Machine vision-based %PP trait drives GWAS of inner shell color in Hyriopsis cumingii

Inner shell color, particularly purple nacre, is a key determinant of pearl quality in Hyriopsis cumingii, a cornerstone of China's freshwater pearl industry. We conducted a genome-wide association study (GWAS) on 590 F4-generation individuals using custom liquid SNP arrays: a 30 K array targeting 30,000 SNPs and a 95 K array that includes these 30 K SNPs plus additional loci. After stringent quality control, 25,900 (30 K) and 82,585 (95 K) SNPs were analyzed, identifying significant associations with seven inner shell color traits—L*, a*, b*, dE, C*, h*, and a novel machine vision-derived purple pixel percentage (%PP)—on chromosomes 17 and 15. The %PP trait enhances GWAS precision by quantifying purple nacre coverage. Notably, the 95 K array and 30 K array showed partial SNP overlap, with significant loci under ${-\log }_{10}\left(0.05/N\right)$ and ${-\log }_{10}\left(1/N\right)$ thresholds forming congruent and divergent sets, reflecting the interplay between SNP density and threshold settings. Key SNPs, including LG17_17440155 and LG17_17816229, were validated in 130 independent H. cumingii individuals, confirming their associations with L*, a*, C*, and dE for LG17_17440155, and with L*, a*, C*, and %PP for LG17_17816229. Candidate genes, including pksM, pks1, and SLC family members (SLC13A2_3_5, SLC6A8, etc.), were validated in an independent F2 population (n = 463), implicating pigment synthesis and transport pathways; validation of the combined significant SNPs from both arrays further confirmed their reliability, with ${-\log }_{10}\left(0.05/N\right)$ demonstrating higher credibility and reduced false positives. These findings provide genetic markers for selective breeding and deepen insights into molluscan pigmentation genetics.