| Testudines belong to the Vertebrate Phylum Reptiles and are one of the oldest reptiles in the world.Their ancestors can be traced back to the Odontochelys semitestacea 200 million years ago.Testudines are usually divided into two branches,Pleurodira and Cryptodira.Its species have a wide range of distribution and a huge pedigree.Chromosome numbers vary widely among Testudines(2n=26-68),suggesting extensive chromosomal rearrangements in turtles.The sex determination mechanism of Testudines is divided into temperature-dependent sex determination and genetic sex determination,and the sex determination master gene has not yet been identified in Testudines.The key to solving the sex determination mechanism of TSD is how to convert the external temperature signal into an electrical signal that can promote gonadal differentiation,in which the transient receptor potential(TRP)gene family plays a key role in this process.In addition,Hox genes play an important role in the development of the reproductive system of Testudines.Therefore,clarifying the phylogeny of Testudines,the evolution of chromosomes and related genes is very important for analyzing the sex determination mechanism of Testudines.In this study,using the published multi-omics data of turtles,the kinship of Testudines,the evolution of autosomes and sex chromosomes were analyzed from the omics level,and then the genetic identification and evolutionary analysis of sex determination master genes,TRP and Hox genes were carried out.The findings of this study are listed below:(1)Phylogenetic analysis of TestudinesFirst,based on the global alignment results of 28 genome sequences of Turtles,a phylogenetic tree was constructed with chicken as the outgroup.Based on the single-copy orthologous gene,the phylogenetic tree was constructed by the method of Concatenation and Coalescence,and the evolutionary relationship was found to be completely the same as that of the whole genome evolutionary tree.By calculating the evolutionary divergence time of species within Testudines,we clarified the evolutionary history of Testudines.The ancestor of Testudines originated 250 million years ago,and diverged into the ancestors of the two branches of Pleurodira and Cryptodira 200 million years ago.Trionychidae ancestors diverged from Trionychia 50 million years ago.(2)Chromosome evolution and origin of sex chromosomes in TestudinesBased on comparative genome research methods,we conducted chromosome evolution studies on 11 turtle species whose assembly quality reached the chromosome level.The results showed that autosomes 7,8,9 and 10 of Durocryptodira were formed by the fusion of two autosomes of its ancestors.The karyotypes of Trionychidae were similar to those of their ancestors.Chromosomal rearrangements directly contributed to the apparent chromosome number differences between Trionychidae and Durocryptodira.In addition,we also found that the sex chromosomes of Trionychidae(GSD)are highly homologous to the chromosome segments of Durocryptodira(currently all Durocryptodira at the chromosome level are TSD)and the red ventral turtle,which proves that the sex chromosomes of Pelodiscus sinensis evolved from autosomes Come.The GO enrichment results of the Z and W sex chromosomes of P.sinensis showed that most of the gene functions on the Z chromosome were directly related to life activities,which confirmed the necessity of the Z chromosome for maintaining biological life activities.While retaining a weak homology with the Z chromosome,the W sex chromosome repeat sequence of P.sinensis accounts for74.53%,which is significantly higher than the repeat sequence content of autosomes and Z chromosomes,which confirms the classic theory of sex chromosome evolution.(3)Evolutionary analysis of sex-related genes in TestudinesWe used PAML branch model and branch site model to test the selection pressure of the vertebrate master sex-determining gene and TRP gene.The results show that most vertebrate sex-determining master genes are absent in Testudines,while all temperature-sensing-related TRP genes are conserved in Testudines.In addition,we found that growth differentiation factor Gdf6 has undergone fast evolution in the Trionychidae ancestral branch,and the androgen receptor Ar has undergone positive selection;TRPV1,TRPV2 and TRPV4 genes have undergone fast evolution in the TSD species ancestral branch.Although Hox genes are also highly conserved in turtle species.However,the deletion of the non-repeated sequence caused the intergenic region of Trionychidae Hox B9-Hox B13 to be 10 kb shorter than that of Durocryptodira.In addition,some Hox genes undergone fast evolution and positive selection in the ancestral branch of Trionychidae.At the same time,branch-specific sequence insertions or deletions generally occurred in the sex-determination-related genes we studied.In summary,this study took Testudines as the research object,using known sex determination-related genes and published Testudines genome data,to construct an accurate Testudines phylogenetic relationship.In addition,we also discovered the characteristics of Testudines chromosome evolution and the origin of sex chromosomes,and deduced that some sex determination-related genes had different evolution rates between TSD and GSD species,and positive selection occurred in specific clades.We comprehensively inferred the differences of the two sex determination modes at the genetic level from both macro and micro levels.This project lays a theoretical foundation for the subsequent deciphering of the sex determination mechanism of turtles,and provides guiding research for the sex control industry of turtles. |
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