【作者】 黄浩；

【导师】 张彦定；

【作者基本信息】 福建师范大学 ， 动物学， 2020， 博士

【摘要】 鸟类是脊椎动物里物种多样性最为丰富的类群之一。鸟类的基因组相对稳定,其染色体数目大部分是40对(2n=80)。然而鸟纲的鹦形目(Psittaciformes)中,染色体核型却呈现多样性,并且发生歧异的时间很短。由于鸟类存在大量的微染色体,其中个别微染色体又具有大量重复序列。所以,传统的测序技术对此几乎无能为力。近年来,随着三代测序技术和Hi-C辅助组装技术的发展,基因组组装可以成功提升到染色体级别,为染色体演化的研究提供了新的契机。目前鹦形目中鹦鹉科(Psittacidae)也仅有虎皮鹦鹉属(Melopsittacus)完成了染色体级别的组装,其他属的鹦鹉尚未有染色体级别基因组信息报道。和尚鹦鹉是鹦鹉科乃至鸟类中染色体数目最少的鹦鹉,本研究采集雌性和尚鹦鹉的血液,采用Pac Bio三代测序配合Illumina二代测序和Hi-C辅助组装策略,进行全基因组组装。测序得到总有效数据量80.2 Gb,subreads平均读长度15.25 Mb。通过Canu软件组装获得Contig级别基因组大小为1.2 Gb,Contig N50达到13 Mb;其次采用Hi-C数据进行染色体级别基因组的辅助组装,获得基因组大小为1.17 Gb,N50达到75.71 Mb。Hi-C热图分析和验证显示,成功获得24条染色体的superscaffolds组装图谱。基因注释结果与鸟类模式物种---原鸡(G allus gallus)接近,表明和尚鹦鹉注释信息较为完整。将和尚鹦鹉基因组数据分别在GO、KEGG、COG和egg NOG等数据库进行了功能注释。注释成功56,746个,占总基因数的97.55%,其中19,549个基因在上述四大数据库中均有注释信息,表明基因功能注释较全面。将注释得到的基因与鸟类模式物种—鸡和其他几种鸟类的转录因子(TFs)比较分析,在和尚鹦鹉基因组中鉴定到了69类,共约1472个转录因子。这些结果表明,成功获取染色体级别的高质量和尚鹦鹉基因组。青绿顶亚马逊鹦鹉是鹦鹉科中另外一个重要种属。目前已经公布有一个基因组草图,但是其未组装到染色体级别。我们对青绿顶亚马逊鹦鹉进行Hi-C文库的构建和测序,对之前的基因组进行了Hi-C基因组辅助组装。使Scaffold N50由原来的12.81 Mb提高到89.04 Mb,提升了6.95倍,并且所有的scaffold都被挂载到35条superscaffold。基因组结构注释结果显示有16,144个可编码蛋白基因,其中有15,433个都能获得功能注释信息,占全部预测基因数的95.60%。尽管鹦鹉的系统发育在近二十年得到广泛的研究,但是其中许多属种的演化关系至今尚不清楚。利用组装成功的和尚鹦鹉与青绿顶亚马逊鹦鹉的基因组数据,结合已发表的3种鹦鹉全基因组序列,对鹦鹉科进化树进行构建,确立了和尚鹦鹉和青绿顶亚马逊鹦鹉的分类地位。进化树显示和尚鹦鹉和青绿顶亚马逊鹦鹉与啄羊鹦鹉、虎皮鹦鹉和太阳锥尾鹦鹉歧异时间在37.2 MYA、26.4 MYA和20.8 MYA,和尚鹦鹉和青绿顶亚马逊鹦鹉彼此歧异时间在19.9 MYA。该系统进化树符合鹦鹉科系统发育的主流理论。本研究比较了具有染色体级别基因组组装的3种鹦鹉与4种外群鸟类的染色体的共线性。观察到在和尚鹦鹉、青绿顶亚马逊鹦鹉和虎皮鹦鹉等3种鹦鹉的基因组中,有3个染色体融合事件是一致的。同时,12个染色体在3种鹦鹉之间发生了独立的染色体重排事件。这显示积极活跃的染色体重排是鹦鹉所特有,并且染色体重排的发生在不同鹦鹉种间具有高度多样性。对鹦鹉科的5个种属鹦鹉进行的CR1(Chicken Repeat 1)序列分析表明,鹦鹉科中CR1数目在全基因组中所占比例高于其他鸟类,但与原鸡接近。我们进而在鹦鹉科鸟类中发现了一个新的CR1元件亚家族,并将其命名为CR1-psi。CR1-psi元件在鹦鹉类所有CR1元件中占50%左右。这表明鹦鹉科在基因组变异上较为活跃。对鹦鹉科的种属进行了基因家族扩张和收缩分析,发现与外群的物种相比,鹦鹉科并没有发生特异性的基因家族扩张现象,但是却有74个基因存特异性丢失的情况。对这些丢失的基因进行GO分析和KEGG分析,没有显著的基因功能注释。从这些基因中发现了2个功能保守的基因CHD1L和PARP3,且这2个基因均与DNA损伤修复和基因组稳定性密切相关。这两个基因的发现,表明保守基因的丢失可能是鹦鹉核型的快速演变的遗传学机制之一。鸟类的性染色体类型为保守的Z/W系统,且其中绝大多数类群中的W染色体已经严重退化。新-性染色体就成为研究鸟类性染色体退化的过程与机制的理想材料。鹦鹉科的性染色体演化情况至今尚不明确。通过鹦鹉性染色体的共线性分析,发现在所有鹦鹉中Chr11的整段序列与Chr Z/Chr W发生融合演化形成新-性染色体。和尚鹦鹉中在Chr11融合插入之后,又可见一个极小的微染色体Chr25被插入融合到性染色体中。这个新-性染色体形成在与青绿顶亚马逊鹦鹉演化歧异的19 MYA之后,说明它是独立且在较近时期发生的。此外,和尚鹦鹉的新-性染色体中插入的Chr11片断已经充分退化,而插入的Chr25片断还未开始退化,这可能成为研究鹦鹉及鸟类性染色体演化进程的新模型。我们继而检测了组装后的Chr Z和Chr W之间的序列相似性。结果显示,在鹦鹉的性染色体重组停止后形成了多个的“演化断层”。S0是所有鸟类共享的演化断层,S1-S3存在于绝大多数今颚总目(Neognathae)鸟类,多数鹦鹉物种中形成了起源于鹦鹉共同祖先的S4演化断层。S4演化断层完全来源于Chr11,由Chr11起源的新-W染色体上保留了约16.9%的原始基因含量,而鸟类共有的旧Chr W仅保留2.6%。在和尚鹦鹉中出现了由Chr25融合形成的新-性染色体。但由于Z-W的分化尚未开始,由Chr25衍生的Z-W新-性染色体差异度极小,因此无法单独组装其Z和W染色体序列。分析S4断层中的转座子序列,发现与Chr W连锁的LTR转座子含量占比已经从约51.99%增加到76.79%。表明LTR可在S4这样的年轻断层中快速传播,而CR1在此断层中的插入则较缓慢;提示Chr W退化早期能有效地选择性抵抗CR1的插入。对鹦鹉雌性特异的neo-W染色体S4断层进行转录组分析。结果表明,Chr W的S4断层上幸存的连锁基因均起源自Z连锁基因,且相对于Chr Z连锁基因来说呈现整体表达下调的情况。仅发现NUTF2、MIER3和PCGF3这3个基因出现了上调。根据以上结果,本研究绘制了和尚鹦鹉新-性染色体演化路线图,诠释了鸟类主要类群中各演化断层的分布和发展情况。综上所述,本研究首次获得了染色体级别的高质量和尚鹦鹉基因组组装和注释。通过对鹦形目的比较基因组学和系统发育研究确定了和尚鹦鹉和青绿顶亚马逊鹦鹉的演化歧异时间,同时发现积极活跃的染色体重排是鹦鹉特有的,并且在不同鹦鹉种间发生的染色体重排具有高度多样性。发现了鹦鹉科新-性染色体的演化断层以及和尚鹦鹉新-性染色体中独特的演化断层,并对鹦鹉的新-性染色体的演化进程进行了解析。这些研究成果将为鸟类多样性、性染色体演化进程和核型演化的研究提供数据基础和新的研究模型。更多还原

【Abstract】 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.更多还原