新疆沙冬青基因组调查测序与基因组大小预测

新疆沙冬青是中国荒漠地区代表性常绿阔叶植物,属于第三纪孑遗植物。其极强的逆境耐受性受到了研究者的广泛关注,但由于缺乏基因组序列,分子生物学研究水平进展缓慢。本研究对新疆沙冬青进行了基因组调查测序,共得到65 Gb大小的双端测序数据。结合基于K-mer分析和流式细胞分析的方法,预测基因组大小、杂合率和GC含量等特征,估计基因组大小为770~787 Mb。测序数据拼接构建得到contigs的N50为684 bp,总读长为0.538 Gb;进一步组装后scaffolds的N50为12.09 kb,总读长为0.602 Gb。对拼接数据进行SSR分子标记预测,共得到151858个SSR,其中二核苷酸重复单元比例最高为56.39%,在二核苷酸重复单元中,AT/TA组成形式占多数。本研究首次报道了荒漠植物新疆沙冬青的基因组特征,为后续基因组学研究提供参考。     

甘薯属耐盐植物马鞍藤基因组大小及特征分析

马鞍藤(Ipomoea pes-caprae(L.)R. Br.)是甘薯的近缘野生种之一,为热带、亚热带海滩生长的多年生藤蔓植物,作为沿海地区的园林绿化植物之一,具有较强的耐盐性。马鞍藤基因组学的研究可为耐盐甘薯种质创制提供信息参考。本试验调查了马鞍藤基因组大小和特征概况,为后续全基因组精细图谱绘制打下基础。研究以已知基因组大小的三裂叶薯(Ipomoea triloba L.)为对照,运用流式细胞术对马鞍藤基因组大小进行初步预估;使用二代高通量测序技术(Illumina Hiseq2500)对马鞍藤基因组进行survey评估,测序深度30×,利用生物信息学方法估算马鞍藤GC含量(即鸟嘌呤和胞嘧啶所占比例)、杂合率、重复序列等基因组概况。结果表明:流式细胞术估算马鞍藤基因组大小为1012.704±17.37 Mb;经全基因组Survey测定获得马鞍藤有效数据为21.71 Gb,基因组大小经修正后估算为1041.65 Mbp;通过K-mer分布曲线估算马鞍藤基因组中重复序列所占比率为74.52%,杂合率为0.99%;经初步组装后,GC平均深度及含量分布存在异常,出现分层的现象,这可能与马鞍藤基因组杂合率较高有关。本试验首次报导甘薯属耐盐植物马鞍藤基因组大小及特征信息,为马鞍藤进一步的全基因组深度测序和甘薯耐盐基因挖掘打下基础。     

中国金缕梅科植物属分布区特点的定量研究

利用ArcGIS软件制作以县为单元的中国金缕梅科植物属的空间分布图,并从图中提取每个属的斑块个数、面积及周长,计算斑块的形状指数、最大斑块指数、Simpson均匀度指数和Shannon均匀度指数,定量分析中国金缕梅科植物各属的空间分布区特点,为金缕梅植物资源的保护、开发和利用提供依据。结果表明:(1)山铜材属、银缕梅属、四药门花属、牛鼻栓属、金缕梅属、壳菜果属均为单种属,其中前三属总面积较小且分布狭窄,后三属的总面积与分布范围均大于前三属;双花木属、山白树属、红花荷属、秀柱花属、半枫荷属、马蹄荷属、蕈树属、水丝梨属、蚊母树属、檵木属、蜡瓣花属、枫香树属均为多种属,随着属的面积增加,其属的分布区范围基本呈递增的趋势,且多种属的数量是单种属的2倍。(2)枫香树属分布区的总面积和最大斑块面积均最大,它能够整体体现出中国金缕梅科植物的空间分布和特点。(3)中国金缕梅科植物属的空间分布类型全部为间断分布,山铜材属、蚊母树属、檵木属和枫香树属呈现主分布区,牛鼻栓属、金缕梅属、壳菜果属、山白树属、半枫荷属、马蹄荷属、蕈树属、水丝梨属和蜡瓣花属呈主次分布区,银缕梅属、四药门花属、双花木属、红花荷属和秀柱花属呈星散分布。     

银缕梅属花形态及其分类学意义

以1995年在江苏宜兴发现的一较大银缕梅居群的花为材料,观察确认金缕梅亚科单种属银缕梅属（ShaniodendronM.B.Deng,H．T．WeietX．QWang）的花序为近头状短稳状花序,由4～7朵花组成,花序内轮4～5朵花,两性;外轮1～2朵花,常为雄花,构成雄全同株。花无柄,无花瓣,花喜常合成浅林状,杯缘及杯背早期簇生长硬毛（hirsute）,花生于初生苞片腋处,初生苞片卵形或阔卵形。雄蕊不定数,5～15枚,花丝长,直立。与其他无花瓣属植物比较表明,银缕梅属与特产里海南岸的Parrotia形态极为相似,主要区别在于本属花萼合生成浅怀状。银缕梅属花形态特征的阐明,对探讨金缕梅亚科无花瓣类群的系统发育具有重要意义。     

宽边黄粉蝶(Eurema hecabe)基因组Survey分析

宽边黄粉蝶(Eurema hecabe)是重要的传粉昆虫,广布于非洲热带区、东洋区、澳洲区及古北区东部,具有较高的学术价值和经济意义。为了深入研究其广泛适应性,确定适合该蝴蝶的全基因组的测序研究策略,我们首先做低覆盖度的基因组Survey测序,然后做大规模的全基因组深度测序。采用第二代高通量的测序技术作为该研究的研究方法,测定了宽边黄粉蝶基因组大小,并利用生物信息学方法估计该种的杂合率、重复序列和GC含量等基因组信息。结果表明:(1)宽边黄粉蝶的基因组大小估计为285.34 Mb,测序深度51×;(2)从K-mer分布曲线发现黄粉蝶基因组有明显的杂合峰,杂合率达1.97%,重复序列比例为35.37%。该研究结果对于揭示宽边黄粉蝶物种的起源和进化及适应性具有重要意义,为宽边黄粉蝶选择全基因组测序策略提供依据。     

蒙古沙冬青基因组调查及SSR分析

蒙古沙冬青是第三纪冰川孑遗物种,具有极强的耐逆性,是研究植物耐逆分子机制的良好材料。利用高通量测序技术和生物信息学工具预测蒙古沙冬青的基因组大小及杂合度,进行SSR分子遗传标记的初步鉴定。蒙古沙冬青基因组大小约为812 Mb,杂合率为0.506%,基因组杂合度较高。对基因组进行拼接,进行SSR分子遗传标记分析,共鉴定183 102个SSR,不同类型核苷酸重复差异较大,其中,二核苷酸重复中的AT/TA含量最高,四核苷酸重复最少,共占总数的1.48%。     

罗汉果全基因组Survey分析

罗汉果是广西特有药用及甜料植物,其主要成分之一甜苷V作为天然、非糖甜味剂,具有广阔的开发前景,但罗汉果目前完全来自于栽培,适生区狭窄,连作障碍严重,加之含量低导致甜苷V生产成本居高不下,严重限制了其应用。为了减少盲目性,在大规模全基因组深度测序之前,先做低覆盖度的基因组Survey测序,评价基因组的大小及复杂程度,以确定适合该植物全基因组的测序研究策略。该研究采用第二代高通量测序技术(Illumina Hiseq TM 2000)首次测定了罗汉果基因组大小,并利用生物信息学方法估计罗汉果杂合率、重复序列和GC含量等基因组信息。结果表明:(1)获得了18.1 Gb罗汉果基因组测序数据,基因组大小估计为344.95 Mb左右,测序深度为52×;(2)从K-mer分布曲线发现罗汉果基因组有明显的杂合峰,杂合率达1.5%,基因组高杂合导致组装的结果中Contig N50和Scaffold N50的长度比预期的要短很多,还造成GC平均深度及含量分布明显异常,存在一个低深度分布区域。基因组主峰后面有微弱的重复峰,说明罗汉果存在较多的重复序列;(3)由于罗汉果存在高杂合率和重复序列较多的特点,该基因组测序分析仅采用全基因组鸟枪法(WGS)策略不合适,为了更好地对全基因组进行序列拼接和组装,可尝试结合采用Fosmid-to-Fosmid或BAC-to-BAC策略。该研究结果对于揭示罗汉果产量、有效成分含量、发育及抗病虫的分子机制,以及通过分子育种来提高甜苷V含量和降低生产成本具有重要意义,为全基因组测序策略的选择提供了依据。     

金缕梅亚科ITS序列分析及其系统学意义

根据金缕梅亚科22属（活塞花属Embolanthera除外）代表种的nrDNA ITS序列数据构建了分子系统树。结合形态解剖证据,金缕梅亚科可分为3个族,即①Ji木族LoropetaleaeZhangtrib.nov.,包括蜡瓣花属Corylopsis、Maingaya、Matudaea、活塞花属、四药门花属Tetrathyrium和Ji木属L;②DicorypheaeZhangtrib.nov.,包括毛枝花属Trichocladus、Dicoryphe、Neostrearia、Ostrearia、Noahdendron、秀柱花属Eustigma、牛鼻栓属Fortunearia、山白树属Sinowilsonia、Molinadendron;③金缕梅族Hamamelideae,包括Fothergilla、金缕梅属Hamamelis、Parrotiopsis、水丝利属Sycopsis、Parrotia、银缕梅属Shaniodendron、蚊母树属Distylium和拟母树属Distyliopsis。     

樟树全基因组调查

樟树是我国特有的珍贵用材和经济树种,富含多糖多酚、萜类等次生代谢物质,是香精香料、油脂化工和医药等的重要原料树种。本研究采用高通量测序技术(Illumina HiseqTM2000)首次测定了樟树基因组大小,并利用生物信息方法估计樟树杂合率、重复序列情况和GC含量等基因组信息,为全基因组测序策略的选择提供依据。主要结论如下:(1)樟树基因组大小粗略估计为760 Mb左右;(2)樟树基因组有较高的杂合率和一定的重复,杂合率约为0.65%;(3)由于樟树杂合率较高,全基因组鸟枪法策略不适合该基因组测序分析,可尝试使用BAC-to-BAC策略或fosmid策略,有利于樟树基因组的序列拼接和组装。     

重庆金佛山自然保护区种子植物区系新资料

报道重庆市南川金佛山自然保护区新记录植物10种,隶属8科9属,其中新分布属6个,新分布科1个。它们分别为樟科的木姜叶润楠Machilus litseifolia S.Lee、金缕梅科的小叶蚊母Distylium buxifolium(Hance)Merr.和小叶银缕梅Parrotia subaequalis(H.T.Chang)R.M.Hao et H.T.Wei、千屈菜科水苋菜Ammannia baccifera Linn.和多花水苋菜Ammannia multiflora Roxb.、桃金娘科的赤楠Syzygium buxifolium Hook.et Arn.、鹿蹄草科喜冬草Chimaphila japonica Miq.、狸藻科的高山捕虫堇Pinguicula alpina L.、菊科的异芒菊Blainvillea acmella(L.)Philipson和毛瓣杓兰Cypripedium fargesii Franch.。     

浙江龙王山银缕梅种群结构和分布格局研究

银缕梅为我国特有珍稀树种,属于国家I级保护植物。本文采用相邻格子样方法和典型样地法研究浙江龙王山自然保护区银缕梅的种群结构和分布格局。结果表明：龙王山银缕梅种群规模偏小,但是其萌生特性明显;其种群共有6个龄级,但幼苗、幼树较少,中龄级的个体偏多,种群更新能力弱,种群结构总体上呈现衰退趋势。在龙王山3个地点的银缕梅种群主要呈集群分布,这与它们生长的生境、繁殖方式密切相关。此外,对该种的生态保护提出了具体建议。     

Genome survey and microsatellite motif identification of Pogonophryne albipinna

The genus Pogonophryne is a speciose group that includes 28 species inhabiting the coastal or deep waters of the Antarctic Southern Ocean. The genus has been divided into five species groups, among which the P. albipinna group is the most deep-living group and is characterized by a lack of spots on the top of the head. Here, we carried out genome survey sequencing of P. albipinna using the Illumina HiSeq platform to estimate the genomic characteristics and identify genome-wide microsatellite motifs. The genome size was predicted to be ∼883.8 Mb by K-mer analysis (K = 25), and the heterozygosity and repeat ratio were 0.289 and 39.03%, respectively. The genome sequences were assembled into 571624 contigs, covering a total length of ∼819.3 Mb with an N50 of 2867 bp. A total of 2217422 simple sequence repeat (SSR) motifs were identified from the assembly data, and the number of repeats decreased as the length and number of repeats increased. These data will provide a useful foundation for the development of new molecular markers for the P. albipinna group as well as for further whole-genome sequencing of P. albipinna.     

崖壁植物太行菊与长裂太行菊全基因组大小及特征分析北大核心CSCD

太行菊(Opisthopappus taihangensis)、长裂太行菊(O.longilobus),为太行山特有多年生崖壁草本植物,菊科(Compositae)重要野生资源,具有较高的经济与生态价值。为确定适合两物种的全基因组测序策略,该研究利用流式细胞法和高通量测序技术,分析两物种基因组大小、杂合率、重复序列及GC含量等信息。结果表明:(1)流式细胞法估算太行菊基因组大小约为2.1 Gb,长裂太行菊基因组大小约为2.4 Gb。(2)高通量测序修正后太行菊基因组大小为3.13 Gb,重复序列比例为84.35%,杂合度为0.99%,GC含量为36.56%;长裂太行菊基因组为3.18 Gb,重复序列比例为83.83%,杂合度为1.17%,GC含量为36.62%。(3)初步组装后GC含量分布及平均深度存在异常,出现分层现象,可能是两物种基因组杂合率较高所致。综上结果表明,太行菊、长裂太行菊均属于高重复、高杂合、大基因组的复杂基因组,建议使用Illumina+PacBio测序组装策略,进行全基因组测序分析。     

Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb

Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15 % below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15 % more DNA than 2C chicken nuclei (with >2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75 % above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57 % above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.     

Assembly and RNA‐free annotation of highly heterozygous genomes: The case of the thick‐billed murre (Uria lomvia)

Thanks to a dramatic reduction in sequencing costs followed by a rapid development of bioinformatics tools, genome assembly and annotation have become accessible to many researchers in recent years. Among tetrapods, birds have genomes that display many features that facilitate their assembly and annotation, such as small genome size, low number of repeats and highly conserved genomic structure. However, we found that high genomic heterozygosity could have a great impact on the quality of the genome assembly of the thick‐billed murre (Uria lomvia), an arctic colonial seabird. In this study, we tested the performance of three genome assemblers, ray /sscape , soapdenovo 2 and platanus , in assembling the highly heterozygous genome of the thick‐billed murre. Our results show that platanus , an assembler specifically designed for heterozygous genomes, outperforms the other two approaches and produces a highly contiguous (N50 = 15.8 Mb) and complete genome assembly (93% presence of genes from the Benchmarking Universal Single Copy Ortholog [BUSCO] gene set). Additionally, we annotated the thick‐billed murre genome using a homology‐based approach that takes advantage of the genomic resources available for birds and other taxa. Our study will be useful for those researchers who are approaching assembly and annotation of highly heterozygous genomes, or genomes of species of conservation concern, and/or who have limited financial resources.     

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 ± 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa , version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.     

昆虫基因组及其大小

昆虫基因组大小是由于基因组各种重复序列在扩增、缺失和分化过程中所致的数量差异造成的。这些差异使得昆虫不同类群间、种间和同种的不同种群间表现出基因组大小的不同。目前有59种昆虫已经列入基因组测序计划, 其中6种昆虫（黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、家蚕Bombyx mori、意大利蜜蜂Apis mellifera、埃及伊蚊Aedes aegypti和赤拟谷盗Tribolium castaneum）的全基因组序列已经报道。有725种昆虫的基因组大小得到了估计, 大小在0.09~16.93 pg （88~16 558 Mb）之间。本文还介绍了昆虫基因组大小的估计方法, 讨论了昆虫基因组大小的变化及其意义。