利用RAPD标记鉴定山葡萄种质

采用修改后的CTAB法获得了高质量的基因组DNA.利用随机扩增多态性即RAPD标记对山葡萄7份种质进行鉴定,用4个引物(从30个引物中筛选)对试材进行PCR扩增,共扩增出30条谱带,平均每条引物产生7.5条谱带,其中21条谱带为多态性谱带,占总谱带数的70%.不同引物扩增的谱带数不同,范围在6～9条之间.利用4个引物扩增出的多态性谱带可以将7份山葡萄种质区分.     

大白菜简单序列重复(SSR)和插入/缺失(InDel)标记的开发及通用性分析

为探讨大白菜基因组序列中SSR位点的分布规律并开发SSR引物,利用SSRHunter软件对大白菜A10(16899818～17299817)的DNA序列进行简单序列重复(SSR)位点查找,共得到394个SSRs,平均每1.02kb出现1个SSR。二核苷酸和三核苷酸重复是最主要的SSR类型,分别占79.44%和18.78%。为了提高SSR标记开发的准确性和通用性,对检索得到的含SSR位点的序列进行了同源比对,选取符合条件的15条SSR序列并设计引物;依据Blast过程中发现的在SSR位点不存在差异而在其侧翼序列中存在插入/缺失(InDel)差异的序列,设计了19条InDel引物。用34对SSR及InDel引物在6个大白菜(Brassica rapassp.pekinesis)材料中进行多态性研究,发现28对引物能扩增出理想的PCR产物,有效扩增率为82.35%,其中27对引物具有多态性,多态性比率为79.41%。为验证SSR引物的真实性,随机对4对SSR引物的部分白菜扩增片段进行了测序,发现100%的片段具有相应的SSR位点。28对SSR和InDel引物在甘蓝(B.oleracea)、油菜(B.napus)和萝卜(Raphanus sativus)品种的有效扩增率分别为85.71%、100%和77.78%,说明新开发的SSR和InDel标记具有较好的多态性和通用性。利用6对引物分析了48份十字花科种质的遗传多样性,结果表明48份材料被明显地区分成白菜和甘蓝组、萝卜组、油菜组3大类群,与传统分类一致。大白菜SSR和InDel标记的开发对于十字花科种质亲缘关系及遗传多样性分析具有重要的应用价值。     

16份石榴RAPD扩增产物的两种电泳方法检测及其序列特征

本实验以16个石榴品种为实验材料,筛选出10个重复性及多态性均较好的引物进行RAPD分析。分别采用琼脂糖凝胶以及聚丙烯酰胺凝胶(PAGE)电泳检测方法对PCR扩增结果进行检测并对其结果进行比较,结果显示,两种电泳方式均能得到较为清晰的扩增条带,且两种电泳方式获得的条带总数及多态性条带数均有所不同,琼脂糖凝胶电泳方法共检测出76条带,其中有43条为多态性谱带,多态性比率为56.4%;而在PAGE电泳方法共检测出123条谱带,多态性谱带数为87条,多态性比率为70.95%。PAGE电泳方法检测出的条带数约为琼脂糖凝胶电泳方法检测出条带数的1.5倍。基于两种电泳方法所得RAPD标记的多态性位点,利用NYSYS软件计算遗传相似系数,并构建遗传关系聚类图,分析结果显示,石榴遗传多样性丰富,两种电泳方法所得聚类结果大致相同,可以利用RAPD分子标记及两种电泳检测方法对不同数量的石榴进行分子水平的品种鉴定和遗传多样性的分析。同时通过对来自几个引物随机挑选的17个片段进行克隆,测序结果显示17个片段都是对应引物的RAPD扩增产物,其中有3条是编码蛋白的基因片段,表明了RAPD不仅扩增基因组上的非编码蛋白序列,同时也可以扩增编...     

鸭脂肪型脂肪酸结合蛋白基因（A-FABP）内含子1序列多态性分析

根据鸭A-FABPmRNA序列和鸡A-FABP基因组序列设计1对引物扩增出鸭脂肪型脂肪酸结合蛋白基因(A-FABP)内含子1序列(GenBank登录号:FJ536257),并根据扩增产物设计4对引物,利用PCR-SSCP对鸭A-FABP基因部分序列进行SNP检测,且对不同鸭群体进行群体遗传学分析.结果表明:克隆序列包含鸭A-FABP基因外显子1、2部分序列和完整的内含子1序列,与鸡A-FABP基因内含子1同源性为75.1%;经SSCP检测,发现引物P1扩增片段有3处碱基突变,引物P3扩增片段有6处碱基突变,这些突变分别产生了3种和8种基因型.在P1位点樱桃谷鸭和苏牧麻鸭偏离了Hardy-Weinberg平衡(P0.05),在P3位点3个群体均符合Hardy-Weinberg平衡(P＞0.05).同时,这些基因型在不同鸭群体中分布存在显著或极显著差异(P<0.05或P<0.01).群体遗传学分析结果显示A-FABP基因在不同鸭群体中具有丰富的单核苷酸多态性,可以进一步作为候选基因来分析其与脂肪性状相关性.     

RAPD和ISSR标记对甜瓜种质遗传多样性的研究

利用RAPD和ISSR两种分子标记技术对37份甜瓜(CucumismeloL.)种质进行了遗传多样性研究.在供试材料中筛选到具有多态性的RAPD引物21个,ISSR引物10个(其中ISSR-2与ISSR-4等量混合组成ISSR-10引物对).RAPD引物共扩增出多态性带106条,多态性条带比率(PPB)为58.62%,平均多态信息量(PIC)为0.47;ISSR引物共扩增出多态性带73条,PPB值为65.51%,平均PIC值为0.53.根据两种标记的结果,采用UPGMA聚类分析,将供试材料分为两大类群野生甜瓜和栽培甜瓜;栽培甜瓜又分为厚皮甜瓜和薄皮甜瓜两大亚群,各野生甜瓜种质之间的遗传距离较大,这与其分类地位基本一致.两种分子标记的分析结果呈极显著正相关(r=0.62>r=0.01).研究表明,RAPD和ISSR标记可用于甜瓜种质遗传多样性的研究.     

绵羊骨形态发生蛋白受体IB基因部分3′非翻译区的多态性分析

根据绵羊骨形态发生蛋白受体IB（bone morphogenetic protein receptor IB, BMPR-IB）基因部分调控区的mRNA序列设计3对引物,采用PCR-SSCP技术检测BMPR-IB基因部分3′非翻译区序列在4个绵羊品种（小尾寒羊、湖羊、特克塞尔和中国美利奴绵羊）中的单核苷酸多态性。结果发现引物P1扩增片段存在多态性,其余2对引物的扩增片段均不存在多态性。对于引物P1,只在湖羊中发现AA和BB两种基因型,其余3个品种均为AA型。测序表明BB型与AA型相比有2个碱基突变（121T→C和195T→C）。     

甘蓝型油菜陕2A细胞质雄性不育的遗传及RAPD标记

本研究以甘蓝型油菜陕 2A细胞质雄性不育系、保持系和F2 分离群体为材料 ,对F2 分离群体的遗传分离情况进行分析 ,结果表明 ,可育与不育株花器存在明显差异 ,符合 3∶1的分离比例 ,因而推断细胞质雄性不育恢复基因受 1对显性基因控制。利用分离群体分组分析法(BSA) ,用 1 0 5个随机引物对细胞质雄性不育恢复基因进行RAPD分析 ,发现有 6个随机引物扩增出多态性差异谱带。引物S62 和S74在可育和不育基因池中扩增出单条特异性谱带所代表的DNA序列 ,很可能与不育系的恢复基因连锁。     

花生栽培种InDel有效标记筛选与评估

为获得花生栽培种中有效的InDel标记,本研究基于花生简化基因组检测到的InDel信息,经PCR扩增以及电泳检测验证引物的有效性与多态性,利用生物统计学软件检测InDel标记的多态性水平。结果表明,在花生基因组上设计的39对引物在覆盖4个植物学类型的21份花生中均可获得扩增产物,其中14对引物具有多态性,为总引物数的35.9%,有3对引物位于基因编码区;Shannon指数为0.191 4~1.295 1,均值为0.579 4;多态性信息量(PIC)为0.090 7~0.674 6,均值为0.349 6。本研究筛选获得的InDeI标记可为花生栽培种遗传图谱构建和分子标记辅助育种提供有效的遗传标记。     

中国96个荔枝种质资源的EST-SSR遗传多样性分析

根据本实验室已获得的荔枝果皮cDNA文库EST序列,通过SSRIT在线检索,从3391条EST序列中,发现305条含有SSR,占整个文库EST的8.99%。利用SSR-ESTs序列共设计100对EST-SSR引物,其中62对在荔枝上有扩增产物,50对有扩增多态性,即具有一定的通用性。接着从96份荔枝种质中选取12个品种的基因组DNA,开展核心引物筛选,共筛选出多态性较好的EST-SSR分子标记30个;这30个EST-SSR分子标记在96份资源共扩出284条带,不同引物的扩增条带在3～18条之间,平均9.47条,其中有282条为多态性带,多态率高达99.30%,每对引物的Nei＇s基因多样度范围为0.186～0.396,香农信息指数范围为0.318～0.558;此外,系统聚类分析结果表明,在相似系数0.5525处,可将96份荔枝种质资源分成了8大类群,该8大类群基本与其生态类型和植物学性状特征相符。在此基础上,还对荔枝的主栽品种和特殊种质进行鉴别,结果表明,该30个EST-SSR分子标记在不同品种间可产生较清晰可辨的多态性差异,为荔枝品种以及种质资源鉴别和鉴定的分子指纹的构建奠定了良好基础。     

利用RAPD和SSR标记分析陆地棉种质资源的遗传多样性*

遗传多样性的量化与分类是收集和利用种质资源的重要前提。利用随机扩增多态性（RAPD）和简单重复序列(SSR)两种分子标记对31份陆地棉(Gossypium hirsutum L.)种质资源进行了遗传多样性分析,其中14份材料最近从美国和伊朗引进。研究的目标是对这些陆地棉种质资源进行遗传聚类分析,为制定引进资源的利用策略提供参考。从有多态性的21个RAPD引物和18对SSR引物中共获得117个多态性位点。利用NTSYS-pc2．10统计分析软件,采用Jaccard′s相似系数UPGMA法进行聚类。结果表明,中国的17份材料部分聚为一类,国外的14份材料大部分聚为另一类,其它材料相间排列。分别对31份材料的相似系数与中国的17份材料的相似系数进行了比较分析,中国材料相对于新引进的国外材料的遗传多样性水平稍高,与国外材料之间的遗传差异较大。这说明扩大不同地区间种质资源的交流和引进种质资源可以丰富本地材料的遗传多样性。     

中国南瓜种质资源农艺性状与RAPD标记分析

本研究利用形态学标记和RAPD分子标记同时对70份来自我国不同地区的中国南瓜种质进行遗传多样性和亲缘关系分析。在所观察的56个有差异的农艺性状中,变异系数从6.60%~262.22%,平均变异系数为37.50%。从150个随机引物中筛选出21个进行RAPD分析,结果表明,在检测到的167条带中有130条具有多态性,多态带比率为73.23%;平均每个引物检测到的条带数多达8条,平均Shannon信息指数(Ⅰ)为0.268。基于农艺性状的聚类分析将70份中国南瓜种质分为七类。基于RAPD标记的聚类分析将70份中国南瓜种质分为五类,其聚类结果与形态特征有一定的相关性。但两种聚类结果均无法从地理来源上进行区分。     

RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Characterization of Some Italian Common Bean (<Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> L.) Landraces by RAPD,Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)₃GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.     

RAPD markers in the analysis of genetic diversity among common bean germplasm from Central Himalaya

A set of 99 common bean germplasm collected from central Himalaya was investigated for their genetic variability using random amplified polymorphic DNA (RAPD) markers. Ten oligonucleotide primers, selected from 60 initially screened, generated 123 amplicon products. Of these, two amplicons were shared by all the accessions whereas 112 were polymorphic at least in two pair wise comparison. Nine unique bands identified were as low as 0.32 kb M.W. to as high as 3.5 kb and were confined to eight collections. All primers produced polymorphic amplicons though the extent of polymorphism varied with each primer. The primer OPF-17 was found to be most powerful and efficient as it generated a total of 17 bands of which 15 were polymorphic. RAPD markers data were analysed statistically using NTSYSpc.2.02e software and a dendrogram was generated using Jaccard’s similarity coefficient. The similarity coefficient values varied from 0.19 to 0.91. Grouping analysis revealed the categorization of 99 germplasm into 12 major branches with different level of similarity. Three branches namely branch 2, 3 and 5 out of 12 had only one accession. Branch 1 which consisted of three accessions was the most divergent as revealed by Jaccard’s similarity coefficient. Branching pattern of the accessions did not show any correlation with morphological data or altitudinal alignment of the accessions.     

大豆种质资源RAPD标记遗传多样性研究

为深入研究并充分利用野生大豆资源,本文利用RAPD分子标记对40份大豆材料加以分析,旨在从DNA分子水平上探索野生大豆、地方品种和育成品种之间的遗传多样性状况。结果表明:50个RAPD引物筛选出具有多态性且扩增条带清晰的引物38个,共检测出407条带,其中多态谱带309条,多态性程度为75.92%。每个引物可扩增出2～14条多态性带,平均产生多态性谱带8.1条;平均多样性指数为2.3377,变幅范围为0.5865～4.2133。遗传相似系数变幅范围为0.44～0.92,平均为0.75。野生大豆的多态比例(94.35%)、多样性指数(2.2336)分别高于育成品种(87.47%、1.7331)和地方品种(83.54%、1.6198)。遗传相似系数为野生大豆(0.6498)地方品种(0.7015)育成品种(0.7177),育成品种与地方品种间为0.6599,育成品种与野生大豆间为0.6487,地方品种与野生大豆间为0.6045。UPGMA聚类分析结果表明,40份大豆材料聚为6类,育成品种和地方品种各自聚为一类,野生大豆聚为4类。野生大豆特异等位基因数远远高于育成品种和地方品种二者的相加之和。本研究从分子水平上揭示了野生大豆与栽培大豆区别明显,宜作为一个独立的种,同时野生大豆变异幅度大,遗传基础广,是大豆育种实践中的优良基因资源。     

Microsatellites and RAPD Markers to Study Genetic Relationships Among Cowpea Breeding Lines and Local Varieties in Senegal

Genetic diversity in local cowpea varieties and breeding lines from Senegal were studied using random amplified polymorphic DNA (RAPD) and microsatellite (SSR) techniques. Among the 61 RAPD primers used, twelve show polymorphism. Fifteen of the 30 microsatellite primer pairs were polymorphic, detecting one to nine alleles per locus. The RAPD and SSR data were analyzed both separately and in combination to assess relationships among genetic lines. Although RAPD provided information on levels of genetic diversity, microsatellite markers are most effective in determining the relationship among cowpea accessions and varieties. The SSR results support the genetic diversification of cowpea in Senegal and underscore their potential in elucidating patterns of germplasm diversity of cowpea in Senegal. An erratum to this article is available at .     

Assessment of genetic diversity in Solanum trilobatum L., an important medicinal plant from South India using RAPD and ISSR markers

Solanum trilobatum L. is an Indian medicinal plant containing rich amount of steroidal glyco-alkoloids that can be used as precursor for commercial steroid production. Two efficient marker systems such as Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used for the first time to assess the genetic diversity across 14 S. trilobatum accessions obtained from five South Indian states. Twenty out of 60 RAPD primers generated 189 distinct bands of which 160 were polymorphic with an average of 8 polymorphic bands per primer. A maximum of up to 15 fragments were amplified with an average of 9.45 bands per primer and the amplicons varied in size between 100 and 3,000 bp. The percentage of polymorphism ranged from 55.5 to 100, with an average of 84.6. ISSR profiling using 7 out of 20 primers amplified 83 bands and the number of amplified fragments varied from 2 to 16 with a size range of 200–1,800 bp. Totally 72 polymorphic bands were obtained using 7 ISSR primers at an average of 10.28 polymorphic bands per primer. Polymorphism percentage varied from 50 to 100 among the selected accessions resulting in an average percentage of polymorphism of 86.7. The PIC values ranged from 0.49 to 0.93 for RAPD and 0.16 to 0.90 for ISSR primers. The study pointed out that ISSR markers were more efficient than RAPD markers in evaluating the degree of genetic variation in S. trilobatum. The UPGMA cluster analysis grouped all Tamil Nadu accessions in one cluster and other state accessions in another cluster. The Principal component analysis also substantiates this clustering pattern. Thus the phylogenetic relationship and a high genetic variation revealed in the present study could provide a baseline data for conservation and improvement of this plant in future. Also the molecular markers identified in this study will be helpful in authentication of this species to prevent adulteration in herbal medicine.     

不同基因型刺梨及其近缘种亲缘关系的RAPD分析*

采用RAPD标记，对刺梨(Rosa roxburghii Tratt)7个基因型及8个近缘种(类型)进行鉴定和亲缘关系分析。结果表明，筛选出的16个随机引物可扩增出137条480bp到3.3kb大小的清晰。DNA片段，其中95条为多态性片段，为总数的69．3％；每个引物平均产生8．6条。由OPB-11、OPAF-16和OPW-02扩增的14条特异DNA片段，可有效地将普通刺梨7个基因型和无籽刺梨区分开来。基于遗传距离矩阵，采用IJPGMA法对15个供试样品的亲缘关系进行了聚类分析，文中还就无籽刺梨及重瓣刺梨的可能来源进行了探讨。     

广西地方葛根种质资源遗传多样性的SCoT分析

为了解广西地方葛根种质资源之间的亲缘关系,本研究以SCoT分子标记对44份广西葛根种质资源的基因组DNA进行PCR扩增,并进行遗传多样性分析。结果表明,25条SCoT引物共扩增出223个条带,其中具有多态性的条带有194条,平均每条引物获得7.76个多态性条带,多态性比例为86.99%。聚类分析表明,44份葛根种质资源的遗传相似系数在0.587~0.982之间。在系数0.65处,44份葛根分为两大类,37号材料单独成为一类;在系数为0.74处,可聚为六类。综上所述,SCoT分子标记适用于葛根种质资源遗传多样性分析,本研究结果为广西葛根种质资源鉴定评价和新品种选育奠定了一定的理论基础。