| As the only extant representatives in the subphylum Cephalochordata of chordates,amphioxi(including the genera Branchiostoma,Asymmetron and Epigonichthys)has many characteristics of invertebrates and vertebrates,which makes it being the “living specimen” and“living fossil” for studying the evolution of invertebrates and the origin of vertebrates.The systematic and in-depth studies of the amphioxus genome are of great significance for understanding the origin and evolution of species.With the released draft genomes of Branchiostoma floridae(B.floridae)in 2008 and Branchiostoma belcheri(B.belcheri)in 2014,researches on the cloning and expression of amphioxus functional genes,and the origin of the immune system have gradually increased.However,due to the limitations of sequencing technology,the previously released genomes have many problems,such as poor sequence continuity,poor assembly of repetitive sequence regions and many gaps,which greatly restricts subsequent genomics,transcriptome and other multi-omics researches.In this study,we assembled the chromosome-level reference genomes of B.belcheri,Branchiostoma japonicum(B.japonicum)and B.floridae using Pac Bio and Hi-C sequencing technologies.Using the newly assembled genomes as reference genomes,we analyzed the chromosomal evolution of Branchiostoma,and employed a whole-genome resequencing strategy to detect the population variation and population historic dynamics of Branchiostoma.The main research results and conclusions are as follows:(1)Chromosome-level genome assembly of B.belcheri,B.japonicum and B.floridae.This study used Pac Bio and Hi-C to assemble a chromosome-level genome of B.japonicum for the first time.The genome was assembled into 385.7 Mb in length,and the N50 of contigs and scaffolds are 4.43 Mb and 19 Mb,respectively.We anchored 97.3% of the genome sequences onto 18 chromosomes,and 98.8% of the BUSCO complete genes were identified in this genome.Additionally,we improved the B.belcheri genome from scaffold-level to chromosome-level,and the upgraded B.belcheri genome was 410.8 Mb in length,with a contig N50 of 2.65 Mb,and a scaffold N50 of 19.9 Mb.We anchored 96.8% of the genome sequences onto 20 chromosomes,and 98.6% of the BUSCO complete genes were identified in this genome.The genome continuity(contig N50)is nearly 60 times higher than that of the previously released genome in 2014(Branchiostoma belcheri v18h27).We also improved the B.floridae genome from scaffold-level to chromosome-level,and the upgraded B.floridae genome was 497.1 Mb in length,with a contig N50 of 1.69 Mb,and a scaffold N50 of 23.9 Mb.We anchored 95.8%of the genome sequences onto 19 chromosomes,and 99.3% of the BUSCO complete genes were identified in this genome.The genome continuity(contig N50)is nearly 32 times higher than that of the previously released genome in 2020(Bfl?VNyy K).Besides,the upgraded genomes have significantly improved in terms of gene annotation,repetitive sequence assembly,and the proportion of gaps.The upgraded genomes at chromosome-levels provide valuable resources for studying the evolution of amphioxus,the evolvement and invertebrates,and the origin of vertebrates,and can be served as reference genmones with more precision for immunology and omics researches.(2)Genomic reasearch on species divergence of amphioxus.Based on the intraspecific synteny analysis of three Branchiostoma species,we found that the genome of Branchiostoma has not experienced any large-scale genome duplication,gene loss,gene acceleration,and large-scale changes in hundreds of millions of years,and it retains many original characteristics of the ancestral genome.Based on the interspecific synteny analysis of three Branchiostoma species and the synteny analysis between B.belcheri and chicken,we found that the chordate ancestors(2n=34)experienced three chromosomal breakage events in the process of differentiating into amphioxus ancestors(2n=40).Subsequently,one chromosome fusion occurred in the ancestral genome of amphioxus(2n=40)when it evolved into B.floridae(2n=38)at 112 Mya,and two chromosomal fusions occurred when it evolved into B.japonicum(2n=36)at 80 Mya,while B.belcheri retains 20 chromosomes of its ancestors.So,B.belcheri can be used as an ideal model organism for studying the evolution of invertebrates and the origin of vertebrates.(3)Population variation detection of B.belcheri,B.japonicum and B.floridae.Using the assembled genomes as the reference,we performed a whole-genome resequencing analysis for57 Branchiostoma species,including 2 B.belcheri populations,2 B.japonicum populations,and 1 B.floridae population.Additionally,we also downloaded 10 resequencing data of Branchiostoma species from NCBI.With a total of 67 resequencing data,we generated the population variation maps of three Branchiostoma species.We detected 30.76 million SNPs and 7.59 million Indels in the B.belcheri population,31.76 million SNPs and 7.93 million Indels in the B.japonicum population,and 26.86 million SNPs and 6.37 million Indels in the B.floridae population.(4)Functional annotation of variations in intracellular digestion-associated genes of Branchiostoma.The epithelial cells of the Branchiostoma diverticulum can directly phagocytize and effectively digest,absorb and utilize algal cells and other tiny food particles.This study identified and annotated 210 non-synonymous SNPs in intracellular digestionassociated genes in Branchiostoma,of which 129 belong to rare mutation,providing important gene editing sites to solve algae pollution through genetic engineering.Besides,we found that the highly expressed intracellular digestion-associated immune genes contained more nonsynonymous SNPs than the highly expressed digestive and hydrolytic genes,which indicates that the epithelial cells of the Branchiostoma diverticulum need more variations in immune genes to adapt to a large number of microbes in food when digesting food particles.(5)Inference of demographic histories of B.belcheri,B.japonicum and B.floridae populations during the Pleistocene.By analyzing the impact of different sequencing depths on the PSMC results,it was found that the best threshold of sequencing depth for the PSMC analysis of Branchiostoma and other highly heterozygous species was 25×.The Pleistocene population dynamics of three Branchiostoma populations are slightly different due to the different sea temperatures required for reproduction,but in general,the historical population of Branchiostoma first experienced a long-term population expansion during the early Pleistocene,and then gradually contracted in the middle of the Pleistocene,and finally formed a population bottleneck in the last glacial maximum about 20,000 to 30,000 years ago.Besides,we found that the Xiamen and Zhanjiang B.belcheri populations most probably started to diverge after the first glaciation of the Pleistocene,while the Japan and Xiamen B.japonicum diverged about400,000 years ago.The research on the demographic histories of Branchiostoma populations not only provides an important reference for the PSMC analysis of other invertebrates and highly heterozygous species,but also helps us understand the adaptability of Branchiostoma under different climatic fluctuations and reminds us to pay more attention to protecting the habitat of Branchiostoma. |
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