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（英文）

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。     

草鱼Ⅰ型微卫星标记的发掘及其多态性检测

表达序列标签(EST)是发掘Ⅰ型微卫星标记的重要资源。研究运用生物信息学方法,从草鱼头肾组织3027条EST序列中搜索到322个微卫星位点,占整个EST数据库的10.6%。其中,二核苷酸重复位点151个(46.9%),三核苷酸重复位点137个(42.5%),四、五、六核苷酸重复位点较少;在二核苷酸重复位点中,AC/GT重复位点最为丰富,占二核苷酸重复位点总数的50.3%,AG/CT重复次之,占二核苷酸重复位点总数的40.4%,AT和GC重复较少。10个微卫星位点的多态性检测结果显示,4个位点在草鱼测试群体中呈多态性,多态性位点的平均多态信息含量(PIC)和平均遗传杂合度(H)分别为0.5236和0.5441,其中,2个多态性位点的PIC值大于0.5,呈现高度多态性特征。Ⅰ型微卫星标记将为草鱼遗传连锁图谱构建和QTL分析提供有效的基因分子标记。       

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。     

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.     

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。     

家蚕第12连锁群EST-SSR标记的筛选和分析

为了探究家蚕Bombyx mori EST-SSR标记的多态性, 对检索获得的家蚕第12连锁群的4 465条EST序列进行了分析, 整理和拼接后得到581条非冗余EST序列, 总长度约为480 kb。其中, 有122条序列中共检测到154个EST-SSR, 占所研究的EST序列的2.73%, 平均每3.12 kb 含有一个EST-SSR。在所检测的EST-SSR中, 三核苷酸和四核苷酸重复是主导类型, 分别占总数的36.36%和28.57%,大部分表现为Perfect形式; 核苷酸重复平均长度约为16.2 bp, 最长为30 bp。进一步进行同源性分析, 发现有26条序列可以在NCBI中检索到同源序列, 在这些序列中一共含有40个SSR, 其中14个(35.0%)位于5′-UTR, 11个(27.5%)位于3′-UTR, 15个(37.5%)位于CDS区。根据筛选到的微卫星序列设计11对引物, 其中8对引物有扩增产物, 且条带清晰; 应用引物ES1204对8个家蚕品种进行PCR扩增都呈现多态性。结果说明通过家蚕EST数据库发掘SSR标记是一条可行的途径。     

橡胶树EST-SSR标记的开发与应用

通过对橡胶树10 778条EST序列进行拼接, 共得到Unigene 3 090条, 从中发掘出分布于353条Unigene的430个EST-SSR位点, SSR发生频率为11.42%, 平均分布距离为3.93 kb。在橡胶树EST-SSR中, 二核苷酸和三核苷酸重复是主要的重复类型, 分别占63.49%、32.09%。TC/AG、CT/GA和CTT/GAA、AAG/TTC、AGA/TCT是二、三核苷酸的优势重复类型。利用PRIMER5.0设计引物148对, 随机挑选21对引物进行合成, 并用其中15对扩增较好的引物, 通过变性聚丙烯酰胺凝胶结合银染的方法对44个橡胶树无性系进行了遗传多样性检测, 构建了聚类分析树状图。研究结果表明, 从橡胶树EST中开发SSR标记是有效、可行的, 文章所开发的EST-SSR引物可用于橡胶树遗传分析研究。     

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.     

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。     

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。     

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 and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 × PB217 population

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.     

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.     

Characteristics, development and mapping of Gossypium hirsutum derived EST-SSRs in allotetraploid cotton

In order to construct a saturated genetic map and facilitate marker-assisted selection (MAS) breeding, it is necessary to enhance the current reservoir of known molecular markers in Gossypium. Microsatellites or simple sequence repeats (SSRs) occur in expressed sequence tags (EST) in plants (Kantety et al., Plant Mol Biol 48:501–510, 2002). Many ESTs are publicly available now and represent a good tool in developing EST-SSRs. From 13,505 ESTs developed from our two cotton fiber/ovule cDNA libraries constructed for Upland cotton, 966 (7.15%) contained one or more SSRs and from them, 489 EST-SSR primer pairs were developed. Among the EST-SSRs, 59.1% are trinucleotides, followed by dinucleotides (30%), tetranucleotides (6.4%), pentanucleotides (1.8%), and hexanucleotides (2.7%). AT/TA (18.4%) is the most frequent repeat, followed by CTT/GAA (5.3%), AG/TC (5.1%), AGA/TCT (4.9%), AGT/TCA (4.5%), and AAG/TTC (4.5%). One hundred and thirty EST-SSR loci were produced from 114 informative EST-SSR primer pairs, which generated polymorphism between our two mapping parents. Of these, 123 were integrated on our allotetraploid cotton genetic map, based on the cross [(TM-1×Hai7124)TM-1]. EST-SSR markers were distributed over 20 chromosomes and 6 linkage groups in the map. These EST-SSR markers can be used in genetic mapping, identification of quantitative trait loci (QTLs), and comparative genomics studies of cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Zhiguo Han and Changbiao Wang contributed equally to this work.     

银杏EST序列中微卫星的分布特征

本文利用从NCBI下载的21 590条银杏EST序列,分析了银杏(表达序列标签微卫星)EST-SSR在银杏EST序列的分布和比较了在不同长度EST序列中的SSR特性.在剔除冗余和低质量序列后,得到总长为5 708.385 kb的无冗余EST序列7 961条,发现了405个EST序列(5.09%)含有475个SSR,长度400-1000 bp的EST序列含SSR位点数为445个,占SSR总数的93.68%.二核苷酸和三核苷酸基元类型是银杏EST-SSR的主要类型,分别占SSR总数的73.89%和24.00%,最常见的SSR基元是:(AT)_n、(AG)_n、(AC)_n、(AAG)_n和(AAT)_n.通过对银杏EST序列中SSR位点信息的发掘分析,为有针对性地设计EST-SSR引物,开发银杏EST-SSR分子标记奠定基础.     

Development,Characterization, and Cross Species/Genera Transferability of Novel EST-SSR Markers in Lentil,with Their Molecular Applications

Lentil is nutritionally important crop for human diet and enriched with quality protein, complex carbohydrates, fibers, essential minerals, and vitamins. However, genetic improvement of lentil is hampered largely due to unattributed and unexploited genetic and genomic resources. To administer genomic resources in lentil, we have identified 9949 EST-SSR loci from lentil RNA Seq data and validated 50 of them using 234 genotypes representing various Lens species and 34 accessions of 12 different legumes. Out of 50 EST-SSRs, 46 were polymorphic with polymorphic information content (PIC) ranging from 0.16–0.74. The transferability of these markers exhibited varied levels from 45.1 to 71.3% across the cultivated/wild species of lentil and from 10.8 to 54.3% across the twelve legume genera. On the basis of total identified EST-SSRs, mononucleotide (51%) repeat proportion was high followed by trinucleotide (30%) and dinucleotide (14%) repeat. Population structure and cluster analysis classified all the studied genotypes into 4 groups. However, principal coordinate analysis (PCA) was able to group genotypes based on their area of collection. Annotation of all the 46 polymorphic marker sequences revealed that most of the markers linked to genes involved in metabolism of plants. Further, polymorphic markers were also used for linkage mapping in F₃ population where 4 markers were found to be linked with a map distance of 72.5 cM. The newly developed markers represent an impressive tool for characterization of germplasm, genetic linkage mapping, phylogenetic studies, as well as to determine disparity in taxonomic status of subspecies of the genus Lens.

     

Comparative population genetic analysis of bocaccio rockfish Sebastes paucispinis using anonymous and gene-associated simple sequence repeat loci

Comparative population genetic analyses of traditional and emergent molecular markers aid in determining appropriate use of new technologies. The bocaccio rockfish Sebastes paucispinis is a high gene-flow marine species off the west coast of North America that experienced strong population decline over the past 3 decades. We used 18 anonymous and 13 gene-associated simple sequence repeat (SSR) loci (expressed sequence tag [EST]-SSRs) to characterize range-wide population structure with temporal replicates. No F(ST)-outliers were detected using the LOSITAN program, suggesting that neither balancing nor divergent selection affected the loci surveyed. Consistent hierarchical structuring of populations by geography or year class was not detected regardless of marker class. The EST-SSRs were less variable than the anonymous SSRs, but no correlation between F(ST) and variation or marker class was observed. General linear model analysis showed that low EST-SSR variation was attributable to low mean repeat number. Comparative genomic analysis with Gasterosteus aculeatus, Takifugu rubripes, and Oryzias latipes showed consistently lower repeat number in EST-SSRs than SSR loci that were not in ESTs. Purifying selection likely imposed functional constraints on EST-SSRs resulting in low repeat numbers that affected diversity estimates but did not affect the observed pattern of population structure.     

Aligned genetic linkage maps of apple rootstock cultivar ‘JM7’ and Malus sieboldii ‘Sanashi 63’ constructed with novel EST-SSRs

Identification of markers associated with genes of interest and quantitative trait loci (QTLs), combined with high-density genetic linkage maps, can help reduce labor and costs by enabling marker-assisted selection (MAS). In this study, a dwarfing apple rootstock cultivar ??JM7?? (Malus prunifolia × Malus pumila ??Malling 9??) and wild apple Malus sieboldii ??Sanashi 63?? (section Sorbomalus) were used for constructing genetic linkage maps. Here, a species from section Sorbomalus was used for the first time as a target species in a genome-wide mapping study. We also developed and mapped 137 novel-expressed sequence tag-simple sequence repeat (EST-SSR) markers. The genetic linkage maps of ??JM7?? and ??Sanashi 63?? consisted of 415 and 310 loci and spanned 998.0 and 981.8?cM, respectively, comparable to the reference map of Malus × domestica ??Discovery??. A BLASTN search revealed that all of the EST-SSR sequences used in this study exhibited very high homology to one or more previously characterized apple genome contigs. Although the most homologous contigs of 89 EST-SSRs were located within the same linkage groups (LGs) identified by mapping analysis, the other 48 EST-SSRs were aligned into contigs positioned in different LGs than those identified by mapping. When search criteria were expanded to include the five most homologous contigs of each EST-SSR, at least one of the top five contigs for 15 of these 48 EST-SSRs corresponded to the LG obtained by mapping. The maps of ??JM7?? and ??Sanashi 63?? may be useful for analyzing important rootstock characteristics and identifying markers for MAS.     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.