蜜蜂EST中的微卫星分析

为加速分子标记在蜜蜂遗传、进化与行为等方面的利用,分析了简单重复序列(Simple Sequence Repeats,SSRs)在蜜蜂EST中的分布频率与密度。所分析的蜜蜂EST数据集包含15869条序列,总长为7．9Mb。结果显示,蜜蜂ESTs中SSRs的频率为1／0．52kb,其中6碱基重复基序占总SSRs的45．0％,是最丰富的重复单元,而2、1、3、4与5碱基重复基序分别占总SSRs的17．9％、14．1％、11．6％、9．2％和2．2％。同时,在各种SSRs重复单元中,富含A碱基的重复单元占据优势地位,如：A、AT、AG、AC、AAT、AAG、AAC、AAAT、AAAG、AAAAG、AAAAT、AATAT、AAAAAG和AAAAAT重复基序,而富含G碱基的重复单元在基因编码区中含量较低。进一步分析显示：蜜蜂SSRs在冗余与非冗余EST数据集中的分布频率与密度相似,仅存在极小的偏差,表明可从现有的部分ESTs数据中方便地获取有效的微卫星标记。     

用EST-SSR检测不同年代小麦育成品种基因多样性的变化趋势

EST—SSRs是一种基于基因表达序列有关基因功能的“真质”标记,其多态性直接反映了基因的多样性。本研究采用37对小麦EST—SSRs引物检测了42份我国不同年代小麦选育品种相关基因位点多样性的变化趋势。结果表明：基于表达序列设计的小麦EST—SSRs引物具有较好的扩增效果,其中37对引物共检测到134个位点,绝大多数引物有2～5个等位变异;在相似系数为0.9的水平上这些引物可将除燕大1817和旱选3号外的40份材料区分开来;供试的42份小麦选育品种的基因多样性从20世纪60年代以前到90年代呈递增趋势,但不同年代的增幅不同：20世纪60～80年代增长最快,80年代至90年代增长十分缓慢;同时,材料间相似系数变化呈下降趋势,60年代以前的选育品种相似性最高,70年代最低,70年代以后至90年代略有增加,但远低于60年代以前,这与引人大量的外来品种的育种策略有关。     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

中国地方品种香猪的肌肉特异组织表达序列标签（ESTs）的分析

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列。在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs。对这10个已知、未知ESTs进行开放阅读框预测并进行BlastX分析,没有找到高度同源的氨基酸序列。对上述EST所对应的基因功能分析结果表明,除去27．27％的EST未能分类外,克隆到的EST大多来自与基因／蛋白的表达调控相关的基因（占45．46％）。来自具有其他功能的基因的EST依次是细胞代谢占10．10％、细胞结构／迁移占10．10％、细胞／机体防御占5．05％和细胞信号／传导占2．02％。没有发现和细胞分裂相关的已知功能基因。本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础。     

柔嫩艾美尔球虫EST序列中SSR的获取及分析

对柔嫩艾美尔球虫EST—SSR进行生物信息学分析,共获取Eimeria tenella EST序列34074条,总长度为16．45Mb,小于12bpSSR的ESTs达7651条,从中获得SSR序列19576条、总长度为0．35Mb,EST—SSRs的频率是48．00％,平均相隔S40bp出现一个长度不小于12bp的SSR。在E．tenella的核苷酸重复基元中,2、3、4、5、6和7bp重复序列在基因组中出现的种类分别有11种472条、49种14710条、31种525条、13种25条、21种43条和15种400条,3碱基重复序列是最丰富的重复单元,占总数的75．14％。各种SSRs中富含G、C碱基的重复单元以GCA出现频率最多（28．63％）,次为AGC（17．59％）,GCT（8．76％）,TGC（7．62％）,CTG（7．15％）。     

茶树EST-SSRs分布特征及标记开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288条茶树(Camellia sinensis) ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2.10 kb出现一个SSR。在2-6 bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高(51.97%),其次是三核苷酸(19.55%)。对所有的重复基元类型进行统计分析发现, 所占比例最高的是AG/CT(47.74%),其次分别是AT/TA(4.73%)和AAG/CTT(4.73%)。利用Prime 5 软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93.5%。这些EST-SSRs将有助于茶树基因组学方面的研究。     

中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

抗口蹄疫病毒相关基因的筛选及分析

口蹄疫是一种烈性传染病,其广泛流行给社会造成了巨大经济损失。为了研究口蹄疫灭活疫苗免疫的分子机制,同时也为抗口蹄疫病毒药物的研制奠定基础,本研究应用mRNA差异显示技术,以PK-15细胞为材料,系统比较了口蹄疫疫苗刺激组（A组）和正常的PK-15细胞（B组）的基因表达情况,回收差异片段,经二次扩增并纯化后,得到30条ESTs。将30条ESTs采用以地高辛标记的反向Northern点杂交鉴定,将阳性条带克隆测序,筛选出8条ESTs,编号E1~E8,应用BLASTn工具将8条ESTs对核酸数据库nr和dbEST中所有序列进行了同源性分析,其中E1,E2分别与猪的热休克蛋白基因、猪的MHCⅠ类基因同源,序列相似性都达到100%。E3,E4,E5,E7分别与已有核酸数据库中的基因克隆或EST具有较高同源性,为已知的EST,但功能未知;E6,E8在数据库中没有发现与其相似性较高的序列,为新的EST。应用数据库资源将E5、E7进行电子延伸后,将延伸序列进行开放阅读框分析,又经TBLASTx分析发现E7蛋白质序列与猪的精氨酸酶Ⅰ类蛋白序列有很高同源性。将E1、E2、E4、E5序列进行了基因表达谱分析,对E6、E8用BLASTx工具对非冗余蛋白质数据库nr进行了相似性搜索,在其他物种中找到了相似的基因序列。本研究筛选出的热休克蛋白基因、MHCⅠ类基因、精氨酸酶Ⅰ类基因和其他未知功能基因可以作为抗口蹄疫病毒研究中的侯选基因,其具体的功能有待今后进一步研究。     

鲤鱼多态性EST标记的筛选与特性分析

表达序列标签(Expressed sequence tag,EST)标记在基因组作图和分子标记辅助育种研究中具有重要价值.筛选和开发可用于遗传作图的鲤鱼多态性EST标记,对研究鲤鱼的基因组结构、遗传多样性研究和遗传育种有重要意义.本研究根据GenBank数据库中鲤鱼EST序列设计了67对EST引物,有47对在鲤鱼基因组DNA中成功扩增得到稳定的特异性条带,经单链构象多态性(SSCP)分析,12对(25.5%)引物扩增的EST在1个鲤鱼回交家系中具有多态性,其中6个为鲤鱼功能基因,5个与斑马鱼(Danio rerio)功能基因具较高相似性,1个与斑点叉尾(Ictalurus punctatus)功能基因具较高相似性.这些多态性的EST在鲤鱼二倍体家系和单倍体群体中的等位基因分离均符合孟德尔规律(1∶1或3∶1),单倍体中的SSCP条带数目为二倍体的二分之一.选用其中4对多态性的EST引物对洞庭湖鲤鱼进行初步群体遗传分析,结果显示基因多样性(H)为0.437,远低于微卫星标记所揭示的该群体基因多样性(接近于1).结果表明,多态性的EST-SSCP标记尽管遗传变异性较低,但是作为来源于编码区的Ⅰ型遗传标记,在遗传作图和种群遗传适应性等研究中有较好的应用潜力.     

An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.     

普通小麦SSR和EST-SSR引物对冰草通用性的比较分析

选用定位于普通小麦7个部分同源群的534对SSR引物和351对EST-SSR引物分别对普通小麦品种‘Fukuho’和四倍体冰草‘Z559’的基因组DNA进行扩增,结果显示:有475对(89.0%)SSR引物和314对(89.5%)EST-SSR引物对‘Fukuho’能有效扩增,226对(42.3%)SSR和258对(73.5%)EST-SSR引物对‘Z559’能有效扩增,表明小麦EST-SSR对冰草的通用性明显高于SSR;扩增强带比率SSR和EST-SSR引物分别为76.1%、84.1%,说明小麦EST-SSR在冰草上扩增带的质量亦优于SSR。选择上述在‘Fukuho’和‘Z559’基因组DNA之间有多态性扩增且带谱清晰的SSR和EST-SSR引物各60对,对‘Fukuho’、‘中国春’、‘北京8号’和二、四、六倍体冰草‘Z804’、‘Z559’、‘Z1075’的基因组DNA再行PCR扩增,结果显示,40对(66.7%)SSR和22对(36.7%)EST-SSR引物在‘Fukuho’、‘中国春’和‘北京8号’间扩增产物表现多态性,且前者高于后者;50对(83.3%)SSR和52对(86.7%)EST-SSR引物在冰草‘Z804’、‘Z559’和‘Z1075’间扩增产物表现多态性,两者相当。通用性、多态性和扩增强带比率综合比较表明,普通小麦EST-SSR和SSR经筛选虽都能转用于冰草,但两者相比EST-SSR更优。     

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs)

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.     

High transferability of bread wheat EST-derived SSRs to other cereals

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In this study, 300 primer pairs were designed from 265 wheat ESTs that contain microsatellites in order to develop new markers for wheat. Their level of transferability in eight related species [Triticum durum, T. monococcum, Aegilops speltoides, Ae. tauschii, rye (Secale cereale), barley (Hordeum vulgare), Agropyron elongatum and rice (Oryza sativa)] was assessed. In total, 240 primer pairs (80%) gave an amplification product on wheat, and 177 were assigned to wheat chromosomes using aneuploid lines. Transferability to closely related Triticeae species ranged from 76.7% for Ae. tauschii to 90.4% for T. durum and was lower for more distant relatives such as barley (50.4%) or rice (28.3%). No clear putative function could be assigned to the genes from which the simple sequence repeats (SSRs) were developed, even though most of them were located inside ORFs. blast analysis of the EST sequences against the 12 rice pseudo-molecules showed that the EST-SSRs are mainly located in the telomeric regions and that the wheat ESTs have the highest similarity to genes on rice chromosomes 2, 3 and 5. Interestingly, most of the SSRs giving an amplification product on barley or rice had a repeated motif similar to the one found in wheat, suggesting a common ancestral origin. Our results indicate that wheat EST-SSRs show a high level of transferability across distantly related species, thereby providing additional markers for comparative mapping and for following gene introgressions from wild species and carrying out evolutionary studies.     

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 of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

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.     

Identification of microsatellites in cattle unigenes

To identify EST-SSR molecular markers, 41,986 cattle UniGene sequences from NCBI were mined for analyzing SSRs. A total of 1,831 SSRs were identified from 1,666 ESTs, which represented an average density of 19.88 kb per SSR. The frequency of EST-SSRs was 4.0%. The dinucleotide repeat motif was the most abundant SSR, accounting for 54%, followed by 22%, 13%, 7% and 4%, respec-tively, for tri-, hexa-, penta- and tetra-nucleotide repeats. Depending upon the length of the repeat unit, the length of microsatellites varied from 14 to 86 bp. Among the di- and tri-nucleotide repeats, AC/TG (57%) and AGC (12%) were the most abundant type. Annotation of EST-SSRs was also carried out. Three hundred primer pairs were randomly designed using Prime Premier 5.0 program and Oligo 5.0 for further experimental validation.