Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

基于AFLP技术的枣树主栽品种 亲缘关系的研究

试验取30个枣树优良主栽品种进行AFLP分子标记,并进行遗传聚类分析,以期获得不同枣树品种之间的亲缘关系特征。从64对引物组合中筛选出9对重复性和稳定性好的引物组合,总共扩增出399条清晰条带,即对枣品种基因组399个位点进行了检测,其中共有位点85个,多态性位点314个,多态性百分率78.70%,可鉴别出所有供试品种。UPGMA聚类表明,30个枣树品种的相似系数在0.4654-0.8231之间,平均相似系数为0.6223,在相似系数0.666处,可将供试的30个枣树品种分为6类,为枣树品种分类及亲缘关系分析提供了有力参考。     

Italian rice varieties: historical data, molecular markers and pedigrees to reveal their genetic relationships

Two molecular marker approaches [amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR)] were employed to study genomic relationship among 96 rice cultivars. These included most of the best reputed Italian accessions. AFLP produced 461 fragments, 248 (53%) of which were polymorphic, SSR produced four to 11 alleles in the 12 genomic loci investigated. Genomic similarity was estimated independently for the two molecular marker techniques. Both AFLP and SSR dendrograms agree in splitting the cultivars into two main clusters: a small one, comprising four exotic accessions, and a larger one which could be split into four subgroups. These were also analysed on the basis of historical and pedigree information. This is the first report on the application of DNA polymorphism analysis to reveal genomic relationship among cultivated Italian rice germplasm. Results will be useful for breeding programmes.     

黄瓜种质资源遗传多样性的AFLP分析

对23份不同来源的黄瓜材料进行了AFLP分析。18对引物组合共扩增出543条谱带,其中,多态性带为106条,多态率为19.5%。UPGA分类结果将23份黄瓜材料划分为三大类群:第1类群为野生型黄瓜的聚类;第2类群为美国类型黄瓜、荷兰类型黄瓜以及具有荷兰血统的黄瓜的聚类;第3类群中包含所有中国类型黄瓜和2个前苏联类型黄瓜。遗传相似性分析和聚类结果表明:野生黄瓜和栽培黄瓜之间的亲缘关系较远,AFLP标记的分类结果与材料的主要性状特点基本一致。     

葡萄炭疽病菌SRAP遗传多样性分析

探讨葡萄炭疽病菌的变异和群体结构,为进一步深入研究葡萄炭疽病的发生、流行及防控技术提供理论依据。采用SRAP分子标记技术对不同地区的25个葡萄炭疽菌(Colletotrichum gloeosporioides)菌株进行遗传多样性分析。利用4个菌株从100对引物组合中筛选出6对扩增条带多样性丰富、稳定性较好的引物组合;对供试的25个菌株进行SRAP扩增,共得到164条清晰可辨的条带,其中多态性条带为156条,多态性比率为95.12%;利用NTSYS-2.1软件进行病原菌的聚类分析,其相似性系数在0.61~0.95之间。不同地区葡萄炭疽菌的亲缘关系较近,遗传多样性丰富,但各菌株间存在遗传差异,且菌株之间的差异与地理来源无明显相关性。     

甘蔗常用亲本及优良组合双亲间遗传相似性分析

采用18对SSR引物和15对AFLP引物对我国196份甘蔗常用亲本的遗传相似性进行分析,分别获得438和1192个标记,多态性位点比例分别是87.60%,80.80%,平均为84.2%;亲本遗传相似系数变幅介于0.476～0.9099之间,总平均值为0.6673,遗传差异处于中等水平的亲本占了大多数,而差异较小和较大的都只是少数;45个甘蔗品种亲系的遗传相似系数分布在0.5687～0.6723,平均值为0.6723,91.11%甘蔗品种亲系的遗传相似系数集中在0.6100～0.7300之间,较理想的甘蔗杂交组合亲本间的遗传相似系数应为0.6723±0.0600;聚类分析表明,以遗传相似系数阈值约为0.700划分,育出45个品种所使用的30个亲本可分为9大类,在不同类型间选择亲本搭配的组合更容易选育出优良品种.     

甘蔗品种的AFLP和SSR标记鉴定及其应用

品种遗传多样性和指纹图谱是育种、品种权保护和新品种推广等工作的重要参考和依据。本研究选用38个来自国家甘蔗品种区域试验的甘蔗新品种(系), 以9对AFLP标记扩增出348个位点, 多态性位点248个, 多态性比率为72.26%; 15对SSR标记扩增出180个位点, 多态性位点176个, 多态性比率为97.78%。38个新品种(系)的遗传相似系数分布在0.668~0.847之间, 其箱线图分布特征显示, 其中的FN、MT、YZ、YG、GT等系列品种(系)遗传基础相似。通过UPGMA聚类表明, 可在遗传相似系数为0.732处将38个甘蔗新品种(系)划分为2个群体, 其中福农09-2201和桂糖06-1492作为一个子群体最先被划分出来, 它们在群体中的异质性较强; 另外, 在遗传相似性系数为0.770处划分出一个子群体a, 其中包含参照品种ROC22、福农07-3206、福农40、海蔗22、桂糖09-12、柳城07-150等品种(系)。ROC22具有广适应性和高产高糖等优良特性, 子群体a中的另外几个品种(系)则更有可能拥有这些特性, 具有更高的推广潜力。本研究选择60个SSR位点构建了38个甘蔗新品种(系)的指纹图谱, 对品种鉴定及品种权的保护具有重要作用, 可望直接应用于指导甘蔗种质资源的遗传多样性评价和分子指纹图谱鉴定, 并将为这些品种(系)推广布局或作为杂交亲本利用提供参考和借鉴。     

刺槐Genomic-SSR与EST-SSR遗传差异性分析

[目的]对刺槐Genomic-SSR与EST-SSR的遗传差异性进行研究,为今后刺槐遗传多样性分析等育种相关研究中合理选用不同来源的SSR分子标记奠定基础。[方法]选取来自美国四个不同采集地的种子育出的12个刺槐个体,试剂盒提取DNA后分别利用9对Genomic-SSR引物和9对EST-SSR引物进行扩增,并采取毛细管电泳检测扩增产物。利用所得条带信息及相关软件对两种SSR分子标记进行多态性、遗传相似系数相关性以及聚类等方面的比较分析。[结果]刺槐Genomic-SSR平均检测到的条带数为6.0、Shannon多样性指数为1.3833、观测杂合度为0.5749、期望杂合度为0.6832；EST-SSR平均检测到的条带数为5.1、Shannon多样性指数为1.2711、观测杂合度为0.5648、期望杂合度为0.6526。由Genomic-SSR计算得到的个体间的遗传相似系数以及聚类结果与两种SSR标记综合计算得到的遗传相似系数和聚类结果更为相似。[结论]刺槐Genomic-SSR与EST-SSR存在一定的遗传差异性,但差异并不显著；刺槐Genomic-SSR能更加准确地揭示基因型之间的遗传关系；刺槐EST-SSR具有相对较强的保守性。     

Utility of AFLP markers for the assessment of genetic diversity within Brassica nigra germplasm

Genetic diversity of 18 Brassica nigra accessions was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two B. rapa and two B. juncea accessions were selected as outliers in the study. Eight AFLP primer combinations generated a total of 426 bands, of which 79% were polymorphic. The UPGMA method was employed to construct a dendrogram based on the Jaccard's similarity coefficient. The accessions of B. rapa separated from those of B. nigra at a genetic similarity coefficient of 0.27 while those of B. juncea did so at 0.5. The genetic similarity coefficients within the B. nigra accessions ranged from 0.58 to 0.86. Based on these coefficients it was concluded that the B. nigra accessions show high levels of genetic variation. These results have significant implications in the crop improvement programmes for the agronomically important crop B. juncea, an amphidiploid of B. nigra and B. rapa. Two incorrectly labelled B. nigra accessions were also identified. These accessions were found to cluster with those of B. juncea accessions. This result demonstrates the great value of AFLP markers in the management of genebanks.     

Genetic diversity among selected Argentinean garlic clones (Allium sativum L.) using AFLP (Amplified Fragment Length Polymorphism)

Genetic diversity of eight selected Argentinean garlic clones (Allium sativum L.) were investigated at the DNA level with the amplified fragment length polymorphism DNA (AFLP) procedure. A total of 405 unambiguous bands were identified by six primer combinations of EcoR I +3 and Mse I +3, of those, 398 showed a clear polymorphism, representing 98% of the total bands. A presence/absence matrix was constructed with the polymorphic bands, and a dendrogram was obtained from it with the UPGMA method. The accessions showed different levels of similarity ranging between 0.24 and 0.97, using the coefficient of Jaccard. The dendrogram showed six arbitrary groups. Accessions typically considered as different clones show similarities between 0.97 and 0.495. The garlic clones were clustered according to the physiological group and bulb color. We could detect an association between AFLP and the geographical origin of the clones. The potential use of AFLP could allow not only the differentiation among species, but also between botanical varieties and well-defined ecotype groups. This is the first report of the use of AFLP to characterize Argentinean garlic clones. This revised version was published online in August 2006 with corrections to the Cover Date.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

甘蓝品种的AFLP指纹鉴别图谱分析

本试验采用AFLP技术分析了来自全国9个栽培地区的44个甘蓝主栽品种,共筛选了40对E 3/M 3引物组合,多态性条带的数量从0条到15条不等。其中引物组合E-AAC/M-CTA是甘蓝品种中多态性最高的引物,有15条多态性条带,多态性条带的百分率为30%,但该引物不足以区分44个供试的甘蓝品种。同时筛选了11组E 2/M 3对引物组合,其中引物组合E-AG/M-CTC产生了13条清晰的多态性条带。以E-AAC/M-CTA和E-AG/M-CTC这两对引物组合的多态性条带构建了44份甘蓝品种材料的指纹图谱,此指纹图谱可以将供试的44份材料一一区分。     

哺鸡竹亲缘关系的AFLP和SRAP分析

本研究采用AFLP和SRAP分子标记技术对9个哺鸡竹类竹种(含2个变型)的亲缘关系进行分析。研究结果表明:12对AFLP引物共扩增出359条带,多态性条带比率为49.7%,相似系数变化范围在0.779～0.978之间;24对SRAP引物共扩增出258条带,多态性条带比率为64.0%,相似系数变化范围在0.721～0.958之间。UPGMA聚类分析表明,AFLP和SRAP标记均将哺鸡竹分成2大类:第Ⅰ类为富阳乌哺鸡竹、花哺鸡竹、黄纹竹、黄秆乌哺鸡竹、毛壳花哺鸡竹、乌哺鸡竹、白哺鸡竹;第Ⅱ类为云和乌哺鸡竹、红哺鸡竹。对这两种标记相似性系数进行相关性分析,AFLP与SRAP的结果呈极显著相关(r=0.934),但SRAP标记比AFLP标记可在竹类及其种下等级检测到更大的遗传变异。     

Genomic analysis in three Hylocereus species and their progeny: evidence for introgressive hybridization and gene flow

Interest in vine cacti of the genus Hylocereus (Cactaceae) has grown markedly due to their high economic potential as exotic fruit crops. Thus, we investigate the genomic and genetic characteristics of 18 accessions belonging to three Hylocereus species, from which were produced eight progeny from self-pollination and 51 interspecific-homoploid and -interploid hybrids. We reported ploidy estimation, allele frequencies, polymorphic information content (PIC) and genetic relationships observed among the Hylocereus species and their progeny. The progeny were diploid, triploid, tetraploid, pentaploid, or hexaploid. Each primer combination used in this work amplified different sets of restriction fragments ranging from 74 to 102 bands. Among the total number of bands observed for the Hylocereus accessions and their progeny, 97.5 and 98.1 %, respectively, were polymorphic. The variability of PIC between primers, species and hybrids suggested high heterozygosity and gene flow between them. In addition, amplified fragment length polymorphism (AFLP) markers were used to successfully identify one of the H. megalanthus accessions beforehand as the unknown male progenitor of the allotriploid S-75. AFLP markers demonstrate the efficacy for assessing genetic relationships and introgression; and provide strong support for the pursuit of additional breeding programs of these fruit crop.     

AFLP analysis of introgression in coffee cultivars (Coffea arabica L.) derived from a natural interspecific hybrid

S.288 an offspring of a putative spontaneous interspecific hybrid between tetraploid Coffea Arabica (2n = 4x = 44) and diploid C. liberica(2n = 2x = 22) and 17 arabica coffee introgression lines (representing F₂ and F₄) derived from the cross S.288 x Kent arabica were evaluated for introgression of C. liberica genetic material. In all, 36 AFLP primer combinations were used in the analysis. The AFLP profiles of introgressed lines were compared to five accessions each of C. arabica and C. liberica. A total of 137 polymorphic bands were scored among the 29 accessions analysed. The introgressed genotypes exhibited 102 marker bands consisting of 65 additional bands and 37missing bands associated with introgression of C. liberica genetic material. C. liberica accessions of EA group (C. liberica var liberica of Guinean origin) seemed to be the likely progenitor in the origin of natural hybrid. Analysis of genetic relationships in the introgressed lines suggested that introgression was limited to few fragments. Segregation and wide variation in number of marker fragments in the F₂ and F₄progenies were attributed to chromosome recombinations. The differences in the level of introgression between introgressed parent, F₂ and F₄ groups was not pronounced. Therefore the alien genetic material appeared to be fixed and there was no elimination or counter-selection over generations, from introgressed parent to F₄.In C. arabica accessions, only 35 polymorphic bands were seen confirming the low genetic diversity. On the contrary, although representing a small amount of alien genome introgression, the Liberica-introgressed genotypes provided notable genetic diversity. Considering the fact that the diploid species of Coffea constitute a valuable source of genetic diversity, the potential implications of variability generated by Liberica-introgressed genotypes in C. arabica breeding are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

羊耳蒜遗传多样性研究中AFLP反应体系的建立与初步应用

为研究羊耳蒜种质资源遗传多样性,建立并初步应用羊耳蒜AFLP反应体系。以羊耳蒜幼嫩叶片为材料,采用常规SDS法提取基因组DNA,通过优化AFLP反应体系中的几个关键因素,建立适合羊耳蒜的AFLP银染反应体系,并利用筛选出的引物组合对羊耳蒜的遗传多样性进行初步研究。经EcoRⅠ和MseⅠ酶组合37℃酶切3 h后可将500 ng基因组DNA完全切开;酶切产物和接头经16℃连接过夜后,用带有1个选择性碱基的预扩引物和带有3个选择性碱基的选扩引物分别进行扩增,扩增产物经变性聚丙烯酰胺凝胶电泳分离,银染,能够得到清晰的扩增图谱。3对引物组合对8个羊耳蒜种群共185个体的扩增共得到221条条带,其中多态性条带195条,多态性条带百分率为88.24%。本研究结果表明建立的AFLP反应体系可用于羊耳蒜种质资源遗传多样性研究。     

Pedigree, RAPD and simplified AFLP-based assessment of genetic relationships among Avena sativa L. cultivars

Two molecular marker techniques: RAPD and simplified ^PstIAFLP have been compared in order to decide on, which technique is better suited to genetic characterization of oat (Avena sativa L.) cultivars. It was investigated, if the same pattern of variability is revealed by two approaches and whether the observed molecular variability reflects pedigree-based relationships. Polymorphic RAPD and ^PstIAFLP markers were sufficient to distinguish all analysed cultivars, demonstrating the usefulness of both methods for cultivar identification. Genetic similarity estimates derived from RAPD, simplified ^PstIAFLP and combined RAPD and ^PstIAFLP data were compared with coefficients of parentage (COP). Molecular markers-based mean genetic similarities were considerably greater than mean COP value. Correlation coefficients between COP and genetic similarities calculated from RAPD, ^PstIAFLP and combined molecular data were very low and not significant. A better correlation (0.50) was found between similarity estimates derived from RAPD and ^PstIAFLP markers. Four separate dendrograms were constructed based on pedigree and molecular analyses using a neighbor-joining algorithm (NJ). The dendrograms were compared and found to be topologically different. The results of this study showed, that both molecular techniques can be conveniently used for genetic characterization of oat cultivars, however ^PstIAFLP would be the method of choice due to the higher efficiency and reproducibility. This revised version was published online in August 2006 with corrections to the Cover Date.