Development of EST SSR markers and analysis of genetic diversity in Zingiber species

Microsatellite markers have been proven as one of the most powerful markers, which can accurately access the level of genetic diversity for phylogenetic analysis. In the present study microsatellite primers were developed from the expressed sequence tags of Zingiber species available in the db EST database of NCBI. A total of 726 class 1 microsatellites were identified from the non redundant dataset of 12,019 unigenes. The frequency of identified SSR (simple sequence repeat) motifs was 72.5 SSR/1Mbp (one SSR/13.77Kbp) of Zingiber transcriptome genome. The most abundant microsatellite repeat was mononucleotides (46.8%), followed by trinucleotides (21.5%). The most frequent microsatellite motif was (A/T) (43.1%) then AG/CT (6%) followed by AGG/CCT (5.2%). Primers were designed for 188 class 1microsatellite repeats. A set of 22 EST SSR primers were short-listed based on the functional characters and utilized for genetic diversity analysis along with eight ginger genomic SSR primers. Ten Zingiber species viz., Z.capitatum, Z.ligulatum, Z.nimmonii, Z.neesanum, Z.odoriferum, Z.officinale, Z.roseum, Z.spectabile,Z.zerumbet and Zingiber sp. from Arunachal Pradesh were used for cross species amplification. 18 out of 30 SSR primers tested were amplified in the expected size range and 10 SSR markers were polymorphic across the 10 Zingiber species studied. The number of alleles generated ranged from 2 to 4 per locus. The dendrogram elucidated the genetic diversity utilizing these markers between different species of Zingiber and grouped them into two major clusters. The first cluster consisted of all the species collected from India and Z. spectabile was out grouped to second cluster. The study demonstrated that the newly developed EST SSR markers were informative and will be added to the repository of microsatellite markers for studying the genome architecture of Zingiber.