Genome-wide association study reveals the role of Lox4 in shell morphology of Mulinia lateralis

The shell, a type of biological exoskeleton, is a critical feature of bivalves, serving key functions including protection, locomotion, and structural support. Although bivalve shell morphology exhibits rich and complex variability, research on the genetic basis of this diversity remains limited. The dwarf surf clam, Mulinia lateralis , with its rapid growth and short generation interval, serves as an ideal model organism for bivalves, displaying notable shell morphology diversity. In this study, we performed high-throughput sequencing on 180 individuals, obtaining 32,318 SNPs to investigate the genetic basis of shell morphology variation in M. lateralis . The heritability of shell morphological traits was high, with values ranging from 0.439 to 0.814. Through genome-wide association study (GWAS), we identified 20 single nucleotide polymorphisms (SNPs) associated with shell morphology traits and screened 16 candidate genes, such as Lox4 , AJM1-like , and Luc7l . These genes are implicated in the determination of an anteroposterior (A-P) body plan and the regulation of cytoskeletal structure and growth. Of particular interest is Lox4 , which is prominently expressed during the trochophore stage and in the adult mantle tissue, playing a crucial role in shell formation in bivalves. Sanger sequencing confirmed that the SNP Chr15-22361661 located in the Lox4 gene exhibited significant dimorphism in the validation population. Individuals with the AA-genotype exhibited a diminished short-long beak length ratio (SLBR) trait alongside an upregulation of Lox4 expression ( p ​< ​0.05). RNA interference (RNAi) experiments further revealed that the inhibition of Lox4 expression altered shell morphology, leading to an increase in SLBR ( p ​< ​0.05). Additionally, the inhibition of Lox4 expression resulted in a downregulation of Lox2 expression, whereas the suppression of Lox2 did not affect the expression of Lox4 . These results suggest the presence of a unidirectional regulatory cascade wherein Lox4 modulates Lox2 to influence shell morphology. These findings reveal genetic variations associated with shell morphology in M. lateralis and highlight the potential regulatory roles of Lox4 and homeobox clusters on bivalve shell morphology, which enhance our understanding of the genetic basis of shell morphological diversity in bivalves and provide novel insights into how they adapt to diverse lifestyles. • Four traits were developed to quantify the shell morphology of Mulinia lateralis. • Shell morphological traits showed high heritability (0.439–0.814). • GWAS identified 20 SNPs and 16 genes associated with shell morphological traits. • Lox4 plays a key role in regulating the shell morphology of M. lateralis. • A unidirectional Lox4 - Lox2 regulatory cascade influences shell morphology.