RAPD技术在生物遗传多样性研究中的应用

遗传多样性的检测可在不同水平上进行 ,最初人们对遗传多样性的检测是从形态学开始的 ,进入 2 0世纪80年代 ,分子生物学与分子克隆技术的发展带来了一系列检测遗传多样性更为直接的方法。目前 DNA水平的分析方法已成为最有效的遗传分析方法 ,原则上可以做到对任何基因组中任何片段进行分析。本文对 RAPD技术及其在生物遗传多样性研究中的应用作一综述。1　 RAPD技术随机扩增多态 DNA标记 (Random amplified poly-morphic DNA,RAPD)技术是 Williams和 Welsh等于1990年同时建立的一项 DNA多态性检测新技术 ,是一项建立在 PCR基础…     

玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散,研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响,结果表明：花粉悬液浓度在4μg/50μl以上,用10碱基随机引物均能扩增出较清晰的PCR条带,且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异,利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应,较叶片等植物组织用常规法提取DNA快速、简便、廉价,可有效地应用于以PCR为基础的植物基因组DNA变异,农艺性状的RAPD标记和分子标记辅助育种的研究。     

随机扩增多态DNA技术在遗传育种中的应用

RAPD是一项可以在没有任何分子生物学研究的情况下找出DNA的多态性的技术。本综述了RAPD技术在遗传多样性研究、分类及亲缘关系分析、品种(杂种)鉴定、杂种优势预测、构建遗传图谱、基因定位及基于图谱的克隆,分子标记辅助育种等方面的应用;还总结了RAPD技术的原理、优点、缺点及对策。     

动物种群遗传多态性研究中的PCR技术

基因组DNA的变异是种群遗传多态性研究的基础。PCR技术可以在反应管内经济快速地扩增特定DNA序列,在动物种群遗传多态性研究中的应用主要包括三个方面：(1)种群遗传多态位点的检测;(2)基因定位或利用已经定位的单拷贝基因设计染色体位点特异的分子标记;(3)与DNA测序技术相结合,高效经济地获取特定基因座位的全部遗传变异。     

RAPD技术在植物遗传育种上的应用

RAPD技术以其快速、简便、高效等优点,已广泛应用于多个学科、领域。本文综述了RAPD技术在植物遗传育种上的应用,如遗传多样性研究、分子标记辅助育种、品种(杂种)真实性鉴定、基因定位、构建遗传图谱等。     

RAPD技术在植物遗传育种研究中的应用进展

RAPD技术是一种随机扩增多态性DNA的方法,操作简单、快捷且经济,可从分子水平提供直接的遗传证据。RAPD技术在植物遗传育种中的应用如下：1）遗传图谱的构建;2）分子标记辅助选择育种;3）外源染色体片段的鉴定和标记;4）遗传关系与遗传多样性的研究;5）体细胞杂种的鉴定。     

一种新的分子标记方法－随机微卫星扩增多态DNA (RMAPD)

随机微卫星扩增多态DNA（RMAPD）是利用随机引物和微卫星的上游或下游引物一起作为该扩增的引物,在Taq DNA聚合酶、MgCl2、dNTPs和模板DNA等共同作用下进行PCR扩增的一种新型分子标记方法。其核心是RMAPD引物的有效性问题。通过对西农萨能奶山羊群体RMAPD电泳检测、数据统计分析及验证实验等证明RMAPD的引物是有效的。通过与微卫星和RAPD标记比较,发现RMAPD标记在扩增引物、扩增程序和重复性等方面区别于微卫星和RAPD标记;它是RAPD标记的一种广义的延伸,但又不完全等同于RAPD标记。因此,确定RMAPD是一种新的分子标记方法。该方法也具有DNA标记的特点,在群体遗传结构和亲缘关系分析以及标记辅助选择等遗传育种领域具有广阔的应用前景。     

RAPD技术及其在动物遗传育种中的应用

RAPD技术是在PCR基础上发展起来的一种DNA多态性检测技术,已广泛应用于基因组研究的各个领域。本文概述了RAPD反应的原理、特点,总结了其在遗传多样性检测、亲缘关系鉴定、遗传连锁分析和数量性状的辅助标记选择等方面的应用,并肯定了RAPD在动物遗传育种领域的应用前景。     

DNA分子标记在实验动物遗传分析中的应用

DNA分子标记技术很多,基本都是建立在RFLP、PCR和重复顺序的基础上的。本文重点介绍了限制性片段长度多态性（RFLP）标记、随机扩增多态性DNA（RAPD）标记、微卫星DNA（STR）标记、DNA指纹（DFP）标记、扩增片段长度多态性（AFLP）标记等几种重要的DNA分子标记技术的定义、结构、分布、组成、保守性、优点及丰富的多态性等。并重点介绍了微卫星DNA（STR）标记在分子遗传监测、遗传多样性分析和遗传血缘关系及个体识别等领域的应用。     

PCR技术在植物病原物和植物抗病研究中的应用

着重结合植物病原物的群体遗传、毒性变异和分子检测,以及植物抗病机制和抗病基因定位等领域,综述了聚合酶链反应技术在植物病理研究中的应用现状和潜力。     

A PCR-based molecular marker applicable for marker-assisted selection for anthracnose disease resistance in lupin breeding

Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F(8)derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program.     

拟南芥NPR1基因的克隆与表达载体的构建

NPR1基因为植物抗病基因表达和系统获得性抗性中的一个关键基因。该文以DNA PCR扩增的方法,从拟南芥基因组DNA中克隆出NPR1基因,通过序列分析,所克隆的 NPR1 基因与报道的基因序列完全一致。将其构建成植物表达载体,为今后植物抗病基因工程的开展奠定了基础。     

Molecular tagging of gene conferring leaf blight resistance using microsatellites in sorghum [Sorghum bicolor (L.) Moench

Resistance to leaf blight in sorghum [Sorghum bicolor (L.) Moench] accession G-118 was found to segregate as a single dominant trait in a cross to susceptible cultivar, HC-136. Molecular marker(s) linked to the locus for disease resistance was identified using simple sequence repeat (SSR) markers coupled with bulk segregant analysis. Genomic DNA from the parental cultivars and bulks were screened by PCR amplification with 50 simple sequence repeat primer pairs. Out of these, 38 SSR primers produced polymorphism between parents. After screening of these 38 SSRs with resistant and susceptible bulk, one SSR primer, Xtxp 309 produced a unique band of approximately 700 bp only in resistant parent and resistant bulk and a unique band of 450 bp only in susceptible parent and susceptible bulk. Upon screening with individual resistant and susceptible recombinant inbred lines (RILs), marker Xtxp 309 produced amplification in 23 of the 26 resistant RILs and no amplification was produced in any of the 25 susceptible RILs. The same marker Xtxp 309 produced amplification in 21 of the susceptible RILs and 3 of the resistant RILs of 450 bp band. This was found to be located at a distance of 3.12 cM away from the locus governing resistance to leaf blight which was considered to be closely linked and 7.95 cM away from the locus governing susceptibility to leaf blight. This marker may prove useful in MAS for gene introgression, plant genetic diagnostics and gene pyramiding for resistance via genetic transformation for disease resistance in plants.     

Isolation and molecular analysis of R-gene in resistant Zingiber officinale (ginger) varieties against Fusarium oxysporum f.sp. zingiberi

Marker assisted selection (MAS) of resistant varieties is a reliable and faster method of selecting the right varieties for cultivation. The aim of the present study is to find the genes responsible for resistance in highly resistant varieties. In the present work we report the presence of a Resistance (R) gene of CC-NBS-LRR class of plant resistance genes. Both direct PCR amplification from genomic DNA as well as cDNAs, yielded a 0.6 kb DNA sequence indicating the absence of an intron. Sequence analysis of the PCR amplicon obtained from the genomic DNA showed very high homology to R-genes. An interesting observation from the present study is the presence of the R-gene in only resistant varieties. Neither the partially resistant or susceptible varieties showed the presence of this gene sequence. This in turn raises interesting questions on the evolution of these ginger varieties. The cloned R-genes provide a new resource of molecular markers for rapid identification of fusarium yellows resistant ginger varieties.     

林木抗病基因工程研究进展

植物抗病性是当前植物病理学中研究的热点和难点之一。着重讨论植物抗病机制、抗病基因的转化方法及其在林木抗病基因工程中的应用情况,并对现阶段林木抗病基因工程中存在的主要问题和应用前景进行了讨论。     

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F₈ recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F₈ population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of “false positives” (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.     

与西瓜野生种质抗枯萎病基因连锁的RAPD标记

运用ＲＡＰＤ技术,采用混合分组分析（ｂｕｌｋｅｄｓｅｇｒｅｇａｎｔａｎａｌｙｓｉｓ,ＢＳＡ）方法进行了西瓜（Ｃｉｔｒｕｌｌｕｓｌａｎａｔｕｓ（Ｔｈｕｎｂ．）Ｍａｎｓｆｅｌｄｖａｒ． ｃｉｔｒｏｉｄｅｓ） 野生种质ＰＩ２９６３４１ 抗枯萎病基因连锁的分子标记研究。研究结果表明：西瓜野生种质Ｐ１２９６３４１ 抗枯萎病生理小种１ 的抗性由单显性基因控制,ＲＡＰＤ标记ＯＰＰＯＬ／７００ 与其抗病基因连锁,其遗传距离为３０ ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。这为进行抗病分子标记辅助选择,以及最终定位与克隆其抗病基因打下了良好基础。     

Identification of a RAPD Marker Linked to Fusarium Wilt Resistant Gene in Wild Watermelon Germplasm (Citrullus lanatus var. citroides

Random amplified polymorphic DNA(RAPD) was employed to detect a molecular marker linked to Fusarium wilt resistant gene in the wild watermelon ( Citrullus lanatus (Thunb.) Mansfeld var. citroides ) germplasm P1296341. The resistance to race 1 Fusarium wilt of PI296341 was controlled by one dominant gene. A RAPD marker OPPO1/700 was proved to be linked to the resistant gene. The genetic distance is 3.0 cM (centimorgan). This work has provided a solid basis for molecular marker-assisted selection (MAS) for disease resistance, and made location and cloning of disease resistant genes possible.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.