| Amphioxus is affiliated with the phylum Chordata,the subphylum Cephalochordata,and has been living on earth for over 500 million years with the name " living fossil ".Amphioxus is regarded as an overabundant group that evolved from invertebrates to vertebrates and represents the direct ancestor of vertebrates.In addition,amphioxus has a genome size of only 400 M and has not undergone two rounds of whole genome duplication in vertebrates.Amphioxus has no complex and functionally redundant gene families and possesses the most basic set of coding genes for chordates.Amphioxus is the best model to study vertebrate gene chromosome evolution,gene family origin,and organ type evolution.Therefore,it is of great interest to carry out a genomic evolution study focusing on amphioxus.The current genomes of amphioxus have been published for the Branchiostoma floridae(B.floridae)and the Branchiostoma belcheri(B.belcheri).The genome of Branchiostoma japonicum(B.japonicum),which was common in China,had not been reported.The heterozygosity of three amphioxus species was estimated to be between 3.2%and 4.2%,with the highest heterozygosity in the animals.The low quality of the genome assemblies of these two types of amphioxus resulted from the high degree of genome heterozygosity in amphioxus.In order to solve this genomic difficulty,we used the strategy of 'Trio binning' genome assembly for the assembly of high-quality amphioxus genomes.First of all,we used the advantages of artificial breeding of amphioxus in our laboratory.We performed artificial in vitro fertilization experiments on laboratory bred B.floridae,B.belcheri,and B.Japonicum,which were grouped separately.We found that B.floridae with both B.belcheri and B.japonicum produced normal zygotes and that individuals were able to grow and develop.However,there is reproductive isolation between B.belcheri and B.Japonicum,the eggs cannot be fertilized normally.We further sequenced two amphioxus hybrids(B.floridae X B.belcheri and B.floridae X B.Japonicum)by Pacbio and Illumina,respectively.As a result,there are significant differences in chromosome sequences among different species of amphioxus.Therefore,we split the Pacbio data of hybrid amphioxus into the data of the corresponding two sets of species based on the sequencing data of Illumina of amphioxus for the three homozygous species.The assembly then proceeds.This way,the assembly of the genome was performed,which overcame the problem of high heterozygosity of amphioxus.Finally,we obtained the chromosome level haplotype genomes of B.floridae,B.belcheri and B.japonicum by contig assembly and Hi-C assisted assembly of the genomes,respectively.Compared with the published results of short read sequencing of the amphioxus genome assembly,the scaffold N50 length of the genome was increased by more than 200-fold,which greatly reduced the assembly errors due to repetitive sequences in the genome.Genome integrity exceeded 97% as determined by inspection and evaluation with BUSCO software.The number of Gaps on the chromosomes of B.belcheri and B.japonicum was less than three,approximately six in B.floridae.This is sufficient to be comparable to,or even less than,vertebrate reference genomes.Meanwhile,telomere sequences were assembled from the genomes of three amphioxus species.And we verified the presence of telomere sequences by fluorescence in situ hybridization experiments(FISH).In summary,this part of the work involved obtaining extremely high quality genomes of three species of amphioxus.Provided a foundation for bioinformatics analysis using the genome of amphioxus.Further,we selected seven vertebrate model organisms along with three amphioxus species for the prediction of species divergence time.It was found that strong interspecific differentiation occurred among the three species of amphioxus.However,the three species of amphioxus differ only slightly in appearance,and this interspecies differentiation is presumed to occur in the chromosomes of the genome.We analyzed the chromosome fusion events among three species of amphioxus.B.belcheri was found to have 20 gene linkage groups.It fused twice on the chromosomes of amphioxus japonicus.Specifically,linkage group 4 and 19 were fused,and linkage group 13 and 17 were fused.In B.floridae,there was a fusion between linkage group 16 and linkage group 20.Since B.belcheri possesses the highest number of chromosomes,it represents the ancestral form of amphioxus.In addition,the chicken is representative of vertebrate chromosomes because it exhibits the lowest chromosomal evolutionary lineage specificity and the lowest gene duplication rate among vertebrates.Therefore,we aligned the genome of B.belcheri to chicken.Reconstruction of karyotypes of chordate ancestors through chordate linkage groups(CLG).It was elucidated which chromosomes among the 20 chromosomes of B.belcheri formed17 chordate linkage groups by chromosome fusion.On the other hand,we performed genetic contraction and expansion analysis of amphioxus and human,mouse,zebrafish,and chicken,the representative model species of vertebrates.We have analysed the horizontal paralogs of genes that undergo replication in amphioxus in the evolution of vertebrate genes.This indicated that although two rounds of genome amplification have not occurred in amphioxus,genome ploidy has begun within its genome.The phenomenon of gene duplication that emerged over the evolutionary course and continued thereafter in vertebrate genomes across two rounds of whole genome duplication.We also analysed the proportion of segmental duplication of chromosomes in the amphioxus genome.Segmental duplication of chromosomes was found to be higher in all three species of amphioxus than in four vertebrate species of human,mouse,chicken and zebrafish.The GO functions of these genes that underwent chromosomal segmental duplication were enriched in the GO function entries of G protein receptor activity,complex protein kinase activity and nucleic acid binding function.Finally,we have analysed the most evolutionarily conserved Hox gene family in vertebrates.Through sequence alignment,we have mapped the evolution of the Hox gene family of amphioxus to the vertebrate Hox gene family.For a long time,the research on sex had been one of the hot topics in life science.Sex determination extends throughout the evolutionary history of chordates,and evolutionary biologists have argued that 'sex was the queen of question in evolutionary biology'.The mechanisms of sex determination were mainly of two types.One was environmental factor determinant and the other was genetic determinant.Our laboratory determined that sex determination in amphioxus was independent of environment during long-term amphioxus rearing.On the basis of the three chromosome-level amphioxus genomes assembled,we selected 30 male and female individuals of each of three amphioxus species for GWAS analysis.We screened a 16 Mb interval of female-specific SNPs on chromosome 18 of B.floridae,a 2Mb interval of female-specific SNPs on chromosome 3 of B.japonicus,and a3 Mb interval of female-specific SNPs on chromosome 3 of B.belcheri,respectively.Further,we sequenced the sex SNP loci of three species of amphioxus by PCR amplification and demonstrated that the sex determination of amphioxus is ZW type.We built on this by crossing and pairing female B.floridae(ZW)with male B.japonicus(ZZ).The W karyotype of B.floridae was successfully disentangled by sequencing female hybrids of the progeny.In the study of gonadal development in amphioxus,we have carefully documented the gonadal developmental history of three species of amphioxus.Moreover,we collected immature and mature testis and ovary of three species of amphioxus for transcriptome sequencing.Having resolved the sex-linked regions of three amphioxus species,we were interested in what genes were present on the sex-linked regions of three amphioxus species and whether these genes existed in common between the three species.We screened for a candidate gene from the sex-linked region of B.japonicus,which was present in both B.floridae and B.belcheri,but not in the sex-linked region,and also for a homologue in vertebrates.We further analysed the expression of this gene in gonadal transcriptome data from three species of amphioxus.Excitingly,the gene was specifically highly expressed in immature and mature testis and lowly expressed in immature and mature ovary of three amphioxus species,exhibiting tissue specificity of the testis.We finally demonstrated by RT-PCR and in situ hybridization that this gene is also specifically expressed in the spermatozoa.We used 10 reported vertebrate sex-determining genes in the sex-linked region of three amphioxus species for homologous sequence alignment,and found that none of them were present in the sex-linked region of three amphioxus species.Meanwhile,none of the currently identified vertebrate upstream sex-determining genes,such as the Dmrt1 gene,the Amh gene and the Rspo1 gene,have homologous sequences in the genomes of three amphioxus species.We further analysed the expression of these 10 genes in amphioxus testis and ovary transcriptome data and found that only the Foxl2 gene and the Sf1 gene were sex-biased.Expressed Foxl2 specificity in testis and Sf1 specificity in ovary.Together,these results suggest that amphioxus and vertebrates have each evolved their own pathways of sex determination.In summary,we have obtained high-quality genomes of three species of amphioxus by hybrid sequencing.The analysis of interspecific chromosome fusion in amphioxus and chromosome evolution with ancestral vertebrate forms was also carried out.It was discovered that although amphioxus has not undergone two rounds of genome duplication,genome ploidy has begun within its genome.We have mapped the evolution of the Hox gene family in amphioxus to the vertebrate Hox gene family.Finally,it was demonstrated that the sex-determining type of amphioxus was ZW.We screened for a gene that was specifically expressed in the testis.The conserved sex-linked region genes and vertebrate sex-related genes were analysed to clarify that amphioxus and vertebrates have evolved their own sex-determining pathways. |
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