An efficient method for developing polymorphic microsatellite markers from high-throughput transcriptome sequencing: a case study of hexaploid oil-tea camellia (Camellia oleifera)

Abstract

The bottleneck of microsatellite marker development is to determine polymorphisms of microsatellite markers. A large amount of microsatellites can be detected via high-throughput sequencing. However, most previous studies didn’t fully use the high-throughput sequencing data to predict number of alleles at microsatellite loci. Instead, laborious experiments were performed for manually screening microsatellite loci, finding out number of alleles at each microsatellite loci and selecting those with polymorphisms for marker development. In this study, we improved the method for efficient development of polymorphic microsatellite markers from high-throughput transcriptome sequencing, using hexaploid oil-tea camellia as a case study. Leaf transcriptomes were sequenced of eight wild oil-tea camellia samples at different altitudes in Jinggang and Lu Mountains, China. Microsatellites were directly identified in the sequencing reads and primers were designed. Strategies were designed to filtering duplicate and multi-locus markers. For each marker, number of alleles cross samples was predicted and length of the potentially amplifiable sequence was estimated. 153 predicted polymorphic markers were selected and empirically validated in the eight samples. Sixty five markers (42%) were polymorphic (2–12 alleles) and 31 (20%) were highly polymorphic (6–12 alleles). The empirical number of alleles was generally higher than the predicted number of alleles but they were significantly correlated. The predicted allele length was among the empirical allele length range. Compared with most previous studies, the method shows a higher efficiency for developing polymorphic markers and filtering duplicate and multi-locus markers. The polymorphic microsatellite markers developed can be used for analyzing the genetic diversity of oil-tea camellia.