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。     

木麻黄EST序列中SSR的分布特征及有效SSR标记的筛选

从NCBI的EST数据库中获得的木麻黄EST序列共有34 752条,进行拼接后得到全长7 278.578 kb的非冗余序列(Unigene)12 062条,并从中检索得到分布于353条Unigene的367个SSR位点,SSR检出率为2.93%,平均分布距离为19.83 kb,包括39种重复基序类型。其中,以二核苷酸和三核苷酸为主要类型,在总SSRs中所占比例分别为57.77%和34.60%;而二核苷酸重复基序中,AG/CT所占比例最高,为93.87%;在三核苷酸重复基序中AAG/CTT所占比例最高,为44.09%。对检索出的EST-SSR位点设计得到97对引物,其中32对为可有效扩增引物。Blastx分析发现77.3%的含SSR位点的EST序列与非冗余蛋白序列数据库中功能序列具有同源性,而功能已知的序列中葡萄来源的序列占有最大比例(10.4%)。GO功能分类发现,含有SSR位点的EST序列中有47.3%至少具有1个GO注释,归入细胞组分的序列最多,而其中细胞质和细胞核的功能项所占比例较大。     

三种人参属植物的EST-SSR信息分析及其在三七中的应用

为了解人参属植物EST中SSR分布特点及其在三七SSR标记中的应用,利用生物信息学方法,用primer 3软件对dbEST数据库中人参属植物人参、西洋参和三七的EST序列进行搜索,发现人参属植物EST-SSR出现频率为11.54%,平均每4.39 kb出现1个SSR.人参属植物单核苷酸重复基元占主导地位,其次为三核苷酸重复基元和二核苷酸重复基元,分别占总SSR的54.48%、17.31%和16.36%.人参属EST-SSR的优势类型为A/T和AT/TA,分别占54.73%和8.21%.根据人参属植物EST中的SSR设计48对引物,在合适的PCR扩增体系下,用5个三七样品的DNA为模板进行PCR扩增,引物有效扩增率85.42%,其中多态性引物占可扩增引物的70.73%.结果表明,利用人参属EST序列开发三七EST-SSR标记是可行的.     

番红花EST资源的SSR信息分析

从NCBI公共数据库下载得到6745条番红花EST,通过前处理得到全长为612.01 kb的无冗余EST 1431条.在这些序列中搜索出108个SSR,分布于103条EST中,出现频率为7.55%.这些EST-SSR的平均分布距离是5.67 kb.二核苷酸重复和三核苷酸重复是番红花主要的重复类型,分别占总EST-SSR...     

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。     

甘薯与牵牛EST资源的SSR信息分析

为挖掘番薯（Ipomoea）属EST-SSR资源,从NCBI数据库下载23406条甘薯（Ipomoea batatas (L.) Lam.）EST和62282条牵牛（Ipomoea nil (L.) Roth）EST,利用生物信息学软件预处理、去冗余、拼接处理后得到12812条无冗余的甘薯EST（6.70 Mb）和28422条牵牛唯一序列（17.19 Mb）。对这些序列进行SSR搜索,在甘薯上获得328个SSR位点,发生频率为2.56%;牵牛上筛选到962个SSR位点,出现频率为3.38%。甘薯和牵牛EST-SSR具有多个共同特征：在SSR位点中,主要是二核苷酸重复类型,其次是三核苷酸重复;在二核苷酸重复中,出现最多的重复基序为AG/CT,其次是AT/AT;在三核苷酸重复中,主要基序是AAG/CCT;SSR位点的长度主要集中在20~22 bp。结果表明,这些搜索出的EST-SSR重复基序类型丰富、多态性潜能高,具有较高的开发和利用价值。     

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。     

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.     

陆地棉EST长度多态性与其SSR分布特征相关性分析

目的:分析陆地棉EST长度多态性与其SSR分布特征的相关性。方法:从NCBI公共数据库下载陆地棉EST序列,应用SSRIT搜索SSR,分析20 000条无冗余的EST序列。结果:在剔除低质量和冗余的序列后,得到全长为7 363.878kb的无冗余EST序列7 322条,其中含有SSR位点的EST序列数520条,占被分析EST比例的2.60%。长度在400bp以下的EST序列含SSR的比例为1.46%;长度在400bp以上的EST序列含SSR的比例为8.94%。在1～6bp的重复基元中,二核苷酸重复基元的SSR重复频率最高,占总数的63.46%,其次是三核苷酸,占总数的34.04%。二核苷酸类型(AG)n、(AT)n和三核苷酸类型(AAG)n、(ACC)n、(ACT)n、(AAT)n是SSR的主要重复基元。结论:棉花EST-SSR可用于棉花分子标记,为有针对性设计陆地棉EST-SSR引物奠定基础。     

美洲南瓜转录组SSR信息分析及其分子标记开发

该文对美洲南瓜(Cucurbita pepo L.)开展转录组测序分析,共获得83 650条Unigene,利用MISA软件搜索1 Kb以上的15 356条Unigene,共检测出7 478个SSR位点,分布于5 786条Unigene中,出现频率为48.7%,平均分布距离为4.08 Kb。优势重复基序为单核苷酸、二核苷酸和三核苷酸,分别占总SSR的47.90%、20.57%和22.36%。二核苷酸重复基序中以AG/CT为优势重复基序,三核苷酸重复基序以AAG/CTT为主。利用Primer 3.0共设计出5 786对SSR引物。从114对有效扩增引物中随机选择50对引物,对28个美洲南瓜种质进行多态性验证分析,其中35对(占70%)引物表现稳定可重复的多态性。利用UPGMA作图,将28份供试材料分为2类。利用美洲南瓜转录组数据进行SSR标记开发能获得较高频率的SSR位点,且类型丰富,为美洲南瓜遗传多样性分析和遗传图谱构建提供更丰富可靠的标记选择。     

皂荚EST-SSR分子标记开发与分析评价

本研究旨在开发皂荚EST-SSR分子标记,为今后皂荚种质资源评价与分析提供基础。首先通过对已公布的皂荚转录组数据进行拼接得到41003个Unigenes,总长度70.4 Mb,平均长度1716 bp,N50为2533 bp。进一步在7009个Unigenes中检测到8494个EST-SSR位点,其中1200条Unigenes含有2个及以上的SSR位点,复合型SSRs有369个。针对所有EST-SSR位点设计得到6494条特异性引物,随机挑选60个位点进行试验验证与分析,其中44对引物可扩增出特异性片段,17对具有多态性,PIC值范围为0.195~0.742,均值为0.501,且大部分多态性位点位于UTR区域。通过对皂荚近缘种进行跨种PCR扩增试验,结果显示在开发的44对有效引物中,9个近缘种美国皂荚、日本皂荚、绒毛皂荚、滇皂荚、华南皂荚、小果皂荚、野皂荚、肥皂荚和美国肥皂荚分别有32、31、23、24、7、40、25、18和18对引物可获得有效扩增片段,说明EST-SSR标记在皂荚近缘种之间具有良好的通用性。研究表明利用转录组数据挖掘的EST-SSR位点具有扩增稳定、多态性良好、近缘种间通用等优点,是林木物种开发分子标记的有效途径之一。     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

基于陆地棉茎尖转录组序列的genic-SSR标记的开发与评价

利用软件MISA和SSR Locator 对陆地棉（Gossypium hirsutum L.）Coker312茎尖转录组测序得到的73515条Unigene序列进行分析,查找到4507个SSR位点,分布于4039条转录本序列中,SSR出现频率为5.5%,非编码区的SSR位点要显著高于编码区（2853/1654）。SSR重复基元中,三核苷酸重复基元占主导地位（51.03%）,且AAG/CTT （20.08%）类型最多;其次是二核苷酸重复基元（28.76%）,主要为AG/CT（55.47%）。利用引物批量设计软件共开发1569对SSR引物,在陆地棉TM-1、海岛棉3-79 (G. barbadense L.)、阿非利加棉(G. herbaceum L.)、雷蒙德氏棉(G. raimondii Ulbrich.)四个棉种中进行初步评价,其中1117对引物能在四个棉种间扩增出稳定的条带,扩增率为71.2%。选择650对能稳定扩增出条带的引物,在涉及5个棉种（增加了亚洲棉（G. arboreum L.））的13份材料中进一步筛选,83对引物能在13份材料中扩增出特异性条带, PIC变幅在0.121～0.648之间,平均值为0.422,其中80对引物能在7份陆地棉材料中扩增出特异性条带,平均PIC值为0.336;挑选5对在本实验室作图亲本鲁棉研22和鲁原343间有明显多态的引物进行连锁分析,其中4对成功连锁到本实验室构建的遗传图谱上。本研究丰富了棉属genic-SSR的数量,为棉属遗传作图、性状关联分析提供了更多引物选择。     

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.     

Development, characterization, and cross-species/genera transferability of SSR markers for rubber tree (Hevea brasiliensis)

Genomic simple sequence repeat (SSR) markers are particularly valuable in studies of genetic diversity, evolution, genetic linkage map construction, quantitative trait loci tagging, and marker-assisted selection because of their multi-allelic nature, reproducibility, co-dominant inheritance, high abundance, and extensive genome coverage. The traditional methods of SSR marker development, such as genomic-SSR hybrid screening and microsatellite enrichment, have the disadvantages of high cost and complex operation. The selectively amplified microsatellite method is less costly and highly efficient as well as being simple and convenient. In this study, 252 sequences with SSRs were cloned from the rubber tree (Hevea brasiliensis) genome from which 258 SSR loci were obtained. The average repeat number was six. There were only 10 (3.9%) mononucleotide, trinucleotide, and pentanucleotide repeats, whereas the remaining 248 (96.1%) were dinucleotide repeats, including 128 (49.6%) GT/CA repeats, 118 (45.7%) GA/CT repeats, and 2 (0.8%) AT/TA repeats. A total of 126 primer pairs (see ESM) were successfully designed of which 36 primer pairs generated polymorphic products from 12 accessions of the cultivated species, 4 related species, and 3 species of the family Euphorbiaceae. In addition, investigations based on four genomic SSRs (GAR4, ACR22, CTR25, and GTR28) by cloning and sequencing provided evidence for cross-species/genera applicability, and homologous sequences were obtained from the rubber tree and Euphorbiaceae. Further analysis about the variation of the flanking regions of the four markers was carried out.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Genome-Wide Characterization of Simple Sequence Repeat (SSR) Loci in Chinese Jujube and Jujube SSR Primer Transferability

Chinese jujube (Ziziphus jujuba), an economically important species in the Rhamnaceae family, is a popular fruit tree in Asia. Here, we surveyed and characterized simple sequence repeats (SSRs) in the jujube genome. A total of 436,676 SSR loci were identified, with an average distance of 0.93 Kb between the loci. A large proportion of the SSRs included mononucleotide, dinucleotide and trinucleotide repeat motifs, which accounted for 64.87%, 24.40%, and 8.74% of all repeats, respectively. Among the mononucleotide repeats, A/T was the most common, whereas AT/TA was the most common dinucleotide repeat. A total of 30,565 primer pairs were successfully designed and screened using a series of criteria. Moreover, 725 of 1,000 randomly selected primer pairs were effective among 6 cultivars, and 511 of these primer pairs were polymorphic. Sequencing the amplicons of two SSRs across three jujube cultivars revealed variations in the repeats. The transferability of jujube SSR primers proved that 35/64 SSRs could be transferred across family boundary. Using jujube SSR primers, clustering analysis results from 15 species were highly consistent with the Angiosperm Phylogeny Group (APGIII) System. The genome-wide characterization of SSRs in Chinese jujube is very valuable for whole-genome characterization and marker-assisted selection in jujube breeding. In addition, the transferability of jujube SSR primers could provide a solid foundation for their further utilization.     

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。     

亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。