基于Solexa高通量测序的唐古特红景天(Rhodiola algida)微卫星信息分析

利用所获得的Solexa高通量唐古特红景天转录组拼接EST序列进行微卫星位点的挖掘分析,期望为红景天属SSR标记的开发提供生物信息学依据。在得到的6552条EST序列中,三碱基最多,占总EST序列的41.50%;单核苷酸和二核苷酸重复类型的SSR含量相似,分别为27.76%和24.76%;二至六碱基微卫星分布密度与其对应的SSR含量成正比。在单核苷酸重复类型中,T和A重复类型最多,分别为总SSR的14.91%、12.70%,而G和C重复类型则很少;在二核苷酸重复类型中,AG重复类型最多,占总SSR的5.60%,GA和TC重复类型次之,分别为4.75%、4.72%;在三核苷酸重复类型中,GAA重复类型最多,为总SSR的1.85%,GAT次之,为1.79%,TTC、TCT、TCA、GGA、GCT、GAG重复类型间的SSR数相差不大;四、五、六核苷酸重复类型则很少。除五、六核苷酸重复类型外,其长度变化与其对应的重复类型碱基长度成反比;同种重复类型中,微卫星的长度与其对应的SSR数成反比。     

藏羚羊基因组微卫星分析与初步验证

为明确藏羚羊Pantholops hodgsoni核DNA中微卫星分布情况和特征,利用MISA工具对藏羚羊基因组进行微卫星扫描。在全长2 696.89 Mb的藏羚羊基因组中,搜索到723 135个微卫星座位,其中完全型微卫星有675 809个。6种重复类型中,单核苷酸重复最多,有471 142个,占69.72%;其次是二核苷酸、三核苷酸,分别为88 832个和86 658个,占13.14%和12.82%;六核苷酸最少,仅215个,约占0.03%。以藏羚羊基因组DNA为模板对微卫星座位进行验证,在100个微卫星座位中筛选到8个具有多态性的微卫星座位,多态比例约为8%。本研究将为研究藏羚羊微卫星标记、群体遗传多样性、藏羚羊保护生物学提供基础。     

极度濒危植物巧家五针松基因组微卫星特征分析

巧家五针松是世界极度濒危植物,对其SSR引物的开发有助于其遗传学研究以及物种的保护。本研究通过Illumina高通量测序技术获得巧家五针松全基因组序列,并以MISA软件查找得到2 651个微卫星序列,其中单核苷酸重复最多,可能预示了其悠久的进化历史。不同重复类型中,A/T含量显著高于G/C;在不同长度重复单元中,二核苷酸重复微卫星长度变异程度最高;各重复类型微卫星长度与微卫星出现的频率成反比。获得的微卫星序列能够满足巧家五针松的种群遗传学研究,而且反映了该物种的偏好性及对应的潜在功能,并且对该物种的保护提供了资料。     

埃博拉病毒基因组中微卫星序列的分布分析

微卫星或简单重复序列(simple sequence repeat, SSR)在真核和原核生物以及病毒基因组中普遍存在,并被广泛用于遗传与进化研究。本研究从NCBI中下载埃博拉病毒属的四个不同种的埃博拉病毒全基因组序列,筛选36条作为实验材料,利用IMEx在线提取软件提取SSRs,用Python编程统计数据,从而分析SSRs在埃博拉病毒全基因组序列中的分布情况。分析得出,埃博拉病毒基因组序列中二型SSRs含量最为丰富,其次是一型SSRs,三型SSRs有少量,四型SSRs则更少,没有发现五型和六型SSRs。在更深入的分析中得出在埃博拉病毒属四个种中,含A/T碱基的SSRs含量远远大于含C/G碱基的SSRs。分析得出一型SSRs中(A)n/(T)n远多于(G)n/(C)n,二型SSRs中不存在(GC/CG)n,三型中也不存在(GGC/CGG/GCG/CCG/CGC/GCC) n。上述发现可能跟埃博拉病毒的致病机理有密切联系。通过对埃博拉病毒基因组序列中SSRs的分析,为研究埃博拉病毒的变异情况及致病机制提供更多参考。     

二斑叶螨和肩突硬蜱基因组微卫星分布规律研究

本研究利用已公布的二斑叶螨Tetranychus urticae和肩突硬蜱Ixodes scapularis的全基因组测序结果,对其基因组中的微卫星序列进行了系统分析和比较。结果表明:在二斑叶螨基因组中共找到微卫星7934个,出现频率为1/11.45kb,占全基因组碱基总数的0.16%,其中三碱基重复微卫星最多,占总数的72.83%;在肩突硬蜱基因组中共找到550629个微卫星,出现频率为1/3.21kb,占全基因组碱基总数的0.57%,其中单碱基重复微卫星最多,占总数的73.74%。另外,肩突硬蜱基因组中微卫星的重复次数普遍高于二斑叶螨基因组中微卫星的重复次数,二斑叶螨基因组中微卫星的GC含量(34.10%)明显高于肩突硬蜱(24.35%)。微卫星家族方面,二斑叶螨基因组倾向拥有更多的唯一序列(P<0.0001)。A、T、AG、TC、TG、GAT、ATTT、AATA是两个物种共有的常见核心重复序列。     

草鱼Ⅰ型微卫星标记的发掘及其多态性检测

表达序列标签(EST)是发掘Ⅰ型微卫星标记的重要资源。研究运用生物信息学方法,从草鱼头肾组织3027条EST序列中搜索到322个微卫星位点,占整个EST数据库的10.6%。其中,二核苷酸重复位点151个(46.9%),三核苷酸重复位点137个(42.5%),四、五、六核苷酸重复位点较少;在二核苷酸重复位点中,AC/GT重复位点最为丰富,占二核苷酸重复位点总数的50.3%,AG/CT重复次之,占二核苷酸重复位点总数的40.4%,AT和GC重复较少。10个微卫星位点的多态性检测结果显示,4个位点在草鱼测试群体中呈多态性,多态性位点的平均多态信息含量(PIC)和平均遗传杂合度(H)分别为0.5236和0.5441,其中,2个多态性位点的PIC值大于0.5,呈现高度多态性特征。Ⅰ型微卫星标记将为草鱼遗传连锁图谱构建和QTL分析提供有效的基因分子标记。       

大黄鱼微卫星标记的开发及其遗传方式分析

采用FIASCO方法构建大黄鱼(AC)n微卫星富集文库,从文库中随机挑选90个白色克隆,经过菌液PCR筛选得到60(66.7%)个阳性克隆进行测序,其中有56个克隆(93.3%)含有CA/GT重复数大于5的微卫星序列。56个微卫星序列中,二核苷酸微卫星51个(91.1%),三核苷酸微卫星5个;二核苷酸重复中有48个为(AC)n重复,占二核苷酸总数的94.1%。根据Weber的微卫星分类规则,完美型占75.0%,非完美型占8.9%,复合型微卫星占16.1%。共设计引物52对,在1个大黄鱼家系中35对引物所在位点具有多态性,28个(80.0%)位点子代基因型为1∶1∶1∶1(AB×CD/AB×AC)分离类型,6个位点属1∶1分离类型,1个位点属1∶2∶1(AB×AB)分离类型。35个位点中有32个位点的分离符合孟德尔分离比(P>0.05),另外3个位点(LYC0137、LYC0139、LYC0152)明显偏离1∶1或者1∶1∶1∶1的孟德尔分离比(P<0.05)。本研究开发的微卫星标记为大黄鱼微卫星遗传连锁图谱构建以及群体遗传学、分子进化和系统发育等研究提供了有用的分子工具。     

利用RNA-seq技术分析棘腹蛙编码区微卫星特征

棘腹蛙Paa boulengeri的遗传研究和基因组信息比较匮乏,致使可有效利用的分子标记非常有限。以棘腹蛙RNA-seq高通量测序数据为基础进行微卫星分子标记的大规模发掘和特征分析,结果显示:在121.6 Mb的棘腹蛙转录组序列中发现微卫星位点3165个,包含于3034条Contig序列中。在筛选到的1~6碱基重复核心的微卫星中,单碱基重复核心的比例最高,之后为三碱基、二碱基、四碱基、六碱基和五碱基重复核心,分别占29.0%、25.2%、21.7%、10.0%、10.0%和3.0%。其中A/T、AC/GT、AGG/CCT、ACAT/ATCT、AAAAT/ATTTT和AAAAAG/CTTTTT分别是单碱基、二碱基、三碱基、四碱基、五碱基、六碱基重复类型中对应的优势重复单元。棘腹蛙编码区微卫星多为重复长度小于24 bp的短序列,长度大于24 bp的微卫星仅占总数的0.92%。对编码区微卫星的侧翼序列分析发现,微卫星侧翼序列的GC含量显著低于转录组整体GC含量,且在含有微卫星上下游侧翼序列的Contig中,71.9%的序列可以设计特异引物扩增出含有微卫星序列的位点。研究结果为棘腹蛙的遗传研究和分子系统地理学研究提供了丰富的序列信息和标记资源。     

功能基因中的微卫星序列

微卫星序列广泛存在真核生物和一些原核生物的基因组中,它在基因组中的分布并不是随机的。不同重复拷贝类别在基因组中存在种属间和碱基组成的特异性,各种优势的重复序列类型不同。此外,基因中在编码区和非编码区的分布也表现出种属和碱基组成差异。这种差异显示了微卫星序列起源进化的复杂性,也反映了基因中微卫星序列的生物学功能。功能基因为遗传学工作者提供了一个联系表型和基因型的手段,研究功能基因中的微卫星序列不仅在绘制精细遗传图谱、筛选重要农艺性状基因、物种进化等问题上都有着重要的作用,而且在疾病治疗有潜在的应用价值。本文主要阐述了微卫星序列的形成机制、基因中微卫星序列的分布以及功能基因中微卫星序列的生物学作用,并指出了目前实践中的一些问题。     

天麻基因组微卫星特征分析与分子标记开发

该研究基于第二代测序技术建立了天麻的基因文库,筛选微卫星序列,并对微卫星位点的类型、丰度、长度、偏好性等进行了分析与比较;并为60条重复次数高的微卫星序列设计了引物,运用4个种群80个样本进行了PCR扩增和聚丙烯酰胺凝胶电泳检测。结果表明:(1)天麻基因组测序得到61 048条基因序列,检测出微卫星位点12 107个,其中二核苷酸重复最多、长度变异大。(2)设计的60对微卫星引物中的20对能扩增出清晰条带且有多态性,每个位点的复等位基因数(N_a)在4~14之间,平均为8.40;多态性信息含量(PIC)平均为0.77。该研究开发的天麻微卫星分子标记为开展天麻遗传学研究及种质资源鉴定等工作奠定了基础。     

Genome-wide characterization and analysis of microsatellite sequences in camelid species

Microsatellites or simple sequence repeats (SSRs) are among the genetic markers most widely utilized in research. This includes applications in numerous fields such as genetic conservation, paternity testing, and molecular breeding. Though ordered draft genome assemblies of camels have been announced, including for the Arabian camel, systemic analysis of camel SSRs is still limited. The identification and development of informative and robust molecular SSR markers are essential for marker assisted breeding programs and paternity testing. Here we searched and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites for draft genome sequences of the Camelidae. We analyzed and compared the occurrence, relative abundance, relative density, and guanine-cytosine (GC) content in four taxonomically different camelid species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. A total of 546762, 544494, 547974, and 437815 SSRs were mined, respectively. Mononucleotide SSRs were the most frequent in the four genomes, followed in descending order by di-, tetra-, tri-, penta-, and hexanucleotide SSRs. GC content was highest in dinucleotide SSRs and lowest in mononucleotide SSRs. Our results provide further evidence that SSRs are more abundant in noncoding regions than in coding regions. Similar distributions of microsatellites were found in all four species, which indicates that the pattern of microsatellites is conserved in family Camelidae.     

Genome wide survey of microsatellites in ssDNA viruses infecting vertebrates

Microsatellites or Simple Sequence Repeats (SSRs) are tandem iterations of one to six base pairs, non-randomly distributed throughout prokaryotic and eukaryotic genomes. Limited knowledge is available about distribution of microsatellites in single stranded DNA (ssDNA) viruses, particularly vertebrate infecting viruses. We studied microsatellite distribution in 118 ssDNA virus genomes belonging to three families of vertebrate infecting viruses namely Circoviridae, Parvoviridae, and Anelloviridae, and found that microsatellites constitute an important component of these virus genomes. Mononucleotide repeats were predominant followed by dinucleotide and trinucleotide repeats. A strong positive relationship existed between number of mononucleotide repeats and genome size among all the three virus families. A similar relationship existed for the occurrence of DTTPH (di-, tri-, tetra-, penta- and hexa-nucleotide) repeats in the families Anelloviridae and Parvoviridae only. Relative abundance and relative density of mononucleotide repeats showed a strong positive relationship with genome size in Circoviridae and Parvoviridae. However, in the case of DTTPH repeats, these features showed a strong relationship with genome size in Circoviridae only. On the other hand, relative microsatellite abundance and relative density of mononucleotide repeats were negatively correlated with GC content (%) in Parvoviridae genomes. On the basis of available annotations, our analysis revealed maximum occurrence of mononucleotide as well as DTTPH repeats in the coding regions of these virus genomes. Interestingly, after normalizing the length of the coding and non-coding regions of each virus genome, we found relative density of microsatellites much higher in the non-coding regions. We understand that the present study will help in the better characterization of the stability, genome organization and evolution of these virus classes and may provide useful leads to decipher the etiopathogenesis of these viruses.     

Mononucleotide repeats represent an important source of polymorphic microsatellite markers in Aspergillus nidulans

In fungi, microsatellites occur less frequently throughout the genome and tend to be less polymorphic compared with other organisms. Most studies that develop microsatellites for fungi focus on dinucleotide and trinucleotide repeats, and thus mononucleotide repeats, which are much more abundant in fungal genomes, may represent an overlooked resource. This study examined the relative probabilities of polymorphism in mononucleotide, dinucleotide and trinucleotide repeats in Aspergillus nidulans. As previously found, the probability of polymorphism increased with increasing number of repeating units. Dinucleotide and trinucleotide repeats had higher probabilities of polymorphism than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats within the genome. Mononucleotide microsatellites with 20 or more repeating units have a probability of polymorphism similar to dinucleotide and trinucleotide microsatellites, and therefore, consideration of mononucleotide repeats will substantially increase the number of potential markers available.     

Genome-Wide Comparative Analyses of Microsatellites in Papaya

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic and universally distributed in eukaryotes. SSRs have been used extensively as sequence tagged markers in genetic studies. Recently, the functional and evolutionary importance of SSRs has received considerable attention. Here we report the mining and characterization of the SSRs in papaya genome. We analyzed SSRs from 277.4 Mb of whole genome shotgun (WGS) sequences, 51.2 Mb bacterial artificial chromosome (BAC) end sequences (BES), and 13.4 Mb expressed sequence tag (EST) sequences. The papaya SSR density was one SSR per 0.7 kb of DNA sequence in the WGS, which was higher than that in BES and EST sequences. SSR abundance was dramatically reduced as the repeat length increased. According to SSR motif length, dinucleotide repeats were the most common motif in class I, whereas hexanucleotides were the most copious in class II SSRs. The tri- and hexanucleotide repeats of both classes were greater in EST sequences compared to genomic sequences. In class I SSR, AT and AAT were the most frequent motifs in BES and WGS sequences. By contrast, AG and AAG were the most abundant in EST sequences. For SSR marker development, 9,860 primer pairs were surveyed for amplification and polymorphism. Successful amplification and polymorphic rates were 66.6% and 17.6%, respectively. The highest polymorphic rates were achieved by AT, AG, and ATG motifs. The genome wide analysis of microsatellites revealed their frequency and distribution in papaya genome, which varies among plant genomes. This complete set of SSRs markers throughout the genome will assist diverse genetic studies in papaya and related species.     

红原鸡全基因组中微卫星分布规律研究

本文对红原鸡Gallus gallus全基因组中微卫星数量及分布规律进行了分析,查找到l～6个碱基重复类型的微卫星序列共282728个,约占全基因组序列(1.1Gb)的0.49%,分布频率为1/3.89kb,微卫星序列的长度主要在12～70个碱基长度范围内。第1、2、3条染色体上微卫星分布频率较高,而32号染色体上无微卫星分布。不同类型微卫星中,单碱基重复类型数目最多,为184192个,占总数的65.1%;其次是四、二、三、五、六碱基重复单元序列,分别占到总数的12.8%、9.7%、7.2%、4.6%、0.8%。T、A、AT、GTTT、AAAC、G、C、ATTT、AC、GT、AAAT、ATT、AAC、AAT、GTT、AG、CT、CTTT、AAAG、GTTTT、AAACA、AAGG、CCTT是红原鸡基因组中最主要的微卫星重复类型。本研究为红原鸡微卫星标记的分离筛选、遗传多样性的研究以及不同物种微卫星的比较分析奠定了基础。     

蜜蜂EST中的微卫星分析

为加速分子标记在蜜蜂遗传、进化与行为等方面的利用,分析了简单重复序列(Simple Sequence Repeats,SSRs)在蜜蜂EST中的分布频率与密度。所分析的蜜蜂EST数据集包含15869条序列,总长为7．9Mb。结果显示,蜜蜂ESTs中SSRs的频率为1／0．52kb,其中6碱基重复基序占总SSRs的45．0％,是最丰富的重复单元,而2、1、3、4与5碱基重复基序分别占总SSRs的17．9％、14．1％、11．6％、9．2％和2．2％。同时,在各种SSRs重复单元中,富含A碱基的重复单元占据优势地位,如：A、AT、AG、AC、AAT、AAG、AAC、AAAT、AAAG、AAAAG、AAAAT、AATAT、AAAAAG和AAAAAT重复基序,而富含G碱基的重复单元在基因编码区中含量较低。进一步分析显示：蜜蜂SSRs在冗余与非冗余EST数据集中的分布频率与密度相似,仅存在极小的偏差,表明可从现有的部分ESTs数据中方便地获取有效的微卫星标记。     

In silico analysis of SSRs in mitochondrial genomes of plants

Simple sequence repeats (SSRs) or microsatellites constitute a countable portion of genomes. However, the significance of SSRs in organelle genomes has not been completely understood. The availability of organelle genome sequences allows us to understand the organization of SSRs in their genic and intergenic regions. In the current study we surveyed the patterns of SSRs in mitochondrial genomes of different taxa of plants. A total of 16 mitochondrial genomes, from algae to angiosperms, have been considered to analyze the pattern of simple sequence repeats present in them. Based on study, the mononucleotide repeats of A/T were found to be more prevalent in mitochondrial genomes over other repeat types. The dinucleotides repeats, TA/AT, were the second most numerous, whereas tri-, tetra-, and pentanucleotide repeats were in less number and present in intronic or intergenic portions only. Mononucleotide repeats prevailed in protein-coding exonic portions of all organisms. These results indicates that microsatellite pattern in mitochondrial genomes is different from nuclear genomes and also focuses on organization and diversity at SSR locuses in mitochondrial genomes. This is the novel report of microsatellite polymorphism in plant mitochondrion on whole genome level.     

The analysis of microsatellites and compound microsatellites in 56 complete genomes of Herpesvirales

Simple sequence repeats (SSRs), or microsatellites, are special DNA/RNA sequences with repeated unit of 1–6 bp. The genomes of Herpesvirales have many repeating structures, which is an excellent system to study the evolution and roles of microsatellites and compound microsatellites in viruses. Therefore, 56 genomes of Herpesvirales were selected and the occurrence, composition and complexity of different repeats were investigated in the genomes. A total of 63,939 microsatellites and 5825 compound microsatellites were extracted from 56 genomes. It found that GC content has a significant strong correlation with both the counts of microsatellites (CM) and the counts of compound microsatellites (CCM). However, genome size has a moderate correlation only with CM and almost no correlation with CCM. The compound microsatellites occurring in genic regions are obviously more than that in intergenic regions. In general, the number of compound microsatellite decreases with the increase of complexity (C) (the count of individual microsatellites being part of a compound microsatellite) and the complexity hardly exceeds C = 4. The vast majority of compound microsatellites exist in intergenic regions, when C ≥ 10. The distributions of SSRs tend to be organism-specific rather than host-specific in herpesvirus genomes. The diversity of microsatellites and compound microsatellites may be helpful for a better understanding of the viral genetic diversity, genotyping, and evolutionary biology in herpesviruses genomes.     

Microsatellites in palm (Arecaceae) sequences

Microsatellites are the most promising co-dominant markers, widely distributed throughout the genome. Identification of these repeating genomic subsets is a tedious and iterative process making computational approaches highly useful for solving this biological problem. Here 38,083 microsatellites were localized in palm sequences. A total of 2, 97,023 sequences retrieved from public domains were used for this study. The sequences were unstained using the tool Seqclean and consequently clustered using CAP3. SSRs are located in the sequences using the microsatellite search tool, MISA. Repeats were detected in 33,309 sequences and more than one SSR had appeared in 3,943 sequences. In the present study, dinucleotide repeats (49%) were found to be more abundant followed by mononucleotide (30%) and trinucleotide (19%). Also among the dinucleotides, AG/GA/TC/CT motifs (55.8%) are predominantly repeating within the palm sequences. Thus in future this study will lead to the development of specific algorithm for mining SSRs exclusively for palms.