Whole-genome resequencing reveals the single nucleotide polymorphisms associated with shell shape in Crassostrea gigas

Highlights

• We performed descriptive statistics and correlation analysis of shell related traits in long and round oyster shell group.

• 925 candidate single nucleotide polymorphism sites and 593 candidate genes were screened by genome resequencing.

• GO and KEGG enrichment were carried out using candidate genes, and the related terms were dug out.

• Two SNPs were identified contributing to shell shape differentiation and allele C was a favourable genotype of round group.

• Ten differentially expressed genes were verified, indicating that different shell shape oysters may vary in stress responses.

Abstract

Shell shape is an economically important trait in bivalves, as it can directly affect consumer behaviour. However, there have been few studies on the genes related to shell growth and shape. The Pacific oyster, Crassostrea gigas, is one of the most important aquaculture species worldwide, and studies on its shell shape could significantly increase its economic value. In this study, the shell-length-to-shell-height ratio (SLSH) was defined as the phenotype of interest. Descriptive statistics and correlation analyses of shell-related traits in the long (low phenotypic) and round (high phenotypic) groups of Crassostrea gigas are presented. Whole-genome resequencing, using individuals from these two groups, was performed to screen candidate single nucleotide polymorphisms (SNPs) and genes. A total of 925 SNPs and 593 genes associated with shell shape were found. SNaPshot was used for genotyping validation in an independent population: two SNPs were significantly related to shell shape differences, and the minor C allele of SNP g26854_18684937 favoured the growth of round shells. Further, ten genes were identified as significantly differentially expressed in the extreme shell shape groups of the independent population using real-time PCR, and the results showed that different shell shape may vary in environmental stress responses. This is the first study to use whole-genome resequencing to identify shell shape-related sites in bivalves. These results not only provide a reference for genetic research on oyster shell shape but also contribute to the knowledge base for future selective breeding of bivalves.

Introduction

The Pacific oyster, Crassostrea gigas, is one of the most widely cultivated and economically valuable bivalves worldwide (Guo, 2009). In 2018, Pacific oyster production in China reached 5.14 million tons, accounting for 36% of the China's total marine shellfish aquaculture production (Tan et al., 2021). Oyster shell appearance is an important economic trait that can impact consumer behaviour, as different shell shapes influence the popularity of certain oyster varieties (Mizuta and Wikfors, 2019): generally, oysters with thick, deep and wide shells are most popular in the mainstream market (Kube et al., 2011).

Growth-related traits, including shell height (SH), shell length (SL), shell width (SW) and shell whole weight (WW), and shape-related traits, such as the shell-width-to-shell-height ratio (SWSH) and the shell-length-to-shell-height ratio (SLSH), are secondary traits of bivalve shell shape (He et al., 2021). Previous studies have found that shell-related traits are heritable (Mizuta and Wikfors, 2019); thus, it is possible to selectively breed for them. The heritability (h²) of oyster SH ranges from 0.21 ± 0.03 to 0.49 ± 0.25, SL ranges from 0.19 ± 0.03 to 0.36 ± 0.19, SW ranges from 0.13 ± 0.03 to 0.45 ± 0.23 and the SLSH reaches 0.4 (Feng et al., 2015; Gutierrez et al., 2018; Wang et al., 2012; Vu et al., 2020; Ward et al., 2005). Numerous single nucleotide polymorphisms (SNPs) and genes related to shell growth and shape have been identified using genome-wide association studies (GWAS), quantitative trait loci (QTL) mapping and omics analyses (Hao et al., 2018; He et al., 2021; Li et al., 2018; Wang et al., 2016; Zhang et al., 2019). Most of these genes are associated with shell growth and formation; for example, gene E2F3, a positive regulator of shell and soft tissue growth, was identified using a GWAS on composite shell growth traits of the Yesso scallop (Patinopecten yessoensis) (Ning et al., 2019). Proteins, such as nacrein, aspein and PU14 have been determined through proteomics as contributors to pearl oyster shell biomineralisation (Ji et al., 2021; Jones et al., 2014; Li et al., 2017a; Zhang and Zhang, 2006; Zhang and He, 2011). In Crassostrea gigas, the genes Rpsa, Trim3, Tnn, Ky, Fndc2, SLC7A9 and Ankrd44 have been found to be associated with shell traits (e.g., SLSH, SWSH and SL) using GWAS (He et al., 2021). However, the majority of shell-related SNPs and genes that have been screened using RNA sequencing or QTL mapping have not yet been verified (Li et al., 2018; Wang et al., 2016). In addition, due to the complexity of the oyster rearing environments (e.g., water movement, biofouling, predator influence, etc.) and the difficulty of constructing experimental materials (e.g., mass mortality in summer, etc.), there have been few studies on shell shape.

With the development of sequencing techniques, whole-genome resequencing has become a popular method for identifying candidate sites and genes, especially for important traits that have not received much research attention. The method of combining individuals with extreme phenotypes into a single sample, as is done during bulked segregant analysis, makes whole-genome resequencing cost-effective and reliable (Tan et al., 2021). Moreover, SNaPshot and high-resolution melting are genotyping methods that are widely used for medium- or small-throughput sequencing, and are powerful tools for screening SNPs located in target regions (Liu et al., 2019; She et al., 2018).

This study focused on SLSH as the shell shape phenotype of interest. The low phenotypic group of SLSH was defined as the long group (C) and the high phenotypic group was defined as the round group (Y). To explore the genetic differences between different shell shapes of Crassostrea gigas, whole-genome resequencing was conducted to screen candidate SNPs and genes using individuals from these two groups, which represent the extremes of the shell shape phenotypes. Moreover, SNaPshot and real-time PCR were used for genotyping and gene expressed validation in an independent population, respectively. This study, not only provides a genetic basis for shell shape research, but is also an important knowledge base for the future selective breeding of bivalves.