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

INTRODUCTION

The pearl oyster, Pinctada fucata, is an important commercial marine shellfish that is cultured for producing saltwater pearls in China, Japan, and Australia (Yu and Chu, 2006). It is also an important animal model for investigating biomineralization (i.e., scientific, medical, and commercial applications) and evolutionary biology (Jones et al., 2013). Pearl quality has recently decreased in both China and Japan. One possible reason is that the growth performance of P. fucata is hampered by inbreeding during aquaculture (Wada and Komaru, 1996; Qiu et al., 2014).

Genetic markers are powerful genetics study tools, particularly for genetic mapping and trait improvement (Huang et al., 2014a). Because of their abundance, value, and efficiency, single nucleotide polymorphisms (SNPs) have become the most powerful marker system for genetic research (Gomez-Uchida et al., 2014). Compared to non-coding genomic markers, SNPs developed from functional genes may be responsible for traits of commercial interest in this species, such as growth, reproduction, and resistance (Gao et al., 2013; Klinbunga et al., 2015; Ranjan et al., 2015). Transcriptome sequencing with next-generation sequencing technologies could provide extensive resources for large-scale gene-associated SNP mining (Grabherr et al., 2011). High-resolution melting (HRM) has proven to be a simple, low-cost, and highly sensitive technique to detect SNPs, and to profile genetic variation within polymerase chain reaction (PCR) amplicons (Cui et al., 2013).

In this study, the genetic diversity and structure of a wild population of P. fucata from South China were examined. A total of 119 polymorphic SNPs from the transcriptome sequence were successfully isolated by HRM analysis, which can contribute to P. fucata genetics and breeding studies.

MATERIAL AND METHODS

RESULTS AND DISCUSSION

Small-amplicon HRM assays (SA-HRMAs) provide a rapid, inexpensive, and high-throughput closed-tube method for genotyping (Smith et al., 2010). To ensure SA-HRMA accuracy, we used three criteria: 1) SA-HRMA amplicons were no more than 100 bp long, which ensured that homozygous genotypes of alleles were easily distinguished; 2) only one SNP was present in each amplicon; and 3) high- and low-temperature controls were added for each amplicon, which decreased melting temperature variations attributable to the instrument or solution chemistry and corrected melting profiles (Seipp et al., 2007). An improved two-step SA-HRM method for Pacific oyster (Crassostrea gigas) SNP validation has been shown to be efficient and economical (Wang et al., 2013, 2015), and this method was successfully used to validate 119 polymorphic SNPs from P. fucata transcriptome data, demonstrating that it is feasible in shellfish.

A subset of 468 primers was randomly designed to validate the SNP predictions. No amplification products were seen in 66 sets of primers, and introns were found in genomic DNA but not the transcriptome. If the primer flanked, or was located in, an intron, the intervening fragment could not be amplified. 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).

All of the SNP-containing unigenes were annotated with the corresponding top best BLASTx hits, and 88 SNPs were annotated though BLASTx in the Nr database (Table 1). Of these, heat-shock protein 70 is expressed in response to changes in temperature, bacterial infection, or pH. Its main function is to promote protein folding, and thereby prevent the cellular accumulation of non-native proteins (Mymrikov et al., 2011). F-box proteins are an expanding family of eukaryotic proteins, characterized by an approximately 40-amino-acid motif (Cenciarelli et al., 1999). F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes, in which they bind substrates for ubiquitin-mediated proteolysis (Kipreos and Pagano, 2000). Fatty acid-binding proteins participate in lipid uptake, transport, and homeostasis (Bayır et al., 2015). Sox9 (SRY-related HMG-domain-containing transcription factor 9) and cullin-3-B play important roles in testis development (Bergstrom et al., 2000; Lu et al., 2005). Among the 229 well-amplified SNPs, 119 (51.97%) were polymorphic in 8 P. fucata individuals, according to the SA-HRMA (Table 1). Seventy-five SNPs were genotyped as transitions, including 40 A/G and 35 C/T, and 44 were genotyped as the transversions 11 A/C, 18 A/T, 6 C/G, and 9 G/T. According to ORF Finder, 90 SNPs were located in the ORF, including 16 non-synonymous SNPs and 74 synonymous SNPs; 12 SNPs were located in the 3'-untranslated region (UTR), and 1 was located in the 5'-UTR. SNPs within a coding sequence may change a protein’s amino acid sequence and structure, thus influencing its functions (Gao et al., 2014; An et al., 2015). The post-transcriptional regulation of gene expression is crucial for many physiological processes. SNPs within UTRs may have consequences for gene splicing, expression, and regulation (Malodobra-Mazur et al., 2016; Xu et al., 2016). SNPs developed from functional genes may be used in association studies, which could genetically improve species. For example, some SNPs are associated with growth traits in the pearl oyster (Shi et al., 2014), and SNPs screened from the myostatin gene are associated with growth traits in the scallop and carp (Wang et al., 2010; Guo et al., 2011; Liu et al., 2012; Sun et al., 2012). All of the annotation unigenes and their SNPs may be useful for studying the commercial traits of P. fucata, such as growth, resistance, and reproduction.

Twenty-five SNPs were successfully used to test the genetic diversity of 40 wild P. fucata from Liusha Bay, China (Table 3). All of the SNP loci had intermediate PIC values (0.25 < PIC < 0.5), with a mean of 0.3336. The H_O was 0.0417-0.6042 and the H_E was 0.2945-0.5053. Li et al. (2016) used SNP loci to analyze the genetic diversity of P. fucata individuals from three families, and obtained PIC values of 0.2435, 0.2479, and 0.2977. Huang et al. (2014a) used SNP loci to study the genetic diversity of a wild P. fucata population in Shenzhen, China, and reported MAF, H_O, and H_E values of 0.0642-0.4375, 0.1282-0.4872, and 0.1215-0.4984, respectively. These findings indicate that the Liusha population genetic diversity is higher than that in culture or in the Shenzhen population. Table 3

Summary of 25 single nucleotide polymorphisms in wild Pinctada fucata individuals.

Figure 1

Genotyping results using high-resolution melting with a small amplicon. a. PF_SNP9, homozygotes (GG and CC) and heterozygotes (GC) are represented by red, blue, and gray curves, respectively. b. PF_SNP98, homozygotes (AA and CC) and heterozygotes (CA) are represented by gray, blue, and red curves, respectively.