new variety for general cultivation.