| Aves are one of the most divers e groups of vertebrates.The genome s of birds are relatively stable,with most of their chromosomes having 40 pairs(2n=80).However,drastic diversity of the karyotypes appear s in the Psittaciformes,within a recent period of divergence.B irds have a large n umber of micro-chromosomes and high proportion of repetitive sequences in some of the micro-chromosomes.Therefore,conventional sequencing technologies have limited power in assembling the complete sequence of the genomes.In recent years,with the developmen t of third-generation sequencing technology and Hi-C assisted assembly technolog y,the genome can be successfully upgraded to the chromosome level,providing a new opportunity for the research of karyotype evolution.Currently only Melopsittacus undulatus in the Psittacidae of Psittaciformes has a chromosome-level assembly,and the chromosome-level genome information has been unavailable for an y other parrot.The My M is the parrot with the least number of chromosomes in Psittacidae even in Aves,Collected the blood of a female My M,and performed the third-generation sequencing(TGS)on the Pac Bio platform and also combined with the next-generation sequencing(NGS)on the Illumina platform and H i-C assisted genome assembly strategies to acquire the My M genome informa tion.The total valid data acquired by the TGS sequencing is 80.2 Gb,and the N50 of subreads is 20.01 Mb.Contig level assembly was carried out by Canu,and the genom e size of the contig was 1.2 Gb,and the N50 of contig reached 13 Mb.T hen,used H i-C to assist the scaffolding,and the genome size was1.17 Gb,and the N50 reached 75.71 Mb.Through Hi-C contact map,the contigs were successfully clustered into 24 superscaffolds assembly map.The gene annotations have shown that the number of annotated genes is close to that of the avian model species —Gallus ga llu s,indicating that My M annotation information is almost complete.My M genome data were annotated forfunctional terms with GO,KEGG,COG and egg NOG databases.The results sh owed that 56,746 were successfully annotated,accounting for97.55% of the total number of gene models.There were 19,549 genes with annotation information in the above four databases.B y comparing and analyzing the transcription factors(T Fs)of Gallus gallus and several other birds,69 types of TE s in the My M genome have been identified,with a total of about 1,472.These results show that the chromosome-level and high-quality genome sequence information of My M have been successfully acquired.The A m A is ano ther important species in the Parrot order.A draft genome of A m A has been published,but it has not been assembled to the chromosome level.A library of H iC sequencing was constructed for an A m A.After sequencing,the H i-C assisted assembly based on the previous draft genome has been performed.Finally,we have acquired high-quality genome sequence information of A m A successfull y at the chromosome-level.Scaffold N50 was increased from 12.81 Mb to 89.04 Mb,an increased of 6.95 times.All scaffolds were mount ed on 35 superscaffolds.The genome annotation results showed that there were 16,144 protein-encoding genes and 15,433 of those can be annotated,accounting for 95.60% of all predicted genes.Although the phylogen y of parrots has been extensively studied in the past two decades,the evolutionary relationship of many genera and species of Psittacidae is still unclear.We u sed the genome data of the successfully assembled M y M and Am A,combined with the published genome sequences of the three parrots,to reconstruc t the phylogenetic tree of parrots,and also determined phylogenetic positions of My M and Am A.The ph ylogenetic tree shows that M y M and A m A diverged from Nestor nota bilis,Me lop sitta cus undu la tus and A ra tinga solstitia lis at 37.2 MYA,26.4 MYA and20.8 MYA,respectivel y,and My M and A m A diverged 19.9 MYA.This phylogenetic tree agrees with the mainstream theor ies of Psittacidae phylogenetics.Then,the collinearity of parrot genomes and other bird genomes has been compared.In the genomes of the My M,the A m A,a nd the budgerigar,there are 3 chromosomal fusion events that are shared by all of them.Moreover,12 chromosomes had independent chromosomal rearrangements b etween the 3 species of parrots.This shows that active chromosomal rearrangements are unique to parr ots,and there are highly diverse among the parrots.It has been performed that CR1(Chicken Repeat 1)sequence analysis on 5 species of parrots in the psittacidae,and found that the proportion of CR1 in the Psittacidae in the whole genome is higher than oth er birds,but close to the Gallus gallu.A novel CR1 in parrots has been identified,named CR1-psi,accounting for about half of the CR1 content in parrots.This indicates that Psittacidae was m ore active in genome variations.Analysis of expansion s and contraction s of gene families across bird species showed that there was no specific gene family expansion in parrots,but there are 74 genes with specific loss in parrots.However,GO analysis and KEGG analysis of the se lost genes using the Gallus gallu s orthologous genes did not show significant functional enrichment.B y comparative anal ysis on the synten y these genes,2 conserved genes CHD1 L and PARP3 were found to be lost in parrots,both of which are closely related to DNA damage repair and genome stabil ity.T he discovery of these two genes indicates that the loss of conserved genes ma y be one of the genetic mechanisms of the rapid evolution of parrot karyotypes.The sex chromosome type of birds is the conservative Z/W system,and the W chromosomes in most groups of birds have been degenerated,Neo-sex chromosomes have become ideal materials for studying the process and mechanism of bird sex chromosomes degradation.Analyzed the collinearity of sex chromosomes and found that the fusion between the entire sequence of C hr11 and Chr Z/Chr W have been fused,leading to the formation of a neo-sex chromosome in all parrots.Following the fusion of Chr11 in My M,a mi cro-chromosome---Chr25 was fused into the sex chromosome s.This neo-sex chromosome appeared within 19 MYA after the divergence from A m A,indicating independent and recent formation of neo-sex chromosome.In addition,the fused fragment of Chr11 in the neo-sex chromosome of parrots has been full y degraded,but the inserted fragment of Chr25 in M y M has not yet begun the degenerat ion process.This ma y become a new model for the study of the evolution ary process of the sex chromosomes of parrots and birds.Then,we analyzed the sequence similarity between the assembled Chr Z and Chr W.The results showed that after the stop of recombination between the sex chromosomes in parrots,multiple "evolutionary strata " were formed.Stratum S0 is an evolutionar y strata shared by all birds;S1-S3 exist in most birds of the Neognathae;most the stratum S4 originated at the common ances tor of the parrot s.S tratum S4 is entirely derived from Chr11 and retains about 16.9% of the original gene content on the ne o-W chromosome,while the evolutionary strata shared by birds only retains 2.6 %;In the My M,the unique neo-sex chromosomes appear ed b y the fusion of C hr25.Since the differentiation of Z-W has not yet begun,there were almost no difference between the neo-Z chromosome and neo-W chromosome that derived from Chr25.S o we were unable to assemble the neo-Z or neo-W chromosome sequences separat el y.Analyzed the transposon sequence in the S4 stratum and found that the content of Chr W-linked transposon LTR has increased from about 51.99 % to 76.79%.It demonstrated that LTR can spread rapidl y in young stratum like S4,C onversel y,the insertion of CR1 in this stratum was slow.It can effectivel y and selectively resist the insertion of CR1 in the early stage of Chr W degradation.Transcriptome analysis was further done for the S4 strata of the female-specific neo-W chromosome of the parrot.The results showed that the surviving linked genes on the S4 strata of Chr W all derived from Chr Z-linked genes.The overall expression was down-regulated compared to Chr Z-linked genes while only three genes NUTF2,MIER3 and PCGF3 were up-regulated.According to the above results,a new-sex chromosome evolution roadmap for Myiopsitta monach us was drawn based on the research results,which explained the distribution and development of the evolutionary strata in the main groups of birds.In summary,the high-quality genome assembly and annotation of Myiopsitta monachus have been successfull y acquired at the chromosome level for the first time;With co mparative genomics and ph ylogenetic studies of the parrots,the divergence time between the Myiopsitta monachus and the Amazona aestiva has been determined.It was found that active chromosomal rearrangements are unique to parrots,and t he chromosomal rearrangements that occur between different parrot species are highly diverse.In this paper,we reported the evolutionary strata in the neo-sex chromosomes of P sittacidae and the unique evolutionar y strata in the neo-sex chromosomes of the My M.The evolution process of the neo-sex chromosome of the parrot was also analyzed.These results will provide a da ta basis for the study of the diversity,the evolution of sex chromosomes and the mechanism of sex determination of Aves. |
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