Development and validation of sex-specific markers in Piaractus mesopotamicus

The development of genetic markers for genomic screening is essential for addressing more accurate breeding programs in aquaculture and to ascertain the genetic architecture of productive traits, particularly the mechanisms underlying sex determination in fish. Sex determination systems display huge variation in fish, ranging from those strictly determined by genetic factors to others influenced by environmental ones. Understanding the genetic basis of sex determination is important in aquaculture, because economically valuable traits are usually associated with sex. Piaractus mesopotamicus, popularly known as pacu, is a freshwater neotropical native fish of major importance for South American aquaculture. Despite its commercial importance, genomic information about this species is limited. Available information indicates that genetic breeding programs could be essential for improving production in pacu and to adjust to market demands. Growth rate is currently the main target for pacu farming and it shows sex dimorphism, with females growing faster than males. Since pacu lacks morphological sex dimorphism, it would be useful to develop a simple cost-effective technique for sexual identification. In this study, we searched for genomic regions including markers associated with sex which allowed designing a simple PCR protocol for sex identification. First, we assembled de novo a reference genome for P. mesopotamicus from a male. Then, we performed a genome-wide association study (GWAS) by re-sequencing 59 females and 74 males taking the assembled genome as a reference. More than 12 millions variable sites were identified, mostly single nucleotide polymorphisms (SNP). After filtering SNPs by p-value, we selected four contigs that contained those pre-selected sites, to which a read coverage analysis was performed. Thus, we were able to identify one indel (insertion / deletion) associated with sex and two regions that only were observed in male genomes. Finally, three pairs of primers spanning those regions were designed and tested, obtaining sex diagnostic bands of females and males. These markers were validated on a large sample, confirming their usefulness for sexual identification in pacu.