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.     

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。     

漆树EST-SSR引物开发

利用NCBI数据库进行漆树EST-SSR引物开发,从NCBI数据库中共下载漆树EST序列87 856条。利用MISA软件进行序列处理、拼接及聚类后,从87 856条漆树EST序列中拼接组装成3 979条非冗余序列,含SSR位点的EST序列出现频率占EST序列总数的4.5%,从3 979条非冗余序列中检测到487个SSRs微卫星位点,出现频率为12.2%。这些SSR位点中,三核苷酸和二核苷酸重复基元所占比例较高。采用Primer5.0软件,共成功设计50对EST-SSR引物,50对EST-SSR引物在25个漆树个体上均能扩增出清晰的电泳条带,其中18对引物检测出了多态性条带,扩增率达36%。     

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。     

中间锦鸡儿转录组EST-SSR标记系统性识别与引物筛选

旨在对中间锦鸡儿转录组数据库EST信息进行SSR系统性识别和初步验证,为进一步SSR分子标记开发提供依据。对Hi Seq2000测序技术获得的中间锦鸡儿转录组Unigenes进行SSR位点搜索,共获得45 706个SSR位点,出现频率为10.38%,平均4.30kb出现一个SSR位点。SSR重复类型以单核苷酸重复序列基元为主,所占比例为56.47%;二核苷酸、三核苷酸重复序列基元的数量所占比例分别是20.56%和21.04%;其他数量的基元所占比例仅为1.9%。多核苷酸重复类型中最多的为2核苷酸重复AG/CT;其次为3核苷酸重复AAG/CTT。针对EST-SSR位点随机挑选了150对引物,通过琼脂糖凝胶电泳进行PCR验证,其中有79对能获得扩增条带,21对引物扩增出单一条带,比例为14.0%。     

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。     

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。     

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.     

油菜EST-SSR标记的建立

在油菜17987条非冗余EST中,共发掘出了2083个EST-SSR,分布于2443条EST中,发生频率是13.58%,平均分布距离为4.34kb。在油菜EST-SSR中,二、三核苷酸重复是主要的重复类型,二者出现的频率相近,占总SSR的89.05%。AG/CT和AAG/CTT是二、三核苷酸中的优势重复类型,分别占二、三核苷酸重复类型的84.31%和37.71%。进一步设计了23对SSR引物,通过梯度PCR试验确定了各引物的适宜退火温度,并利用非变性聚丙烯酰胺凝胶银染对这些引物在10个油菜品种中的扩增情况和多态性进行了检测。有21对引物显示扩增,引物可用率为91.30%;有12对引物显现出多态性,占可扩增引物的57.14%。本研究结果证明根据油菜EST建立SSR标记是有效、可行的。     

Identification and characterization of SSRs from soybean (Glycine max) ESTs

Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies.     

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.)

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.     

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.)

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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。     

Identifying Novel Polymorphic Microsatellites from Cultivated Flax (Linum usitatissimum L.) Following Data Mining

One of the major concerns in genetic characterization and breeding of cultivated flax is the lack of informative microsatellite markers (SSRs). In this regard, the development of SSRs using molecular methods might be time-consuming, laborious, and expensive. On the other hand, using bioinformatics to mine sequences in public databases enables a cost-effective discovery of SSRs. A total of 3,242 Linum usitatissimum genomic sequences were surveyed for the identification of SSRs. Among them, 118 non-redundant sequences containing repeats were selected for designing primers. The most abundant motifs were tri- (72.4%) and dinudeotide (16.6%), within which AGG/CCT and AG/CT were predominant. Primers were tested for polymorphism in 60 L. usitatissimum cultivars/accessions including 57 linseed and three fiber flax. Eighty-eight pairs gave amplifications within the expected size range while 60 pairs were found to be polymorphic. The mean number of alleles amplified per primer was 3.0 (range, 2–8; 180 total alleles). The mean polymorphism information content (PIC) value was 0.39 (range, 0.06–0.87), and the highest average PIC was observed in dinucleotide SSRs (0.41). The SSR data mining presented here demonstrates the usefulness of in silico development of microsatellites. These novel genomic SSR markers could be used in genetic diversity studies, the development of genetic linkage maps, quantitative trait loci mapping, association mapping, and marker-assisted selection.