Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.     

Genetic Diversity of Pyrenophora teres Isolates as Detected by AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to analyse mainly 83 Czech isolates of Pyrenophora teres, P. graminea, P. tritici‐repentis and Helminthosporium sativum. Each species had distinct AFLP profiles. Using 19 primer combinations 948 polymorphic bands were detected. All main clusters in dendrogram correspond to the studied species. Even the two forms of P. teres–P. teres f. teres (PTT) and P. teres f. maculata (PTM) – formed different clusters. Genetic diversity, with regard to the locality and the year of the sample's collection, was analysed separately within the AFLP‐based dendrogram cluster of PTT and PTM. Unweighted pair‐group method (UPGMA) analysis of the 37 isolates of PTT and 30 isolates of PTM, using 469 polymorphic bands, showed that the variability seemed to have been influenced more by the year of sampling than by the geographic origin of the isolate. The presence of intermediate haplotypes with a relatively high number of shared markers between the two groups indicated that hybridization between the forms of P. teres could happen, but it is probably often overlapped by selection pressure or genetic drift.     

灰背栎遗传多样性和遗传结构的AFLP指纹分析

用AFLP方法对灰背栎 (Quercussenescens) 8个居群进行了遗传多样性、居群遗传结构研究。TFPGA软件分析两组引物组合共产生 12 5个位点 ,其中 94个为多态位点 ,多态位点百分率为 75 2 % ,发现灰背栎居群的遗传变异水平有随着海拔升高而遗传多样性下降的趋势。Arliquin 2 0 0 0中的AMOVA分析表明灰背栎居群间分化大 ,分化指数达 φst=0 2 95 6。用PAUP软件对所有个体间的遗传关系进行了聚类分析     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles

AIMS: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. METHODS AND RESULTS: Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters. CONCLUSIONS: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.     

橡胶树AFLP银染体系的建立和优化

目的:扩增片段长度多态性(AFLP)为遗传图谱的构建及育种的辅助选择提供了有力的工具。建立一套适合橡胶树的AFLP技术优化体系。方法:以197个GT1×IAN873橡胶树杂交群体为材料,通过对影响AFLP的多种关键因素如模板DNA质量、酶切连接体系、酶切连接反应时间、预扩增体系、选择性体系的分析进行研究。结果与结论:找出一套适于热带植物基因组DNA提取及橡胶树AFLP技术。用改进的CTAB法,经多次抽提纯化,用细玻璃棒挑出DNA得到高质量的模板DNA;酶切连接时DNA模板为250ng,反应体系采用各3U的EcoRⅠ/MseⅡ/T4连接酶,反应时间为9h;预扩增模板为稀释1/2的酶切连接产物,用量为1μL;选择性扩增要用稀释至1/20的预扩增产物。     

三例小麦细胞质雄性不育系线粒体DNA的扩增片段长度多态性分析

细胞质雄性不育是小麦杂种优势利用的重要途径,为了鉴定3例小麦雄性不育系的细胞质类型,对其线粒体DNA(mtDNA)进行扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)分析。文中利用差速离心法和不连续蔗糖密度梯度超速离心法提取纯化小麦线粒体。结果表明:通过该提取方法获得的mtDNA,其质量和纯度能够满足PCR反应和遗传学分析。在64对选扩引物中,筛选到了4对特异性引物,其中引物E1/M7在ms(Kots)-90-110不育系扩增出3条特异条带;引物E4/M2在ms(Ven)-90-110不育系扩增出2条特异条带;引物E7/M6在ms(S)-90-110不育系中扩增出2条特异条带;引物E6/M4在ms(Kots)-90-110不育系中扩增出2条特异条带。这些特异引物可以用来作为鉴定具有粘果山羊草Aegilops kotschyi、偏凸山羊草Ae.ventricosa、斯卑尔脱小麦Triticum spelta 3类不育细胞质型小麦雄性不育系的细胞质分子标记,为研究小麦细胞质雄性不育机理奠定了分子基础。     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

Genetic Diversity of Rice False Smut Fungus, Ustilaginoidea virens and its Pronounced Differentiation of Populations in North China

Rice false smut caused by Ustilaginoidea virens is an important constraint affecting rice yield and quality in Asia. In China, rice false smut is especially severe in the japonica rice-growing areas in the North China. Nothing is known neither about the diversity of the pathogen in this region nor about the characteristic of its local population. In this study, 110 U. virens isolates sampled from Liaoning and Beijing of North China were analysed using amplified fragment length polymorphism (AFLP) markers to primarily understand the genetic diversity of this pathogen and its population characteristics. At the genetic distance of 0.32, all isolates were divided into two groups. Group A consists of two subgroups differentiated at the genetic distance of 0.55, subgroup 1 included all isolates from Liaoning with an average genetic similarity over 0.82 and subgroup 2 included 27 isolates from Beijing with similarity of 0.74. Group B consists of 28 isolates from Beijing having a diversity of 0.054. The isolates from the Liaoning province, where rice false smut has generated different take-all epidemic for 20 years, showed a genetic diversity of 0.305, which was approximately equally distributed within and among populations. Whereas genetic diversity was 0.458 among isolates from Beijing, an extremely high level of genetic differentiation among 55 isolates was observed in this disease hotspot. Our results suggested that the populations among different locations where sexual stage of the pathogen was rare to be found within ecological region were similar, and the variation of this pathogen has mainly arisen via asexual mechanisms. The migration through human activities in breeding perhaps provides a means of transporting the pathogen from one region to another.     

Molecular Variability of Mycosphaerella graminicola as Detected by RAPD Markers

A total of 90 isolates of Mycosphaerella graminicola, the cause of septoria tritici leaf blotch of wheat, were tested for DNA polymorphism using 15 decamer random primers. There was a high level of genetic variability among isolates. In 131 random amplified polymorphic DNA (RAPD) fragments, which were produced, 96% were polymorphic. Based on multilocus analysis, 40 different molecular phenotypes were detected. These molecular phenotypes were randomly distributed among sampling sites, suggesting that no clonal structure existed in the population. Cluster analysis showed that the maximum similarity value among isolates was approximately 81% and no identical isolates were detected, indicating that every isolate was a unique genotype. The high degree of DNA polymorphism, the large number of different molecular phenotypes, their random distribution and the results of the cluster analysis all suggested that sexual reproduction has a major role in the genetic structure of M. graminicola in western Canada. The presence of sexual reproduction provides the opportunity for development of new virulent genotypes in the population and suggests that the pathogen may adapt rapidly to any race‐specific sources of resistance. Therefore, when breeding for resistance to M. graminicola, emphasis should be placed on use of non‐race‐specific resistance.     

AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较

AFLP和RAPD标记技术是近年来发展最快的基于PCR基础上的两种DNA标记技术,本文比较了两种标记技术在我国栉孔扇贝群体遗传多样性研究中的应用。共筛选20个RAPD引物和7个AFLP引物组合,检测到AFLP标记的有效等位基因数和平均多态信息量稍低于RAPD标记,但AFLP标记在每单位分析中扩增到的野生和养殖群体的多态性条带数(23．8,24．8)分别高于RAPD标记(5．6,5．6),AFLP多态性检测效率显著高于RAPD标记。AFLP和RAPD两种标记技术所揭示的野生种群与养殖群体间的近交系数、遗传距离两项指标均表明,我国栉孔扇贝养殖群体和野生种群之间尚未出现明显的遗传分化。研究结果表明：RAPD和AFLP这两种标记技术均可用于栉孔扇贝遗传多样性的分析,其分析结果是一致的。     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estimation of Genetic Variability of Vigna radiata Cultivars by RAPD Analysis

DNA was isolated from 14 cultivars of Vigna radiata (L.) Wilczek and subjected to RAPD analysis using 14 random decamer primers. These cultivars revealed polymorphism with respect to RAPD markers and were subjected to hierarchical cluster analysis. A dendrogram was prepared based on these data. Analysis of banding patterns confirmed that two strongly aromatic cultivars IC1, IC4, were closely linked. But another aromatic cultivar, B1, formed a separate cluster. The high yielding cultivars were closely related to B1. The phylogenetic tree constructed by the neighbour joining method showed that RAPD results were correlated with morphological characters like plant height, leaf and seed size, seed colour, etc. This revised version was published online in July 2006 with corrections to the Cover Date.     

基于果蝇DNA甲基化的AFLP体系的建立及优化

以黑腹果蝇(Drosophila melanogaster)Canton-S品系为材料,采用改良SDS法提取高质量DNA,对连接、预扩增及选择性扩增进行分析,建立适用于果蝇基因组DNA甲基化多态性研究的AFLP优化体系:1)10μL连接体系加T4连接酶1 U,AluⅠ接头50 pmol,EcoR Ⅰ接头5 pmol,4℃反应12 h;2)25μL预扩增体系含Mg2+0.2 mmol/L,dNTPs 0.15 mmol/L,模板1.0μL,Taq DNA聚合酶2 U,E-00 50 ng,A-00 50 ng;3)25μL选择性扩增体系含Mg2+0.1 mmol/L,dNTPs 0.15 mmol/L,模板2.0μL,Taq DNA酶1.5 U、E+340 ng,A+3 40 ng.该体系稳定性高、重复性好,适用于果蝇基因组DNA甲基化多态性研究.     

Optimisation of DNA extraction for AFLP analysis of mycorrhizal fungi of terrestrial orchids caladeniinae and drakaeinae

Published DNA extraction methods present a number of problems when applied to mycorrhizal fungi of native Australian terrestrial orchids. Grinding with liquid nitrogen shears the DNA, and other pulverisation methods yield too little DNA. We found that freezing the fungal sample with liquid nitrogen, with no grinding, followed by the Qiagen DNeasy extraction procedure produced good yields of high-molecular-weight DNA. The DNA was then used for amplified fragment length polymorphism (AFLP) fingerprinting. Good fingerprints were produced by restriction withEcoRI/MseI enzymes, the use of preamplification primer mix II (for small genomes), and a 2-base extensionMseI primer (m-cc) with 3-base extensionEcoRI primers in the selective amplification. This protocol may be of general utility for other fungi with similarly fragile DNA.     

Variation in Heavy Metal Accumulation and Genetic Diversity at a Regional Scale Among Metallicolous and Non-Metallicolous Populations of the Facultative Metallophyte Biscutella laevigata subsp. laevigata

Biscutella laevigata is a facultative metallophyte, with populations on non-metalliferous and metalliferous soils. Some of its metallicolous populations have been shown to hyperaccumulate thallium or lead in nature. Only Tl hyperaccumulation has been experimentally confirmed. We aimed to compare the patterns of metal (hyper)accumulation and genetic diversity among populations of B. laevigata subsp. laevigata in NE Italy.

None of the populations exhibited foliar hyperaccumulation of Cu, Zn, or Pb. The root-to-shoot accumulation rates for these metals were unchanged or decreased rather than enhanced in the metallicolous populations, in comparison with the non-metallicolous ones. Hyperaccumulation of Tl was confined to the population of the Cave del Predil mine. This population was genetically very distinct from the others, as demonstrated by AFLP-based cluster analysis. The two other mine populations did not surpass the threshold for Tl hyperaccumulation, but showed enhanced foliar Tl concentrations and root-to-shoot translocation rates, in comparison with the non-metallicolous populations. Genetic analysis suggested that adaptation to metalliferous soil must have been independently evolved in the metallicolous populations.     

Detection and Identification of Aster Yellows (16SrI) Phytoplasma in Peach Trees in Jordan by RFLP Analysis of PCR-Amplified Products (16S rDNAs)

Aster yellows phytoplasma were detected, for the first time, in peach trees in Al‐Jubiha and Homret Al‐Sahen area. Leaves of infected trees showed yellow or reddish, irregular water‐soaked blotches. Discoloured areas become dry and brittle and the dead tissues dropped out. Under severe infections, leaves fall down and fruits dropped prematurely. Phytoplasmas were detected from all symptomatic peach trees by polymerase chain reaction (PCR) using universal phytoplasmas primers P1/P7 followed by R16F2/R2. No amplification products were obtained from templates of asymptomatic peaches. PCR products (1.2 kb) used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease AluI, HpaII, KpnI and RsaI produced the same restriction profiles for all samples, and they were identical with those of American aster yellows (16SrI) phytoplasma strain. This paper is the first report on aster yellows phytoplasma affecting peach trees in Jordan.     

亚欧美栗疫病菌群体的遗传多样性

从 12 0个随机引物中筛选出条带清晰、主带明显、重复性好的 9个引物 ,对来自不同地域和寄主的 7个群体的 14 2个栗疫病菌菌株进行 RAPD分析。 9个引物共扩增出条带 12 4条 ,其中多态性条带 111条 ,多态性比率为 89.5 2 %。利用 Popgen3.2软件对供试群体进行遗传多样性分析和 UPGMA聚类。结果表明 ,中国地区 4个群体间的遗传相似性较大 ,与美国、意大利和日本群体间的相似性较小 ;美国和意大利群体间的遗传相似性较大 ,且它们与日本群体间的相似性大于与中国群体间的相似性。病原菌群体的遗传变异率为 0 .2 35 1,其中在地区水平上 ,82 .34%由群体内的变异引起 ,17.6 6 %由群体间的差异引起 ,群体间的基因流动值为 2 .3311;而在寄主水平上 ,则 79.4 2 %由群体内的变异引起 ,2 0 .5 8%由群体间的差异引起 ,群体间的基因流动值为 1.92 97     

Genetic differences between Escherichia coli O26 strains isolated in Brazil and in other countries

Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup -- encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains -- was evaluated through random amplified polymorphic DNA (RAPD) analysis. Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters. RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H(-) serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data. The epidemiological significance of these data is discussed.