Local W-like Haplotype Architecture in the Candidate Sex-Determining Region of Black Tiger Shrimp

While female heterogamety in black tiger shrimp (Penaeus monodon) is consistent with a ZW/ZZ system, the sequence organization of its sex-associated region remains unclear. In this study, we examined the candidate sex-determining region on chromosome 19 (2.71–2.89 Mb) using PacBio HiFi reads from a marker-supported ZW female, genotypes from 200 full-sib offspring, local assembly, and public long reads. Three phase blocks overlapped this interval. Sex-associated SNPs assigned W-like and Z-like alleles in two blocks, while the central block remained unoriented. The first block contained gene24, a partial dmd-3-related gene model, and a 54 bp insertion in gene25 intron 1 (INS00000105; INS54). This insertion, supported by 17 W-like HiFi reads and two local assemblers, was called in 100/100 female offspring and 1/99 called males; one male was uncalled. Sanger reads confirmed the insertion and both flanks in three selected market-sourced females and the no-insertion junction in three selected males. Public reads from a Thai female reproduced the insertion-bearing structure, whereas Australian male reads supported the alternative structure. Separate Thai and Chinese long reads crossed the two phase gaps in their respective samples. Together, these results define local female-associated sequence differences and targets for functional investigation, rather than complete W and Z chromosome sequences.