Prof. Songlin Chen and colleagues from Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences, working jointly with BGI-Shenzhen and other institutes, have successfully decoded the first flatfish genome (half-smooth tongue sole, Cynoglossus semilaevis). This is the first fine mapping of fish genome in China, providing insights into ZW sex chromosome evolution, sex determination, and adaptation to a benthic lifestyle. Moreover, the data generated in this project has laid a foundation for the genetic breeding of tongue sole. The paper has been published in the latest issue of Nature Genetics, March 2014 (46(3): 253-260. DOI 10.1038/ng.2890).
As a warm-water demersal fish, half-smooth tongue sole is a valuable marine fish which is common in offshore area of China. Its sex determination belongs to ZW/ZZ type, with female harboring the huge sex chromosome (W chromosome). Correspondingly, the female fishes are 2-4 times larger than the male ones, which shows so far the biggest sexual difference in fish. Since the origin and evolution of vertebrate sex chromosomes have been a hot issue in biology, the remarkable difference of sex chromosome and male/female phenotypes allows tongue sole as the ideal model to study the evolution of vertebrate sex chromosomes and phenotypic differentiation.
In this study, the researchers used de novo sequencing and assembled the genomes of one male (ZZ) and one female (ZW) tongue sole, respectively. Based on the difference of sequencing depth of Z/W linked-scaffolds between female and male, together with the high-resolution genetic map constructed by SSR and SNP, they assembled the Z and W chromosome of tongue sole at high quality. Study suggested that, because of well-differentiated sex chromosome, W and Z chromosomes contained abundant transposable elements (W: 29.94%; Z: 13.13%), while the ratio in autosome is only 4.33%. Concurrently, the ratio of pseudo-genes in W chromosome is the highest (19.74%), followed by Z chromosome (3.54%) and autosome (2.48%). Using the Z-W homologous genes, they estimated that the age of the tongue sole sex chromosome pair is relatively young (about 30 million years), which suggests a rather fast evolution of the tongue sole sex chromosomes.
To ascertain the evolutionary trajectory of the tongue sole genome, the researchers addressed the phylogenetic branching and divergence time by global alignment of tongue sole, tetraodon, medaka, and zebrafish, using human and chicken as outgroups. They found that the sex chromosomes of tongue sole are derived from the same ancestor as chicken, but not as any known fishes. Notably, the sex chromosome of tongue sole and chicken showed convergent evolution, sex chromosomes in both species are rich in repetitive DNA, Z chromosome of tongue sole exhibited partial dosage compensation in female as that of chicken. Moreover, genetic characteristic of sex-reverted fish implied that Z chromosome is particularly important for the inheritance of sex determination. Furthermore, one Z chromosome linked gene dmrt1, which is specifically expressed in male and required for testis development, shows the features of male-determining gene that is compatible with its similar function in birds.
The male tongue sole are smaller and grow slowly, so the high male proportion in the population affects their commercial quality, reduces the fishermen’s interest, and limits its fishery and aquaculture development. Thus it is an urgent issue to identify the mechanism for high male proportion and develop high- or full-female fry. By comparing female/male whole genomes and analyzing gender-specific microsatellite markers, a new technique based on microsatellite markers could distinguish ZZ male, ZW female and WW super-female with 100% accuracy, solving the problem of distinguishing ZW females and WW super-females. With this method, researchers found that physiologically female accounts for only 10-30%. After the analyses of hereditary ratio and physiological ratio in different families, they found that more than 90% of pseudo-male offspring sex-reverted into a pseudo-male. Whole-genome methylation sequencing revealed that all second-generation pseudomales had inherited the Z chromosome from their sex-reversed fathers and retained the paternal methylation pattern, implying that trans-generational inheritance of DNA methylation status at certain loci on the Z chromosome is particularly important for the inheritance of sex reversal. So the reason why there is 70-90% male, but only 10-30% female in breeding population is, THE OFFSPRING OF PSEUDOMALE IS PRONE TO BECOME PSEUDOMALE. In addition, because W-sperm is absent in ZW pseudo-male, WW super-female was not successfully obtained by crossing ZW female and ZW pseudo-male. This observation provides theoretical basis and technical support for studying tongue sole sex determination and developing fry with high-female proportion.
Flatfish are characterized by a transition from pelagic to benthic habitats when they metamorphose from free swimming larvae to bottom-dwelling fish, accompanied by a series of biological and phenotypic changes, such as eye migration, displacement of internal organs, the difference of pigmentation and the sensitivity of light. By comparing the genomes and transcriptomes of pelagic and benthic fish, the researchers revealed that the differentially expressed genes between these two developmental stages are enriched in genes potentially involved in adaptation to a benthic lifestyle. They also identified 15 positively selected genes, which have putative roles in metamorphosis. In addition, they found that the expression levels of rod pigment (rh1) and cone pigment (lws1) genes, which are responsible for scotopic vision and long-wavelength-sensitivity, respectively, were significantly up-regulated in benthic stages, whereas the expression of the middle-wavelength-sensitive pigment gene (rh2) was significantly up-regulated in pelagic stages. Moreover, they also found that several crystallin genes (crybb2 and crybb3) were lost or became pseudogenes. They speculate that these changes coincide with the tongue sole visual degeneration after shifting to the benthic environment with weak light conditions. As compensation, tongue sole have developed a strong lateral-line sense organ and especially, the papillae - a specific mechanoreceptor sense organ during evolution.
Regarding this study, Songlin Chen said: “Half-smooth tongue sole genome is the first sequenced flatfish genome in the world, opening up the genomic era of flounder culture. This map has profound effects, not only on illustrating the mechanism of sex determination, growth, reproduction, development, disease-resistance, metamorphosis, and ecological adaptation, but also on investigating sex chromosome evolution and sex determination mechanism in other vertebrates. It will establish the technique for tongue sole sex determination and genome-wide selective breeding, settle the platform for obtaining new breeds with high productivity, high female ratio and disease resistance, so this map has important theoretical significance and practical value”.
