越橘属植物EST-SSR分子标记开发及遗传多样性分析

以84份越橘种质为材料,从NCBI公共数据库下载22 402条越橘属(Vaccinium)EST序列,通过CAP3组装软件将EST序列拼接成11 541条unigene序列,其中2 076条unigene序列含有2 679个SSR位点。二核苷酸和三核苷酸重复是主要的SSR类型,约占SSR总数的96.01%。利用Primer Premier 5.0软件设计81对引物,其中55对引物在供试越橘种质中扩增出理想的PCR产物,55对引物均有多态性。聚类分析结果显示,在遗传相似系数为0.70时,可以将供试越橘种质分成两大类。越橘EST-SSR标记可以用于种质鉴定与遗传多样性分析。     

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。     

三年桐EST-SSR标记的开发与种质遗传多样性分析

以三年桐品种对年桐发育中的种子为材料,构建c DNA文库,获得3202个原始EST序列,经过去除冗余和低质量序列后,获得1047条非冗余单一序列,利用MISA软件从其中的173个非冗余EST序列中搜索得到212个SSR位点。其中,二核苷酸重复类型为主导(68.39%),其次是三核苷酸重复类型(25.94%),其优势基元为AG/CT(43.87%)、AT/AT(19.34%)和AGC/GCT(5.66%)。针对可设计引物的68个序列进行EST-SSR引物设计,结合PCR扩增和数据分析,鉴定出14对多态性标记,用该14对引物对169份三年桐种质资源进行遗传多样性检测,共得到41个等位基因(Na),平均每对引物扩增出2.93个等位基因,期望杂合度(He)变幅为0.08~0.63,平均值为0.33,多态性信息含量(PIC)变幅为0.08~0.56,平均值为0.3,表明14对EST-SSR标记多态性较高,能较好地反映三年桐种质的遗传多样性。三年桐种质资源按群体进行非加权配对算术平均法(UPGMA)聚类分析,群体间的相似度变幅为0.9604~0.9986,遗传距离变幅为0.0022~0.0404,表明三年桐群体间的遗传多样性相对较低,亲缘关系较近。对169份三年桐种质资源个体进行聚类分析,结果显示不同地理种源的遗传关系缺乏明显的地理结构。     

高丹草EST-SSR标记的开发及其遗传多样性

对NCBI数据库中210 878条高粱EST序列进行处理, 得到57 498条无冗余EST序列, 经SSR搜索, 发现3 338个SSR分布于3 116条EST序列中, 分布频率为1/11.28 kb, 包括215种基元重复类型。其中三核苷酸重复最高, 占68.33%, 二核苷酸重复占17.97%。3 338条SSR序列中有1 694条序列能够设计出引物, 所占比例为50.75%。选取14对引物进行合成, 对50份高丹草、7份高粱和3份苏丹草材料进行了EST-SSR扩增, 共检测到72个等位变异, 平均每对引物检测出5.14个基因位点。每对引物多态性指数范围为0.54~0.93, 遗传距离的变化范围0.1646~0.6398。结果显示：供试材料具有较丰富的遗传多样性, 根据EST-SSR数据的聚类分析, 将供试材料按亲缘关系远近分为5大类, 来源相同的品种大致聚在一类, 呈现出一定的地域性分布规律。同时发现4个特异分子标记, 其中引物D1763只对314A和白壳苏丹草杂交后代GB-4-2高丹草审定品种产生特异性, 此标记已作为该材料的特异性标记用于种质资源的鉴定中, 同时表明, EST-SSR标记是高丹草遗传多样性及特异性研究的一种有效方法。     

菠菜性别相关 EST-SSR 标记的开发及应用

为了明确菠菜EST序列中SSR的总体特点,开发菠菜EST-SSR引物;为利用EST-SSR引物进行菠菜性别相关特异序列的克隆奠定基础,本文从NCBI上获得1093条EST,利用在线软件SSRIT检测所含SSR序列,并进行分析。共检索出68条SSR序列,分布于64条EST中,检出率为6.22%,包括22种重复基元。其中二核苷酸重复基元的EST-SSR占主导地位,占总SSR数目的32.3%。利用在线引物设计软件Primer3.0设计了7对EST-SSR引物,在适合的PCR反应体系下,分别以雌、雄菠菜DNA基因组为模板,对设计的EST-SSR引物进行筛选,结果显示以EST序列HS097148设计的一对引物从菠菜雌雄基因组中扩增出一条雄性特异的条带,表明通过菠菜EST-SSR引物获得菠菜性别相关特异序列是可行的。     

梅EST-SSR标记的开发及利用

利用MISA软件对10 123条梅EST序列进行SSR位点查找,得到含SSR位点的序列935条,SSR位点1233个,平均每100条EST序列中含有12.18个SSR位点.2核苷酸、3核苷酸重复是最主要的重复类型,分别占35.52%和41.36%.设计了40对EST-SSR引物并进行扩增,有24对引物能扩增出理想的PCR 产物,其中17对引物具有较好的扩增多态性.测序后发现13对引物中有73.08%的片段具有相应的SSR位点,对杏DNA指纹中部分谱带的测序结果也证明了是梅扩增出的相应的SSR位点.根据本研究含有SSR位点的测序结果推算,从梅EST中开发真实SSR位点的数目为901.随机选择13对引物对杏和梅进行DNA指纹构建与遗传多样性分析,结果发现,来源于梅的EST-SSR引物在杏中有很高的通用性,这些引物把梅和杏分成了两大群体,说明他们是遗传差异明显的两种植物.     

EST-SSR及其在植物基因组学研究中的应用

数量迅速增加的表达序列标签已经成为开发分子标记的重要资源。EST—SSR是基于表达序列标签开发微卫星的一种新型分子标记,与基因组SSR相比,EST-SSR具有在植物物种之间可转移性的优点。目前,EST—SSR被广泛应用于植物基因组学研究如遗传图谱构建、比较作图、遗传多样性评价、种质鉴定、系统发育与进化研究等方面。该文介绍了EST—SSR原理、引物开发、实验方法,并对其物种间通用性以及其在植物基因组研究中的应用进行了评述。     

麦红吸浆虫唾腺EST-SSRs的信息分析及分子标记筛选

昆虫EST资源库的扩充为开发新的分子标记提供了宝贵的资源。本研究对NCBI的EST数据库中来源于麦红吸浆虫Sitodiplosis mosellana唾腺的1 217条EST序列进行了unigene组装、 SSR信息分析和EST-SSR分子标记筛选。结果表明： 在1 047个unigenes中共找到141个SSR位点, 分布于106个（10.12%）unigenes中, 平均每3.49 kb出现一个SSR位点。在1~6碱基重复基元中, 1~3碱基是主要重复类型, 占总SSR的97.16%以上。A/T（31.21%）, AC/GT（15.60%）和AAC/GTT（9.22%）分别是单、 双和三碱基中占优势的重复基元类型。利用Primer Premier 5.0软件对查找的EST SSRs进行引物设计, 并以麦红吸浆虫基因组DNA为模板, 对从中选出的26对SSR引物进行多态性检测。结果有20对（76.92%）引物能扩增出清晰的目的条带, 并且其中9对（45%）引物表现出多态性。多态性分析结果表明, 从9对EST-SSR引物中, 共检测到51个等位基因, 平均每个位点含有等位基因数为5.67, 平均期望杂合度为0.65, 平均多态信息含量为0.60。本研究能够为今后麦红吸浆虫的种群遗传结构与遗传多样性研究提供帮助。     

华仁杏EST-SSR标记引物设计与开发

随着生物科技的进步,ESTs（表达序列标签）已经成为开发SSR（简单重复序列）标记的重要资源。本文利用NCBI公共数据库下载蔷薇科EST序列22 458条,使用SSRHunter1.3软件进行了SSR搜索,从中获得22 527条SSR,应用Primer5.0软件设计并经由Oligo7.0软件检测,共得到61对EST-SSR引物。利用这些引物对8个华仁杏品种进行了PCR扩增及检测,得到10对能产生清晰多态性条带的EST-SSR标记,标明了10对引物的序列,为进一步开展华仁杏SSR分子标记辅助育种研究奠定了基础。     

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。     

EST derived SSR markers for comparative mapping in wheat and rice

Structural and functional relationships between the genomes of hexaploid wheat (Triticum aestivum L.) (2n=6x=42) and rice (Oryza sativa L.) (2n=2x=24) were evaluated using linkage maps supplemented with simple sequence repeat (SSR) loci obtained from publicly available expressed sequence tags (ESTs). EST-SSR markers were developed using two main strategies to design primers for each gene: (1) primer design for multiple species based on supercluster analysis, and (2) species-specific primer design. Amplification was more consistent using the species-specific primer design for each gene. Forty-four percent of the primers designed specifically for wheat sequences were successful in amplifying DNA from both species. Existing genetic linkage maps were enhanced for the wheat and rice genomes using orthologous loci amplified with 58 EST-SSR markers obtained from both wheat and rice ESTs. The PCR-based anchor loci identified by these EST-SSR markers support previous patterns of conservation between wheat and rice genomes; however, there was a high frequency of interrupted colinearity. In addition, multiple loci amplified by these primers made the comparative analysis more difficult. Enhanced comparative maps of wheat and rice provide a useful tool for interpreting and transferring molecular, genetic, and breeding information between these two important species. These EST-SSR markers are particularly useful for constructing comparative framework maps for different species, because they amplify closely related genes to provide anchor points across species.Communicated by R. Hagemann     

从公共数据库中利用数据挖掘的方法开发15条黄金鲈EST-SSR标记

黄金鲈是美国中西部地区重要的淡水经济型鱼类。TypeⅠ型标记对于比较作图和遗传学研究有相当重要的作用, 但是黄金鲈的TypeⅠ标记数量相当少。研究通过数据挖掘的方法从公共数据库中挖掘黄金鲈的TypeⅠ型即EST-SSR标记。实验结果分析了21968条EST序列, 约14.4%的序列包含不同类型的核心重复单元。CA重复是最丰富的二碱基重复单元。从中挑选62条包含SSR的EST序列设计引物, 筛选得到15对多态的微卫星位点。群体遗传学参数显示15对EST-SSR位点的等位基因数范围为4—19(平均数为9), 观测杂合度和期望杂合度的范围分别为0.103—0.929和0.116—0.934, 有4个位点偏离HWE平衡。研究得到的EST-SSR标记适用于黄金鲈的群体遗传学和基因组作图研究。     

Transferability and utility of white oat (Avena sativa) microsatellite markers for genetic studies in black oat (Avena strigosa)

Preservation and use of wild oat species germplasm are essential for further improvement of cultivated oats. We analyzed the transferability and utility of cultivated (white) oat Avena sativa (AACCDD genome) microsatellite markers for genetic studies of black oat A. strigosa (A(s)A(s) genome) genotypes. The DNA of each black oat genotype was extracted from young leaves and amplified by PCR using 24 microsatellite primers developed from white oat. The PCR products were separated on 3% agarose gel. Eighteen microsatellite primer pairs amplified consistent products and 15 of these were polymorphic in A. strigosa, demonstrating a high degree of transferability. Microsatellite primer pairs AM3, AM4, AM21, AM23, AM30, and AM35 consistently amplified alleles only in A. sativa, which indicates that they are putative loci for either the C or D genomes of Avena. Using the data generated by the 15 polymorphic primer pairs, it was possible to separate 40 genotypes of the 44 that we studied. The four genotypes that could not be separated are probably replicates. We conclude that A. sativa microsatellites have a high transferability index and are a valuable resource for genetic studies and characterization of A. strigosa genotypes.     

Use of Comparative Genomics to Develop EST-SSRs for Red Drum (Sciaenops ocellatus)

Microsatellites physically linked to expressed sequence tags (EST-SSRs) are an important resource for linkage mapping and comparative genomics, and data mining in publicly available EST databases is a common strategy for EST-SSR discovery. At present, many species lack species-specific EST sequence data needed for the efficient characterization of EST-SSRs. This paper describes the discovery and development of EST-SSRs for red drum (Sciaenops ocellatus), an estuarine-dependent sciaenid species of economic importance in the USA and elsewhere, using a phylogenetically informed, comparative genomics approach to primer design. The approach entailed comparing existing genomic resources from species closely allied phylogenetically to red drum, with resources from more distantly related outgroup species. By taking into account the degree to which flanking regions are conserved across taxa, the efficiency of PCR primer design was increased greatly. The amplification success rate for primers designed for red drum was 100?% when using EST libraries from confamilial species and 92?% when using an EST library from a species in the same suborder. The primers developed also amplified EST-SSRs in a wide range of perciform fishes, suggesting potential use in comparative genomics. This study demonstrates that EST-SSRs can be efficiently developed for an organism when limited species-specific data are available by exploiting genomic resources from well-studied species, even those at extended phylogenetic distances.     

Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat.

Expressed sequence tags (ESTs) are a valuable source of molecular markers. To enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in hexaploid wheat (Triticum aestivum L.), over 260,000 ESTs from 5 different grass species were analyzed and 5418 SSR-containing sequences were identified. Using sequence similarity analysis, 156 cross-species superclusters and 138 singletons were used to develop primer pairs, which were then tested on the genomic DNA of barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and wheat. Three-hundred sixty-eight primer pairs produced PCR amplicons from at least one species and 227 primer pairs amplified DNA from two or more species. EST-SSR sequences containing dinucleotide motifs were significantly more polymorphic (74%) than those containing trinucleotides (56%), and polymorphism was similar for markers in both coding and 5' untranslated (UTR) regions. Out of 112 EST-SSR markers, 90 identified 149 loci that were integrated into a reference wheat genetic map. These loci were distributed on 19 of the 21 wheat chromosomes and were clustered in the distal chromosomal regions. Multiple-loci were detected by 39% of the primer pairs. Of the 90 mapped ESTs, putative functions for 22 were identified using BLASTX queries. In addition, 80 EST-SSR markers (104 loci) were located to chromosomes using nullisomic-tetrasomic lines. The enhanced map from this study provides a basis for comparative mapping using orthologous and PCR-based markers and for identification of expressed genes possibly affecting important traits in wheat.     

EST-SSRs characterization and in-silico alignments with linkage map SSR loci in grape (Vitis L.) genome

11,581 grape (Vitis L.) EST-SSRs were produced and characterized from a total of 381,609 grape ESTs. Among the EST-SSRs, the tri repeat (5,560, 45.4%) represented the most abundant class of microsatellites in grape EST. Most of grape EST-SSR motifs fall within 18-24 bps in length. The EST-SSRs tri-repeats occurred a higher percentage in 5??-end (59.3%) than in 3??-end (48.3%). And EST-SSR tri-repeats had abundant codon repeats for putative amino acid runs as Proline, Arginine in grape ESTs. To better utilizing these markers, 142 of newly developed and validated EST SSR loci as well as 223 linkage map SSR loci were in silico aligned and mapped in grape genome. The orders of these SSR loci in the chromosomal physical locations and in the linkage groups were compared, and about twenty linkage map loci positions were switched or rearranged in grape genome. The EST-SSR markers extended the linkage map in grape genome. The method of in silico mapping reported in this study provided an initial collection for grape mapping resources. This approach offers great opportunities to understand the genetic variations in nucleotide sequences differences in physical map, and genetic recombination in linkage maps, as well as benefits for markers enrichment in a specific grape genome region for fine mapping or QTL mapping.     

Development of EST-SSR markers and their utility in revealing cryptic diversity in safflower (Carthamus tinctorius L.)

With the aim of developing additional genomic resources in safflower, a set of 41,011 ESTs of safflower were mined for the presence of SSRs. 18,773 SSR containing ESTs (SSR-ESTs) were identified and were analyzed to remove redundant sequences leading to identification of 8,810 non-redundant SSR-ESTs (categorized into 6104 singletons and 2,706 contigs) having 13,085 non-redundant SSRs. The average number of non-redundant SSRs per EST was 0.32 and they predominantly consisted of dinucleotide (57.7 %), and trinucleotide (37.7 %) repeat motifs. 500 primer pairs were designed for the non-redundant EST-SSRs of which, 151 were tested. 60 markers which gave robust amplicons, were validated in a set of 19 Carthamus lines. A subset of EST-SSR markers, having average polymorphism information content (PIC) ≥0.4 could precisely elucidate the pedigree relatedness among these lines. Further, these markers exhibited high cross-species transferability to five other wild species of Carthamus. The markers reported here would be a valuable addition to existing safflower marker resources aiding in hastening its improvement.