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。     

甜瓜EST序列中微卫星的分布特征

GenBank中35547条甜瓜EST经去冗余处理后,得到总长度为250.3Mb的无冗余EST34438条。这些序列中有2813个微卫星简单重复序列（Simple sequence repeat,SSR）,分布于2107条EST中,出现频率为8.16%,平均分布距离为8.90kb。三核苷酸重复是主导重复类型,占SSR总数的47.14%;其次是二核苷酸和单核苷酸重复,分别占SSR总数的20.72%和16.99%。AAG/TTC是优势重复基元,占微卫星总数的29.26%,AG/CT和A/T分别占14.61%和16.25%。在所有的SSR中,重复次数为4～10次的占70.32%,长度为12～20bp的占51.12%。并对这些SSR的多态性潜能进行了评价。     

鲫鱼(Carassius auratus)表达序列标签资源的SSR构成与分布分析

分析鲫鱼EST资源的SSR信息,为开发EST-SSR标记奠定基础.从GenBank中获得鲫鱼EST序列,然后用Sequencher 4.8软件进行序列拼接得到Uni-EST序列,再通过SciRoKo 3.4软件扫描Uni-EST序列中的SSR,最后得出EST-SSR的分布特征、频率和重复基元类型等特征.通过搜索共获得9 230条鲫鱼EST原始序列,通过使用计算机软件进行预处理共得到全长为3.81×106 bp的无冗余Uni-EST 7 092条.在这些序列中共搜索出597个SSR位点,分布在545条Uni-EST序列中,发生频率为8.13％,EST-SSR的平均长度为(19.34±6.23) bp,平均每Mb含156.55个SSR位点.单核苷酸重复在鲫鱼EST-SSR中占主导地位,发生频率为39.53％,其次为二核苷酸重复,发生频率为36.68％以及三核苷酸重复的15.41％.在所有非单核苷酸重复基元中,AC基元出现频率最高,其次为AG.设计出引物404对.最后得出结论鲫鱼EST中SSR出现的频率较高,并且类型较为丰富,为进行遗传多样性分析和重要经济性状筛选等方面的研究提供了基础和指导.     

甘薯EST资源的SSR信息分析

从NCBI公共数据库下载获得22371条甘薯EST序列,去除低质量的和冗余的序列后,得到总长为5．09×10^3kb的9204条唯一序列。从这些序列中搜索到总共436个SSR位点,平均相距11．68kb出现一个SSR。这些SSR的出现频率和平均长度分别为4．4％和24．28bp。在2-6bp的重复基元中,六核苷酸重复基元出现频率最（30．96％）,其次是三核苷酸重复基元（29．59％）和二核苷酸重复基元（24．54％）。出现最多的重复基元是AG／CT（16．28％）,其次是AAG／CTT（11．01％）。     

褐飞虱EST资源的微卫星信息分析

表达序列标签（expressed sequence tags,ESTs）是开发微卫星标记的一个重要的资源。褐飞虱Nilaparvata lugens （Stål） EST序列的公布为开发EST-SSRs提供了宝贵的数据资源,本研究利用生物信息学对NCBI公共数据库中的37 398条褐飞虱ESTs序列进行EST-SSRs特征分析,得到全长为7 619 324 kb的无冗余EST 9 852条。按照3个不同的查找标准在这些序列中搜索SSR。查找结果显示：褐飞虱EST-SSRs主要重复基元以1～3碱基为主,占总EST-SSR的95％以上。在单碱基重复基元中,A/T是占优势的重复基元,在二相重复类型中,AG/CT重复基元出现的频率最多,而AAG/CTT是三相重复中占绝对优势的重复基元。在褐飞虱EST-SSRs中未查找到GC重复基元。以100 bp为参照,在3种查找标准下含有SSR的EST序列中两端侧翼序列均≥100 bp的序列分别为738,89和42个。通过分析褐飞虱EST-SSRs标记可以为褐飞虱和近缘种的SSR标记的开发提供信息,同时通过分析褐飞虱EST-SSRs的分布频率和分布特征可以为昆虫EST-SSRs的研究提供借鉴和参考。     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

人参EST资源的SSR信息分析

从7055条人参EST序列中搜索出791个SSR,其出现频率为11．21％,平均长度为21．37bp,平均分布频率为1／5．7kb。二核苷酸重复是主要的重复类型,占全部EST-SSR的56．89％,其次是三核苷酸重复的占全部SSR的21．11％。AT、GAA是二核苷酸和三核苷酸中出现次数最多的重复基元类型,分别占28．89％和10．18％。     

甘薯与牵牛EST资源的SSR信息分析

为挖掘番薯（Ipomoea）属EST-SSR资源,从NCBI数据库下载23406条甘薯（Ipomoea batatas (L.) Lam.）EST和62282条牵牛（Ipomoea nil (L.) Roth）EST,利用生物信息学软件预处理、去冗余、拼接处理后得到12812条无冗余的甘薯EST（6.70 Mb）和28422条牵牛唯一序列（17.19 Mb）。对这些序列进行SSR搜索,在甘薯上获得328个SSR位点,发生频率为2.56%;牵牛上筛选到962个SSR位点,出现频率为3.38%。甘薯和牵牛EST-SSR具有多个共同特征：在SSR位点中,主要是二核苷酸重复类型,其次是三核苷酸重复;在二核苷酸重复中,出现最多的重复基序为AG/CT,其次是AT/AT;在三核苷酸重复中,主要基序是AAG/CCT;SSR位点的长度主要集中在20~22 bp。结果表明,这些搜索出的EST-SSR重复基序类型丰富、多态性潜能高,具有较高的开发和利用价值。     

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。     

芦笋EST资源的SSR信息分析

为了在芦笋中开发EST-SSR功能性标记,对来源于NCBI公共数据库的8590条芦笋(AsparagusofficinalisL.)EST序列进行简单重复序列SSR搜索。剔除冗余序列,得到非冗余序列8377条。在非冗余序列中共挖掘出469个EST-SSR,平均相隔14.80kb出现1个SSR。在所有的重复基序中,二核苷酸重复基序的SSR所占比例最高40.51%(190/469),其次是三核苷酸34.97%(164/469),六核苷酸21.11%(99/469)。在所有基序里,CT/AG出现的频率最高有62次,占全部重复基序的13.22%(62/469)。选取含SSR的EST序列30条,并利用primer5软件设计引物,进行SSR位点的扩增,其中27对引物扩增产物,24对有较清晰可靠的目标扩增条带,占引物数的80%,且所检测出的芦笋等位基因数量较丰富,平均4.93个/对。这些EST-SSR标记的开发将有助于芦笋群体遗传多样性、遗传图谱构建、基因定位、分子标记和系谱分析等方面的研究。     

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。     

Genome-Wide Comparative Analysis of Microsatellites in Pineapple

Pineapple (Ananas comosus (L.) Merrill) is the second most important tropical fruit in term of international trade. The availability of whole genomic sequences and expressed sequence tags (ESTs) offers an opportunity to identify and characterize microsatellite or simple sequence repeat (SSR) markers in pineapple. A total of 278,245 SSRs and 41,962 SSRs with an overall density of 728.57 SSRs/Mb and 619.37 SSRs/Mb were mined from genomic and ESTs sequences, respectively. 5′-untranslated regions (5′-UTRs) had the greatest amount of SSRs, 3.6–5.2 fold higher SSR density than other regions. For repeat length, 12 bp was the predominant repeat length in both assembled genome and ESTs. Class I SSRs were underrepresented compared with class II SSRs. For motif length, dinucleotide repeats were the most abundant in genomic sequences, whereas trinucleotides were the most common motif in ESTs. Tri- and hexanucleotides of total SSRs were more prevalent in ESTs than in the whole genome. The SSR frequency decreased dramatically as repeat times increased. AT was the most frequent single motif across the entire genome while AG was the most abundant motif in ESTs. Across six examined plant species, the pineapple genome displayed the highest density, substantially more than the second-place cucumber. Annotation and expression analyses were also conducted for genes containing SSRs. This thorough analysis of SSR markers in pineapple provided valuable information on the frequency and distribution of SSRs in the pineapple genome. This genomic resource will expedite genomic research and pineapple improvement.     

Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species.

Simple sequence repeat (SSR) markers are widely used in many plant and animal genomes due to their abundance, hypervariability, and suitability for high-throughput analysis. Development of SSR markers using molecular methods is time consuming, laborious, and expensive. Use of computational approaches to mine ever-increasing sequences such as expressed sequence tags (ESTs) in public databases permits rapid and economical discovery of SSRs. Most of such efforts to date focused on mining SSRs from monocotyledonous ESTs. In this study, we have computationally mined and examined the abundance of SSRs in more than 1.54 million ESTs belonging to 55 dicotyledonous species. The frequency of ESTs containing SSRs among species ranged from 2.65% to 16.82%. Dinucleotide repeats were found to be the most abundant followed by tri- or mono-nucleotide repeats. The motifs A/T, AG/GA/CT/TC, and AAG/AGA/GAA/CTT/TTC/TCT were the predominant mono-, di-, and tri-nucleotide SSRs, respectively. Most of the mononucleotide SSRs contained 15-25 repeats, whereas the majority of the di- and tri-nucleotide SSRs contained 5-10 repeats. The comprehensive SSR survey data presented here demonstrates the potential of in silico mining of ESTs for rapid development of SSR markers for genetic analysis and applications in dicotyledonous crops.     

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.     

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.     

亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。