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。     

红尾蚺和原矛头蝮基因组微卫星分布特征比较分析

本研究分析比较了红尾蚺Boa constrictor和原矛头蝮Protobothrops mucrosquamatus基因组微卫星的分布特征,通过MISA分别鉴定出398 860个和422 364个微卫星,其长度分别为8 550 741 bp和12 243 226 bp,分别占基因组序列总长度的0.59%和0.73%,在各自基因组中的丰度分别为275.46个/Mbp和252.33个/Mbp。红尾蚺基因组中单碱基重复类型微卫星最多,其次是四碱基、二碱基、三碱基、五碱基和六碱基,最丰富的5种微卫星类型是A、AC、AAAT、AG、AAT;原矛头蝮基因组中单碱基重复类型微卫星最多,其次是三碱基、四碱基、二碱基、五碱基和六碱基,最丰富的5种微卫星类型是A、AAT、AC、C、AAAT。红尾蚺和原矛头蝮微卫星在基因组不同区域丰度不同,基因间区丰度最高,其次是内含子和外显子,编码区微卫星丰度最低,表明编码区微卫星受到的选择压力最大。红尾蚺和原矛头蝮在基因中微卫星丰度分布的位置特征相似,即微卫星在基因上下游500 bp丰度最高,在内含子次之,在外显子最低。红尾蚺和原矛头蝮基因编码区所有6种重复类型微卫星中,三碱基重复类型占绝对优势。红尾蚺和原矛头蝮基因组中含有微卫星的编码序列分别有1 480条和1 397条,被GO注释的分别有736条和733条。它们的GO功能归类结果类似,但是与其他物种相比存在种系差异。本研究结果为后续开发这2种蛇的高质量微卫星标记提供了方便,也为进一步探索这些微卫星在它们基因组中的生物学功能提供了有意义的基础数据。     

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。     

家蚕全基因组微卫星分布规律及其生物信息学分析

本研究利用MSDB v2.4软件以及生物信息学方法获取了家蚕全基因组的完整型SSRs序列,并对其分布规律进行比较分析。家蚕全基因组中SSRs总数量为141 311个,相对丰度为209.01 No/Mb,总长度为2.41 Mb,全基因组SSRs六种碱基重复类型的数量和密度分布模式为:单碱基四碱基三碱基二碱基五碱基六碱基,说明全基因以单碱基为主要碱基类型,六种碱基类型中五碱基SSRs G-C含量最高。对全基因组3'非翻译区(3'UTR)、5'非翻译区(5'UTR)、编码区(CDs)、内含子区(Introns)和基因间隔区(Intergenics)等不同区域SSRs分析表明,Introns区SSRs数量最高,为125 178个,最小的是5'UTR,为278个,其数量大小顺序为IntronsIntergenics3'UTRCDs5'UTR。5个不同区域的SSRs的碱基的总计数差异较大,编码区总计数最大的是三碱基,而其他4个区域最多的是单碱基。分别对5个区域SSRs中六种重复拷贝类别进行统计分析,碱基总计数(或频率)最多的分别是A;AC、AG、AT;AAT、CCG;AAAT、AAAC;AAATC、AAACT和TAAGTT、GAATTT、AATTAA,Introns和Intergenics区的重复类型总计数显著高于3'UTR、CDs和5'UTR。各重复类型拷贝数分布范围为4～100,主要集中在4～30之间。这为进一步系统分析家蚕SSRs分子标记筛选和遗传分析打下基础。     

绿尾虹雉全基因组微卫星分布规律研究

分析了绿尾虹雉Lophophorus lhuysii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了注释分析。结果显示,在绿尾虹雉1. 01 Gb的全基因组中,1～6个碱基重复类型的完美型微卫星序列共292 430个,总长度5 465 549 bp,相对丰度为290. 47个/Mb,占全基因组的0. 54%,序列长度主要为10～43 bp。不同类型的微卫星中,单碱基重复类型数量最多,长度为3 535 260 bp,占71. 75%,其次是四碱基(611 568 bp,9. 99%)、二碱基(376 944 bp,7. 07%)、三碱基(335 742 bp,6. 38%)、五碱基(500 615 bp,3. 93%)和六碱基(105 420 bp,0. 88%)重复类型。在绿尾虹雉全基因组中,数目最多的10种优势微卫星分别是:A、C、AAAC、AT、AAAT、AC、AAT、AAC、AG、AAAAC,共计占90. 20%,表现出明显A偏倚。分布于外显子的微卫星有2 816个,内含子的有101 791个,基因间区的有187 823个。外显子的微卫星分布于1 314个编码基因中。GO注释分析发现,这些编码基因主要与细胞组分有关,富集前10的条目主要与代谢、转录和合成过程有关。KEGG富集最显著的通路是黏着连接通路。位于外显子的微卫星移码突变可能会造成基因突变,进而可能会影响绿尾虹雉对环境信号的处理。本研究为绿尾虹雉的微卫星筛选和进一步的遗传多样性、功能研究提供了数据基础,从分子角度为绿尾虹雉的保护提供了基础信息。     

4种河鲀全基因组微卫星分布特征分析

本研究利用MISA软件对四种河鲀全基因组中的微卫星进行筛选并分析.结果如下:在红鳍东方鲀(Takifugu rubripes)(391.49 Mb)、菊黄东方鲀(T.flavidus)(366.29 Mb)、双斑东方鲀(T.bimaculatus)(371.68 Mb)及黑青斑河鲀(Tetraodon nigroviridis)(342.40 Mb)全基因组中,分别筛选出142 885个、135 009个、147 549个和179 703个完整型微卫星.相对丰度分别为365个/Mb,369个/Mb,397个/Mb和525个/Mb.微卫星总长度分别为2 876 322 bp,2 689 710 bp,3 140 445 bp 和3 615 069 bp,分别占基因组序列总长度的0.73％,0.73％,0.84％和1.06％.在1～6个不同碱基重复类型完整型微卫星中,四种河鲀的6种碱基类型数目排序是一致的.均是单碱基重复数目最多,然后依次是二碱基、三碱基、四碱基、五碱基和六碱基.其中AC,A,C,AG,AGG,AT,AAT和AAC是四种河鲀共有的常见核心重复类别.东方鲀属(Takifugu)三种河鲀基因组微卫星分布特征极为相似,分析红鳍东方鲀和双斑东方鲀的遗传距离可能更为接近.鲀属(Tetraodon)黑青斑河鲀与其他三种东方鲀属河鲀除部分微卫星特征相似外,在微卫星总数、微卫星相对丰度和密度、部分碱基类型数目及类别方面和东方鲀属差距较大.这可能与两属鱼类地理分布及进行滑动复制的碱基组成有关,推测东方鲀属和鲀属基因组可能具有独特的进化机制.本研究为多种河鲀基因组特征分析、多种河鲀微卫星引物设计、不同属种河鲀遗传距离及亲缘关系的探究等奠定了基础.     

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。     

牛科动物转录组微卫星变异分析

微卫星(simples equencere peats, SSRs)广泛分布于生物基因组中,是最常用的分子标记之一。本研究利用生物信息学方法搜索和统计了7种牛科动物转录组中完整型SSRs序列,揭示不同重复类型SSRs变异水平,并对其生物信息学特征进行比较分析。在牛科动物转录组中,牛、绵羊和山羊SSRs总丰度较高,三者基本一致(157.98, 157.09 vs 158.52个/Mb);其次是瘤牛和水牛(140.70 vs 129.63个/Mb);牦牛微卫星总丰度较低(96.99个/Mb),藏羚羊微卫星总丰度最低(63.71个/Mb)。牛科动物转录组中SSRs所占比例普遍偏低,范围为0.156%～0.753%,三碱基SSRs占主导地位,并且以(CCG)n和(AGG)n为优势重复基元,这可能与转录组编码区三联体密码子高度保守有关。在这7种牛科动物转录组中,相同单碱基至六碱基SSRs重复拷贝数(repeat copy number, RCN)分布高度一致。通过牛科动物转录组SSRs变异系数(coefficient of variability, CV)分析表明,单碱基SSRs和二碱基SSRs重复拷贝数变异水平较高,其次RCN变异模式如下:三碱基SSRs四碱基SSRs六碱基SSRs五碱基SSRs,六碱基SSRs重复拷贝数变异水平有稍微增加的趋势。本研究为牛科动物SSR标记的开发、遗传多样性评估和遗传育种提供科学依据。     

中华按蚊全基因组微卫星的鉴定、特征及分布规律

【目的】中华按蚊Anopheles sinensis是我国及东南亚重要的传疟媒介。本研究在全基因组上鉴定和分析中华按蚊微卫星并注释微卫星相关基因的功能,为遗传分子标记的筛选提供依据,也为昆虫微卫星比较基因组学进一步研究提供基础。【方法】用MISA程序鉴定中华按蚊基因组微卫星;用Excel 2010统计微卫星长度,结合微卫星序列信息编写Perl脚本计算微卫星碱基含量;结合微卫星位置信息编写Perl脚本定位微卫星出现的基因区域,并对基因区的微卫星进行GO功能注释;运用WEGO比较中华按蚊和冈比亚按蚊An.gambiae含微卫星相关基因功能注释。【结果】共鉴定出105 981个微卫星,出现的密度是365.5个/Mb。其中100 391个(94.7%)微卫星是完整型微卫星,其余5 590个(5.3%)是复合型微卫星。单碱基微卫星最为丰富,共58 837个,占总微卫星数量的55.5%,其余依次是二碱基(30 345个,占28.6%)、三碱基(15 104个,占14.3%)、四碱基(1 530个,占1.4%)、五碱基(121个,占0.1%)和六碱基(44个,少于0.1%)微卫星。(A)n为最主要的微卫星,其次是(AC)n,(AG)n,(C)n,(AGC)n,(ATC)n,(ACG)n和(ACC)n,数量都在2 000个以上。中华按蚊基因组微卫星长度以10~20 bp为主(87.1%)。这些微卫星的AT含量(63%)明显高于GC含量(37%),仅三碱基微卫星的GC含量(53%)略高于AT含量(47%)。90 632个微卫星(85%)分布在基因间区,15 349个(15%)微卫星分布在基因区。在基因区,2 782个(3%)微卫星分布在外显子区,12 567个(12%)分布在内含子区。GO注释比较中华按蚊和冈比亚按蚊含微卫星的基因,发现这两个物种各小类基因所占总基因数的百分比基本一致,但电子传递类(electron carrier)基因在中华按蚊所占百分比(0.9%)明显高于冈比亚按蚊(0.1%)。【结论】这是蚊虫中首个在全基因组上系统的微卫星研究工作,为进一步通过微卫星作为分子标记开展中华按蚊种群遗传学、遗传变异、功能基因的遗传定位和调控机制研究奠定了基础,也为昆虫微卫星的多样性和进化研究积累了科学素材。     

大鼠基因组微卫星分布特征分析

大鼠(Rattus norvegicus)是重要的生物模型动物,已被广泛应用于医学和药学研究。利用生物信息学方法对大鼠基因组外显子、基因间隔区以及内含子微卫星进行了搜索与分析,并对外显子微卫星所在的基因进行了定位,以及通过Blast2Go和KAAS程序进行功能分类注释。分析表明:(1)不同区域微卫星数量上,基因间隔区(1 104 149)内含子(806 024)外显子(4 665),外显子不同区域微卫星数量表现为CDS3'UTR5'UTR;(2)外显子中出现最多的重复拷贝类别是A,其次是AAG、AGC,三碱基和四碱基微卫星在外显子微卫星中最多,占56.7%;基因间隔区微卫星和内含子均以二碱基数量最多,两者出现最多重复拷贝类别均是AC,其次均是A、AG;(3)外显子区、基因间隔区和内含子区不同重复类型重复次数均以最小重复次数,微卫星数量最多;(4)外显子微卫星序列2 330条,位于1 203个基因上,GO注释表明涉及细胞成分(cellular component)占46.9%、生物过程(biological process)占39.3%和分子功能(molecular function)占13.8%;(5)KEGG通路分析表明,与人类疾病相关的基因最多(431),其次是参与机体系统相关的基因(387),和细胞过程相关基因最少(158)。本研究将为进一步分析大鼠微卫星功能及微卫星分子标记提供理论依据。     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775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。     

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。     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.     

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at and/or . The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

An unusually low microsatellite mutation rate in Dictyostelium discoideum, an organism with unusually abundant microsatellites

The genome of the social amoeba Dictyostelium discoideum is known to have a very high density of microsatellite repeats, including thousands of triplet microsatellite repeats in coding regions that apparently code for long runs of single amino acids. We used a mutation accumulation study to see if unusually high microsatellite mutation rates contribute to this pattern. There was a modest bias toward mutations that increase repeat number, but because upward mutations were smaller than downward ones, this did not lead to a net average increase in size. Longer microsatellites had higher mutation rates than shorter ones, but did not show greater directional bias. The most striking finding is that the overall mutation rate is the lowest reported for microsatellites: approximately 1 x 10(-6) for 10 dinucleotide loci and 6 x 10(-6) for 52 trinucleotide loci (which were longer). High microsatellite mutation rates therefore do not explain the high incidence of microsatellites. The causal relation may in fact be reversed, with low mutation rates evolving to protect against deleterious fitness effects of mutation at the numerous microsatellites.     

Development and diversity of Andean-derived, gene-based microsatellites for common bean (Phaseolus vulgaris L.)

Background  

Gene-based (genic) microsatellites are a useful tool for plant genetics and simple sequence repeat loci can often be found in coding regions of the genome. While EST sequencing can be used to discover genic microsatellites, direct screening of cDNA libraries for repeat motifs can save on overall sequencing costs. The objective of this research was to screen a large cDNA library from and Andean common bean genotype for six di-nucleotide and tri-nucleotide repeat motifs through a filter hybridization approach and to develop microsatellite markers from positive clones.     

Map and analysis of microsatellites in the genome of <Emphasis Type="Italic">Populus</Emphasis>: The first sequenced perennial plant

We mapped and analyzed the microsatellites throughout 284295605 base pairs of the unambiguously assembled sequence scaffolds along 19 chromosomes of the haploid poplar genome. Totally, we found 150985 SSRs with repeat unit lengths between 2 and 5 bp. The established microsatellite physical map demonstrated that SSRs were distributed relatively evenly across the genome of Populus. On average, These SSRs occurred every 1883 bp within the poplar genome and the SSR densities in intergenic regions, introns, exons and UTRs were 85.4%, 10.7%, 2.7% and 1.2%, respectively. We took di-, tri-, tetra-and pentamers as the four classes of repeat units and found that the density of each class of SSRs decreased with the repeat unit lengths except for the tetranucleotide repeats. It was noteworthy that the length diversification of microsatellite sequences was negatively correlated with their repeat unit length and the SSRs with shorter repeat units gained repeats faster than the SSRs with longer repeat units. We also found that the GC content of poplar sequence significantly correlated with densities of SSRs with uneven repeat unit lengths (tri-and penta-), but had no significant correlation with densities of SSRs with even repeat unit lengths (di-and tetra-). In poplar genome, there were evidences that the occurrence of different microsatellites was under selection and the GC content in SSR sequences was found to significantly relate to the functional importance of microsatellites.     

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.