RAPD技术及其在微生物学方面的应用

198 0年 ,Ｂｏｔｓｅｉｎ提出ＤＮＡ限制性片段长度多态性 (ＲＦＬＰ)可以作为遗传标记 ,从此开创了直接应用ＤＮＡ多态的新阶段。 80年代后 ,ＤＮＡ多聚酶链式反应 (ＰＣＲ)的发展 ,使直接扩增ＤＮＡ的多态性成为可能 ,并在此基础上产生了许多种新型分子标记 ,诸如扩增片段多态性 (ＡＬＦＲ)、串联重复序列(ＶＮＴＲ)、单链构型多态性 (ＰＣＲ ＳＳＣＰ)、序列特异扩增区域 (ＳＣＡＲ)、随机扩增多态性ＤＮＡ(ＲＡＰＤ)等。而ＲＡＰＤ是较为突出的一种。ＲＡＰＤ是由Ｗｉｌｌｉａｍｓ和Ｗｅｌｓｈ在 1 990年各自独立发现的一种ＤＮＡ多态检…     

西瓜抗枯萎病育种分子标辅助选择的研究

将西瓜野生种质ＰＩ２９６３４１抗枯萎病生理小种１的抗性基因连锁的ＲＡＰＤ标记ＯＰＰ０１．７００进行克隆、测序,Ｓｏｕｔｈｅｒｎ杂交证明此标记为１个单拷贝,并转化为ＳＣＡＲ标记,简化了ＳＣＡＲ扩增产物的检测技术。上述技术在抗病转育后代造反中得到了很好的应用,初步建立了西瓜抗枯萎病育种分子标记辅助选择技术系统。     

小麦抗白粉病基因Pm21的分子鉴定和标记辅助选择

利用小麦抗白粉病基因Ｐｍ２１的ＲＡＰＤ标记、ＳＣＡＲ标记和荧光源位杂交技术对小麦抗病育种材料中的抗白粉病Ｐｍ２１基因进行了分子鉴定和标记辅助选择。     

DNA分子标记在柑桔中的应用

ＤＮＡ分子标记是最为理想的遗传标记 ,依其多态性检出所用的分子生物学技术 ,大致可分为Ｓｏｕｔｈｅｍ杂交技术为核心的分子标记和ＰＣＲ技术为核心的分子标记。前者的代表性技术有ＲＥＬＰ(ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ)和ＤＮＡ指纹技术 (ＤＮＡｆｉｎｇｅｒｐｒｉｎｔｉｎｇｔｅｃｈｎｉｑｕｅｓ)。后者的代表性技术有ＲＡＰＤ(ｒａｎｄｏｍａｍｐｌｉｆｉｅｄｐｏｌｙｍｏｒｐｈｉｃＤＮＡ)、ＳＣＡＲ(ｓｅｑｕｅｎｃｅｃｈａｒａｃ ｔｅｒｉｓｔｉｃａｍｐｌｉｆｉｅｄｒｅｇｉｏｎ)…     

利用RFLP、SSR、AFLP和RAPD标记分析玉米自交系遗传多样性的比较研究

利用 ＲＦＬＰ、ＳＳＲ．ＡＦＬＰ和ＲＡＰＤ ４种分子标记方法研究了 １５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性信息量（ＰＩＣ）最大（０．５４）,ＡＦＬＰ标记位点最小（０．３６）,但ＡＦＬＰ标记具有最高的多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似系数相关性显著,比较相关系数表明 ＲＡＰＤ可靠性较低。依据 ４种分子标记结果将 １５个供试自交系划分为塘四平头、旅大红骨、兰卡斯特、瑞德和ＰＮ共５个类群,与系谱分析基本一致。认为ＳＳＲ和ＲＦＬＰ两种分子标记方法适合进行玉米种质遗传多样性的研究。     

利用RFLP,SSR,AFLP和RAPD标记分析玉米自效系遗传多样性的比较研究

利用ＲＦＬＰ、ＳＳＲ、ＡＦＬＰ和ＲＡＰＤ４种分子标记方法研究了１５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６７６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似自交系划分     

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。     

大鼠RAPD标记的观察

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）技术,分析ＳＤ和Ｗｉｓｔａｒ二种大鼠的基因多态性,探讨用ＲＡＰＤ标记鉴别二种大鼠及其血标本实验中的认证,结果表明,二种大鼠表现出了各自不同的多态性ＲＡＰＤ标记,作为大鼠的分子标记,可在基因水平区别二种大鼠,故认为是一种大鼠研究的分子依据。     

葡萄感霜霉病基因RAPD标记的序列分析

利用Ｗｉｚａｒｄ ＤＮＡ ｃｌｅａｎ－ｕｐ ｓｙｓｔｅｍ纯化葡萄感霜霉病基因ＲＡＰＤ遗传标记的ＤＮＡ片段,用细菌质粒ｐＧＥＭ Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该片段,采用自动荧光ＤＮＡ测序仪对片段的核苷酸组成进行双向测序。来自欧洲葡萄粉红玫瑰的葡萄感霜霉病基因ＲＡＰＤ标记由８３５对核苷酸及其特定序列组成。所获的感霜霉病基因ＲＡＰＤ标记可以作为合成探针的基础,用于葡萄抗病育种过程中的早期选择及品种对霜     

黑麦1R染色体的微切微克隆研究

显微分离出黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体,用ＣｏｈｅｓｉｖｅａｄａｐｔｅｒｓｓｉｎｇｌｅｐｒｉｍｅｒＰＣＲ（ＣＡＳＰＰＣＲ）方法进行体外扩增,以ＤＩＧ１１ｄＵＴＰ标记扩增产物为探针,进行Ｓｏｕｔｈｅｒｎ分子杂交,结果表明扩增产物来自黑麦１Ｒ染色体。用１／１０体积的连接物转化Ｅ．ｃｏｌｉＤＨ５α,获得１００００多个重组菌落。经酶切分析,克隆子的插入片段为２５０～５００ｂｐ,为进一步筛选１Ｒ染色体的分子标记打下了基础     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerpringting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Linkage mapping of the Mediterranean cypress, Cupressus sempervirens, based on molecular and morphological markers

Gene mapping for a Cupressus species is presented for the first time. Two linkage maps for the Mediterranean cypress (Cupressus sempervirens) varieties, C. sempervirens var. horizontalis and C. sempervirens var. pyramidalis, were constructed following the pseudo-testcross mapping strategy and employing RAPD, SCAR and morphological markers. A total of 427 loci (425 RAPDs, two SCARs) representing parents and F(1) progeny were screened for polymorphism with 32 random decamer and two SCAR primers. A morphological marker defined as "crown form" was also included. Of 274 polymorphic loci, the 188 that presented Mendelian inheritance formed the mapping dataset. Of these loci, 30% were mapped into seven linkage groups for the horizontalis (maternal) and four linkage groups for the pyramidalis (paternal) map. The putative "crown form" locus was included in a linkage group of both maps. The horizontalis and the pyramidalis maps covered 160.1 and 144.5 cM, respectively, while genome length was estimated to be 1696 cM for the former variety and 1373 cM for the latter. The four RAPD markers most tightly linked to crown form were cloned and converted to SCARs. Each of the cloned RAPD markers yielded two to three different sequences behaving as co-migrating fragments. Two SCAR markers, SC-D05(432) and SC-D09(667), produced amplified bands of the expected sizes and maintained linkage with the appropriate phenotype, but to a lesser extent compared to their original RAPD counterparts. These linkage maps represent a first step towards the localization of QTLs and genes controlling crown form and other polygenic traits in cypress.     

Development of SCARs by direct sequencing of RAPD products: a practical tool for the introgression and marker-assisted selection of wheat

RAPD markers generated by mixtures of two different primers were developed for octoploid × Tritordeum (amphiploid Hordeum chilense × Triticum aestivum) and its parents. Addition lines were used to identify 21 specific RAPD markers for the H. chilense chromosomes detectable in a wheat background. Ten RAPD bands were selected and eight of them were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step. The methodology overcomes some of the pitfalls associated with the election of the right clones when developing SCARs from RAPD markers. The SCARs generated have maintained both the chromosome specificity and the possibility of detection in a wheat background. This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers for both the characterization of the introgression of H. chilense in bread and durum wheat, as well as the efficient and reliable screening of tritordeum lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of two quantitative trait loci for resistance to ascochyta blight in an intra-specific cross of chickpea (Cicer arietinum L.): development of SCAR markers associated with resistance

Two quantitative trait loci (QTLs), (QTL_AR1 and QTL_AR2) associated with resistance to ascochyta blight, caused by Ascochyta rabiei, have been identified in a recombinant inbred line population derived from a cross of kabuli×desi chickpea. The population was evaluated in two cropping seasons under field conditions and the QTLs were found to be located in two different linkage groups (LG4a and LG4b). LG4b was saturated with RAPD markers and four of them associated with resistance were sequenced to give sequence characterized amplified regions (SCARs) that segregated with QTL_AR2. This QTL explained 21% of the total phenotypic variation. However, QTL_AR1, located in LG4a, explained around 34% of the total phenotypic variation in reaction to ascochyta blight when scored in the second cropping season. This LG4a region only includes a few markers, the flower colour locus (B/b), STMS GAA47, a RAPD marker and an inter-simple-sequence-repeat and corresponds with a previously reported QTL. From the four SCARs tagging QTL_AR2, SCAR (SCY17₅₉₀) was co-dominant, and the other three were dominant. All SCARs segregated in a 1:1 (presence:absence) ratio and the scoring co-segregated with their respective RAPD markers. QTL_AR2 on LG4b was mapped in a highly saturated genomic region covering a genetic distance of 0.8 cM with a cluster of nine markers (three SCARs, two sequence-tagged microsatellite sites (STMS) and four RAPDs). Two of the four SCARs showed significant alignment with genes or proteins related to disease resistance in other species and one of them (SCK13₆₀₃) was sited in the highly saturated region linked to QTL_AR2. STMS TA72 and TA146 located in LG4b were described in previous maps where QTL for blight resistance were also localized in both inter and intraspecific crosses. These findings may improve the precision of molecular breeding for QTL_AR2 as they will allow the choice of as much polymorphism as possible in any population and could be the starting point for finding a candidate resistant gene for ascochyta blight resistance in chickpea.     

Rapid mapping of two genes for resistance to downy mildew from Lactuca serriola to existing clusters of resistance genes

Two resistances to downy mildew derived from Lactuca serriola were characterized genetically and mapped using molecular markers. Classical genetic analysis suggested monogenic inheritance; however, the presence of multiple, tightly-linked genes in each case could not be eliminated. Therefore, they were designated resistance factors R17 and R18. Analysis with molecular markers known to be linked to clusters of resistance genes quickly revealed linkage of R18 to the major cluster of resistance genes and provided six linked markers, three RAPD (Random Amplified Polymorphic DNA) markers and three codominant SCAR (Sequence Characterized Amplified Region) markers. The mapping of R17 required the screening of arbitrary RAPD markers using bulked segregant analysis; this provided five linked markers, three of which segregated in the basic mapping population. This demonstrated loose linkage to a second cluster of resistance genes and provided additional linked markers. Two RAPD markers linked to R17 were converted into SCARs. The identification of reliable PCR-based markers flanking each gene will aid in selection and in combining these resistance genes with others.     

Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Development of SCAR Markers for Germplasm Characterisation in Olive Tree (Olea europea L.)

A set of 14 SCAR markers were developed starting from RAPD, AFLP and SAMPL analysis of several olive germplasm accessions. Eight RAPD, two AFLP and four SAMPL fragments were converted into dominant and codominant SCARs by cloning and sequencing the selected fragments. The markers obtained were evaluated on forty different olive cultivars from different Italian production areas (mainly from Liguria). The combined use of these SCARs made possible to univocally identify 26 cultivars while the remaining 14 will require the development of further markers since most of them are placed in a main group containing six genetically similar cultivars (among which Frantoio and Taggiasca) and four minor groups containing two cultivars each. A total of 31 different haplotypes were identified and the analysis of several individual plants indicated no intra-cultivar variability. Considering the SCAR polymorphism two alleles were scored for each markers with the only exception of markers IGPS3 and IGPS4 showing 4 alleles with 7 recognised groups and 5 alleles with 4 groups, respectively. Though less polymorphic in comparison with other markers like SSRs, the developed SCARs proved useful in genotype identification. In addition, they could potentially be used for breeding applications and forensic analysis. An erratum to this article is available at .     

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。