Sex chromosome and sex locus characterization in the goldfish, Carassius auratus

Abstract

38Background: Goldfish is an important model for various areas of research, including neural 39 development and behavior and a species of significant importance in aquaculture, especially as 40 an ornamental species. It has a male heterogametic (XX/XY) sex determination system that 41 relies on both genetic and environmental factors, with high temperatures being able to produce 42 female-to-male sex reversal. Little, however, is currently known on the molecular basis of 43 genetic sex determination in this important cyprinid model. We used sequencing approaches to 44 better characterize sex determination and sex-chromosomes in goldfish. 45 Results:Our results confirmed that sex determination in goldfish is a mix of environmental 46 and genetic factors and that its sex determination system is male heterogametic (XX/XY). 47Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we 48 characterized sex-linked polymorphisms and developed male specific genetic markers. These 49 male specific markers were used to distinguish sex-reversed XX neomales from XY males and 50 to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing 51 temperature (18°C), for which sex reversal has been previously shown to be close to zero. We 52 also characterized a relatively large non-recombining region (~11.7 Mb) on goldfish linkage 53 group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This 54 large LG22 region harbors 373 genes, including a single candidate as a potential master sex 55 gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms 56 were detected in the goldfish amh gene or its 5 kb proximal promoter sequence. 57 Conclusions:These results show that goldfish have a relatively large sex locus on LG22, which 58 is likely the goldfish Y chromosome. The presence of a few XX males even at low temperature 59 also suggests that other environmental factors in addition to temperature could trigger female-60 to-male sex reversal. Finally, we also developed sex-linked genetic markers in goldfish, which 61 will be important for future research on sex determination and aquaculture applications in this 62 species. 63 assembly 65 66 3 BACKGROUND 67Goldfish, Carassius auratus, is a domesticated fish species originating from central Asia and 68 China that has been introduced throughout the world. Goldfish belongs to the Cyprinidae family 69 and is considered as an important fish model for research in endocrinology [1,2], 70 developmental biology [3,4] or fish pathology [5]. Thanks to the recent availability of a whole 71

RADSex: a computational workflow to study sex determination using Restriction Site-Associated DNA Sequencing data

“…In such cases when there is very little differentiation between the sex chromosomes, the fragmented resolution of RAD-Seq means that the the likelihood to obtain RAD sequences from the sex locus can be very low. Although this problem might be overcome by increasing the number of RAD-seq reads, for example by using a restriction enzyme that cuts more frequently, genome-wide approaches like pooled sequencing of males and females (Feron et al, 2020;Gammerdinger, Conte, Baroiller, D'Cotta, & Kocher, 2016;Wen et al, 2019) or individual whole genome sequencing (Star et al, 2016) may be necessary to identify the sex-determining region. Second, a complete absence of sex-biased markers can reflect a complex sex determination system, for instance a polygenic system or a system involving a strong environmental effect that would weaken the association between genetic markers and phenotypic sex.…”

“…For such systems, pooled sequencing may not be well-suited because male and female pools will each contain heterogeneous genotypes, and individual sequencing remains expensive despite decreasing sequencing costs. A hybrid strategy using both RAD-Seq and pooled sequencing may prove the most efficient for these complex sex-determination systems, as demonstrated in the case of the goldfish (Wen et al, 2019) , a species with a strong thermal effect on sex determination (Goto-Kazeto et al, 2006) . Finally, in some species sex is determined entirely by environmental factors and therefore does not involve any genetic mechanism (Martínez-Juárez & Moreno-Mendoza, 2019) .…”

RADSex: A computational workflow to study sex determination using restriction site‐associated DNA sequencing data

“…For such systems, pooled sequencing may not be well-suited because male and female pools will each contain heterogeneous genotypes, and individual sequencing remains expensive despite decreasing sequencing costs. A hybrid strategy using both RAD-Seq and pooled sequencing may prove the most efficient for these complex sex-determination systems, as demonstrated in the case of the goldfish (Wen et al, 2019) , a species with a strong thermal effect on sex determination (Goto-Kazeto et al, 2006) . Finally, in some species sex is determined entirely by environmental factors and therefore does not involve any genetic mechanism (Martínez-Juárez & Moreno-Mendoza, 2019) .…”

“…In such cases with very little differentiation between the sex chromosomes, the fragmented resolution of RAD-Seq means that the likelihood of obtaining RAD sequences from the sex locus can be very low. Although this problem might be overcome by increasing the number of RAD-Seq reads, for example by using a restriction enzyme that cuts more frequently, genome-wide approaches such as pooled sequencing of males and females (Feron et al, 2020;Gammerdinger et al, 2016;Wen et al, 2019) or individual whole genome sequencing (Star et al, 2016) may be necessary to identify the sex-determining region. Second, a complete absence of sex-biased markers can reflect a complex sex determination system, for instance (i) a polygenic system, (ii) strong polymorphism on the sex chromosomes leading to multiple W or Y haplotypes each shared by only a fraction of individuals from the heterogametic sex, or (iii) a system involving a strong environmental effect that would weaken the association between genetic markers and phenotypic sex.…”

Evolution of Tandemly Arranged Repetitive DNAs in Three Species of Cyprinoidei with Different Ploidy Levels

“…The authors first suggested that micro-B chromosomes play a role in the sex determination of C. gibelio, and then demonstrated the presence of the SET domain containing gene (setdm) on these elements . However, the most recent data indicated the presence of a male-determining gene on CGI22 [Wen et al, 2019]. This may reflect the variability of sex-determining systems in the species, as different populations have evolved specific sex-determining mechanisms with setdm modulating sex determination.…”