DNA分子标记在果树遗传学研究上的应用

本文综述了近年来DNA分子标记在果树种质资源研究、分子遗传图谱构建、 基因标记、辅助选择育种等方面研究的应用。 Abstract:This paper reviewed the recent progress of the application of DNA molecular markers in various aspects of fruit tree genetics including germplasm reseach,genetic mapping,genetic tagging,marker asisted selection.     

DNA分子标记在番茄遗传育种研究中的应用

本文综述了DNA分子标记在番茄遗传图谱构建、番茄种质资源研究与品种纯度的鉴定、番茄基因分子标记研究及番茄基因图位克隆方面的应用研究进展。 Abstract:This paper reviewed the recent advance of the application of DNA molecular marker in various aspects of tomato breeding including genetic map construction,germplasm research and purity control of cultivars,identification markers linked to important genes and map-based gene cloning.     

偏分离分子标记的作图方法

对取自MAPMAKER软件小鼠F2群体(含333个体)的5个RFLP连锁标记数据作了共显性分子标记偏分离的分析。先确定选择类型的方程组(配子或合子),随后采用Newton-Raphson迭代法估算标记间的重组值。在构建分子标记遗传图谱时,如果两个相邻标记均存在偏分离,最好采用纳入偏分离因子的估算方法。在估计F2群体标记间偏分离重组距离上,用连续χ2检测方法比传统χ2检测更为准确。Abstract The comparative analysis of segregation distortions of the codominant markers data presented in software MAPMAKER are made, where five RFLPs markers involve in a mouse F2 population with 333 individuals. The successive χ2 test begins with the determinations of gametic or zygotic selection types, followed by the estimation of recombination fractions between two markers with the Newton-Raphson iteration method. It is better to use the molecular marker showing segregation distortion for constructing a genetic map, in the case of seriously skew segregation between both the adjoining markers. The successive χ2 test provides better accuracy than that of classical χ2 test for the estimation of the recombination values in F2 population with segregation distortion.     

单核苷酸多态性在作物遗传及改良中的应用

单核苷酸多态性（single nucleotide polymorphism,SNP）是等位基因间序列差异最为普遍的类型,可作为一种高通量的遗传标记。已建立了PCR扩增目标序列及其产物测序和电子SNP（eSNP）等多种发现和检测SNP的方法。玉米和大豆等作物也已开展了SNP分析。一些栽培作物种质的多样性不断减少,其结果使连锁不平衡（linkage disequilibrium,LD）增加,这有利于目的基因座上SNP单元型（haplotype）与表型的相关性分析。SNP已在作物基因作图及其整合、分子标记辅助育种和功能基因组学等领域展示了广泛的应用价值。 Abstract:Single nucleotide polymorphism(SNP) is the most common type of sequence difference between alleles,which can be used as a kind of high-throughput genetic marker.Several different routes have been developed to discover and identify SNP.These include the direct sequencing of PCR amplicons,electronic SNP(eSNP) and so on.SNP assays have been made in many crop species such as maize and soybean.The elite germplasm of some crops have been narrowed in genetic diversity,increasing the amount of linkage disequilibrium(LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes.SNP analysis has been broadly used in the field of plant gene mapping,integration of genetic and physical maps,DNA marker-assisted breeding and functional genomics.     

相关序列扩增多态性(SRAP)标记及其应用研究进展

SRAP是一项基于PCR技术的分子标记技术,利用其独特的引物设计对基因组的开放阅读框(ORFs)进行特异扩增,利用个体以及物种的内含子、启动子和间隔序列的不同,产生基于内含子和外显子的SRAP多态性。阐述了SRAP的原理和流程,详细论述了SRAP标记目前在植物遗传多样性、作物品种鉴定、遗传图谱构建等方面的研究进展及应用前景。     

PCR扩增近交系大鼠微卫星位点DNA多态性的研究

本实验选取大鼠7条染色体上的微卫星位点合成了10对引物,利用聚合酶链反应(PCR)扩增技术对国内北京和哈尔滨等4家单位提供的6个品系(SHR、SHRSP、LEW、RCS、WKY和F344)的8个近交系大鼠群体进行了DNA多态性分析的研究.结果表明:9个微卫星位点具有显著多态性;不同品系个体之间具有多态性;同一群体不同个体之间除SHR(哈)的SMST位点和WKY(哈)的AGT位点出现一定的差异外,其他均没有差异;不同地区同一品系的不同个体之间也存在一定的差异.该方法能有效地对近交系与杂交系、品系与品系、品系与亚系加以区分.因此,本实验为开展近交系大鼠遗传作图、基因定位和为实验动物的遗传背景监测提供可靠的信息,为大鼠遗传基因的研究提供了一个快捷简便、特异准确的方法。 Abstract:In the experiment,It were selected that 20 primers were assigned on 7 chromosomes of inbred rat.DNA polymorphism of 8 colonies from 6 inbred rat strains(SHR、SHRSP、WKY、LEW、RCS、F344)were studied in national Beijing and Harbin using PCR-analyzed microsatellites.The results indicated that there were remarkable polymorphism in 9 microsatellite loci;There is a polymorphism among the various rat strains;and no polymorphism in the same rat strains except SMST locus of SHR(Harbin)and AGT locus of WKY(Harbin)and there is a little difference among the same inbred rat strains in different areas.The method of PCR-analyzed microsatellites can be used for distinguishing between inbred and outbred、different strains、strain and substrain.and it provides a lot of information for genetic mapping,gene location and heredity probe of the inbred rat strains,and a speedy、convenient method for genetic research of the inbred rat.     

小麦叶片直接用于PCR和RAPD反应的方法

本研究以经过碱处理的小麦叶片直接作为PCR和RAPD反应的模板,并应用于小麦转基因目标性状的跟踪检测、品种多态性分析等方面。该方法具有快速、简便等特点,尤其是在受测群体较大时,此种方法更显得经济有效。 Abstract:In this paper we introduce a new method of PCR and RAPD reaction by using a small quantity of wheat leaf tissue with alkali treatment.It can be used either to detect target gene or to analyze polymorphism of wheat varieties.This method is simple and reliable,especially for a large-scale detection.     

DNA多态性分析技术研究进展

随着现代分子生物学的迅速发展,DNA多态性技术在很多领域得到了应用,本文简单总结了DNA多态性分析技术的发展,并展望了DNA多态性技术的应用前景。     

水稻一多拷贝微卫星DNA多态性分析

用一个多拷贝微卫星 DNA标记分析了238份栽培水稻的遗传多样性及遗传多样性从农家品种到现代栽培品种的动态变化。共检测出16种长度变异类型和32种表现型,但长度变异类型数、表现型数和表现型多样性水平在农家品种和现代栽培品种间没有显著差别。该标记在遗传资源评价和DNA指纹图谱建设中有重要的应用价值,也是研究环境或人工对多拷贝基因选择作用的一个良好模型。 Abstract:The genetic diversity of 238 cultivated rice and its difference between landraces and modern cultivars have been assayed using a multiple copy microsatellite DNA marker.Although 16 length variants and 32 phenotypes were found in all accessions,no significant differences in number of length variants,number of phenotypes and phenotypic diversity were detected between landraces and modern cultivars.This marker is of great value in evaluating genetic germplasm and constructing DNA fingerprints of rice and provides an elite model for tracing effectiveness of natural and artificial selection on multiple copy genes.     

AFLP分析中多态性扩增产物的回收、克隆及鉴定

本研究在摸索和优化了水稻AFLP分析体系的基础上,发展了多态性AFLP产物的高效克隆方法。特异AFLP扩增产物直接从变性聚丙烯酰胺凝胶上分离纯化,再经过一至二轮PCR扩增,即可高效地克隆于pGEM-Teasy vector系统中。本实验利用该方法成功地克隆了水稻温敏核不育等位突变系546 0S和5460F间的4个多态性AFLP产物,Southern bloting分析证明其中3个产物在水稻基因组中为单拷贝序列,另一个为低拷贝序列。AFLP技术强有力的多态性检出能力再结合多态性扩增产物的高效克隆方法,为寻找与目标基因紧密连锁的分子标记提供了有力工具。 Abstracts:An efficient method for cloning DNA fragment from denaturing polyacrylamide gels was developed to allow the isolation of specific bands obtained from amplified fragment length polymorphism(AFLP)products.After isolation and purification from the thin denaturing polyacrylamide gels,specific AFLP products were successfully cloned after one or two rounds of PCR reamplification.Using this method 4 polymorphic AFLP products between a pair of rice allelic lines differing for thermo-sensitive genic male sterile(TGMS)ene were cloned and it was confirmed that 3 of the AFLP products represented single copy sequences and the other 1 represented low copy sequence in rice genome.     

大麦基因组中的微卫星标记及其应用

微卫星是以少数几个核苷酸为单位多次串联重复的DNA序列,是一种简单序列重复(simple sequence repeats,SSR),两侧一般是保守序列。由于它具有多态性高、共显性、容易用PCR检测和结果稳定可靠等特点,因此是一种十分理想的分子标记。大麦的微卫星DNA随机分布于基因组中,平均每一个微卫星基因座有3～18个等位基因,最高可达37个。SSR标记已广泛用于分子遗传图谱的构建、遗传多样性研究、种质鉴定、主要性状基因的定位及分子标记辅助选择育种等。大多数SSR标记集中在着丝粒附近区域,1HL、5HL和6HS明显缺乏SSR标记。大麦的SSR标记还有待进一步的开发。 Microsatellite Markers and Applications in the Barley Genome FENG Zong-yun1,2,3,ZHANG Yi-zheng1,LING Hong-qing3 1.College of Life Sciences,Sichuan University,Chengdu 610065,China; 2.College of Agronomy,Sichuan Agricultural University,Ya'an 625014,China; 3.The State Key Laboratory of Plant Cell & Chromosome Engineering,Institute of Genetics & Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China Abstract:Microsatellites,also called simple sequence repeats (SSR),are simple,tandemly repeated DNA sequences with a repeat length of a few base pairs,and are very ideally used as molecular markers because of their abundance,high level of polymorphism,co-dominance and ease of assay with the polymerase chain reaction (PCR) by selecting primers as the conserved DNA sequences flanking the SSRs,as well as better stability.The experiments showed that SSRs are randomly distributed throughout the barley genome,and there are 3~18 alleles at a single SSR locus,up to 37 alleles/locus.SSR markers have being widely applied in the construction of molecular genetic map,the study of genetic diversity,the identification of germplasm,gene mapping for important traits and molecular marker-assisted selection.Meanwhile,most of markers are strongly clustered around the centromeric regions of all seven linkage groups.As a result of the clustering,genome coverage with SSRs remains incomplete with an obvious lack of markers on the long arms of chromosomes 1H and 5H and short arm of chromosome 6H.Therefore,it is very potential and necessary to further develop SSR markers in barley. Key words：barley;microsatellite marker;simple sequence repeats;genetic diversity;molecular mapping     

Detection of Transgenes in Crop Plants Using Molecular Beacon Assays

Molecular beacons are oligonucleotide probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary targets, they undergo a conformational transition that turns on their fluorescence. These probes recognise their targets with higher specificity than linear probes and can easily discriminate targets that differ from one another by a single nucleotide. As a model system to test the applicability of molecular beacons in crop plants, we have designed a molecular beacon to detect the bar transgene in barley. Results from this experiment indicate that molecular beacons can be successfully employed in detecting transgenes, simultaneously combining the benefits of being highly reproducible and sensitive. The molecular beacon assay is suitable for diagnostics, simultaneously being employed in the development of rapid DNA-based assays for analysing single nucleotide polymorphisms (SNPs).     

基于数字PCR的单分子DNA定量技术研究进展

数字PCR是一项针对单分子目标DNA的绝对定量技术．该技术是将含有DNA模板的反应溶液分配到大量独立的反应室中并且发生扩增反应,通过统计反应室中的阳性信号来定量DNA的拷贝数．DNA样品在反应室中随机和独立分布是单分子成功扩增和准确定量DNA拷贝数的关键因素．本文综述了数字PCR的发展历史、数字PCR与实时荧光定量PCR的区别,以及数字PCR在临床诊断、转基因成分定量、单细胞基因表达、环境微生物检测和下一代测序等方面的最新进展,并展望了该技术的应用前景．     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

大麦类钙调磷酸酶B互作蛋白激酶基因克隆及表达分析

采用RT-PCR法从大麦抗旱品种‘甘啤7号’中克隆了1个类钙调磷酸酶B互作蛋白激酶(CIPK)基因HvCIPK1(GenBank登录号为JX679077)。序列分析表明,该基因全长1 359bp,编码的蛋白含有452个氨基酸,分子量为51.05kD,等电点为9.13。推断具有植物CIPK家族典型的功能结构域,在N端有一特异的催化结构域,该结构域在保守的氨基酸DFG和APE之间含有一个催化所需的活性环;同时C端区域存在一个独特的24个氨基酸组成的调节域,即NAF结构域。荧光定量分析结果表明,在PEG、NaCl和ABA处理下,HvCIPK1基因在大麦幼苗叶片中表达显著上调。推测HvCIPK1基因参与多种逆境信号的转导,可能在大麦的多种非生物胁迫响应中起着重要的作用。     

Degradation of Barley Glucan and Lichenan by a Bacillus pumilus Enzyme

A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases.     

赤眼蜂分子鉴定研究进展

赤眼蜂是害虫生物防治中的重要天敌资源。该属种类繁多,已报道有200余种,其蜂种的正确鉴定与选择是影响其田间防效的重要因素。依赖雄成蜂外生殖器形态特征的传统赤眼蜂分类鉴定技术不仅对专业技术要求高、耗时费力,而且无法用于孤雌产雌品系的种类鉴定以及近缘种的区分。分子鉴定技术可以通过选择合适的分子标记,为赤眼蜂鉴定提供准确、便捷、高效的方法。本文对赤眼蜂分子鉴定中分子标记的选择及常用分子鉴定方法进行了综述,并提出了未来可能的发展方向。     

利用RAPD标记分析大麦种质资源的遗传多样性

利用RAPD标记对19份西藏近缘野生大麦材料、33份我国不同省市的地方品种以及8份国外引进大麦品种共60份大麦种质资源的遗传多样性进行检测.结果表明材料间遗传差异明显.32个RAPD引物中,有25个引物(占78.13%)可扩增出清晰且具多态性的条带,另外7个引物能扩增出1～3条清晰但无多态性的条带.每个引物可扩增出1～8条多态性带,平均为3.72条.32个引物共产生119条DNA片段,其中87条具有多态性,多态性比率(PPB)为73.11%,平均多态信息量(PIC)为0.434;每个位点平均有效等位基因数(Ne)为2.304;材料间遗传相似系数GS变化范围为0.757～0.981,平均值为0.871.19份来源于西藏的近缘野生大麦材料间GS值变幅为0.818～0.969,平均为0.892;33份我国栽培大麦地方品种间的GS值变化范围为0.783～0.981,平均为0.879;8份分别来自8个国家的栽培大麦品种间的GS值变幅为0.820～0.956,平均为0.882.根据RAPD标记分析的结果,对60份大麦种质资源进行聚类分析,在平均GS值0.871水平上60份大麦材料可聚为5类,聚类结果能在一定程度上反应材料的地理分布关系,但某些相同地理来源的材料也较分散地分布在整个聚类树中.本研究从分子水平上进一步证明了我国栽培大麦丰富的遗传多样性,是世界栽培大麦的遗传多样性中心之一.