Genetic sex determination by W and Z chromosomes has developed independently in different groups of organisms. To better understand the evolution of sex chromosomes and the plasticity of sex-determination mechanisms, we sequenced the whole genomes of a male (ZZ) and a female (ZW) half-smooth tongue sole (Cynoglossus semilaevis). In addition to insights into adaptation to a benthic lifestyle, we find that the sex chromosomes of these fish are derived from the same ancestral vertebrate protochromosome as the avian W and Z chromosomes. Notably, the same gene on the Z chromosome, dmrt1, which is the male-determining gene in birds, showed convergent evolution of features that are compatible with a similar function in tongue sole. Comparison of the relatively young tongue sole sex chromosomes with those of mammals and birds identified events that occurred during the early phase of sex-chromosome evolution. Pertinent to the current debate about heterogametic sex-chromosome decay, we find that massive gene loss occurred in the wake of sex-chromosome 'birth'.

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

The present study aims to uncover the relationship between the phenotypic sex and genotypic sex and the growth rate of male and female half-smooth tongue sole and to select the excellent families with high female proportion.In this paper,we firstly established 22 half-smooth tongue sole families which were cultured in the same pond.Secondly,we identified the offspring's phenotypic sex and genotypic sex of 5 normal male families and 5 neo-male families.This study shows that there are significant differences not only in growth rate but also in proportion of male to female.Two fast-growing families(No.16 and No.61)whose relative weight gain rate were 0.81 g/d and 0.56 g/d and 4 relatively fast-growing families(No.21,No.28,No.57 and No.63),the relative weight gain rate ranges from 0.48 g/d to 0.53 g/d were selected;12 commonly-growing and 4 slow-growing families were selected also.The average ratios of genotypic female and phenotypic female of the 5 normal male(ZZ)families were 51.87% and 42.94%,and the ratio of genotypic female reversing to phenotypic male was 14.08%.Instead,the average genotypic female and phenotypic female ratio of the 5 neo-male(ZW)families was 49.34% and 4.45%,respectively,and the ratio of genotypic female reversing to phenotypic male was 91.41%.In the present study we found for the first time that there were significant differences in phenotypic sex ratio between different families.We also found that the genotypic females in neo-male families were more easily reversed to phenotypic males,which increased the number of phenotypic males in cultured populations.It initially showed that the families with high female proportion and fast growing rate could be selected via family selection,which could provide theories and new technical methods for the breeding and the production of high quality seeding of half-smooth tongue sole.

本研究克隆了半滑舌鳎（Cynoglossus semilaevis）AMH基因并对其进行表达分析。该基因cDNA序列开放阅读框长为1 563 bp,编码蛋白含520个氨基酸。该蛋白含有TGF-β超家族的特征序列,包含1个AMH-N区域和TGF-β结构域,并在C端生物活性区含有9个保守的半胱氨酸残基。系统进化分析表明,半滑舌鳎AMH和所有鱼类AMH聚为一簇,与鲽形目相似性最高。实时定量结果表明,AMH基因在半滑舌鳎不同组织中有差异表达,在正常雄鱼和伪雄鱼性腺中表达量最高。胚后不同时期性腺的实时定量结果表明,性腺分化之前AMH在精巢中相对表达量较低,70 dph（days post hatching）达到最高峰,而在卵巢中呈现先升高后下降的趋势,预示AMH基因可能在半滑舌鳎性腺发育中起重要作用。较正常雄鱼后代而言,AMH基因在伪雄鱼后代的雄鱼和伪雄鱼性腺中的表达都有升高的趋势,而在雌鱼中无明显差异,也预示其可能参与半滑舌鳎的性反转过程。

采用RACE方法克隆了半滑舌鳎（Cynoglossus semilaevis）PKR基因（CsPKR）,获得了CsPKR全长cDNA序列为2907bp,其中包含1959bp的开放阅读框,100bp的5′非编码区和848bp的3′非编码区。保守结构域分析显示推导的CsPKR氨基酸在N端存在两个特异的dsRBD（dsRNA binding domain dsRBD）结构域,在C端具有多个保守的激酶活性位点,包括ATP结合位点和底物配体结合位点,以及活性环结构A-loop。系统进化树分析显示鱼类、两栖类、鸟类及哺乳类的PKR具有共同的进化起源,CsPKR与牙鲆PKR的亲缘关系最为密切。荧光定量PCR结果显示,CsPKR基因在健康鱼多个组织中广泛表达,在脑中的表达最高,头肾中表达最低。经鳗弧菌和淋巴囊肿病毒分别感染后,CsPKR基因在免疫相关组织中呈现上调表达趋势,其中感染鳗弧菌12h后在血液中表达量较对照组上升了9.28倍,在感染淋巴囊肿病毒24h后,在肝脏中的表达达到最大,为对照组的9.97倍。以上结果暗示CsPKR基因在半滑舌鳎响应细菌和病毒免疫应答中起重要作用。

Anti-Mullerian hormone(AMH),also known as Mullerian inhibiting substance(MIS),is a glycoprotein belonging to the transforming growth factor β superfamily,which plays a major role during reproductive development in vertebrates.We isolated and characterized AMH cDNA in the half-smooth tongue-sole,Cynoglossus semilaevis.The open reading frame(ORF) of this gene spans a region of 1 563 bp and codes 520 amino acids. Sequence alignment analysis revealed that the AMH of half-smooth tongue-sole and pleuronectiformes shared 61.7% homology.We evaluated the pattern of AMH mRNA expression in 14 tissues of the half-smooth tongue-sole.Expression level was highest in male and neo-male gonads,followed by the blood,skin,and brain. AMH expression was low prior to 70 dph(days post hatching) but was highest in the testis 70 dph.In the ovary, AMH expression increased then decreased,suggesting it plays a role during reproductive development.Furthermore, in contrast with normal offspring,the gonad of male and neo-male non-normal offspring exhibited an increased in expression,but there was no change in females.This suggests that the AMH gene is required for sex reversal.

We detected the genetic sex and the occurrence of sex reversal in eight normal families and their male parents in the half-smooth tongue sole(Cynoglossus semilaevis Gunther) using the SSR female-specific marker scaffold 1128_343 and a gonad slice.The proportion of females in eight normal families(28,30,38,39,40,44,57, and 69) ranged from 37.93 to 55.00%.The genetic sex of four of the eight normal families(28,39,44,57) was detected twice,and the proportion of femalesdid not exibit significant difference between two tests(P0.05).We detected both the genetic sex and physiological sex in four of the eight normal families(28,39,44,and 57) and in all the male parents.The proportion of physiological females ranged from 18.75%.46.88% among the four families, and was lower than the proportion of genetic females.Our results suggest there are inter-family differences in the proportion of females undergoing sex-reversal.The present study also suggests that a significant number of male parent fishes(28.42%) are neo-males.Our results suggest that sex reversal is a natural phenomenon in the half-smooth tongue sole breeding population.Furthermore,we reveal the pedigrees of different physiological spawner ratio have significant differences.Our observations provide a theoretical basis for developing methods to culturing families that have a high proportion of female larvae.

[Objective]This research aimed to understand the cultured genetic gender proportion and the female vs. male gonadal development process of Cynoglossus semilaevis groups in order to provide reference for high proportion female breeding. [Method]The sex linkage microsatellite markers scaffold1128_343 was used to detect the juvenile female proportion variation characteristics of the two groups of half-smooth tough sole; combined with the genetic sex identification and paraffin section technique, the gonad sexual maturity development was also observed through tracking the two groups from 25 days old to 2 years old. [Result]From the results of genetic sex identification performed on the two breeding groups of half-smooth tough sole aged 12 to 76 days,the average genetic female percentage of group I and group II was respectively 50.00 and 50.23%, which coincided with the 1∶1 distribution. The female and male gonadal structure of early half-smooth tough sole gonad development (25 days old) had no significant difference, but starting from the age of 56 days, the female and male gonadal structure started to display visible differences. At 76 days of age, the female and male gonadal structure showed obvious visible differences. At 106 days of age, the female and male gonads appeared to be obviously differentiated, which, though the gonadal biopsy meothd, the female and male gender could be identified. [Conclusion]The female vs. male ratio of Cynoglossus semilaevis accorded with the 1∶1 distribution. The female gonad development and differentiation were faster than those of the male gonad development, but the development processes were more complex.

Female and male linkage maps were constructed using SSR markers based on a full sub-family of the tongue sole(Cynoglossus semilaevis).A total of 320 markers were generated in two parents and 92 F1 progenies of the mapping family by using 320 screened SSR primer combinations containing 112 segregation distortion mark-ers(P0.05).The female linkage map included 21 linkage groups and 242 pairs of SSR markers spanning 1311.9 cM with the average marker interval of 4.9 cM and the observed coverage was 83.3%.The male linkage map in-cluded 21 linkage groups and pairs of 218 SSR markers spanning 1 316.2 cM with the average marker interval of 5.5 cM and the observed coverage was 82.0%.The genetic linkage maps are very significant for QTL analyses marker-assisted selection(MAS) breeding programs for economically important traits and sex control in C.semilaevis.