The determining regions, specific genes and markers of sex in Pyropia haitanensis (Bangiales, Rhodophyta)

Genetic studies have confirmed that the sex of Pyropia haitanensis belongs to the genotypic determination. However, the genes involved in the sex determination have not yet been studied. In this study, two double haploid (DH) populations of the male and female were established using the sex-sectored blades produced by a crossing experiment of P. haitanensis, and the sex-linked regions (SLRs) were identified by BSA-Seq, which were located on chromosome 4. HiFi sequencing was performed on the SLRs of female and male for reassembly and gene annotations, and the results showed that the SLRs of female and male contained 93 and 94 genes with sizes of 0.32 and 0.31 Mb, respectively. In the SLRs of female and male, there was a sex-determining region (SDR) containing 5 and 3 genes, respectively. In addition, gene conversion of the SDRs resulted in non-Mendelian genetic segregation with a segregation ratio of male: female to be 4:0, 3:1, 1:3 or 0:4. Two female-specific genes (PhF00071 and PhF00072) were found in the female SDR. Gene cloning revealed that the full-length CDS of PhF00071 was 816 bp, encoding a protein containing an RWP-RK domain; while the full-length CDS of PhF00072 was 858 bp, encoding a protein that did not contain any known domains. qRT-PCR revealed that the expression levels of the two female-specific genes were significantly increased during the formation of female germ cells (P < 0.05). In addition, primers were designed using the specific sequences of female and male SDRs, and the genomic DNA of female, male and heterozygous strains were amplified by PCR. The results proved that only one specific band could appear in female or male strains, while both female and male-specific bands appeared in heterozygous strains. This study lays a foundation for further elucidating the mechanisms of sex determination and sexual maturity in P. haitanensis.