基于Roche 454 GS FLX高通量测序的叶城沙蜥基因组微卫星特征分析

叶城沙蜥Phrynocephalus axillaris是我国特有的一种小型爬行动物,广泛分布于新疆塔里木盆地、吐鲁番-哈密盆地和甘肃敦煌盆地。本研究利用Roche 454 GS FLX高通量测序技术进行叶城沙蜥微卫星位点筛选,获得了91 190条高质量序列。用Krait搜索微卫星位点,共得到1～6个碱基重复类型的完美型微卫星序列29 890个。不同类型微卫星中,单碱基重复类型数目最多,有14 630个,占总数的48. 95%,其次是二碱基,约占28. 60%,四碱基、三碱基、五碱基和六碱基分别占10. 73%、10. 48%、0. 92%和0. 32%。二碱基微卫星中AC重复类型数量最多,三碱基、四碱基、五碱基和六碱基中分别是ATC、AAAT、AAAAT和AATCCC。叶城沙蜥完美型微卫星中数量最多的11种重复拷贝类型分别为C、A、AC、AG、AAAT、ATC、AT、AAT、ATAG、AGG和AAC。本研究深化了对叶城沙蜥基因组的了解,并为以后开发和筛选大量高质量微卫星标记提供了数据支持,也为利用微卫星标记研究叶城沙蜥种群遗传结构和谱系地理模式奠定了基础。     

基于454 GS FLX高通量测序的南疆沙蜥微卫星特征分析及其候选引物设计

南疆沙蜥Phrynocephalus forsythii是我国特有的一种小型爬行动物,分布于塔里木盆地。利用Roche 454 GS FLX高通量测序对该物种基因组测序,获得了55 909条高质量序列。利用Krait搜索并初步统计和分析基因组微卫星序列,共得到1～6个碱基重复类型的完美型微卫星12 109个。不同类型微卫星中,四碱基重复类型数目最多,有4 037个,约占总数的33.34%,其次是二碱基,约占总数的28.09%,再是三碱基、单碱基、五碱基和六碱基,分别约占总数的18.72%、13.91%、4.48%和1.46%。单碱基微卫星中C最多,二碱基微卫星中AC最多,三碱基、四碱基、五碱基和六碱基中最多的分别是AAC、AAAT、AAAAT和AACCCT。AC、AAAT、C、AG、A、AAC、AAT、AAAC、ACC和ACG是数量最多的10种重复拷贝类别。挑选部分三、四碱基重复类型的微卫星序列设计了100对可用于后续对南疆沙蜥微卫星标记开发的候选引物。本研究开启了对南疆沙蜥基因组微卫星特征的了解,为利用微卫星标记研究南疆沙蜥种群遗传结构奠定了基础。     

基于454 GS FLX高通量测序的四川山鹧鸪基因组微卫星特征分析

为了有效地保护四川山鹧鸪Arborophila rufipectus这一中国特有珍稀濒危鸟类,本研究采用454 GS FLX对该物种基因组测序,首次利用微卫星搜索软件搜索并初步统计和分析基因组微卫星序列,共搜索到l~6个碱基重复类型的完美型微卫星335 263个。不同类型微卫星中,单碱基重复类型数目最多,为197 913个,约占总数的59.03%,其次是四碱基、三碱基、六碱基、二碱基和五碱基,分别约占微卫星总数的13.59%、8.39%、7.55%、6.49%和4.95%。单碱基微卫星中A重复类型数量最多,两碱基中AC最多,三碱基中AAC,四碱基中AAAC最多,五碱基中AAACA最多,六碱基中AGGGTT最多。A、AGGGTT、AAAC、AC、AAAT、AAC、C、AG、AAAG、AAACA、AAT、AGG、AT、AGC、AAGG、CCG是在所搜索到的四川山鹧鸪微卫星中数量最多的16种重复拷贝类型。本研究深化了对四川山鹧鸪基因组的认识和了解,并为以后开发和筛选大量高质量微卫星标记提供数据支持,也为利用微卫星标记研究更加有效地保护和管理四川山鹧鸪这一珍稀濒危动物奠定了基础。     

大熊猫和北极熊基因组微卫星分布特征比较分析

本研究比较分析了大熊猫和北极熊全基因组序列中的1~6碱基重复的完美型微卫星序列的分布特征,通过微卫星序列搜索和统计软件MSDB分析分别得到855 018和936 238个微卫星序列,其长度总和分别是14 919 240 bp和18 434 348 bp;分别占基因组大小的0.64%和0.79%,大熊猫和北极熊基因组总丰度分别是371.8个/Mb和405.6个/Mb,二者基因组中微卫星都是单碱基重复的最多,其次是二碱基、四碱基、三碱基和五碱基,六碱基重复类型的数量最少。大熊猫和北极熊含量最丰富的重复拷贝类别主要有A、AC、AG、AAAT、AAAG、AT和C等。本研究为后续开发和筛选大量高质量的熊科物种微卫星标记提供了数据支持。     

林麝全基因组微卫星分布规律研究

林麝Moschus berezovskii是中国重要的资源动物,也是国家Ⅰ级重点保护野生动物。本研究使用生物信息学方法,分析林麝全基因组中完美型微卫星的分布特征。在林麝2.53 Gb的基因组序列中,共搜索到665 524个完美型微卫星,总长度为11 517 784 bp,占基因组序列总长度的0.42%,总丰度为244个/Mb。林麝基因组中,单碱基微卫星序列数量最多,为221 058个,约占总微卫星数的33.22%,丰度为81.05个/Mb,然后依次为二碱基、五碱基、三碱基、四碱基、六碱基重复类型微卫星。林麝基因组中数目最多的10种微卫星类别依次为:A、AACTG、AGC、AC、AT、AG、AAAT、AAC、AAT和AAAC,占所有基因组微卫星的93.2%,表现出明显的A、T偏好。林麝基因组微卫星序列分布研究表明,其在外显子(2 530个)上的分布数量远低于内含子(200 906个)和基因间隔区(454 596个),与前人关于微卫星在非编码区的分布多于编码区的结论一致。本研究为深入研究林麝基因组特征及筛选更多优良微卫星标记提供了基础数据。     

德国小蠊全基因组中微卫星分布规律

【目的】分析德国小蠊Blattella germanica全基因组中微卫星的数量和分布规律,并对外显子中含有微卫星的基因进行功能注释。【方法】使用微卫星搜索软件查找德国小蠊基因组中微卫星的数量、重复次数以及所有微卫星的位置信息,编写Python脚本对微卫星进行定位,并通过Blast2Go和KASS程序对外显子中含有微卫星的基因进行功能注释。【结果】共找到1~6碱基重复类型的微卫星序列604 386个,总长度15 301 255 bp,约占全基因组序列(约2.04 Gb)的0.75%,分布频率为1/3.37 kb,微卫星序列的长度主要在12~60个碱基长度范围内。不同类型的微卫星中,三碱基(226 876)重复类型微卫星数量最多,占微卫星总数的37.54%;四碱基(150 355)重复类型次之,占微卫星总数的24.88%;其余依次是单碱基(141 167)、二碱基(60 877)、五碱基(21 570)和六碱基(3 541)重复类型,分别占微卫星总数的23.36%,10.07%,3.57%和0.59%。出现最多的重复拷贝类别有:ATT,AAT,A,T,AAAT,ATTT和AT,共411 789个微卫星,占微卫星总数的68.13%,这7种类别的微卫星数量均大于30 000个。共有2 372个微卫星在外显子上,它们分别位于1 481个基因上。GO功能注释结果表明,其中434条归类于细胞组分(cellular component),402条归类于分子功能(molecular function),660条归类于生物学过程(biological process)。KEGG通路分析结果表明,与新陈代谢相关的基因最多(380个),其次是与机体系统相关的(276个),与遗传信息进程相关的基因最少(92个)。【结论】本研究为进一步系统深入分析德国小蠊微卫星功能及微卫星分子标记筛选打下了基础。     

诸氏鲻虾虎鱼转录组序列中微卫星标记的初步筛选及特征分析

旨在为大规模开发诸氏鲻虾虎鱼微卫星标记,采用高通量测序技术,对诸氏鲻虾虎鱼肝脏转录组进行了测序。结果共获得47 979条Unigenes,利用微卫星查找程序在47 979条Unigenes中共获得6 225个微卫星位点(12.97%),平均每7.02 kb就出现1个微卫星位点。6 225个微卫星位点由226种重复基序组成,主要分布在三、四和五碱基重复类型中。在数量上,单碱基重复类型微卫星位点最多,占42.49%,二碱基和三碱基重复类型所占比例相似,分别为25.22%和26.27%,四、五、六重复类型较少,合计占6.03%。单碱基重复序列中最多的类型为A/T,二碱基重复序列中以AG/CT重复单元为主,三碱基重复序列中以AGC/TCG为优势类型。挑选部分二、三和四单元重复类型微卫星序列,共设计76对引物,可稳定扩增出目的条带的有55对,其中32对具有多态性。结果表明,利用诸氏鲻虾虎鱼转录组数据可快速大量开发微卫星标记。     

细梢小卷蛾微卫星富集文库的构建与分析

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库.方法:提取细梢小卷蛾基因组DNA,经限制性内切酶Rsa Ⅰ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库:结果:对100个克隆进行随机测序,获得98个微卫星序列,其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库.结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础.     

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。     

基于转录组数据高通量发掘黄粉甲微卫星引物（英文）

黄粉虫Tenebrio molitor作为理想的模式研究生物, 虽然已围绕该昆虫在多个研究领域开展了诸多研究, 但是有关其分子和遗传方面的研究仍知之甚少。为此, 本研究基于前期构建的黄粉甲转录组数据库, 成功发掘获得1 249个微卫星序列。其中, 单碱基或三碱基序重复列最多, 分别占44.44%和41.15%; A/T型重复序列出现频率最高, 占42.70%。除单核苷酸重复序列外, 重复单元的重复次数以5次最多, 占30.90%。基于鉴定获得的微卫星序列, 共设计获得1 004对微卫星引物, 而且每对引物还设计了5对替代引物。研究获得的微卫星引物将有助于今后开展黄粉甲功能和比较基因组学方面的研究。     

勒氏笛鲷微卫星位点的筛选及特征分析

采用PCR法快速筛选勒氏笛鲷(Lutjanus russelli)基因组文库, 以获得(CA)_n微卫星位点。勒氏笛鲷基因组DNA经限制性内切酶HaeⅢ+ DraⅠ双酶切后, 连接T-载体克隆, 构建基因组文库。以通用引物M13+/-与重复序列引物(CA)15对基因组文库进行筛选, 二次筛选后得到121个可能含有微卫星位点的阳性克隆。进行序列测定, 共获得53个CA(n≥7)重复序列, 重复次数主要分布于7~15(80.77%)。在所得微卫星序列中, 重复单元除CA外, 还观察到单碱基、三碱基、四碱基、五碱基重复单元。根据侧翼序列设计48对引物, 通过优化PCR反应条件, 可获得清晰可重复的目的条带。研究旨在为勒氏笛鲷遗传多样性研究及遗传图谱的构建等奠定基础, 为勒氏笛鲷资源的合理开发利用提供参考。     

Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid; single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.     

Systematic screening of sheep skin cDNA libraries for microsatellite sequences

65,000 sheep skin cDNA clones were gridded in high density on to nylon membranes and screened for (CA)n and (GA)n repeat containing clones. 296 dinucleotide repeat-containing clones were identified with approximately 85% non-redundancy. Clones were single-pass 5' sequenced and we compared the Expressed Sequence Tag (EST) sequences to the Swiss-Prot database to ascertain their identity and/or putative function. We then aligned the ESTs against the human genomic sequence to determine the locations of human orthologous sequences. Finally, we developed a subset of polymorphic microsatellite markers and positioned them on the ovine linkage map.     

Polymorphic microsatellites in a mangrove species,Rhizophora mangle L. (Rhizophoraceae)

Twenty‐six microsatellite loci were isolated and characterized from the mangrove species Rhizophora mangle using (GT)_n and (CT)_n repeats. Eighty‐four per cent of the clones contained microsatellite sequences; the most common dinucleotides were the (GA/CT) and (CA/GT) repeats. Ten primers were selected to investigate the polymorphism among individuals of R. mangle from two natural populations of the Colombian Pacific Coast. The observed heterozygosity per locus varied from 0.20 to 0.80, the power of discrimination was 0.32–0.84 and the power of exclusion was 0.03–0.75. This set of microsatellites offers an efficient tool for population genetics studies on this species.     

Fifty microsatellite markers for Japanese quail

A Japanese quail genomic library enriched for (CA/GT)n simple sequence repeats was screened and positive clones were sequenced. Fifty original microsatellite sequences were isolated that consisted mainly of perfect repeats of the dinucleotide (CA/GT)n motif and a corresponding number of polymerase chain reaction (PCR) primer pairs complementary to unique DNA sequences flanking the microsatellite repeats were designed to detect the repeats. Forty-six percent (23 of 50) of the markers revealed polymorphism in two unrelated quail individuals (one male and one female) randomly sampled from a population of wild quail origin. All 50 primer pairs were tested in the PCR for their ability to amplify chicken genomic DNA. Amplification products were obtained for 14 (28.0%) of the markers at the annealing temperature optimized for quail. These results provide an opportunity to begin characterizing the quail genome for the development of a genetic map for this economically valuable species and the eventual construction of a comparative genetic map in Phasianidae, which comprises a number of agriculturally important species of poultry.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Isolation, cloning and characterisation of motifs containing (GA/TC)n repeats isolated from vetch, Vicia bithynica

Microsatellites are widely distributed in plant genomes and comprise unstable regions that undergo mutational changes at rates much greater than that observed for non-repetitive sequences. They demonstrate intrinsic genetic instability, manifested as frequent length changes due to insertions or deletions of repeat units. Detailed analysis of 1600 clones containing genomic sequences of Vicia bithynica revealed the presence of microsatellite repeats in its genome. Based on the screening of a partial DNA library of plasmids, 13 clones harbouring (GA/TC)n tracts of various lengths of repeated motif were identified for further analysis of their internal sequence organization. Sequence analyses revealed the precise length, number of repeats, interruptions within tracts, as well as sequence composition flanking the repeat motifs. Representative plasmids containing different lengths of (GA/TC)n embedded in their original flanking sequence were used to investigate the genetic stability of the repeats. In the study presented herein, we employed a well characterised and tractable bacterial genetic system. Recultivations of Escherichia coli harbouring plasmids containing (GA/TC)n inserts demonstrated that the genetic instability of (GA/TC)n microsatellites depends highly on their length (number of repeats). These observations are in agreement with similar studies performed on repetitive sequences from humans and other organisms.