AFLP标记技术在鉴定甘蓝种子真实性及品种纯度中的应用

利用AFLP方法对在农业生产上大面积推广使用的5个甘蓝杂交组合及其10个亲本共15个材料进行了分析研究,得到了清晰的DNA扩增指纹图谱.在AFLP分析中,共采用了32个EcoRI+3/MseI+3引物组合,每个引物组合扩增出的条带数在43～62条之间,平均为54.5条.并从这32个引物组合中筛选出一个引物组合,能够用来对供试5个甘蓝杂交种进行真实性及品种纯度鉴定.从而证明了AFLP技术在甘蓝种子真实性及品种纯度鉴定中的可行性.该技术应用于甘蓝种子真实性及品种纯度鉴定在国内尚属首次,其稳定性重复性好,检测分辨率高,很适合甘蓝种子真实性及品种纯度鉴定.是今后种子真实性及品种纯度鉴定的发展方向,应加大其研究力度.     

洋桔梗品种遗传多样性的AFLP分析

该研究以洋桔梗(Eustoma grandiflorum)2个品种‘玛丽艾基粉色’和‘圣剑白底紫边’为试材,提取叶片DNA,经过EcoRⅠ/MseⅠ双酶切、连接、预扩增、选择性扩增,建立了洋桔梗的AFLP最佳反应体系;并以64个常用引物组合进行扩增,得到154个多态性条带,从中筛选出扩增条带较多且多态性较好的4个引物组合(E-ACA/M-CTC,E-ACC/M-CAC,E-AGC/M-CTT,E-ACT/M-CAG),其多态位点百分率均值为24.36%。利用上述4个引物组合,以最佳反应体系为基础,构建了7个常见洋桔梗品种的AFLP指纹图谱,统计7个品种各4个引物组合在1 000~300bp区间7个区段的扩增条带,并将各个品种的AFLP指纹图谱转换成各品种4组7位数构成的28位特异数字指纹,极大地方便了种质比较及鉴定;7个品种间的遗传相似系数介于0.683 5~0.860 8之间,平均值为0.774 6。研究结果为进一步进行洋桔梗的种质研究及利用奠定了基础。     

10种冬青属植物遗传多样性RAPD和AFLPs分析

采用RAPD和AFLP技术,对10种冬青属植物基因组进行DNA片段扩增,以研究该属种间遗传多样性.结果表明:在RAPD分析中,通过对100种10个碱基随机引物的筛选,发现11种引物能得到多态性较高扩增产物,11种引物共扩增出301条多态性条带,多态率为98.63%.在AFLP分析中,3对选择性引物组合均扩增出了丰富的多态性片段.利用RAPD和AFLP技术分析,结果按UPGMA类平均法进行聚类,聚类结果显示冬青和代茶冬青,木姜冬青和浙江冬青以及光枝刺缘冬青与毛枝三花冬青之间的亲缘关系最近.     

利用AFLP技术鉴定凤凰单丛古茶树种质资源

本文采用AFLP技术对34个凤凰单丛古茶树资源进行了种质鉴定分析.结果显示,筛选出多态性较高的5对引物中,引物组合E41M42、E41M39和E41M33可鉴别出供试的全部资源,鉴别效率为100%;有23份资源具有特异带,占供试材料的67.6%.由此研究表明,AFLP技术可以通过特异带、特异的谱带类型、不同引物提供谱带的组合将风凰单丛古茶树资源区分开来,这对保护凤凰单丛古茶树资源的育种产权、登录新品种、鉴定和检测其种子与苗木的真实性和纯度具有重要的现实意义.     

AFLP标记技术在鉴定甘蓝种子真实性及品种纯度的应用

利用AFLP方法对在农业生产上大面积推广使用的5个甘蓝杂交组合及其10个亲本共15个材料进行了分析研究,得到了清晰的DNA扩增指纹图谱,在AFLP分析中,共采用了32个EcoRI 3/MseI 3引物组合,每个引物组合扩增出的条带数在43－62条之间,平均为54．5条,并从这32个引物组合中筛选出一个引物组合,能够用来对供试5个甘蓝杂交种进行真实性及品种纯度鉴定。从而证明了AFLP技术在甘蓝种子真实性及品种纯度鉴定中的可行性。该技术应用于甘蓝种子真实性及品种纯度鉴定在国内尚属首次,其稳定性重复性好,检测分辨率高,很适合甘蓝种子真实性及品种纯度鉴定,是今后种子真实性及品种纯度鉴定的发展方向,应加大其研究力度。     

26种冬青属植物遗传多样性分析

以26种冬青属植物种质资源为研究材料,利用RAPD和AFLP技术对基因组DNA进行扩增,以研究其物种间遗传多样性以及亲缘关系.结果表明:在RAPD分析中,从400条10个碱基的寡核苷酸引物中筛选出反应稳定、扩增性强、重复性好的引物20个,共扩增出312条多态性条带,多态率为95.41%;聚类分析显示26种冬青属植物间,布利奥特夫人枸骨叶冬青和黄果在AFLP分析中,10对选择性引物组合均扩增出了丰富的多态性片段,共扩增出350条谱带,其中336条具有多态性,占95.96%.综合RAPD和AFLP聚类结果,枸骨、无刺枸骨和日拉斯纳尔逊枸骨的亲缘关系较近,钝齿冬青、金宝石钝齿冬青和龟甲冬青三者的亲缘关系较近,可为冬青属植物的杂交育种与种质创新提供理论依据.     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

猕猴桃AFLP分析体系的建立

从64对AFLP分析引物中随机选取了19对引物组合。经过跑小板的聚丙烯酰胺凝胶,银染显带AFLP条带,从中筛选出可做荧光的8对引物。再从中选取了扩增位点丰富,带型质量好,分辨率较高,条带信号强度一致性好,条带分布均匀,且条带较完整的4对引物：E-AAC＋M-CAC、E-AAG＋M-CTG、E-AAC＋M-CAG、EAAC＋M-CTA进行荧光跑带、读带。共在156个位点上扩增出条带,4对引物共扩增出多态性条带132条,多态性比例平均为84．62％,4对引物对10份猕猴桃材料的区分率达100％。说明该4对引物用于猕猴桃属植物的AFLP分析是可行的。     

RAPD标记鉴定水稻3种不同细胞质雄性不育类型的杂交种及其亲本

利用RAPD引物对3种不同细胞质雄性不育类型的杂交水稻组合及其亲本共21个材料进行了DNA多态性分析。从264个随机引物中筛选出具有非常明显多态性的引物25个, 对25个引物在3种不同细胞质不育类型的杂交组合及其亲本间的DNA扩增多态性差异进行比较,最终选出具有不同类型间特异性扩增带的引物7个,利用这些特异性扩增带能有效地区分和鉴定目前在生产上大面积种植和推广,或者是具有应用潜力的3种不同细胞质雄性不育类型—野败型(WA)、红莲型(HL)和包台型(BT))—的6种杂交水稻组合及其亲本。     

三倍体白杨杂种无性系指纹图谱的构建

目的:构建三倍体白杨杂种无性系指纹图谱,鉴定三倍体白杨杂种无性系。方法:分离纯化三倍体白杨杂种DNA模板,采用扩增片段长度多态性(AFLP)分子标记技术构建三倍体白杨杂种无性系指纹图谱。结果:从64对引物组合中筛选出M-CTA/E-CAG、M-CAC/E-CCA、M-ACT/E-CTC和M-CTT/E-CTG等4对多态性较高的引物组合,并应用该引物组合对21个三倍体白杨杂种无性系进行了AFLP分析,构建了21个三倍体白杨杂种无性系指纹图谱。结论:构建无性系指纹图谱是鉴别三倍体白杨杂种无性系的有效方法,能够有效鉴别21个三倍体白杨杂种无性系。本研究为品种鉴定及新品种权保护奠定了基础。     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers’ genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8 bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1 bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.     

Analysis of genetic diversity among different geographical populations and determination of biotypes of Bemisia tabaci in China

Abstract:  Analysis of the genetic diversity among 27 different geographical populations of Bemisia tabaci and determination of biotypes of B. tabaci in China based on amplified fragment-length polymorphism (AFLP) and the mitochondrial cytochrome oxidase I (mtDNA COI) gene sequences were conducted. In AFLP assay, the use of five primer combinations selected from 64 primer combinations allowed the identification of 229 polymorphic bands (97.03%) from 60 to 500 bp, suggesting abundant genetic diversity among different geographical populations of B. tabaci. To further identify biotypes of B. tabaci in China, the mtDNA COI gene sequences of nine representative populations from China, Israel and Spain were obtained. Molecular phylogenetic tree based on AFLP and mtDNA COI gene analyses revealed the presence, in China, of at least four different genetic groups of B. tabaci. B biotype, Q biotype and two non-B/Q biotype. B biotype was distributed nationwide. Q biotype was present only in the local region of China including the YunNan province and BeiJing city. This was also the first report about the invasion of Q biotype into China. Of the other two non-B/Q biotype groups, one was found in ShanDong and HeBei provinces, and another in ZheJiang province. The non-B/Q biotype ZheJiang population showed very high similarity with another Asian population India-IW ( AF110704 ) in mtDNA COI sequences and was possibly a Chinese indigenous population. The close monitoring of the Q biotype in locales of China where commercial plants were exported or imported, is now essential to avoid the further accidental distribution of the Q biotype.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Evaluation of RAPD, ISSR and AFLP Markers for Characterization of the Loose Smut Fungus Ustilago tritici

Random Amplified Polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) profiling were evaluated for assessing the extent of genetic variation among the isolates of Ustilago tritici (Pers.) Rostr., which causes the loose smut disease of wheat.Thirty random decamer primers, six random primer pairs, four SSR primers such as (GACA)₄, (GATA)₄, (CAA)₅ and (GTG)₅ and nine combinations of AFLP selective primers were used to characterize nine isolates of the fungus. These isolates were collected from infected earheads of seven commercial wheat cultivars grown at eight different locations in Haryana, which is a major wheat growing state in the North-West Plain Zone of India. The RAPD and ISSR primers generated 21 0 scorable amplified fragments, all of which were monomorphic among the isolates.The AFLP primer combinations generated 239 fragments out of which 193 were polymorphic. All the isolates could be precisely differentiated from each other employing AFLP and grouped into two distinct clusters.The molecular classification partly corresponded with geographic distribution and host origin of the isolates. AFLP profiling was found superior to RAPD and ISSR and can be effectively utilized for further characterization of loose smut pathogen.     

Evaluation of amplified fragment length polymorphism and simple sequence repeats for tomato germplasm fingerprinting: utility for grouping closely related traditional cultivars.

Cultivated tomato (Solanum lycopersicum L.) germplasm shows limited genetic variation. Many DNA marker systems have been used for genetic diversity studies in wild and cultivated tomatoes, but their usefulness for characterizing phenotypic differences among very closely related cultivars remains uncertain. We have used 19 selected simple sequence repeat (SSR) markers and 7 amplified fragment length polymorphism (AFLP) primer combinations to characterize 48 cultivars of tomato, mainly traditional cultivars from the south-east of Spain. The main types were Solanum lycopersicum L. 'Muchamiel', 'De la pera', and 'Moruno'. The robustness of the dendrograms and the discrimination power reached with each marker type were similar. Unique fingerprinting even of the most closely related tomato cultivars could be obtained using a combination of some SSR and AFLP markers. A better grouping of the 'Muchamiel' cultivars was observed with SSR markers, whereas the grouping of cultivars of 'De la pera' type was best achieved with AFLPs. However, both types of markers adequately grouped cultivars of the main types, confirming the utility of SSR and AFLP markers for the identification of traditional cultivars of tomato.     

河南地方野生小鼠与近交系小鼠的扩增片段长度多态性分析

目的 从分子水平上阐明河南省兰考地区捕获的野生小鼠(LK)与4个小鼠品系(B6,BALB/c,DDK,PWK)之间的遗传差异及遗传关系,从而进一步确定该种野生小鼠的种属及培育野生来源近交系小鼠品系.方法 利用25对引物,对野生小鼠及4个小鼠品系进行扩增片段长度多态性(AFLP)分析.结果 检测到2035条扩增条带,多态...     

AFLP markers for the study of rice biodiversity

 AFLP was used as a DNA fingerprinting technique in rice (Oryza sativa L.) germplasm analysis. The high efficiency and random coverage of AFLP markers were established. With only five combinations of primers and RFLP anchors, a framework linkage map was constructed. This map demonstrated that the AFLP markers from a limited number of primers were not confined to any particular regions or chromosomes in the rice genome. To analyse the biodiversity of 57 rice germplasm accessions, we examined 179 polymorphic AFLP markers generated from four primer combinations. Both principal component analysis and cluster analysis were used, and three groups were clearly identified which corresponded to genotypes of Isozyme Groups I, II and VI. The number of markers needed for robust classification of rice germplasm and the diversity between/within the groups was established. Received: 15 July 1997 / Accepted: 29 October 1997