In this study, we constructed a high-density linkage map for L. vannamei using a population from a breeding program. To our knowledge, this is the densest linkage map constructed available for this species using a 50K SNP array.
There are several linkage maps available for this species, most of them with a relatively low number of SNPs and using RAD-seq methods for genotyping (Chen et al., 2022; Huang et al., 2020; Jones et al., 2017). It is known that L. vannamei presents 44 chromosome pairs [33]. However, some linkage maps showed 45 [11], 48 [17] and even 49 [38] LGs. The present linkage map resulted in 15,256 SNPs distributed in the 44 LGs which agrees with the real number of chromosomes in this species. The LG44 presented a significant lower number of SNPs (24) in comparison to the other LGs (197 to 587). Penaeids present unique genomic characteristics including high level of heterozygosity, polyploidization in some species and simple sequence repeats accounting for up to 50% of the genome in some cases [39]. Such genomic characteristics allied to a large genome size (~ 2.6 Gb) has hampered the construction of a high-quality reference genome and expansion of genome resources for L. vannamei. These factors may also have affected the assignation of SNPs to LG44.
The male and female linkage maps were very similar in terms of total length and overall mean density. Still, it is possible to observe larger recombination gaps, also known as “coldspots”, in the male map in comparison to the female. This is evident in the LG31 (the LG found associated with sex determination) where high recombination rate was observed in the female map, while for males, the recombination is more concentrated towards the centromere of the LGs. This pattern is also common in many fish species. Cooney et al. (2021) evaluated the recombination landscape of 61 fish species and found that 42 of them have higher recombination rates in females in comparison to males. Although there is no clear adaptative explanation for such differences between sexes, some hypotheses were proposed, such as the meiotic drive (i.e., alleles have an advantage during the process of female meiosis changing recombination rates) and protection against aneuploidy (i.e., failure of chromosomes to separate during cell division) [41–43]. In crustaceans, a study showed similar recombination rates between males and females for Daphnia pulex [44] which is conflictive with our results in shrimp. Thus, there is no “rule of thumb” in the recombination dynamics between sexes. Recombination studies are scarce in crustaceans, especially in shrimp, and further investigations are necessary to better understand the recombination landscape in this species.
There is an evident interest in understanding the genetic mechanisms of sexual determination in L. vannamei, mainly due to the sexual dimorphism in this species. Females present higher growth rate and mono-sex culture may increase the economic return of shrimp farming [20]. The identification of the specific genetic region and genes involved in sex determination may help to manipulate the proportion of females optimizing mono-sex culture. Association between markers and sex determination were already reported for L. vannamei. Yu et al. (2017) identified a genomic region on LG18 related to sexual determination and a single marker was found completely associated to the trait being a potential SNP to sex characterization in this species. This SNP (Yu_C19299G_A) is present in the 50K SNP array and was the most significant SNP in our study. Perez-Enriquez et al. (2018) also found three markers associated with this trait using RAD-seq method to develop a set of SNPs to characterize Mexican shrimp broodstock populations. The three SNPs were evaluated in the sex identification of a different population using post-PCR high resolution melting method and high accuracy of sex determination was also reported. Another major effect marker was identified by Jones et al. (2020) in which a strong association with sex was reported and a high percentage of males were homozygotic and females heterozygotic for this marker in almost 2,000 animals. Using GWAS and the new high-density LM, we identified, 21 statistically significant SNPs potentially associated with sexual determination in this population at LG31. Yu’s marker was effective in the determination of sex in our dataset confirming the ZZ/ZW determination system and the potential use of this marker in other L. vannamei populations. These results may help to identify a fine mapping of sex determination region as this important marker was not positioned in a high-density genetic map before. The other two markers with higher statistical association to sex determination explained together about 40% of phenotypic variance but with a low ability to determine sex. A possible explanation for this divergence is the high recombination rate observed through the LM. Most likely, this region is under strong cross-over events making it difficult to be detected across multiple families [19]. This can be also visualized in the LD pattern of the most associated SNPs where a low LD was reported among SNPs that are in the LG31.
Of the organisms used in the GWAS, 0.9% (eight females and two males) disagreed the sex determined in the field (phenotype) with the sex assigned using Yu´s marker. It is possible to infer that these were assignment errors, because when considering AX-249531418, the sex determination coincided with Yu´s marker. The absence of females with the GC/TT (Yu_C19299G_B / AX-249531418) genotype is noteworthy, which could be associated with the presence in this genomic region of genes with an epistatic relationship with lethal effects for females. The main effect of sex-lethal mutations is on reproductive aspects [45] which were not evaluated in this study. As a sex-lethal gene (Sxl) was previously described in this species [46], further investigations about the deleterious effect of these SNPs are necessary.
At least three important genes were described in the process of sexual determination in L. vannamei: sex-lethal (Sxl) [46], Pvfem-1 [47] and the 25749_180 loci [21]. The Sxl gene was reported during embryogenesis and gametogenesis and some isoforms were more abundant expressed in male adult testis (Sxl-5 and Sxl-6) while others in the female oocytes (Sxl-2 and Sxl-4). The Pvfem-1 was found in several L. vannamei tissues including: brain, intestine, gonads and hepatopancreas. A natural antisense transcript of Pvfem-1 was present in females and absent in males suggesting a participation in the process of sexual differentiation. Finally, the 25749_180 uncharacterized locus was also reported in the sexual determination for this species. We did not find any of these genes close to the top10 significative SNPs revealed in the GWAS. However, the oplophorus-luciferin 2-monooxygenase and the serine/arginine repetitive matrix protein (SRRM1) genes were intercepted by significant SNPs. The first gene was found downregulated in both PL15 and PL25 stages in gonadal tissues of M. nipponense [34]. Although its specific function is not clear, this gene was found related to metabolic and extra cellular matrix processes in other crustacean species [48, 49]. The second gene was found downregulated in testis and ovary tissues of P. monodon using transcriptomic analysis [36]. The serine-arginine protein was reported differentially expressed in the testis maturation process of the same species [35]. The spermine oxidase gene was found close to a significant SNP. This gene is a precursor of spermidine, a well-known polyamine involved on the regulation of fertility in mammals [50]. The spermine oxidase was described as significantly up-regulated in narrow-clawed crayfish (Pontastacus leptodactylus) suggesting a potential role on the gonads maturation [51]. It was also found differentially expressed on gonadal tissues of L. vannamei individuals (Chen et al., 2022). Unfortunately, the sex-linked SNP identified by Yu et al. (2017) mapped in this study was not found associated to any gene.
Our results helped in the identification of a genomic region associated to sex determination in shrimp using an informative high-density LM. Future research focused on the fine mapping of this sex-determination region may help to disentangle the mechanisms involved on the sexual differentiation and in the development of mono-sex populations in L. vannamei.