Phylogenetic Analysis of Dipterocarps Using Random Amplified Polymorphic DNA Markers

The phylogenetic relationships among 12 species belonging tothree different genera (Shorea, HopeaandAnisoptera) of Dipterocarpaceaewere studied using random amplified polymorphic DNA (RAPD) markers.A modified CTAB DNA extraction protocol was used to obtain tannin-and polysaccharide-free genomic DNA from mature leaves. Clusteranalysis of data from six random primers placed the 12 speciesin three groups corresponding to their respective genera. Fourdistinct nodes ofShoreaspp. and two ofHopeaspp. could be identified.Anisopteramegistocarpaserved as an outgroup, and was unique when comparedto the other genera examined. RAPD profiles of five individualsofH. odoratawith six random primers were identical, suggestingthat there is little intraspecific variation in this species.The RAPD technique can thus be successfully applied for thestudy of phylogenetic relationships of this important groupof tropical timber trees.Copyright 1998 Annals of Botany Company Dipterocarpaceae,Anisopteraspp.,Hopeaspp.,Shoreaspp., RAPD markers, tropical timber trees.     

中国柱花草炭疽病原菌遗传多态性的RAPD分析

在对中国柱花草炭疽病进行广泛调查和病原采样收集的基础上,利用RAPD分子标记技术对43个代表性菌株进行了基因组DNA分析,并与276份国外菌株进行了综合聚类分析。 结果表明所用8个引物的扩增片段位于0.3～2.8kb之间, 菌株间呈现显著的DNA多态性。以柱花草起源中心——南美的柱花草炭疽菌分类为基础,中国柱花草炭疽菌可划分成3大类型即Ⅱ、Ⅲ、Ⅵ类。中国菌株与来自柱花草起源中心——南美的菌株相比之下,其生物多样性和遗传变异性则相对简单。就中国菌株而言海南菌株与广西、广东菌株相比多样性较丰富, 中国柱花草胶孢炭疽菌正在出现种内遗传分化。 从聚类结果看,通常来自于同一个地理区域或同一个寄主基因型的菌株聚成一类, 即同一RAPD聚类组内的菌株通常来自于同一寄主基因型或同一地理区域。说明来自不同寄主基因型或物种的炭疽菌在遗传基因上具有专化性,而地理上隔离的国家或地区的柱花草炭疽病原菌各自具有相对独立的进化途径。     

Analysis of Genetic Diversity amongIxoraCultivars (Rubiaceae) using Random Amplified Polymorphic DNA

We have optimized the genomic DNA extraction method from freshand dry laminas, as well as fresh and dry corolla lobes ofIxoracultivars.Some woody tropical species such as these contain excessiveamounts of phenolic compounds that co-precipitate with DNA resultingin poor or no amplification during the polymerase chain reaction(PCR). Repeated precipitation with CsCl coupled with phenol:chloroformextraction yielded high quality DNA suitable for consistentPCR amplification. The DNA from fresh laminas of 22 cultivarsofIxorawas subjected to random amplified polymorphic DNA (RAPD)analysis. Individual taxa could be identified using specificDNA markers from the RAPD profiles. Cluster analysis of datafrom six primers grouped all 22 cultivars distinctly under twocultivar groups, viz.,IxoraCoccinea andI.Javanica. The percentagegenetic similarity was calculated for all the cultivars basedon the RAPD data. The two cultivar groups and the outgroup plantswere also clearly distinguishable with polar ordination usinga matrix of genetic dissimilarities (one minus similarity).Our data indicate that besides the use of RAPD markers for identificationof particularIxoracultivars within a germplasm collection, thephylogenetic relationships generated by RAPD analysis may beuseful for future breeding programmes. IxoraL. cultivars; Rubiaceae; RAPD fingerprinting; DNA extraction; woody tropical species     

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。     

Genetic Relatedness Among Strains of Heterobasidion annosum as Detected by Random Amplified Polymorphic DNA Markers

Heterobasidion annosum, the casual agent of root and butt rots of trees is one of the most important plant pathogens in temperate coniferous regions of the world. It is comprised of several intersterility groups with different host preferences. Of the H. annosum isolates from various geographical regions belonging to various intersterility groups, 36 showed group-specific banding patterns in RAPD assay. More polymorphisms were found within the S- and F-group isolates than in the P group. Genetic similarities among the isolates were determined by UPGMA clustering revealing the presence of four major clusters: P, S, F, and the North American S. The P group was the most distinctive of all the other groups, while the European S and F and the North American S were more closely related. The P group showed only 20% similarity to the other groups and may be referred to as a subspecies. The North American S group was separate from the European S group. The potential influence of the historical spread of coniferous trees in the isolate genetic differentiation in Europe is discussed.     

Identification of Gremmeniella abietina Races with Random Amplified Polymorphic DNA Markers

Seven random amplified polymorphic DNA (RAPD) markers amplified from four oligonucleotides (10-mers) by the polymerase chain reaction were used to distinguish between the North American and European races of Gremmeniella abietina, the causal agent of Scleroderris canker of conifers. Forty-three isolates of the pathogen from 11 different host species originating from 11 countries, states, and provinces were tested; race designation was consistent with results from immunogenic and soluble-protein assays. By using RAPD markers, it was possible to identify G. abietina races by DNA amplifications directly from fruiting bodies, thus eliminating the need to culture the fungus, as is necessary with immunogenic and soluble-protein assays. Two isolates which had been previously classified as intermediate were clearly identified as belonging to either one of the two races by using RAPD markers. No interracial hybrids were detected in our survey. Patterns of amplification products from the European race in North America were identical to patterns of European isolates, further substantiating that this is an introduced race to the North American continent.     

冬虫夏草(Cordyceps sinensis)的随机扩增多态DNA及其遗传分化

本文对来自青藏高原3个区域5个具有代表性地方的13个冬虫夏草(Cordyceps sinensis[Berk.] Sacc.)样本进行随机扩增多态DNA(RAPD)分析。19个随机引物获得的RAPD谱带清晰并呈现多态,单个引物获得的RAPD片段数在3～10个之间。该19个引物在每个样本中扩增的RAPD片段总数平均约为65个。基于遗传距离分析,受试的13个冬虫夏草样本中,来自同一地方的样本间遗传差异甚微,同一区域不同地方的样本间遗传差异较大,不同区域的样本间遗传差异最大。这说明冬虫夏草地理群体间存在着遗传分化。应用UPGMA和NJ方法构建的分子系统树显示,来自5个地方的冬虫夏草实际上可以归并为显著不同的3个组,对应于样本来源的3个区域,提示RAPD标记在冬虫夏草群体中有显著的地区特异性。我们的结果还表明,冬虫夏草地理群体间的遗传差异度与地理距离呈正相关。因此,RAPD作为有效的遗传标记,可用于研究冬虫夏草的遗传多样性、起源以及系统演化等。     

Use of Random Amplified Polymorphic DNA Markers for Mapping the Chickpea Genome

Three interspecific crosses were developed using Cicer arietinum (ICC 4918) as the female parent and wild Cicer species [C. reticulatum - JM 2100, JM 2106 and C. echinospermum - ICCW 44] as the male parent. Cicer arietinum (ICC 4918) × C. reticulatum (JM 2100) cross produced the largest number of F₂ plants and was chosen for linkage mapping using Random Amplified Polymorphic DNA (RAPD) primers. A partial linkage map was constructed based upon the segregation of 36 RAPD markers obtained by amplification using 35 primers. The linkage map consists of two linkage groups with 17 linked markers covering a total of 464.9 cM. Analyses also revealed association of three morphological traits with linked RAPD markers. Out of seven morphological traits tested for association with linked markers in the segregating plants, four Quantitative trait loci (QTL) were detected for the trait leaf length and three QTLs each for the traits leaf width and erect plant habit.     

Distribution of Mating Types and Genetic Diversity of Ascochyta rabiei Populations in Tunisia Revealed by Mating-type-specific PCR and Random Amplified Polymorphic DNA Markers

The distribution of mating types of Ascochyta rabiei (teleomorph: Didymella rabiei) was determined in Tunisia using a MAT‐specific PCR assay. Among 123 isolates tested, 80% were MAT1‐1 and 20%MAT1‐2. Only MAT1‐1 isolates were present in the Beja and Bizerte regions of Tunisia, whereas both mating types were present in Nabeul, Kef and Jendouba. In the latter three regions, the hypothesis of random mating could not be rejected based on chi‐squared tests of mating‐type ratios (P > 0.05). The lower frequency of the MAT1‐2 coupled with the restricted distribution of this mating type in Tunisia may indicate a recent introduction of MAT1‐2 in Tunisia. This speculation is consistent with the recent (2001) observation of D. rabiei pseudothecia on chickpea debris in Tunisia. Forty isolates representative of the five regions were genetically analysed using 10 random amplified polymorphic DNA (RAPD) primers to provide a preliminary estimate of genetic diversity of the pathogen in Tunisia. Among 129 putative RAPD loci amplified, 81% were polymorphic and 32 unique RAPD fingerprints were detected. A high level of genetic differentiation was detected among subpopulations (G_ST = 0.33). Cluster analyses revealed that isolates from Bizerte, Beja and Jendouba were genetically similar and distinct from isolates sampled in Nabeul and Kef. MAT1‐1 isolates were clustered separately from MAT1‐2 isolates in Jendouba and Nabeul suggesting that recombination may not yet be occurring in these regions despite the occurrence of both mating types in equal frequency in these regions. This lack of recombination between MAT1‐1 and MAT1‐2 also supports the hypothesis of a recent introduction of MAT1‐2 into Tunisia.     

Molecular Analysis of Chickpea (Cicer arietinum L) Cultivars Using AFLP and STMS Markers

Fifteen AFLP and eighteen STMS primer pairs were employed to reveal genetic diversity and relationship in twenty-one cultivars of chickpea (Cicer arietinum L). Fifteen AFLP primer pairs generated 1804 amplicons, out of which 1732 amplicons (96%) were polymorphic and 600 amplicons (∼33%) were genotype specific. Eighteen polymorphic STMS primer pairs generated 64 amplicons with an average of 3.55 amplicons per primer pair. Polymorphic information content (PIC) varied from 0.52 to 1.0 for STMS markers. The genetic similarity between cultivars varied from 0.30 to 0.85 for AFLP and 0.22 to 0.83 for STMS markers. Dendrogram constructed after combining both AFLP and STMS markers data with Bootstrap analysis, grouped all the cultivars into four clusters. Association of varietal type and flower colour was observed as cultivars E 100Ymu and Nabin (Both Desi type and pink flower) clustered together in the dendrogram.     

中国貉随机扩增多态DNA及其亚种分化关系

对来自陕西、云南、越南、安徽和广西等地的８只中国貉（Ｎｙｃｔｅｒｅｕｔｅｓｐｒｏｃｙｏｎｉｄｅｓ）进行随机扩增多态ＤＮＡ分析。应用２８个１０ｂｐ的随机引物,平均每只貉获得的ＲＡＰＤ标记数约为１３０条。遗传距离计算结果显示,中国貉个体间的平均遗传距离指数值为１１．２０％,最大值为１４．９３％,最小值为２．９４％。以赤狐（Ｖｕｌｐｅｓｖｕｌｐｅｓ）为外群,应用ＰＨＹＬＩＰ３．０计算软件包中的ＵＰＧＭＡ和ＮＪ聚类方法构建分子系统树。结果表明,不同地理群体间的中国貉存在遗传分化;中国貉可分为４组：（１）广西貉,（２）安徽貉,（３）陕西貉,（４）云南貉和越南貉。其中安徽貉和广西貉间的关系稍近,陕西貉则与云南貉－越南貉稍近。对合中国貉的形态分类、地理分布、ｍｔＤＮＡ多态分析以及进化遗传学的观点,认为陕西貉、广西貉和安徽貉可能与云南貉－越南貉具有等同的分类地位。     

应用SSR标记分析鹰嘴豆种质资源遗传多样性

种质资源的遗传多样性是育种工作的基础,本研究利用SSR标记对鹰嘴豆资源进行遗传多样性分析,旨在发掘鹰嘴豆资源中丰富的遗传变异。从48对鹰嘴豆SSR引物中筛选出18对核心多态性引物,对96份不同来源的鹰嘴豆种质资源进行SSR标记的遗传多样性分析。结果表明,18对SSR引物共检测到115个等位变异,占总检测位点的52.99%,每对SSR引物可检测出3~10个等位变异,平均6.39个;平均每个位点多态信息量(PIC)为0.8107,变化范围为0.6661~0.8984。Shannon's信息指数平均为1.4769,变化范围为0.0607~1.9584。PGMA聚类结果表明,在遗传相似系数0.59处,可将96份鹰嘴豆资源分为6个类群,类群Ⅰ包含9份资源,类群Ⅱ包含2份资源,类群Ⅲ包含28份资源,类群Ⅳ包含4份资源,类群Ⅴ包含40份资源,类群Ⅵ包含13份资源。本研究对我国鹰嘴豆种质资源的评价与利用、优异基因的挖掘、育种亲本材料的选择等提供科学依据。     

Genetic Mapping of Soybean [Glycine max (L.) Merr.] Using Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. We have adapted the assay to soybeans by using Stoffel Fragment DNA polymerase and by optimizing the reaction conditions. To increase the percentage of RAPD polymorphisms, the DNA template was digested with restriction enzymes before amplification. The combination of twenty-four primers and five DNA template treatments (Undigested, DraI, EcoRI, HindIII, and TaqI digested) revealed 94 polymorphic DNA fragments differing between soybean lines PI437654 and BSR101. Many polymorphic DNA bands were found unreliable or non-scoreable after re-screening of primers and verification of marker-allele segregation with 20 recombinant inbred lines (RILs). However, 28 RAPD markers were consistently polymorphic between the parental lines and followed Mendelian expectations. The use of DNA templates digested with DraI, EcoRI, HindIII or TaqI increased three times the number of RAPD markers compared to undigested DNA template alone. The 28 RAPD markers obtained were further screened with 72 RILs and placed on an existing RFLP map.     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

Intraspecific Variability within Globodera tabacum solanacearum Using Random Amplified Polymorphic DNA

Random amplified polymorphic DNA (RAPDs) were used to investigate the intraspecific variability among 19 geographic isolates of Globodera tabacum solanacearum from eight counties in Virginia and one county in North Carolina. Globodera tabacum tabacum, G. t. virginiae, and the Mexican cyst nematode (MCN) were included as outgroups. Six primers were used and 119 amplification products were observed. Each primer yielded reproducible differences in fragment patterns that differentiated the isolates and species. Hierarchical cluster analysis was performed to illustrate the relatedness among isolates and species. The average Jaccard''s similarity index among isolates of G. t. solanacearum was 74%, possibly representing greater variation than that reported in the literature across different pathotypes of the potato cyst nematode, Globodera pallida, in studies where RAPD were also employed. The RAPD markers described here may be useful for the development of specific primers or probes that could improve the identification of TCN populations. Such improvements in the characterization of TCN genotypes would facilitate the effective deployment of existing and future resistant cultivars to control these economically important pests.     

Suitability of Random Amplified Polymorphic DNA for Genetic Markers in the Aphid Parasitoid, Aphelinus asychis Walker

The repeatability, variability, transmission, and linkage relationships of random amplified polymerphic DNA (RAPD) fragments were examined using six inbred lines of the haplodiploid parasitoid, Aphelinus asychis, originally collected on one date from a single held in southern France. Repeatability of RAPD fragments could be adequately judged using two replicate amplifications of the same individual in the same amplification run. Thirty-one of 136 repeatable fragments generated by 14 primers were polymorphic among lines. Segregation ratios in Fz males did not differ from 1:1 and extrachromosomal transmission was not observed. However, 5 nonparental bands that would increase the apparent number of loci by 16.2% in outbreeding populations were detected in hybrid F₁ females. In addition, linkage analysis indicates that the 31 polymorphic bands represent 19 presence-absence loci and 6 biallelic, fragment length polymorphism (FLP) or FLP-like loci. Four linkage groups were detected. Our main conclusion is that RAPD polymerphisms cannot be used as genetic markers unless information identifying nonparental bands and FLP and FLP-like loci is obtained. This information can be obtained during the course of typical population surveys in haplodiploid species because of male haploidy. In diploid species though, crossing experiments or DNA hybridization tests to establish homology are necessary prior to working with unpedigreed populations.     

赣中南花猪随机扩增多态DNA与群体遗传关系的研究

用RAPD技术检测了泰和冠朝猪、永丰藤田花猪、瑞金三花猪、兴国茶园猪、上犹花猪、万安花猪、乐安花猪等7个赣中南花猪地方类群基因组混合DNA的多态性,经80个随机引物扩增筛选,16个引物产生了共44个多态标记。遗传距离指数计算结果显示：泰和冠朝猪和瑞金三花猪亲缘关系最近,而兴国茶园猪与乐安花猪遗传距离最远。各类群的UPMGA和NJ聚类分析结果与采用生化、免疫和细胞遗传标记分析的结论不尽相同,在此基础上,结合现行分类方法,认为各受试猪群可暂时归并为赣中南花猪类型,但对泰和冠朝猪和万安花猪的品种归属性应作进一步分析。 Abstract: Random amplified polymorphic DNAs (RAPDs) were used to investigate bulked genomic DNA polymorphism of seven pig populations including Taihe Guanchao,Yongfeng Tengtian Spotted,Ruijin Spotted,Xingguo Chayuan,Shangyou Spotted,Wanan Spotted,and Lean Spotted pig.Eighty random primers were screened,of them,16 primers generated polymorphic markers with total number of 44.The genetic distance index matrix indicated that the genetic relationship between Taihe Gaunchao and Ruijin Spotted pig population was the closest,while that between Xingguo Chayuan and Laan Spotted pig population was the farthest.Our molecular phylogenetic trees constructed by UPMGA and NJ methods were in general consistent with that based on biochemical,immunogenetic and cellular genetic markers except for differences in some populations.It was inferred from this study that all pig populations examined could be temporary classified into one breed,however,the population genetic character of Taihe Guanchao and Wanan Spotted pig populations should be further evaluated.