【作者】 汪涛；

【导师】 胡中立；

【作者基本信息】 武汉大学 ， 遗传学， 2022， 硕士

【摘要】 莲(Nelumbo Adans.),起源最早的双子叶植物之一,兼具双子叶植物和单子叶植物的特性。目前,莲仅有两个亚种:亚洲莲(Nelumbo nucifera)和美国莲(Nelumbo lutea)。莲在中国有超过7,000年的栽培历史。莲极具观赏、食用和药用价值。莲花多姿多彩;莲籽、莲藕富含淀粉,香甜可口;莲各部位皆可入药。自从莲基因组得以成功测序以来,莲的组学研究,包括基因组、转录组、甲基组和蛋白组等蓬勃发展,极大地促进了莲的遗传学基础研究,同时也为莲的分子育种提供基础数据。以上研究大多是基于单个参考基因组开展的,但单个基因组并不能代表这个物种所有的遗传多样性,倘若参考基因组上没有样本基因组上的序列,那样本的变异信息就很难被检测。目前已公布的莲的参考基因组均属于中国地区的莲,缺少东南亚、南亚地区以及美洲地区的莲。因此,本研究基于469份来自世界各地不同地区不同品种的莲材料构建了第一个莲泛基因组,以代表莲所有的遗传信息。莲的泛基因组大小一共1,251 Mb,包含了38,656个蛋白编码基因。基于莲泛基因组,对莲驯化和改良过程中的受选择基因进行鉴定并解析莲重要的园艺性状(花色和花型)及生物学适应性。具体研究内容如下:1.利用从公共数据库(NCBI和CNGB)下载的469份约3,447.63 Gb莲全基因组测序数据,包括52份花莲、178份藕莲、55份籽莲、91份亚洲野莲、86份亚洲栽培莲和7份美国莲,采用“map to pan”策略将莲二代测序数据比对到“太空莲3号”参考基因组上构建莲泛基因组。最终,莲泛基因组序列大小为1,251 Mb,包括807 Mb的参考基因组序列和444.67 Mb的非参考基因组序列,泛基因集大小为38,656,包括28,274个参考基因组基因和10,382个非参考基因组基因。根据基因的存在频率,将基因分为四类:Core基因约占56.03%、Softcore基因约占17.54%、Shell基因约占17.59%和Cloud基因约占8.84%。使用基因存在或缺失变异(presence/absence variation,PAV)分析,在花莲、藕莲以及籽莲群体中分别鉴定到了602、1,637和2,094个在驯化与改良过程中的受选择基因。2.基于莲泛基因组,比较了美国莲与亚洲野莲的基因存在频率差异,鉴定到了11,046个PAV相关的差异基因。之后,对这些差异基因进行KEGG富集分析发现它们富集在类黄酮物质合成及相关通路上。莲中红色、粉色花朵的形成主要是和花青素途径基因有关,而黄色花朵的形成机理尚未明确。通过检测了类黄酮合成途径及花青素途径,发现一个查尔酮还原酶(chalcone reductase,CHR)基因存在于所有美国莲样本中而在所有亚洲野莲样本中几乎不存在并且这个基因的催化产物为黄色的类黄酮物质。因此,这个基因可能是美国莲黄色花朵形成的关键基因。另外,结合基于单核苷酸多态性(single nucleotide polymorphism,SNP)的全基因组关联分析(SNP-genome wide association study,SNP-GWAS)、基于PAV的全基因组关联分析(PAV-GWAS)和转录组测序在莲中鉴定到了两个SEPALLATA(SEP)基因,分别是SEP1(Nnuci＿00651)和SEP3(OF17463),这两个基因可能是莲重瓣花形成的关键基因。3.通过改良传统的比较基因组学分析方法使之能够应用于泛基因组,改良后的方法称为“比较泛基因组学分析”。基于莲、拟南芥、水稻、番茄、苹果和向日葵的泛基因组基因集,采用比较泛基因组学分析方法探究基因家族的扩张和收缩类型。莲的38,656个基因聚成了15,317个基因家族,包括5,845个扩张家族、11,395个收缩家族及50个快速进化基因家族。此外,与其它陆生植物相比,还发现莲在活性氧产生与清除通路、角质层蜡质合成通路及淀粉合成通路上的一些基因家族发生了明显地扩张现象。这些基因家族可能与莲的适应性进化有关,帮助莲更好地适应水生环境。更多还原

【Abstract】 Lotus(Nelumbo Adans.),one of the earliest eudicots,exhibits some characteristics of dicotyledons and monocotyledons.At present,there are only two species in Nelumbo:Asain lotus(Nelumbo nucifera)and American lotus(Nelumbo lutea).Lotus cultivated in China with a history of more than 7,000 years.It’s beautiful and colorful flowers,sweet and delicious seeds and rhizomes,and medicinal leaves attach important ornamental,edible and medical values on lotus.Since the genome of lotus was successfully sequenced in 2013,many studies about genome,transcriptome,proteome and methylome have been successfully conducted,greatly promoting genetic discoveries of lotus and providing valuable data for molecular breeding.Notably,these studies above are conducted based on single reference genome while single reference genome cannot represent the full range of genetic diversity of this species.It is very possible that the genomic variations of those accessions whose genome sequence information are absent from reference genome are hard to be identified.Up to now,all published genome sequences of lotus belong to accessions from China without those from America,South Asia and Southeast Asia.Therefore,using genome sequences of 469 phylogenetically and geographically representative lotus accessions,we constructed the lotus pan-genome which represent the full range of genetic diversity of lotus.The total size of lotus pan-genome sequence is1,251 Mb,containing 38,656 protein-coding genes.Further,based on it,we identified the selected genes during lotus domestication and improvement,and explored the genetic mechanism of some important horticultural traits and unique biological adaptation to water environments.These studies are detailedly described as following:1.Based on a total of～3,447.63 Gb whole genome sequencing data of 469 lotus accessions downloaded from public database,including 52 flower lotus,178 rhizome lotus,55 seed lotus,91 wild Asian lotus,86 cultivated lotus and 7 American lotus accessions,we constructed the lotus pan-genome using a“map to pan”strategy through mapping the genome sequence of each accession to“Taikonglian No.3”reference genome.As a result,the lotus pan-genome,including reference and nonreference genome sequence,had a total size of～1,251 Mb(～807 Mb of the‘Taikonglian No.3’reference genome)and contained 38,656 protein-coding genes(28,274 in the‘Taikonglian No.3’reference genome).Further,according to the presence frequency of genes in all accessions,we categorized them into four classes:21,658(56.03%)core genes shared by all detected accessions,6,781(17.54%)softcore genes present in more than 95%accessions,6,798(17.59%)shell genes present in 5%-95%accessions and 3,419(8.84%)cloud genes present in less than 5% accessions.Using gene presence/absence variation(PAV)analysis,we respectively identified 602,1,637 and 2,094 selected genes in flower lotus,rhizome lotus and seed lotus groups.2.By comparing the presence frequency of each gene in wild Asian lotus and American lotus accessions,we identified a total of 11,046 PAV-related different genes.The KEGG enrichment analysis of these genes suggested that they mainly involved in“flavonoids biosynthesis”and its related pathways.In lotus,the red and pink flowers of Asian lotus are closely associated with genes in anthocyanin biosynthesis pathway while the genetic mechanism of yellow flower of American lotus remains unclear.Then,we detected the presence difference between wild Asian lotus and American lotus of those genes in flavonoids biosynthesis pathway and found a chalcone reductase(CHR)gene which was absent in almost all wild Asian lotus samples but present in all American lotus samples.Therefore,this gene may be the key factor which contributes to the yellow petals in American lotus.In addition,by integrating SNP-based genome wide association study(SNP-GWAS),PAV-GWAS and transcriptomic analysis,we identified two SEPALLATA gene(Nnuci＿00651 and OF17463)which might play important roles in biological process associated with double petals in lotus.3.We improved the traditional comparative genomics analyses to make it suitable for pan-genome and named it“comparative pan-genomics analyses”.Using this method,we explored the genome family evolution of six plants,including lotus,apple,rice,Arabidopsis thaliana,sunflower and tomato.In lotus,38,656 protein-coding genes were clustered into 15,317 orthologous groups,including 5,845 expanded gene families,11,395 extracted gene families and 50 rapidly evolving gene families.In addition,we find that some gene families of lotus in starch synthesis,biosynthesis of cuticular wax and reactive oxygen species(ROS)scavenging pathways expand significantly,suggesting that these expanded gene families may be related to adaptative evolution and make it better for lotus to survive in aquatic environments.更多还原