基于转录组测序信息的水生植物莕菜SSR标记开发

利用软件MISA对水生植物莕菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST-SSR位点。在莕菜的EST-SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57.31%和30.87%,其中AG/CT、AAG/CTT分别是二、三重复单元类型的优势重复基元,分别占总SSR的29.76%和8.66%。随机挑选了130对EST-SSR引物对莕菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为 47.44%。这些多态性引物共检测出114个等位基因,每个位点2~6个,平均3.08个。观测杂合度（H_o）及预期杂合度（H_e）分别在0.229~1.000和0.351~0.756之间,多态信息含量PIC值在0.286~0.698之间,平均达0.495。以上研究结果表明,通过莕菜转录组测序产生的EST数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究莕菜的居群遗传多样性及其遗传结构提供了工具。     

西花蓟马EST-SSR信息分析、标记筛选及其与Genomic-SSR的多态性比较

西花蓟马Frankliniella occidentalis是一种世界性入侵昆虫, 近年来传入我国并不断扩散蔓延。基于简单重复序列（simple sequence repeats, SSRs）的西花蓟马种群遗传结构研究对于揭示其传播途径等具有重要的指导价值。本研究对来源于西花蓟马的13 839条EST序列进行了uni-EST组装、 EST-SSR信息分析以及标记筛选, 并比较了EST-SSR与Genomic-SSR在分析遗传多样性方面的差异。结果表明： 在7 707个singlets中共找到2 623个SSR位点, 分布于1 930个uni-EST中, 平均每2.21 kb就出现一个SSR位点。重复单元中, 以单碱基重复单元为主（83.00%）, 其次是四碱基重复单元（11.17%）, 而二、 三、 五和六碱基重复单元所占比例较低（分别为1.41%, 0.80%, 2.02%和0.91%）。设计出的22对EST-SSR引物中, 4对引物能稳定扩增出清晰的目的条带; 荧光标记毛细管电泳发现3对引物表现出多态性。西花蓟马EST-SSR与Genomic-SSR多态性分析表明, 这3对多态性EST-SSR引物揭示的多态信息含量（PIC）为0.48~0.69, 比5对多态性Genomic-SSR引物揭示的PIC（0.88~0.92）略低。本研究结果可为今后更深入开展西花蓟马的种群遗传结构分析提供帮助。     

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。     

基于转录组测序信息的水生植物蕃菜SSR标记开发

利用软件MISA对水生植物蓄菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST—SSR位点。在蓄菜的EST—SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57．31％和30．87％,其中AG／CT、AAG／C1_r分别是二、三重复单元类型的优势重复基元,分别占总SSR的29．76％和8．66％。随机挑选了130对EST-SSR引物对蓄菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为47．44％。这些多态性引物共检测出114个等位基因,每个位点2—6个,平均3．08个。观测杂合度（心）及预期杂合度（He）分别在0．229～1．000和0．351-0．756之间,多态信息含量PIC值在0．286～O．698之间,平均达0．495。以上研究结果表明,通过萏菜转录组测序产生的EsT数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究苔菜的居群遗传多样性及其遗传结构提供了工具。     

三种人参属植物的EST-SSR信息分析及其在三七中的应用

为了解人参属植物EST中SSR分布特点及其在三七SSR标记中的应用,利用生物信息学方法,用primer 3软件对dbEST数据库中人参属植物人参、西洋参和三七的EST序列进行搜索,发现人参属植物EST-SSR出现频率为11.54%,平均每4.39 kb出现1个SSR.人参属植物单核苷酸重复基元占主导地位,其次为三核苷酸重复基元和二核苷酸重复基元,分别占总SSR的54.48%、17.31%和16.36%.人参属EST-SSR的优势类型为A/T和AT/TA,分别占54.73%和8.21%.根据人参属植物EST中的SSR设计48对引物,在合适的PCR扩增体系下,用5个三七样品的DNA为模板进行PCR扩增,引物有效扩增率85.42%,其中多态性引物占可扩增引物的70.73%.结果表明,利用人参属EST序列开发三七EST-SSR标记是可行的.     

Characterization and molecular marker screening of EST-SSRs and their polymorphism compared with Genomic-SSRs in Frankliniella occidentalis ( Thysanoptera: Thripidae)

西花蓟马Frankliniella occidentalis是一种世界性入侵昆虫,近年来传入我国并不断扩散蔓延.基于简单重复序列(simple sequence repeats,SSRs)的西花蓟马种群遗传结构研究对于揭示其传播途径等具有重要的指导价值.本研究对来源于西花蓟马的13839条EST序列进行了uni-EST组装、EST-SSR信息分析以及标记筛选,并比较了EST-SSR与Genomic-SSR在分析遗传多样性方面的差异.结果表明:在7707个singlets中共找到2623个SSR位点,分布于1 930个uni-EST中,平均每2.21 kb就出现一个SSR位点.重复单元中,以单碱基重复单元为主(83.00％),其次是四碱基重复单元(11.17％),而二、三、五和六碱基重复单元所占比例较低(分别为1.41％,0.80％,2.02％和0.91％).设计出的22对EST-SSR引物中,4对引物能稳定扩增出清晰的目的条带;荧光标记毛细管电泳发现3对引物表现出多态性.西花蓟马EST-SSR与Genomic-SSR多态性分析表明,这3对多态性EST-SSR引物揭示的多态信息含量(PIC)为0.48 ～0.69,比5对多态性Genomic-SSR引物揭示的PIC(0.88 ～0.92)略低.本研究结果可为今后更深入开展西花蓟马的种群遗传结构分析提供帮助.     

杂交兰转录组SSR信息分析及EST-SSR标记开发应用

该研究主要开发筛选适用于杂交兰的EST-SSR引物,为杂交兰种质资源评价和遗传变异研究等提供可靠的分子标记。该研究对杂交兰进行转录组高通量测序,挖掘SSR位点和开发EST-SSR标记,并对不同种质的遗传多样性进行分析。结果表明,从31724条杂交兰Unigene中检测出18603个SSR位点,SSR出现频率为58.64%;SSR位点中的主导类型是单核苷酸重复,占总SSR的65.10%,其次是二核苷酸(23.56%)和三核苷酸(10.76%)重复;优势重复基元为A/T、AG/CT、AT/AT和AAG/CTT,分别占总位点的64.72%、13.74%、8.19%和2.51%。利用Primer Premier 5.0共设计了565对SSR引物,从筛选出的64对有效扩增引物中随机选择28对引物,对40份杂交兰种质进行多态性验证与遗传关系分析,其中16对(占57.14%)引物表现出可重复的高多态性,平均多态信息量(PIC)达0.789。基于扩增的多态性SSR信息,40份种质资源可聚为4类,聚类结果与其遗传背景基本一致。该研究印证了转录组测序获得的Unigene是SSR标记开发的有效来源,开发的EST-SSR引物可为杂交兰及近缘种的良种鉴别、遗传图谱构建、分子标记辅助育种及功能基因挖掘等提供有价值的候选标记。     

燕麦EST-SSR标记的开发和利用

为拓展分子标记在燕麦种质资源分析与鉴定中的应用,利用公共数据库中的25376条EST(expressed sequence tags)序列,开展了燕麦EST-SSR功能性标记的开发和利用研究。25376条EST序列经拼接去冗余后获得了11618条序列,从中筛选出含有不同重复基元的SSR且重复次数较多、长度较长的556条EST序列进行引物设计,开发了50对燕麦EST-SSR引物,通过筛选得到40对有效的EST-SSR引物。选取其中4对引物对5个燕麦种质资源进行了PCR扩增及产物测序,结果表明扩增条带多态性是由SSR差异造成的。利用40对ESTSSR引物对15个六倍体燕麦种质资源进行遗传多样性分析,共扩增出89个等位基因,平均每对引物产生2.23个等位基因;UPGMA聚类分析表明,15个六倍体燕麦种质资源在Dice系数为0.93处聚为3支,基本上是按照不同种进行聚类的,在相同种中又根据地理来源分别聚集成支。利用40对EST-SSR引物对31个遗传背景不清的燕麦种质资源进行基因组倍性鉴定,发现这些种质中可能存在有四倍体和二倍体的燕麦新资源。本研究开发的燕麦EST-SSR功能性标记将在燕麦遗传多样性分析、遗传图谱构建及燕麦属内种间基因组鉴定等方面发挥重要作用。     

龙眼顶芽转录组简单重复序列(SSR)标记信息分析及分子标记开发

随着新一代测序技术的发展,大量的转录组数据和表达序列标签(EST)成为开发简单重复序列(SSR)标记的可利用资源。本研究利用MISA软件筛选龙眼(Dimocarpus longan)顶芽转录组数据库序列,从114 445条龙眼转录组unigene序列中发现11 546个SSR位点,SSR出现频率为10.09%。其中1 975条unigene含有两个或两个以上EST-SSR位点,占所有SSR位点的比例为17.10%,SSR出现的平均距离为7.52 kb。从龙眼转录组SSR核苷酸基序类型来看,二核苷酸(52.11%)和三核苷酸(46.15%)出现频率最高,占所有核苷酸出现频率的99.26%。在龙眼转录组SSR中二核苷酸重复基元出现频率最高的是AG/CT(4 250个,占36.81%),三核苷酸重复基元出现频率最高的是AAG/CTT(1 109个,占9.61%)。对含SSR位点的9 571条unigene序列进行引物设计,共设计出了8 347对SSR位点特异引物。随机挑选合成50对EST-SSR引物,以‘石硖’、‘储良’、‘古山2号’、‘立冬本’等四份龙眼材料的基因组DNA为模板对这批引物进行PCR扩增、筛选,结果表明,其中21对引物能产生理想的PCR产物,有效扩增率为42%;16对引物扩增条带具有多态性,占有效引物的76.2%;16对多态性引物共扩增获得50个条带,其中多态性片段21个,每对引物平均产生1.31个多态性片段。     

砂梨EST-SSR引物开发及其应用

利用GenBank和GDR数据库中的995条梨EST序列开发砂梨SSR引物,并根据开发的EST-SSR引物对砂梨'西子绿'×'喜水'F1群体的遗传变异进行分析.结果发现:(1)60个SSR位点分布于54条EST序列中,占整个EST数据库的5.4%,其中二核苷酸重复基元出现频率最高,达51.7%,其次为三核苷酸重复基元占25%.23类重复基序中AT重复基序出现的频率最高,为32.3%.(2)利用开发的25对EST-SSR引物对'西子绿'×'喜水'F1群体的遗传变异分析结果表明,其中9对引物呈现多态性,多态性引物占设计引物的36%;多态性引物扩增产物在F1群体中的等位基因数(No)平均为2,有效等位基因数(Ne)平均为1.932 4,平均杂合度观测值(Ho)和期望杂合度(He)分别为1和0.480 9.     

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。     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Application of functional genomic information to develop efficient EST-SSRs for the chicken (Gallus gallus)

Many years of domestication and breeding have given rise to the wide range of chicken breeds that exist today; however, an increasing number of local chicken breeds are under threat of extinction. A comprehensive characterization of chicken markers (especially type I markers) is needed to monitor and conserve genetic diversity in this species. The explosion of genomics and functional genomics information in recent years has opened new possibilities for the generation of molecular markers. We analyzed a large number of expressed sequence tags (ESTs) to test the possibility of using EST-derived microsatellite markers for investigating the Gallus gallus genome. Chromosomal locations for the majority of these SSRs were predicted. Of the 31,576 unigenes assembled from the 544,150 redundant EST sequences, 1757 SSR markers were discovered on 1544 ESTs, using the SSRLocator software, with an average density of 28.7 kb per SSR. The dimer motifs were the most frequent (46.38%), followed by trimeric (38.58%), tetrameric (10.19%), pentameric (4.5%), and hexameric (<1%) markers. Different from the case for cattle and sheep, AT/TA was the most abundant dimeric repeat, accounting for 41.71% of all dimeric repeats in the chicken ESTs. The EST-SSR distribution was not uniform among the chromosomes; the majority of the EST-SSRs were located on chromosomes GGA2 and GGA10. We found that most of the EST-SSRs are involved in positive regulation of cellular and metabolic processes. This is the first time that EST sequences have been mined to find chicken microsatellites. On average, 3.8% of the G. gallus UniGene sequences could be exploited for development of EST-SSRs, indicating a good source for molecular markers as well as for functional genome analysis.     

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。     

亚麻EST序列中SSR标记的筛选

利用亚麻NCBI数据库中的7 941条亚麻EST序列进行SSR的筛选,共发现222个SSR,占整个EST数据库的2.73%,其中三核苷酸重复单元的EST-SSR占总SSR的72.1%,二核苷酸和四核苷酸二者出现的频率基本相近,分别占总SSR的14.4%和13.5%.AGAA是四核苷酸中的优势重复类型,占四核苷酸重复类型的67.67%.设计的21对EST-SSR引物中有18对在10个亚麻材料中有扩增产物,占设计引物的85%,有14对产物条带比较清晰并具有多态性.基于SSR标记进行聚类分析,可将10个亚麻材料划分为3个组.本研究建立的亚麻SSR标记,为亚麻遗传多样性鉴定、分子作图等研究提供了一种有效的分子标记系统.     

A database of simple sequence repeats from cereal and legume expressed sequence tags mined in silico: survey and evaluation

Simple sequence repeats (SSRs) or microsatellites are an important class of molecular markers for genome analysis and plant breeding applications. In this paper, the SSR distributions within ESTs from the legumes soybean (Glycine max, representing 135.86 Mb), medicago (Medicago truncatula, 121.1 Mb) and lotus (Lotus japonicus, 45.4 Mb) have been studied relative to the distributions in cereals such as sorghum (Sorghum bicolor, 98.9 Mb), rice (Oryza sativa, 143.9 Mb) and maize (Zea mays, 183.7 Mb). The relative abundance, density, composition and putative annotations of di-, tri-, tetra- and penta-nucleotide repeats have been compared and SSR containing ESTs (SSR-ESTs) have been clustered to give a non-redundant set of EST-SSRs, available in a database. Further, a subset of such candidate EST-SSRs from sorghum have been tested for their ability to detect polymorphism between Striga-susceptible, stay-green drought tolerant mapping population parent 'E 36-1' and its Striga-resistant, non-stay-green counterpart 'N13'. Primer sets for 64% of the EST-SSRs tested produced a clear and specific PCR product band and 34% of these detected scorable polymorphism between the N13 and E 36-1 parental lines. Over half of these markers have been genotyped on 94 RILs from the (N13 x E 36-1)-based mapping population, with 42 markers mapping onto the ten sorghum linkage groups. This establishes the value of this database as a resource of molecular markers for practical applications in cereal and legume genetics and breeding. The primer pairs for non-redundant EST-SSRs have been designed and are freely available through the database (http://intranet.icrisat.org/gt1/ssr/ssrdatabase.html).     

Development of EST-SSRs by an efficient FIASCO-based strategy: a case study in rare minnow (Gobiocyrpis rarus)

Type I markers are useful for comparative mapping and other genetic analyses, but relatively difficult to develop. In the present study a microsatellite (SSR)-enriched cDNA library was constructed for the first time using the fast isolation by AFLP of sequences containing repeats (FIASCO) method in a small fish, Chinese rare minnow (Gobiocypris rarus). A total of 97.4% of the expressed sequence tags (ESTs) contained targeted CA-repeats, in which 29 unique EST-SSRs were identified. Ten out of the 28 loci for which primer pairs were designed were polymorphic with alleles ranging from three to seven (mean 4.50). Some of these EST-SSRs can be amplified in other species. These results proved that cDNA-FIASCO is an efficient way to isolate novel EST-SSRs in a fish.     

Development of EST-SSRs by an Efficient FIASCO-Based Strategy: A Case Study in Rare Minnow (Gobiocyrpis Rarus)

Type I markers are useful for comparative mapping and other genetic analyses, but relatively difficult to develop. In the present study a microsatellite (SSR)-enriched cDNA library was constructed for the first time using the fast isolation by AFLP of sequences containing repeats (FIASCO) method in a small fish, Chinese rare minnow (Gobiocypris rarus). A total of 97.4% of the expressed sequence tags (ESTs) contained targeted CA-repeats, in which 29 unique EST-SSRs were identified. Ten out of the 28 loci for which primer pairs were designed were polymorphic with alleles ranging from three to seven (mean 4.50). Some of these EST-SSRs can be amplified in other species. These results proved that cDNA-FIASCO is an efficient way to isolate novel EST-SSRs in a fish.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.