Characterization of gene flow and genetic diversity of interspecific tea (Camellia sinensis (L.) O. Kuntze) hybrids using Simple Sequence Repeat markers

Abstract

Tea [Camellia sinensis (L.) O. Kuntze] is an evergreen, economically important crop in Kenya and globally that is characterized by high genetic variability, from which tea, a popular soft beverage that is widely consumed, is produced. Tea improvement depends on the extent of genetic diversity within the available population and the ability of the tea crop to hybridize freely within the species as well as with closely related ‘wild’ species in the genus Camellia. There are prospects of using interspecific hybridization for introducing desirable traits in tea such as cold hardiness, drought tolerance, and specific characters in chemical components, and disease and pest resistance, among others. However, the contribution of wild species to the cultivated gene pool is presently not well understood. This study characterized genetic diversity and gene flow in interspecific tea hybrids by genotyping SSRs across eight loci and analyzing the levels of relatedness in the population. Twenty SSR markers comprising of five novel EST-SSRs and fifteen adapted microsatellites were initially screened for polymorphisms using three randomly selected interspecific hybrids (i.e. TRFK 570/2, TRFK 688/1, and TRFK 83/1) and one intraspecific cultivar (TRFK 6/8). Of these, eight most informative polymorphic microsatellites were used to study genetic diversity and gene flow in 88 tea accessions comprising interspecific hybrids, parental clones, and wild tea species. DNA was extracted from each genotype and SSR fragments were PCR-amplified, separated on 1.5% agarose gel, and binary data (1= present, 0= absent) scored at the eight loci. The polymorphic information content (PIC) and discriminating power (D) of SSR markers were determined using the iMEC program, whereas genetic diversity in the genotypes was estimated with POPGENE version 1.32. Analysis of molecular variance was performed using GenAlEx 6.5 software while parentage analysis was performed with Cervus 3.0.7. GenStat (15th edition) and Structure 2.3.4 were used to analyze relatedness based on Jaccard's coefficient and genetic structure of the population, respectively. Eight markers were relatively informative, with PIC and D values of 0.40 and 0.30, respectively. Genetic diversity was highest in Genet 3c/2007 population (Ne = 1.9727 and I = 0.6862) and lowest in wild tea population (Ne =1.4320, I =0.4105) that occur as isolated pure wild type groups with reduced genetic exchange with other Camellia species. Among the families, St. 645 was the most diverse (I = 0.64) and St. 31 the least diverse (I = 0.36). The population was only moderately differentiated (FST = 0.0661) across the eight loci, suggesting past genetic exchanges. The close relatedness among the accessions was revealed by neighbor-joining analysis with most hybrids clustering in a manner consistent with known pedigree information. Wild alleles were highest in Genet 3c/1999 hybrids (95%) and lowest in Genet 3c/2005 hybrids (38.9%) demonstrating a relatively high but unequal genetic contribution of wild Camellia species into cultivated tea under natural pollination conditions. Parentage analysis showed multiple and shared paternity among half-sib and full-sib families. As the results demonstrate that EST-SSRs are highly efficient in identifying interspecific tea hybrids, typing more EST-SSR loci could be useful for accurate parental reconstruction in progenies with unknown identity and half-sibs from polycross mating and for the determination of genetic diversity patterns in tea breeding stocks for hybridization breeding.