Development of SNP markers in Crassostrea hongkongensis based on the next-generation sequencing and high resolution melting analysis

Abstract

Novel single nucleotide polymorphisms (SNPs) were screened using next generation sequencing in Hong Kong oyster, Crassostrea hongkongensis. A total of 91,980 putative SNPs were identified through de novo assembly of the 454 transcriptome database, and 83 of these were selected to validate polymorphism. Using high resolution melting analysis, 17 SNP markers were developed successfully with 48 individuals from two wild populations from Shantou and Zhuhai, China. The observed heterozygosities ranged from 0.125 to 0.667 and 0 to 0.708 in the two populations, respectively, while the expected ones ranged from 0.311 to 0.511 and 0.120 to 0.511 in the two samples, respectively. Fifteen SNPs have been confirmed to fit Hardy-Weinberg Equilibrium after Bonferroni correction (p \ 0.003) in both populations. To our knowledge, this is the first set of SNP markers developed in C. hongkongensis, which would provide valuable information in facilitating genetics and breeding studies in the future.Keywords C. hongkongensis Á SNP markers Á Transcriptome sequencing Á High resolution melting The Hong Kong oyster, Crassostrea hongkongensis, one of the economically important aquaculture species endemic to coastal waters of South China Sea, has not only been cultured widely as one of the largest molluscan industries in this region (Lam and Morton 2003), but also an important component of intertidal and sub-tidal communities in estuarine ecosystem. In recent years, however, this aquaculture industry has suffered from seasonal mass mortality as well as the decline of wild seed's quantity and quality. Therefore, genetic improvement is demanded for the sustainable development of C. hongkongensis industry. And meanwhile, the identification of local adaptation across this geographic range is highly required from the perspective of stock management and utilization.Genetic marker is one of powerful tools in genetics studies, particularly, for genetic mapping and trait improvement. Previously, microsatellites were developed in C. hongkongensis ). However, microsatellite has the disadvantage in that its primer sites may not be quite conserved and homoplastic likely due to the high mutation rate. Single nucleotide polymorphisms (SNPs) are considered as the third generation markers due to their abundant genetic variations in terms of microsatellites in the genome (Vignal et al. 2002), and most of them are biallelic polymorphism, although a priori information about allelic variation in the genome is required (Schlötterer 2004). Furthermore, the most attractive feature of SNPs is their possible association with candidate genes involved in biological functions.The methods based on high-throughput sequencing could generate substantial raw data that could be used to develop molecular markers (Li et al. 2012). However, such utilization is still limited in a few of marine bivalves (Jiang et al. 2011;Gallardo-Escárate et al. 2013). Here, we sequenced the transcriptome of C. hongkongensis using next generation sequencing (NGS) technologies (unpubli...

Development of coding single nucleotide polymorphic markers in the pearl oyster Pinctada fucata based on next-generation sequencing and high-resolution melting analysis

“…A total of 173 sets of primers amplified multiple bands, and 229 amplified a clear target band on PAGE. The ratio of primer screening was 48.93%, which is higher than previously reported values of 41.67% (Zhang et al, 2015) and 28.10% (Huang et al, 2014b).…”

Characterization of 87 EST-SNP markers in hard clam Meretrix meretrix using high-resolution melting analysis

“…Since the first report on the application of this technique in barley (Lehmensiek et al 2008), many studies involving SNP detection using HRM in aquatic species have been published (Jiang et al 2011;Wang et al 2013;Cristian et al 2013;Huang et al 2014;Dong et al 2015). In this investigation, we developed 87 EST-SNP markers using HRM analysis, which will contribute to research into the genetics of M. meretrix.…”