红花EST-SSR分子标记开发与初步验证

旨在开发红花EST-SSR标记,为红花分子辅助育种和种质资源评价提供有力工具.先对红花转录组进行测序,选用MISA软件筛选鉴定SSR位点.在云南红花(YH)转录组中,共获得46 016个SSR位点,分布频率为32.09%,平均每3.11 kb有一个SSR.云南红花SSR位点以二,三核苷酸重复基序为主,其优势重复基序分别为AG/CT(44%)和AAG/CTT(24%).SSR位点重复次数相差较大,其中重复为5次比率最高,SSR位点数为9 369(26.63%),其次为6次(8 148, 23.16%).同时,利用Primer3.0进行引物设计,随机筛选27对引物,在60份不同来源红花种质中进行多态性验证,获得12对具有多态性稳定的引物,多态性引物比率为44.4%.结果表明,基于红花转录组序列开发SSR标记是可行的,开发的标记具有稳定扩增性,丰富多态性等优点,开发的SSR标记将有助于红花功能基因挖掘,遗传连锁图谱构建和遗传多样性分析.     

扁玉螺转录组SSR信息分析

采用生物信息学方法分析扁玉螺转录组文库EST序列(SSR)位点,并且设计简单重复序列(SSR)引物,以期为扁玉螺分子标记辅助育种提供有力的工具。对扁玉螺进行转录组测序采用Illumina Hiseq~(TM)高通量测序平台,再利用Micro SAtellite(MISA)工具,分析扁玉螺转录组中SSR的重复基元及其分布频率。利用Primer 3软件设计引物,并且通过SSRFinder筛选出SSR引物。从233 853条Unigenes中找到202 337个符合条件的SSRs,发生频率为45.34%,SSR位点的出现频率为86.53%。SSR位点中单核苷酸重复是主要类型,占总SSRs的63.44%,其次为双核苷酸和三核苷酸,分别为24.12%和6.73%。SSR所包含的重复基元中,单核苷酸重复基元A/T最多,占总SSR的54.15%;其次为双核苷酸AC/GT多,为15.9%。扁玉螺转录组中SSR位点出现频率高,而且类型丰富;大量的SSR分子标记技术有助于扁玉螺分子育种与遗传多样性分析提供更多的依据。     

云南松转录组SSR的分布及其序列特征

分析云南松转录组中的SSR位点信息的分布及其序列特征,为SSR引物的开发做前期工作.从80000条转录组Unigene序列中,搜索到2455个SSR,出现频率为3.07%,分布的平均距离29 kb.SSR以三、二核苷酸占优势,分别占总数42.73%和27.25%,而以四核苷酸的较少,仅占总数的0.98%.重复片段长度12~25bp,其中近90%的集中在12~20bp,平均16.09bp.AT/AT与AGC/CTG和AAG/CTT为二、三核苷酸优势重复基元,分别占基元总数的16.66%、9.65%和8.39%.从2899对引物中随机挑选32对引物用于检测,24对成功扩增.云南松转录组SSR位点出现的频率较高,分布距离较近,基元类型丰富,重复次数较高,具有较高的多态性潜力.云南松转录组产生的Unigene是开发SSR的有效来源,获得的SSR引物可为云南松种质资源的遗传变异研究提供更多的标记.     

青海小海绵羊肚菌M12-10转录组SSR信息分析及其分子标记开发

为明确羊肚菌转录组SSR总体特征、开发出适用于分子育种的SSR引物,对分离自青海大通的小海绵羊肚菌M12-10菌丝体高通量转录组测序,采用MISA分析小海绵羊肚菌M12-10转录组SSR位点信息,利用Primer 3.0设计引物,并选择12对引物将19株不同种、不同采集地的野生羊肚菌进行聚类分析。结果表明:高通量测序组装得到153 326个转录本,100 672个SSR位点分布在20 043条Unigenes基因上;SSR发生频率为19.9%,单核苷酸(9 268,46.0%)和三核苷酸(5 183,25.0%)是主要的重复类型,优势重复基元为A/T(33.0%)、GA/AG(12.0%)和TGC/CAG(40.0%);基序长度集中在10～20bp的比例为73.9%,具有高多态性。基于SSR的20 043条Unigene成功设计了14 894对引物,随机筛选的12对SSR引物中4对引物表现稳定可重复的多态性;遗传相似系数为0.55时,19株羊肚菌菌株分为两类;相似系数为0.84时,19株羊肚菌全部分开。故基于小海绵羊肚菌M12-10转录组数据SSR标记开发是可行有效的,筛选的4对高频率、高多态性的SSR引物将有助于开展种质资源评价和遗传多样性分析。     

基于RNA-seq的崇左金花茶EST-SSR标记开发

本研究分析了崇左金花茶(Camellia chuongtsoensis)转录组水平的EST-SSR特征.从崇左金花茶转录组数据组装获得的35 410个Unigene中,共鉴定出2~7不同核苷酸重复类型的SSR位点7 754个,分别分布于6 394条序列中,其中1 121个Unigene具有两个以上的位点.在转录组中SSR位点出现频率为21.90%,分布密度为1/3.6kb;在所有重复单元中,二碱基微卫星占主要优势,占SSR位点总数的61.3%.利用Primer 3.0设计引物,共计3 303条Unigene成功设计出引物.随机选择10对SSR引物,对一个崇左金花茶群体进行扩增多态性分析,8对引物能够扩增出符合预期大小的PCR片段,其中4对引物成功检测出多态.以上结果表明,转录组数据能够提供丰富的SSR位点,用于快速高效地开发SSR引物,所开发的引物可为崇左金花茶遗传多样性的研究以及种质资源的鉴定与保护等方面提供分子基础.     

基于转录组的曼氏无针乌贼SSR与SNP位点信息分析

利用IlluminaHiseq平台对曼氏无针乌贼进行了转录组测序,采用生物信息学方法分析了转录组序列中的SSR、SNP位点信息并设计了SSR引物。利用MISA工具筛选了转录组测序获得的127 575条Unigene序列,共得到分布于50 626条序列中的SSR位点108 685个,SSR发生率39.68%,出现频率为85.19%。平均每949 bp含有1个SSR位点。在50626条含有SSR位点的Unigene序列中,有25 548条Unigene包含SSR位点数目在2个及以上。其中单碱基重复是EST微卫星序列的主要形式(42.84%),其次是二碱基重复(28.73%),三碱基重复(14.93%)和四碱基重复(12.79%)。在所有微卫星序列所包含的重复单元中,优势重复基元类型为A/T(占总SSRs的42.23%),其次为AT/AT (13.33%),AC/GT (9.32%),AAAG/CTTT(10.00%)。运用Primer premier 5软件共设计了46 520对SSR引物。在12 323条Unigene中发掘出SNP位点64 732个,每条Unigene平均含5.25个SNP位点。其中转换(Transition) 45 975个(71.02%),颠换(Transversion) 18 757个(28.98%)。本研究通过第二代高通量测序技术,结合生物信息学方法对曼氏无针乌贼进行了SSR位点与SNP位点的开发,为其遗传多样性分析、分子辅助育种和增殖放流效果评估等提供了有力研究工具。     

杉木EST-SSR与基因组SSR引物开发

利用已经公布的杉木444条EST序列和未公布的杉木基因组文库中1 142条基因序列,进行引物开发效率的比较。去冗余后,利用MISA 搜索SSR 位点,分别得到109个和39个含有SSR的 位点。杉木EST序列中SSR分布密度为964.58个/Mbp,基因组中平均每Mbp出现1 037.24个SSR。在两个独立来源的数据库序列中,六核苷酸重复均为最多的重复类型,且AT-rich的重复类型占较大比例。AGC/CTG是杉木EST序列和基因组库中最多的三碱基重复,通过Primer 3.0分别设计出SSR引物95对和37对。为考察设计引物在杉木不同种源(群体)中的有效性,取12个种源(个体)的优良个体, 利用随机抽取的10个EST-SSR和8个gSSR(基因组SSR)进行引物筛选,结果表明:EST-SSR和gSSR各有4对引物在12个种源(个体)中表现出明显的多态性,多态率分别为40%和50%。8对多态性的SSR引物共扩增出 25 个多态性等位位点,平均每个引物产生 3.125 个多态性等位位点,平均有效的等位位点为2.399 5,PIC平均值为0.519 1; Hot平均为0.307 4。其中gSSR标记在检测群体间存在较大的分化,4个gSSR比4个EST-SSR扩增出更多的等位位点数、平均等位位点数,以及更大的PIC值。     

基于高通量转录组测序技术的龙头鱼微卫星信息分析

为龙头鱼资源合理开发与保护提供有效分子标记,本研究利用IluminaHiSeq~(TM) 2500高通量测序平台对采自舟山近海的龙头鱼肌肉组织进行转录组测序,从获得的Unigenes序列中挖掘SSR位点信息并分析其分布及特征。结果显示,测序所得29 756条Unigenes中共识别出5 652个完美型SSR位点,序列总长度为86 517 bp,相对丰度为332.97个/Mb。龙头鱼转录组SSR以单碱基和二碱基重复类型居多,分别占SSR总数的67.59%和20.72%。6类完美型SSR共有重复基元148种,重复次数以10次为主,占总SSR的23.23%。SSR序列长度范围在10～92 bp之间,其中序列长度在12 bp及以上的SSR位点的含量为65.73%。综上所述,龙头鱼转录组SSR位点含量丰富、基元重复类型众多、重复次数较高,具有良好的分子标记开发潜力,获得的SSR标记可用于遗传多样性和系统发育关系研究。     

朱顶红转录组SSR标记的开发与遗传多样性研究

为开发朱顶红SSR标记并用于种质资源遗传特征分析,本研究从漳红2号朱顶红转录组共48 355条unigenes中搜索得到11 055个SSR位点,出现频率为25. 13%. SSR类型以一、二、三核苷酸重复为主,占总SSR比例的92. 80%,其中三核苷重复为主要基序类型,占总SSR的43. 73%.设计合成的50对SSR引物中有7对引物在51份朱顶红种质中得到有效性验证,表现扩增条带清晰、多态性好.基于SSR标记进行51份朱顶红种质的遗传多样性分析表明,在遗传相似系为0. 166时可将其分为6个类,来源于相同地域或亲缘种质大多成簇分布.研究结果表明,漳红2号朱顶红转录组测序产生的unigenes信息可作为开发SSR标记的有效来源,获得的大批量SSR标记可为朱顶红及其近缘种的遗传多样性分析和遗传图谱构建提供更加丰富可靠的标记选择.     

雌核发育棕点石斑鱼EST-SSR的开发验证

通过人工雌核发育棕点石斑鱼(Epinephelus fuscogutatus)的转录组序列,共获得了24 359个微卫星位点,棕点石斑鱼转录组的微卫星重复基序具有不同的分布特征,其中以单碱基(34.05%)、二碱基(37.58%)和三碱基(22.75%)为主要的EST-SSR基序重复类型.根据微卫星引物设计原则,随机筛选了93个EST-SSR位点来进行引物合成和多态性检测,最终开发了48个具有多态性的微卫星标记,并在不同棕点石斑鱼群体中进行了初步的验证.结果显示:每个位点等位基因数(Na)为2～22,平均等位基因数为9;观测杂合度(H_0)为0.111～1.000,期望杂合度(He)为0.636～0.940;多态信息含量(PIC)为0.538～0.922个,表明48个EST-SSR位点均表现出高多态性(PIC 0.5).结果表明:基于雌核发育棕点石斑鱼转录组数据,开发微卫星标记是可行的.本研究所开发的具有多态性的微卫星标记可应用于棕点石斑鱼及其近缘种的遗传多样性分析和分子标记辅助育种研究.     

Mining,characterization, and exploitation of EST-derived microsatellites in <Emphasis Type="Italic">Gossypium barbadense</Emphasis>

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of ex-pressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were de-termined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barba-dense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Further-more, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.     

Characterization, development and exploitation of EST-derived microsatellites in Gossypium raimondii Ulbrich

COTTON IS AN IMPORTANT GLOBAL CASH CROP. IN THE RECENTYEARS, MOLECULAR MARKER TECHNOLOGY HAS BEEN WIDELY APPLIED TO SUCH STUDIES ON COTTON AS GENETIC MAP- PING[1―4], VARIETY PURITY DETECTION[5], GENETIC DIVERSITY ANALYSIS[6], MOLECULAR MARKER-ASSISTED BR…     

Development, chromosome location and genetic mapping of EST-SSR markers in wheat

A number of 151695 wheat expression sequence tags （ESTs） that originated from GenBank/dbEST from July 14, 2003 to August 24, 2004 were used to search for simple sequence repeats （SSRs） with motif 2-5 bp, and 2038 simple sequence repeats （EST-SSRs）, which accounted for 1.34% of EST database, were identified. Based on these SSR sequences, 249 EST-SSR primer pairs and 166 amplified clear bands in various wheat cultivars were designed. These EST-SSR markers can be used as new molecular markers in wheat and related species. Using Chinese Spring nulli-tetrasomic lines, 93 EST-SSR primer pairs and 193 EST-SSR loci were located on 19 wheat chromosomes except for 4A and 4B. Forty-three loci were mapped on 11 chromosomes of the genetic framework map previously constructed using recombinant inbred lines.     

Analysis of simple sequence repeats markers derived from Phytophthora sojae expressed sequence tags

Five thousand and eight hundred publicly available expressed sequence tags (ESTs) of Phytophthora sojae were electronically searched and 415 simple sequence repeats (SSRs) were identified in 369 ESTs. The average density of SSRs was one SSR per 8.9 kb of EST sequence screened. The most frequent repeats were trinucleotide repeats (50.1%) and the least frequent were tetranucleotide repeats (8.2%). Forty primer pairs were designed and tested on 5 strains of P. sojae. Thirty-three primer pairs had successful PCR amplifications. Of the 33 functional primer pairs, 28 primer pairs produced characteristic SSR bands of the expected size, and 15 primer pairs (45.5%) detected polymorphism among 5 tested strains of P. sojae. Based on the polymorphisms detected with 20 EST-SSR markers, the 5 tested strains of P. sojae were clustered into 3 groups. In this study, the SSR markers of P. sojae were developed for the first time. These markers could be useful for identification, genetic variation study, and molecular mapping of P. sojae and its relative species.     

33个杨柳品种指纹图谱构建

[目的]构建杨树与柳树优良品种的指纹图谱,对不同品种进行准确鉴定.[方法]利用柳树EST-SSR标记对33个杨树与柳树优良品种进行通用性检测及基因分型,通过核心引物间的组合构建品种指纹图谱.同时采用非加权组平均法进行聚类,分析各品种间亲缘关系.[结果]12个EST-SSR标记共扩增出97条等位片段,每个位点等位基因数5...     

基于SSR标记的丛生竹杂种鉴定、遗传分析和指纹图谱构建

[目的]准确鉴定丛生竹杂交种,分析杂种及其亲本的遗传关系,开发可以利用的指纹图谱.[方法]以孝顺竹(Bambusa multiplex)×麻竹(Dendrocalamus latiflorus)、孝顺竹(B.multiplex)×粉单竹(B.chungii)两个杂交群体为材料,从已公布的竹子SSR引物中随机选取30对引...     

61个观赏海棠品种的SSR指纹图谱构建及遗传多样性分析

【目的】观赏海棠种类繁多且同名异物和同物异名的现象严重,寻找一种从分子角度快速准确鉴定观赏海棠品种的技术手段,弥补依靠表型性状的分类和鉴定的不足,满足观赏海棠的品种资源鉴别需要。【方法】对61个主要观赏海棠品种,利用从苹果属多个种中筛选出的10对SSR引物对其进行PCR扩增检测,分析其遗传多样。【结果】10对SSR引物共扩增出78条条带,平均每个SSR位点扩增出7.8条,多态性条带百分率为100%。61个观赏海棠品种Nei多样性指数(H)和Shannon指数(I)分别为0.205 2和0.348 8,若以共显性标记的方式进行分析,43个海棠品种的Nei多样性指数的变化范围在0.623 9～0.881 8,平均为0.788 3,表现出较丰富的遗传多样性。【结论】最终利用2对SSR引物成功地构建了61个观赏海棠的指纹图谱,为品种资源的鉴别和深入挖掘利用提供依据。     

Screening microsatellite markers from EST sequences of bay scallop Argopecten irradians

Seventy-five simple sequence repeats （SSRs） were identified by the bioinformatic analysis from 5008 expressed sequence tags （ESTs） of Argopecten irradians. Among the SSRs, the number of repeat nucleotide varied from 2 to 6. Dinucleotide and trinueleotide repeat motifs were dominant in EST-SSRs of bay scallop, with a proportion of 80% over the total screened SSRs. Twenty-nine pairs of primer were designed based on the flank sequences of the selected ESTs using the software of Primet 5, and verified under the given PCR reaction condition. Eighteen of the 29 primer pairs resulted in the expected products, while the remaining either failed to produce any fragments or yielded products over expected size. Thirteen of the 18 SSRs, accounting for 72%, were detected to show polymorphism in the examined scallop samples. A preliminary test in this study indicated that the majority of the identified SSRs were informative in the cultured bay scallops, making them suitable for the population and other genetic analysis. EST-SSR markers have more advantages than the traditional genomic-derived SSRs and there is a wide range of application in comparative mapping, functional gene cloning and marker assisted selection. This research provides a reference to the identification of EST-SSRs with relative bioinformatic analysis from aquaculture species, as well as to those with a large number of ESTs.