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.     

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.     

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。     

湿地松转录组SSR分析及EST-SSR标记开发

【目的】湿地松是南方地区优先推广的优质产脂树种。但湿地松分子遗传基础薄弱,基因组序列信息匮乏,影响了湿地松基因组学的深入研究。目前,湿地松分子研究所用的SSR标记主要来自其他近缘种或利用公共数据库中有限的基因序列资源开发的SSR标记,其多态性和通用性较差。为解决这一问题, 笔者根据湿地松转录组测序数据开发EST-SSR位点,并揭示其在转录组序列中的分布类型及特征,为湿地松分子标记辅助育种奠定基础。【方法】利用MISA软件对转录组序列进行SSRs查找和分布特征分析。查找标准参数设置为: 单核苷酸重复>10次,二核苷酸重复>6次,三、四、五、六核苷酸重复>5次。根据SSR位点两端的保守区域,利用Primer3.0设计并随机挑选120对SSR引物,通过琼脂糖电泳和毛细管电泳对来自美国和吉安的113份家系个体进行遗传多样性分析,确定引物多态性。【结果】79 574条unigenes序列中搜索到3 818个SSR位点,出现频率为4.80%,平均18.27 kb出现1个SSR位点,3 373个unigenes含有SSR位点,SSR发生频率(含SSR位点的序列数/搜索序列总数)为4.24%,其中2 980条序列含1个SSR位点,含1个以上SSR位点的序列有393条。在检测到的3 818个SSR标记中,单核苷酸分布最多,其次是二核苷酸和三核苷酸,SSR数量分别占总数的63.54%、19.15%和16.27%,而四、五、六核苷酸重复类型所占比例较小,分别为0.52%、0.13%和0.31%。SSR重复单元的重复次数分布在5~22次之间,除单核苷酸重复外的1 391个SSR中,重复5次的数量最多,为498个(35.80%);重复6次和7次的次之,分别为417个(29.98%)和198个(14.23%);重复10次以上的仅有38个(2.73%)。在检测到的731个二核苷酸重复SSR中,最常出现的重复单元为AT/AT,数量为491个(12.86%),AG/CT和AC/GT两种类型的重复单元出现的次数次之,分别为156(4.09%)和81个(2.12%)。在检测到的621个三核苷酸重复中,AAT/ATT是出现频率最高的单元,共139个(3.64%),其次是AAG/CTT,共122个(3.20%)。3 818个SSR中有24.59%的位置未知,其余的SSR则分布在非编码区域(untranslated region,UTR)或者编码区(coding sequence,CDS)上,分布数量表现为3'UTR>5'UTR>CDS。参试的120对SSR引物,有92对扩增成功(76.78%),其中24对呈现多态(20%)。24对引物(13个二核苷酸重复、7个三核苷酸重复和4个四核苷酸重复)共检测出81个等位基因,每个位点的等位基因数为2~9,平均为3.38个。多态性信息含量(polymorphism information content,PIC)为0.103~0.726,平均为0.349。【结论】通过对湿地松转录组数据的挖掘,共获得 3 818个SSR位点,主要重复单元为AT/AT和AAT/ATT,可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。基于湿地松转录组序列的SSR标记开发是可行的。     

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.     

Development of SSR markers from ESTs of gramineous species and their chromosome location on wheat

A total of 407,663 expressed sequence tags （ESTs） of wheat, barley, maize, rice, and sorghum, obtained from GenBank/dbEST, were used to search for simple sequence repeats （SSRs）. A total of 10,253 EST-SSRs, which accounted for 2.52% of all the ESTs, were identified. Using Primer Premier 5.0, 1367 EST-SSR primer pairs were designed, of which 715 with high quality were synthesized. The 715 primer pairs were tested on wheat, rice, maize, cotton, and soybean under the same PCR conditions, and the effective primer pairs in the five crops were 500 （69.93%）, 383 （53.57%）, 452 （63.22%）, 357 （49.93%）, and 388 （56.27%）, respectively. This indicated a high transferability of EST-SSR markers between far-ranging species. In addition, 139 EST-SSR primer pairs with 240 loci were localized on all the 21 wheat chromosomes by using Chinese Spring nulli-tetrasomic lines of wheat.     

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…     

松属编码区微卫星特征和相应基因功能分析

从GenBank下载453 892条松属EST序列,序列组装后得到20 886条contig。用Sputnik软件从这些contig中查找了2 678个微卫星,其中3碱基重复微卫星占的比例最高,为59.2%,而其他重复长度的微卫星都相对较低,比例分别为:2碱基重复微卫星占12.0%,4碱基重复微卫星占13.3%,5碱基重复微卫星占15.5%。3碱基重复微卫星变化引起的基因读码框改变最小,松属树种基因区3碱基重复微卫星的富集显示了强烈的密码子选择效应。此次研究还对查找到的微卫星进行了引物设计和扩增分析。实验结果显示,设计的微卫星引物在云南松中的扩增成功率是72.9%。从扩增成功的引物中进一步选取了155对引物,对14个松属树种和1个黄杉属树种进行了引物通用性实验分析,结果显示155对引物在14个松属树种间的通用性在71.0%以上,而在黄杉属树种中的通用性只有25.2%。对松属树种中含有微卫星的基因进行了功能分类研究,结果显示基因在是否保留微卫星序列方面有显著分化,微卫星参与了如细胞成分分类的共质体组成、病毒颗粒及病毒颗粒组成、生物节律调控, 以及生长素转运蛋白等生物学过程。     

Development of simple sequence repeats (SSR) markers of ramie and comparison of SSR and inter-SSR marker systems

Ramie (Boehmeria nivea L. ) is an important bast fiber crop. To study genetic background of this species, we isolated and characterized microsatellite markers of ramie. A genomic library containing inserts of rapid amplification of polymorphic DNA (RAPD)fragments was constructed, and screened by PCR amplification using anchored simple sequence repeats as primers. A total of 26 clones were identified as positives, and 13 microsatellite loci were found after sequencing. The polymorphism of these 13 microsatellite loci was examined and the utility of simple sequence repeats (SSR) and inter-SSR (ISSR) marker systems for genetic characterization compared using 19 selected ramie cultivars. Both approaches successfully discriminated the 19 cultivars which differed in the amount of polymorphism detected. The level of polymorphism detected by SSR was 95.0 %, higher than that by ISSR (72.3 % ), but the average polymorphism information content (PIC) of ISSR (0. 651) was higher than that of SSR (0. 441). The higher PIC value of ISSR suggests that ISSR is more efficient for fingerprinting ramie cultivars than SSR markers. However, because the SSR loci are codominant, they are more suitable for determining the homozygosity levels of ramie, constructing linkage map, quantitative trait loci study of complex traits and marker-as-sisted selection.     

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对引物进行筛选、检测和分析。【结果】 发现6对SSR引物(P8、P9、P18、P19、P20、P27)适于孝顺竹×麻竹组合的杂种鉴定和遗传分析,鉴定结果表明34个子代全部为真实杂种,杂种真实率为100%;另外6对SSR引物(P2、P9、P10、P11、P20、P28)则适于孝顺竹×粉单竹组合的杂种鉴定和遗传分析,鉴定结果表明59个子代中有54个真实杂种,杂种真实率为91.5%。孝顺竹×麻竹的杂种遗传了较多的母本位点,而孝顺竹×粉单竹的杂种则遗传了较多的父本位点。利用SSR引物扩增出的位点分别构建了两个杂交群体的指纹图谱,为品种保护提供了保障。【结论】 SSR引物能有效地鉴定丛生竹杂交种的真伪,父母本的遗传位点在不同杂交组合的子代中遗传概率不同,杂交群体指纹图谱的构建可为品种保护提供保障。     

检疫性杂草假高粱及其近似种SSR分子标记的遗传多样性

利用SSR标记对8个假高粱及其近似种样品的遗传多样性进行研究,结果发现:(1)在11对SSR引物中,有5对扩增出多态性,这5对引物共检测到25个多态性位点,每对引物平均能检测到5个多态性位点;(2)根据Nei’s遗传距离分析的聚类树显示:这8个高粱属物种共可分为2个类群,7个亚类群;假高粱和黑高粱、拟高粱和明福1号间的亲缘关系极近;(3)SSR结果揭示出所测8个群体的Gst为0.0864,说明本文研究的Sorghum属8个物种的群体遗传分化程度属于中度。     

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.     

油茶基因组微卫星特征分析

对油茶基因组约10%覆盖度的DNA序列进行微卫星查找,共获得11 344个重复单元长度为1～6碱基的微卫星。在此基础上,通过对这些微卫星序列分析发现:在油茶基因组中长度为二核苷酸的微卫星重复单元最为丰富,占27.1%;在单碱基重复和二碱基重复这两种类型中,最主要的优势重复单元分别是A/T以及AT/TA、AG/TC。三碱基、四碱基、五碱基重复类型中,(AAN)n、(AAAN)n和(AAAAN)n为对应的优势重复单元,这些优势重复单元中富含碱基A和T。油茶基因组中变异程度高的微卫星(长度≥20 bp)约占11.7%。分析还发现,除单核苷酸重复微卫星外,油茶基因组微卫星长度的变异速率与重复单元长度呈负相关,即油茶基因组中长度较短的微卫星变异速率较快,而较长的重复单元变异速度较慢,相对较为稳定。     

微卫星标记在人参和西洋参鉴别中的应用

从GenBank中寻找含有简单重复序列(SSR)位点的人参DNA片段序列,设计SSR引物,挑选PCR扩增条带清晰的引物,对人参和西洋参进行PCR扩增及聚丙烯酰胺凝胶电泳分析,选择能够区别2个种的引物.共有来自8个SSR位点的9对引物能扩增出可区分人参和西洋参的特异性片段,其中7对SSR引物在西洋参中能稳定地扩增出特异性...     

柳杉无性系指纹图谱的构建及遗传多样性分析

【目的】为了构建柳杉无性系的DNA指纹图谱,准确区分柳杉无性系。【方法】利用SSR标记分析了柳杉89个无性系的遗传多样性和指纹图谱。【结果】11对引物共检测出53个等位点,平均每个SSR位点有5个,变化范围为3～6个; 有效等位基因数(N_e)变化范围为1.887 0～4.295 6,平均为3.041 1; Shannon信息指数(I)变化范围为0.774 9～1.556 3,平均为1.208 1。表明SSR标记具有较高的多态性。根据SSR标记特点及引物的顺序,将SSR引物扩增统计的“0”、“1”转换成基因型,通过不同基因型组合,构建了柳杉89个无性系的 DNA 分子指纹图谱,使每个无性系都获得了 1 个 22 位数的指纹图谱号码,同时基于个体间的遗传距离将柳杉89个无性系分为 5 类。【结论】SSR标记适用于柳杉无性系遗传多样性分析及鉴定,柳杉的遗传多态性处于中等偏高水平。     

基于松材线虫全基因组序列的SSR标记开发

为了研究松材线虫在我国的群体遗传关系及传播路径,获得更为稳定的松材线虫分子标记,使用MISA软件对松材线虫全基因组10 432条DNA片段进行搜索,共获得95个gSSR位点。其中,二核苷酸重复出现频率最高,占全部SSR位点的66.3%。依据所有gSSR位点共设计出36对引物,以1份松材线虫DNA pooling(包含我国46个不同地理来源的松材线虫虫株DNA)为模板进行PCR,产物由QIAxcel全自动凝胶电泳分析系统检测,获得17对可能具有多态性的gSSR引物。进一步对这17对引物的PCR产物进行单克隆试验并测序,BioEdit软件拼接比对结果表明,其中9对确实具有多态性。     

美洲黑杨种质材料倍性鉴定

【目的】对美洲黑杨种质资源库保存的种质材料进行倍性评估,并对候选多倍体进行倍性鉴定。【方法】利用9对用于多倍体鉴定的SSR引物检测448份美洲黑杨种质材料,根据每个引物扩增位点的等位基因数目对种质材料的倍性进行评估,基于初步鉴定结果筛选出候选多倍体材料。在此基础上,使用流式细胞仪(FCM)对候选多倍体种质材料的倍性进行鉴定。【结果】分子标记基因型分型统计结果显示,9个SSR位点共扩增出129条多态性条带,每个位点的等位基因数为5(Ploidp-10)~25(Ploidp-07)个,平均每个位点的等位基因数为14.33个。9个SSR位点的多态性信息含量(PIC)变动范围为0.46~0.93,平均PIC值为0.76。在检测的448份美洲黑杨种质材料中,440份美洲黑杨种质材料在9个引物位点上扩增出的等位基因数不多于2个;有8份种质材料在2个引物位点上扩增出了3个不同的等位基因,其中编号为1346、16-42、17-49、18-25的4份种质材料在Ploidp-02引物位点扩增出了3个等位基因,而编号为5-43、5-45、15-14、19-37的另外4份种质材料在Ploidp-04引物位点上也扩增出了3个等位基因。分子检测结果表明:该8份种质材料在对应的染色体区域发生了遗传物质增加现象,为候选三倍体种质材料。用FCM对8份三倍体候选种质材料进行倍性鉴定发现,编号为1346的种质材料为三倍体,其余7份种质材料疑似为发生局部染色片段增加的候选非整倍体。【结论】9对SSR分子标记均具有较高的多态性信息含量,可用于美洲黑杨种质资源多倍体初步筛选,结合FCM检测,可在大量样品中快速筛选出多倍体种质材料。     

豇豆SSR标记在豌豆中的可转移性与应用初探

利用豇豆SSR引物筛选在豌豆具通用性的SSR引物。13对豇豆SSR引物中检测出8对引物在豌豆中可扩增,其中5对SSR引物表现出多态性,进而分析11个豌豆品种和5个豇豆品种的遗传多样性与遗传关系。结果表明:平均等位基因数、平均有效等位基因数、平均Shannon指数,在豌豆品种群中分别为2.00、1.64、0.55,豇豆品种群中分别为2.00、1.80、0.53,上述遗传参数反映出供试豌豆品种和豇豆品种具有中等水平的遗传多样性;利用UPGMA聚类分析,能将豌豆品种群和豇豆品种群分为两类,且每个品种群内的各个体之间能进行区分,尤其是两个豌豆品种亚群的聚类结果与其地理来源基本一致。     

大花序桉群体适应性相关的SSR位点

【目的】检测大花序桉群体与环境适应性相关的基因组位点,为种质资源保护和利用提供分子生物学信息。【方法】利用84个SSR标记(包括29个基因组SSR 和55个EST-SSR)分析引种到广西,来源于澳大利亚昆土兰州北部和南部各7个大花序桉群体,通过Mantel 检验确定群体间是否存在地理隔离;检测群体间分化系数(F_st)离群值的受选择位点;并基于空间分析法检测与群体气候因子显著相关的等位片段,将显著相关位点的序列与NCBI数据库比对进行功能注释。【结果】大花序桉北部与南部群体间存在地理隔离,基于19个气候因子的聚类分析将北方与南方群体分为独立的组,这表明气候因子亦驱动了群体的分化。共检测到F_st离群值的受选择SSR位点39个(46.4%),其中,LOSITAN软件检测到12个正向选择位点和17个平衡选择位点。5个F_st离群值位点的6个等位片段与1个或多个气候因子显著相关(P<0.001),其等位频率在北部与南部群体间差异明显,其中,Embra6-118 bp与最冷月最低气温(T_mcm)显著相关,位点DNA序列的功能注释为碱性螺旋-环-螺旋(basic helix-loop-helix, bHLH)转录因子bHLH155;Embra20-121 bp与最暖季度降水量(P_wq)显著相关,位点的功能注释为蔗糖转运蛋白(sucrose transporters);EUCeSSR676-168 bp与T_mcm、P_wq、年均气温(T_ma)和最暖月最高气温(T_mwm)均显著相关,位点的功能注释为光系统Ⅱ稳定性/组装因子HCF136 (photosystem II stability/assembly factor HCF136);另外两个显著相关位点EUCeSSR298和EUCeSSR1009的功能未知。【结论】大花序桉北部与南部群体的显著分化受长期气候的影响,在第四纪大冰期可能存在北部和南部避难所。与气候因子显著相关的SSR位点在北部和南部群体间的等位频率差异为基于正向选择的气候适应性提供了分子证据。     

春小麦重组自交系及多种SSR标记构建遗传图谱

 以春小麦重组自交系(RIL)宁春4 号×宁春27 号为作图群体,利用SSR 标记构建小麦遗传连锁图谱。结果表明,用1001对SSR 引物选出亲本间表现多态性的引物307 对,多态性频率为30.7%。利用307 对多态性引物对RIL 群体进行分析,共检测到266 个多态性标记位点。通过χ²检测(P<0.05),有147 个SSR 标记表现为偏分离,偏分离率为55.3%,129 个偏向母本宁春4号,其偏分离位点主要分布在B 和D 基因组上。用Mapmaker 3.0 和Mapdraw 2.1 软件将266 个SSR 位点绘制在小麦遗传连锁图上,该图谱覆盖小麦基因组全长2187.79 cM,标记间的平均遗传距离为8.22 cM。