柑橘EST-SSR分子标记分析

对来源于甜橙（Citrus sinensis Osbeck）、枳壳（Poncirus trifoliata Raf．）和其他柑橘非冗余EST数据库的38124条单-基因（Unigene）序列进行了简单重复序列SSRs（Simple Sequence Repeat）搜索,所分析的柑橘非冗余核酸序列总长23．29Mb,从中获得了8218条SSR,其中包括单碱基重复4913条（59．8％）,2碱基重复1419条（17．3％）,3碱基重复1709条（20．8％）,4碱基重复114条（1．39％）,5碱基重复23条（0．28％）,6碱基重复40条（0．49％）。大约每2．8kb长度的单-基因序列中即存在1个SSR,即平均4．6个单-基因中存在1个SSR。随碱基重复单元（motif）的不同,SSR的最大长度在40-105之间,全部重复序列的平均长度为20．9bp。各种SSR（1-,2-,3-,4-,5-,6-核苷酸重复）的发生频率在甜橙和枳壳间非常接近。其中单碱基重复序列是最丰富的重复单元,其次为3碱基重复。在所得的SSR的重复单元中,富含A碱基的重复单元的分布占据优势地位,出现的频率与密度均较高,而富含CG碱基的重复单元出现频率和密度较低。用25对EST-SSR引物对6个柑橘品种的多样性进行了PCR检测,结果表明,所有25对引物在6个柑橘品种间均扩增到多样性条带,证实通过柑橘EST数据库的发掘能够高效地筛选到基因水平的SSR标记。     

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。     

Analysis on Frequency and Density of Microsatellites in Coding Seauences of Several Eukarvotic Genomes

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in th     

蝴蝶兰EST-SSRs分析

对蝴蝶兰属EST序列进行了SSR分析。蝴蝶兰属EST总长为4.5Mb,含有609个SSR。SSR出现频率7.65%,平均距离7.39kb,平均长度为22.17bp。单碱基、二碱基和三碱基重复是主要重复类型,分别占EST-SSR总数的21.67%、40.39%和33.50%。A、AG和CCG分别是单碱基、二碱基和三碱基重复中主导重复基元,分别占96.21%、58.54%和32.25%。设计引物及检测的结果表明,蝴蝶兰EST-SSR标记对兰科其他属植物具有一定的通用性。     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展,初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记,根据花生EST序列开发EST-SSR标记,根据豆科植物序列信息和SSR标记开发花生SSR标记,将SSR标记与其它分子标记结合开发新的DNA标记,以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

有尾噬菌体目中微卫星和复合型微卫星的发生及分析

简单重复序列亦称微卫星,被成功应用于许多真核生物、原核生物和病毒的基因组和进化研究,但是噬菌体中的微卫星目前很少被研究。因此对60条尾病毒目基因组中的微卫星和和复合型微卫星(由两个或两个以上直接相邻的微卫星组成)做综合性分析,在这60个基因组中总共观察到11 874个微卫星和449个复合型微卫星。相关性分析表明微卫星个数与基因组大小成正线性相关(ρ=0.899, P<0.01)。参考序列中的微卫星个数少于对应的随机序列中微卫星个数,这种反常现象主要是因为参考序列含有较少的单核苷酸和二核苷酸重复。A/T和AT/TA重复是单核苷酸和二核苷酸重复中最主要的类型,因此单核苷酸重复中的GC含量明显低于相应的序列中的GC含量;相比之下,微卫星中的二核苷酸和三核苷酸重复的GC含量与对应的参考序列的GC含量无明显区别。尾病毒目基因组中的这些结果与其它生物体基因组存在一定的差别。有助于了解尾病毒目中微卫星的分布、进化和生物学功能。     

Characterization of new SSR‐EST markers in cod,Gadus morhua

For years, fishermen have noted that cod (Gadus morhua) within the Georges Bank Atlantic fishery spawn multiple times annually with peaks in the spring and in the fall. To evaluate the hypothesis that these differences in reproductive behaviour reflect genetically different spawning populations, we identified eight polymorphic simple sequence repeats within expressed sequence tags (ESTs). Previously, only a few genetic markers have been informative for distinguishing between cod populations of the Northwest Atlantic. The development of additional markers from ESTs will be a valuable tool to improve our understanding of population structure in this commercially important species.     

Sequence Divergence of Microsatellites and Phylogeny Analysis in Tetraploid Cotton Species and Their Putative Diploid Ancestors

To determine the level of microsatellite sequence differences and to use the information to construct a phylogenetic relationship for cultivated tetraploid cotton （Gossypium spp.） species and their putative diploid ancestors, 10 genome-derived microsatellite primer pairs were used to amplify eight species, including two tetraploid and six diploid species, in Gossypium. A total of 92 unique amplicons were resolved using polyacrylamide gel electrophoresis. Each amplicon was cloned, sequenced, and analyzed using standard phylogenetic software. Allelic diversities were caused mostly by changes in the number of simple sequence repeat （SSR） motif repeats and only a small proportion resulted from interruption of the SSR motif within the locus for the same genome. The frequency of base substitutions was 0.5%-1.0% in different genomes, with only few indels found. Based on the combined 10 SSR flanking sequence data, the homology of A-genome diploid species averaged 98.9%, even though most of the amplicons were of the same size, and the sequence homology between G. gossypioides （Ulbr.） Standl. and three other D-genome species （G. raimondii Ulbr., G. davidsonii Kell., and G. thurberi Tod.） was 98.5%, 98.6%, and 98.5%, respectively. Phylogenetic trees of the two allotetraploid species and their putative diploid progenitors showed that homoelogous sequences from the A- and D-subgenome were still present in the polyploid subgenomes and they evolved independently. Meanwhile, homoelogous sequence interaction that duplicated loci in the polyploid subgenomes became phylogenetic sisters was also found in the evolutionary history of tetraploid cotton species. The results of the present study suggest that evaluation of SSR variation at the sequence level can be effective in exploring the evolutionary relationships among Gossypuim species.     

烟草EST-SSR位点分析

利用MISA软件对烟草EST公共数据库中的简单重复序列(SSRs)进行了分析。结果表明,在133523条EST序列中,共获得81757条SSR序列,SSRs之间的距离约为0.92 kb。其中,六碱基重复丰度最大,占60.3%,而单碱基、三碱基、四碱基、二碱基和五碱基重复丰度分别为20.0%、11.0%、4.2%、2.8%和1.7%。在单碱基、二碱基、三碱基和四碱基重复模体中,丰度最大的分别是A/T、AG、AAG和AAAT,而CG在编码区内丰度很低。用CAP3软件进行冗余分析表明,在这6种类型的重复模体中,冗余与非冗余的烟草EST之间没有显著差异。在得到的SSR序列中随机选择10个序列设计引物,在7个烟草品种中进行PCR扩增。结果表明,10对引物全部扩增出PCR产物,其中8对引物扩增出预期片段。用这8组扩增出预期片段的PCR产物进行变性PAGE凝胶电泳检测,结果表明,其中有4对引物(EB4、EB5、EB6和EB8)扩增出多态性条带。     

栓皮栎天然群体SSR遗传多样性研究

利用微卫星(SSR)标记对我国4个省内的5个栓皮栎（Quercus variabilis Bl.）天然群体的遗传多样性进行了研究。16对SSR标记揭示了栓皮栎丰富的遗传多样性：等位基因数（A）平均8.4375个,有效等位基因数(Ne)平均为5.9512个,平均期望杂合度(He)0.8059,Nei多样性指数(h)为0.8041。栓皮栎自然分布区中心地带的群体具有较高的遗传多样性,而人为对森林的破坏将降低林木群体的遗传多样性。栓皮栎群体的变异主要来源于群体内,群体间分化较小,遗传分化系数仅为0.0455。此外,栓皮栎群体间的遗传距离与地理距离之间存在显著的正相关。这些遗传信息为栓皮栎遗传多样性的保护和利用提供了一定依据。Abstract: Genetic diversity of five Quercus variabilis natural populations in four provinces of China was studied with microsatellite (SSR) markers. A relatively high level of genetic diversity was detected in Q. variabilis species with 16 polymorphic microsatellite loci. Average number of alleles (A) and effective number of alleles (Ne) were 8.4375 and 5.9512 respectively. The mean expected heterozygosity (He) was 0.8059 and Nei diversity index (h) was 0.8041. Higher diversity was found with the populations from the central range of the species in contrast to those from peripheral areas and human activities might decrease the genetic diversity of populations. The majority of genetic variation occurred within populations, which could be concluded from the low coefficient of genetic differentiation (Fst=0.0455). In addition, significant correlation was found between geographical distance and genetic distance. All these results present a basis to the conservation and utilization of genetic diversity of Quercus variabilis.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

Characterization of SSRs from the American lobster,Homarus americanus

The American lobster (Homarus americanus), a commercially important benthic marine crustacean, is widely distributed along the continental shelf of the western North Atlantic. The population substructure of this species remains poorly understood despite its economic value. Informative markers are required to clarify relationships between local populations. To this end, we developed eight polymorphic short sequence repeats (SSR) for the American lobster, which were derived from expressed sequence tags. Additionally, we tested four SSRs previously identified for the Norway lobster (Nephrops norvegicus L.) for cross‐species utility; only one of these showed polymorphism.     

Development of EST‐SSRs in Cucumis sativus from sequence database

Simple sequence repeat markers derived from expressed sequence tags (EST‐SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. We have identified 20 polymorphic SSR markers from cucumber ESTs deposited in public sequence database. The average allele number was 3.3 per locus, ranging from two to six alleles during screening 20 cucumber genotypes with the mean expected heterozygosity of 0.477. Amplification products were also detected by 13 pairs of primer in Cucumis melo. These informative EST‐SSR markers can be used in cucumber genetic improvement projects.     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.     

Microsatellite markers for Fragaria from ‘Strawberry Festival’ expressed sequence tags

We present 37 microsatellite primer pairs developed from a cDNA library of Fragaria ^xananassa Duch. cv. Strawberry Festival. Polymorphism was high and the number of presumptive alleles of 13 expressed sequence tag–simple sequence repeats (EST–SSRs) in 70 strawberry cultivars ranged from five to 32 per primer pairs, averaging 16.1. Cross‐species amplification was also high and ranged from 89% in Fragaria vesca L. to 100% in the progenitor species of octoploid strawberry, Fragaria chiloensis (L.) Duch. and Fragaria virginiana Duch.     

柑桔BES SSR标记开发及连锁图延伸和加密

为了系统性地开发和拓展柑桔SSR标记,通过对公布的柑桔BAC文库末端序列(BAC-End sequence,BES)进行SSR分析,选择1500个SSR位点设计合成并检测323对引物。结果表明:(1)从总长度为28.1 Mb的46 339条序列中共检测出22 403个SSR位点,约每2条序列就会出现一个SSR位点,发生频率为48%,相当于平均1.25kb的序列中就会出现1个SSR,频率约为柑桔EST的2倍,且不同核心重复序列的SSR发生特点与EST也不同。(2)所合成的323对引物中,有效扩增316对,扩增率约98%,173对表现多态性,总多态性比率约55%,多态性引物中单核苷酸重复类型15对,双核苷酸重复类型100对,三核苷酸及以上重复类型58对,表明柑桔BES中具有较为丰富的多态性SSR标记。(3)结合已发表的遗传作图数据,对总计349个多态性位点进行遗传连锁分析,获得的新遗传连锁图谱共含有9个连锁群、334个SSR标记、总长844.2cM、平均图距2.53cM,延长和加密了先前的图谱。该研究开发的新SSR标记为开展柑桔遗传鉴定分析和遗传图谱构建提供了新的标记来源,加密的遗传图谱为柑桔的基因定位、图位克隆和标记辅助育种等奠定了基础,SSR分析结果也为其他物种SSR标记的开发提供了参考。     

Microsatellite markers for Vaccinium from EST and genomic libraries

We present 30 microsatellite loci isolated from expressed sequence tag (EST) and genomic libraries in Vaccinium corymbosum L. Allele number per locus in 11 tetraploid and one diploid V. corymbosum accessions ranged from two to 15 (mean = 8.16) in 24 single‐locus simple sequence repeats (SSRs). Cross‐species amplification in a panel of 12 species representing nine sections ranged from 30 to 100% (mean = 83%).     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

引进美洲红点鲑群体遗传多样性微卫星的分析

为了解引进种美洲红点鲑种群遗传结构和种质资源现状,本研究利用15个微卫星标记对其养殖群体遗传多样性进行了分析。结果表明:在30个个体中,15对微卫星引物除1对扩增产物为单态外,其余14对在美洲红点鲑群体内扩增均出现了多态,14个多态性位点等位基因数目为3～7不等,共检测到等位基因数为69个,平均有效等位基因数为3.03;期望杂合度在0.540～0.809之间,平均期望杂合度为0.664;多态信息含量在0.360～0.719之间,平均多态信息含量为0.578,表明引进的美洲红点鲑遗传多样性水平较高,具有良好的选育潜力,可以作为良好的育种材料。