植物基因组表达序列标签(EST)计划研究进展

植物表达序列标签(EST)计划是随机挑选cDNA克隆,并对其3′或5′端进行大规模一次性测序,将得到的150～500 bp长度的DNA片段与数据库中的序列进行比较,获得对基因组结构、组织、表达等认识的基因组研究策略.就近年来国际植物EST计划的实施情况、植物EST计划的研究范围、生物信息学在EST研究中的应用、EST数据库及查询、植物EST研究中遇到的问题等方面内容进行了综述.     

EST分子标记开发及在比较基因组学中的应用

数量迅速增加的EST(expressed sequence tags)为分子标记的开发提供了宝贵的资源。与来自于基因组DNA开发的传统标记相比,以EST为基础的分子标记是一种新型分子标记,具有其显著的优势,如开发简便、信息量高和通用性好等,在多方面都有重要的利用价值。本文详细地介绍了目前基于EST开发的5类分子标记以及基于生物信息学方法的开发策略,这些标记包括EST-PCR、EST-SSR、EST-SNP、EST-RFLP和EST-AFLP。此外,对这些标记在比较基因组学研究中的应用进行了评述,包括比较作图、遗传多样性评价及系统发育研究等。     

表达序列标签(EST)在基因组学研究中的应用

表达序列标签 (expressedsequencetags)是一种快捷、高效的揭示基因组信息的方法。本文对EST的产生、概念、技术原理及其在基因组研究中的广泛应用作一详细的介绍     

植物表达序列标签(EST)标记及其应用研究进展

简要介绍了植物表达序列标签(EST)标记的研究现状,并对几种植物中利用EST建立分子标记的几种策略和EST标记在绘制遗传图谱、资源分析、品种鉴定及比较基因组学研究方面的应用等进行了综合评述。     

分子标记及其在海洋动物遗传研究中的应用

分子遗传标记在农业动植物育种和生产上得到了广泛的应用,且取得了可喜的成果,但在水生生物上的应用还处于初始阶段。本文简要介绍了限制性片段长度多态性（RFLP）、随机扩增多态性DNA（RAPD）、扩增片段长度多态性（AFLP）、小卫星DNA和微卫星DNA（或称简单序列重复,SSR）等分子标记的概念、基本原理及其特点,重点介绍了第三代分子标记单核苷酸多态性（SNP）技术。综述了这些分子标记在海洋动物遗传结构分析、亲缘关系鉴定、遗传图谱的构建和标记辅助育种等方面的应用。     

分子标记在百合属植物遗传多样性研究中的应用

介绍分子标记技术及其发展概况,并重点论述几种常见分子标记技术如随机扩增多态性DNA(random amplified polymorphism DNA,RAPD)、简单重复序列间区(inter-simple sequence repeat,ISSR)、扩增片段长度多态性(amplified fragment length polymorphism,AFLP)和内转录间隔区(internal transcribed spacer,ITS)等的基本原理、技术上的优缺点及其在百合属植物遗传多样性研究中的应用现状,同时对分子标记技术在百合属植物遗传多样性研究中的应用前景进行展望.     

亚历山大藻属微卫星标记的筛选

采用生物信息学方法,从塔玛亚历山大藻的ESTs数据库中筛选微卫星(Simple Sequence Repeat, SSR)位点,设计SSR引物,利用毛细管电泳对4株塔玛亚历山大藻(Alexandrium tamarense)、2株相关亚历山大藻(Alexandrium affine)和1株链状亚历山大藻(Alexandrium catenella)进行种内、种间遗传多样性分析,获得以下主要结果:(1)从6830条非冗余的ESTs中共查找到222个SSR,平均每18.5kb就有一个SSR,三核苷酸重复在所有的重复类型中所占比例最大,达到48.2%,重复单元CTG/CAG出现频率最高.进一步筛选出12条SSR引物,用于遗传多样性分析.(2)种内遗传多样性水平整体偏低,多态性引物比率仅为41 67%,Nei's基因多样性平均为0.2130.种间遗传多样性水平较高,多态性引物比率为75%,Nei's基因多样性平均水平为0 4643;另外,种间遗传分化系数较高,平均水平为0.7051.(3)对7个样品的聚类图分析发现,A. tamarense CCMP116 与A. affine CCMP112遗传距离极为接近;A. tamarense ATHK9301、CCMP1598和ATDH01相互聚在一起;A. catenella ACDH01与A. tamarense分属两枝.以上结果表明,SSR标记在亚历山大藻种内遗传多样性分析方面是一种非常有用的工具.     

微卫星DNA标记开发技术进展及其在经济植物研究中的应用

微卫星分子标记技术被广泛应用于分子生物学研究中,具有多态性高、重复性好、共显性表达、杂合度高等特点,在种群遗传多样性、遗传图谱构建等领域发挥不可替代的作用。近年来,随着新一代高通量测序技术的进一步成熟,简单重复序列(simple sequence repeat,SSR)标记的开发技术与应用得到了进一步的发展。现主要对基于高通量测序的微卫星分子标记最新开发技术进行介绍,并从遗传多样性分析、遗传图谱构建、品种鉴定及分子辅助育种等方面,总结近年来SSR标记技术在经济植物研究中的最新应用,最后对SSR技术的应用前景进行展望,以期为利用微卫星技术进行经济植物研究提供参考。     

SCoT分子标记在植物研究中的应用进展

SCo T是一种新型的目的基因分子标记,该标记不仅能获得与性状联系紧密的目的基因,而且能对性状进行跟踪,已被广泛应用于多种植物的研究。本文概述了SCo T标记的原理、引物设计方法及特点,并从PCR反应体系建立与优化、种质资源遗传多样性与亲缘关系分析、种质鉴定与指纹图谱构建、基因差异表达与分子遗传连锁图谱构建等方面总结了SCo T标记的应用进展。同时,对其存在的问题进行了讨论,并展望了该标记的发展应用前景。     

基于珙桐SNP位点的dCAPS标记开发

本研究收集了珙桐、喜树等19份植物材料,运用分子生物学和生物信息学手段比对分析了rpoB,rpoC,matK等8个基因碱基序列上的单核苷酸多态性(SNP)位点,在atpF-atpH基因上找到了合适的SNP位点并设计了dCAPS引物,经PCR扩增和酶切验证后,将SNP转化为衍生型酶切扩增多态性序列(dCAPS)标记,可促进SNP标记在珙桐的遗传图谱构建、基因定位、种质资源鉴定、遗传多样性研究等领域的应用。     

Tomato SNP Discovery by EST Mining and Resequencing

Many economically important crop species are relatively depauparate in genetic diversity (e.g., soybean, peanut, tomato). DNA polymorphism within cultivated tomato has been estimated to be low based on molecular markers. Through mining of more than 148,000 public tomato expressed sequence tags (ESTs) and full-length cDNAs, we identified 764 EST clusters with potential single nucleotide polymorphisms (SNPs) among more than 15 tomato lines. By sequencing regions from 53 of these clusters in two to three lines, we discovered a wealth of nucleotide polymorphism (62 SNPs and 12 indels in 21 Unigenes), resulting in a verification rate of 27.2% (28 of 103 SNPs predicted in EST clusters were verified). We hypothesize that five regions with 1.6–13-fold more diversity relative to other tested regions are associated with introgressions from wild relatives. Identifying polymorphic, expressed genes in the tomato genome will be useful for both tomato improvement and germplasm conservation.     

Genetic Diversity Analysis of Nelumbo Accessions Based on EST SSR Markers

EST-SSR markers were applied to estimate the genetic diversity for 30 accessions of Nelumbo nucifera, 6 accessions of Nelumbo lutea and 14 hybrids between these two species. The 52 of 123 EST SSR markers (423%) were screened and then applied to amplify the 50 Nelumbo accessions. A total of 177 alleles were identified, and the number of alleles per locus and Polymorphic Information Content (PIC) value varied from 2 to 8 with an average of 34 alleles and from 063 (NNFB 1059) to 091 (NNFB 750) with an average value of 079, respectively. Jaccard similarity coefficients of the amplification results were analyzed by NTSYS pc2.11 software and the genetic similarity coefficient was from 024 to 086. The clustering dendrogram constructed by UPGMA method indicated that 50 accessions of Nelumbo could be divided into four major groups at the similarity coefficient of 037. Group I and group II included Nnucifera; group III included the majority of Asian American lotus hybrids; and group IV included Nlutea. In addition, the Asian American hybrids were closer to Nnucifera based on genetic relationship, which is consistent with the traditional classification result and the previous reports.     

基于逆转座子的植物分子标记技术及其应用

逆转座子特异的结构和功能使其作为一种有效的遗传工具应用于许多方面。本文就植物逆座子的组成、类别、以及其作为分子标记体系应用在遗传多样性、种系发生和基因作图等的新进展作一综述。     

毛乌素沙漠南缘沙区不同科蝗虫酯酶同工酶研究

应用垂直板型聚丙烯酰胺凝胶不连续电泳技术 ,分析分布于毛乌素沙漠南缘风沙区蝗总科中5科 1 0属 1 1种EST同工酶的多态现象。研究结果表明 ,同一个体不同体段的酶谱有一定的差异 ,其中以体壁活性最高 ;在不同科属间、同种不同个体间及性别的酶谱也有所差异 ,但这种差异远小于种间差异。     

反转录转座子标记及在作物遗传育种中的应用

反转录转座子通过RNA中间体进行反转录而转座,广泛分布于各种植物基因组中,拷贝数多,异质性高,在种内和种间表现出较高的序列差异性和丰富的插入多态性。针对这些特点,开发出了几种基于反转录转座子的分子标记,如SSAP、RIVPI、RAP、REMAP和RBIP等。由于反转录转座子标记能揭示出丰富的多态性,因而在遗传多样性和系谱研究、遗传连锁图谱构建及性状基因定位等方面得到了应用。随着分离技术的不断改进,获取序列信息更加容易,反转录转座子作为分子标记用于作物遗传育种将具有广阔前景。     

SNP分子标记及其在木本植物遗传育种的应用

木本植物因其生命周期长、基因组杂合度高、基因组较大、遗传背景不清晰等特性,制约了其研究进程。随着现代生物技术的发展,DNA分子标记技术在木本植物研究领域的应用越来越多,其中单核苷酸多态性(SNP)作为第三代分子标记技术以其高效、快速、稳定、可靠等诸多优点得到广泛应用。本文简述SNP标记的特点、开发方法、检测方法及其在木本植物遗传多样性和亲缘关系分析、品种鉴定、连锁图谱构建和辅助育种等方面的研究进展,为更好地应用SNP技术开展木本植物研究提供参考。     

Analysis of gene-derived SNP marker polymorphism in US wheat (Triticum aestivum L.) cultivars

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat (Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes (P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of Flat and Fan-Shaped Fruit in Actinidia chinensis var. chinensis and Actinidia deliciosa

We describe and quantify development of flat and fan-shapedfruit of Actinidia chinensis var. chinensis from inception tomaturity. Flat fruit arise from particularly large and flatfloral meristems. After bract initiation, the terminal flowerremains elliptic in cross section, produces elliptic whorlsof floral organs, and forms a flat-shaped ovary. The allometryof the ovary does not change from inception to maturity. Fan-shapedfruit develop from exceptionally flat floral meristems. Theyresult from postgenital fusion of the terminal flower with oneor two precocious lateral flowers. Timing of the fusion processvaries, resulting in a variable degree of integration of tissues.The fasciated flower has supernumerary floral organs, and isborne on a single pedicel. The histology of mature flat andfan-shaped fruit is described for commercially-grown Actinidiadeliciosa cv. Hayward. Mature flat fruit have a larger maximumdiameter, but are comparable to normal fruit in the minimumdiameter. Flat fruit have more locules and more pericarp tissuethan normal fruit, but these are not causally related to fruitshape. The flat shape can be attributed to differential planesof enlargement of cells in certain regions of the central core.Mature fan-shaped fruit are larger, and have more pericarp,core and locules than normal or flat fruit.Copyright 1994, 1999Academic Press Actinidia chinensis, Actinidia deliciosa, fruit shape, development, anatomy, fusion     

Development of EST-based SNP and InDel markers and their utilization in tetraploid cotton genetic mapping

Background

Availability of molecular markers has proven to be an efficient tool in facilitating progress in plant breeding, which is particularly important in the case of less researched crops such as cotton. Considering the obvious advantages of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), expressed sequence tags (ESTs) were analyzed in silico to identify SNPs and InDels in this study, aiming to develop more molecular markers in cotton.

Results

A total of 1,349 EST-based SNP and InDel markers were developed by comparing ESTs between Gossypium hirsutum and G. barbadense, mining G. hirsutum unigenes, and analyzing 3′ untranslated region (3′UTR) sequences. The marker polymorphisms were investigated using the two parents of the mapping population based on the single-strand conformation polymorphism (SSCP) analysis. Of all the markers, 137 (10.16%) were polymorphic, and revealed 142 loci. Linkage analysis using a BC₁ population mapped 133 loci on the 26 chromosomes. Statistical analysis of base variations in SNPs showed that base transitions accounted for 55.78% of the total base variations and gene ontology indicated that cotton genes varied greatly in harboring SNPs ranging from 1.00 to 24.00 SNPs per gene. Sanger sequencing of three randomly selected SNP markers revealed discrepancy between the in silico predicted sequences and the actual sequencing results.

Conclusions

In silico analysis is a double-edged blade to develop EST-SNP/InDel markers. On the one hand, the designed markers can be well used in tetraploid cotton genetic mapping. And it plays a certain role in revealing transition preference and SNP frequency of cotton genes. On the other hand, the developmental efficiency of markers and polymorphism of designed primers are comparatively low.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1046) contains supplementary material, which is available to authorized users.     

猕猴桃SCoT遗传多样性分析及指纹图谱的构建

利用SCoT标记对32个猕猴桃品种进行了遗传多样性分析。从47个SCoT引物中筛选了11个引物进行PCR扩增,共扩增出185个条带,其中多态性条带180个,多态性比率为97.30%。各引物Nei's基因多样性指数（H）平均为0.238 4,Shannon's信息指数（I）平均为0.377 8。利用UPGMA构建32份猕猴桃种质资源的聚类树状图。在遗传相似系数为0.78处可将32个猕猴桃品种分为5组,聚类结果与形态学分类基本一致。利用4条引物扩增的16个多态性位点构建了32个猕猴桃品种的DNA指纹图谱,可以将32个猕猴桃品种区分并准确鉴定。