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.     

TAIL-PCR的改良及其在分离小麦基因启动子中的应用

在经典TAIL-PCR(Thermal asymmetric interlaced PCR)的基础上, 对其进行了如下四处改进: 用10个碱基的RAPD引物代替16个碱基的随机兼并引物作为PCR中的随机引物; 将较低特异性循环的复性温度由44°C降至29°C; 增加5个高特异性反应循环, 减少5个较低特异性反应循环; 用单引物对第三轮PCR产物进行初步鉴定。利用改进的TAIL-PCR方法分离了小麦X基因的5′未知的侧翼序列, 与GUS基因融合后转入拟南芥, 通过组织化学检测分析表明分离到的5′侧翼序列具有启动子功能, 同时说明改进的TAIL-PCR能更好地应用到较复杂基因启动子的分离。     

TAIL-PCR的改良及其在分离小麦基因启动子中的应用

在经典TAIL-PCR(Thermal asymmetric interlaced PCR)的基础上, 对其进行了如下四处改进: 用10个碱基的RAPD引物代替16个碱基的随机兼并引物作为PCR中的随机引物; 将较低特异性循环的复性温度由44°C降至29°C; 增加5个高特异性反应循环, 减少5个较低特异性反应循环; 用单引物对第三轮PCR产物进行初步鉴定。利用改进的TAIL-PCR方法分离了小麦X基因的5′未知的侧翼序列, 与GUS基因融合后转入拟南芥, 通过组织化学检测分析表明分离到的5′侧翼序列具有启动子功能, 同时说明改进的TAIL-PCR能更好地应用到较复杂基因启动子的分离。     

RAPD技术的特点及其在昆虫分类中的应用

随机扩增的多态性DNA(RAPD)技术,是近年来发展起来的一项DNA分子水平上 的大分子多态检测手段。由于它具有简捷、灵敏、对材料要求不高,取材少、成本低等优点, 备受人们青睐,在遗传学、分子进化、生物分类等领域被广泛地运用。在昆虫分类的过程中, 由于昆虫的种类繁多,形态、生态差异很大,许多在其它领域被广泛运用的分子生物学技术不能在昆虫分类中充分发挥作用。同时在昆虫分类中出现的一些问题却又需要用涉及遗传本质的分子生物学技术进行探讨和研究。本文就RAPD技术的特点及其在解决昆虫分类中的问题时的优势作一简介。     

采用RAPD标记探讨瓣蕊唐松草(毛茛科唐松草属)的遗传多样性及其地理分布格局(英文)

Random amplified polymerphic DNA(RAPD)method was applied to assessg enetic variation and population structure of Thahctrum petalotdeum L(Ranunoulaceae),Two hundred and forty-six individuals from 11 populations of the species were investigated by RAPD profiles Twenty selected RAPD primers generated 125 bands．in which 120 were polymorphic Ther esults revealed a high level of genetic variation(ercentage of polymorphIc bands(PPB was 96％．Nei’s gene diversity(りwas 03502 and shannon’s information index(I) was 0．5199 at the species level) The differentiation among the populations was high(Gst=0．3511)in this species．Result of analyzing of molecularvariance(AMOVA)showedthat38．88％of genetic variance was found among the populations Positive correlation withr r=01945(P=00002)was found between genetic distance and geographic distance amongpo pulations Two populations distributed in the drainage basin of YanELz River affined genedcally and formed one clada and the rest nine populations formed the other clade in both unweighted pair-group method using arithmetic average(UPGMA)trees made by two different method different methods. It was yen/clear that these two populations were very special, andmust be closely related in history, despite the fact that they now share quite weak link to the restpopulations through gene communication.     

采用RAPD标记探讨瓣蕊唐松草(毛茛科唐松草属)的遗传多样性及其地理分布格局

采用随机扩增多态DNA(RAPD)标记对瓣蕊唐松草(Thalictrum petaloideum L.)(毛莨科)11个居群246个体的遗传多样性及居群遗传结构进行了检测.20个随机引物扩增出125个用于分析的条带.结果表明:瓣蕊唐松草在物种水平上具有极高的遗传多样性,多态条带比率(PPB)为96%,Nei的基因多样度(h)为0.3502,Shannon多样性指数(I)为0.519 9;居群间分化比较明显,基因分化系数(GsT)为0.351 1,分子方差分析(AMOVA)表明居群间遗传变异占总遗传变异的38.88%;根据条带频率与平均分类距离和Nei遗传距离分别进行UPGMA聚类,结果基本一致,并且反映出与该种地理分布有一定相关性;长江流域分布的两个居群表现出较高的相似性,并与北方分布的各居群构成独立的两支,显示出这两个居群的特殊性,从而对该物种独特分布区成因是由于冰退回迁造成的观点提供了一定的支持.     

五种品系猪亲缘关系的RAPD分析

应用190个RAPD引物对贵州小型猪,巴马小型猪、西双版纳近交系小耳猪JB和JS亚系、荣昌猪Ⅰ系、长白猪的亲缘关系进行了初步分析,其中39个引物的扩增结果具有非常明显的品系间多态性,将其扩增结果以RAPDistance package Version 1?04程序进行分析,计算相对遗传距离指数1－F,再用NJ法进行聚类分析,构建系统树。结果表明,三种小型猪相互间亲缘关系较近,尤以贵州小型猪与巴马小型猪更近。荣昌猪Ⅰ系和长白猪关系较近,但与小型猪关系较远。 关键词：随机扩增多态DNA;小型猪;亲缘关系 The phylogenetic relationship of five species of pigs including Guizhou miniature pig,Bama pig,Xisuangbanna inbred pig,Rongchang Pig and Landrance was studied by RAPD analysis.Thirty-nine single polymorphic primers were selected out of 190 primers.The amplified fragments of thirty-nine primers were analysed by the RAPDistance package version 1.04 and the phylogenetic tree was constructed using NJ method.The results indicated as the following: three strains of miniature pigs have close phylogenetic relationship and the phylogenetic relationship between Guizhou miniature pig and Bama miniature pig was much closer.Landrance and Rongchang pig have close phylogenetic relationship too,but their phylogenetic relationship is far from the three strains of miniature pigs.     

Analysis of Cultural and Genetic Diversity in Alternaria helianthi and Determination of Pathogenic Variability Using Wild Helianthus Species

The study aims at assessment of morphological, molecular and pathogenic variability of Alternaria helianthi, incitant of leaf blight of sunflower. Morphological characteristics determined for 26 isolates of A. helianthi from India revealed variations in shape of culture, pigmentation, conidial measurements, number of septa and colony growth. The conidia of isolate Ah‐7 were long, while conidia of isolate Ah‐15 were short. Based on cultural characters, isolates were classified into four groups. Genetic variability of the isolates was assessed by random amplified polymorphic DNA analysis. Good polymorphism was observed and cluster analysis indicated presence of six genetically distinct groups among the isolates. The isolates Ah‐1, Ah‐7 and Ah‐14 were genetically distinct. Resistant sources are not available in cultivated sunflower, while wild Helianthus species possess resistance to multiple stresses. We evaluated reaction of wild Helianthus species to isolates of A. helianthi. Among wild Helianthus species, H. tuberosus followed by H. occidentalis showed moderately to highly resistant reaction to all the isolates and recorded less disease incidence. The species H. argophyllus followed by H. laevigatus showed more disease incidence. The cultivated sunflower recorded susceptible reaction to most of the isolates and recorded high disease incidence. The isolates differed significantly for pathogenic reaction and were grouped into three pathogenicity groups; low, medium and high. Six isolates induced <20% disease incidence and were included in the low pathogenicity group. Majority of isolates were in the medium pathogenicity group. Six isolates i.e. Ah‐9, Ah‐10, Ah‐18, Ah‐20, Ah‐24 and Ah‐26 induced more than 50% disease incidence and were considered high pathogenicity group. Our results demonstrate the existence of considerable variation in resistance of Helianthus species to A. helianthi and also in morphological and genetic characters of A. helianthi isolates prevalent in India.     

Genetic diversity in Isoetes yunguiensis, a rare and endangered endemic fern in China

Isoetes yunguiensis is an endangered and endemic fern in China.Field survey indicated that only one population and no more than 50 individuals occur in the wild.The genetic variation of 46 individuals from the population remaining at Pingha (Guizhou Province,China)was assessed by Random Amplified Polymorphic DNA (RAPD)fingerprinting.Twelve primers were screened from sixty ten-bp arbitrary primers,and a total of 95 DNA fragments were scored.Of these,62.1%were polymorphic loci,which indicated that high level genetic variation existed in the natural population.The accumulation of genetic variation in the history of the taxon and the apparent minimal reduction effect on genetic diversity following destruction of habitat might be responsible for the high level genetic diversity presently remaining in the I.yunguiensis population.However,with the continuing decrease of population size,the genetic diversity will gradually be lost.We suggest that the materials from the extant population should be used for re-establishment of the populations.     

Identification and Assessing the Cultivars of Laminaria Lamx.(Phaeophyceae) with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breed ing in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity of the endangered species Rosa rugosa Thunb. in China and implications for conservation strategies

Rosa rugosa Thunb. is one of the dominant and important shrub species in estuary dunes and shingle beaches of northern China. However, its area of distribution, the number of populations, and the size of each population have decreased rapidly in the past two decades because of habitat degradation and loss. Random amplified polymorphic DNA markers were used to determine the genetic diversity of four remaining large natural populations of R. rugosa and to discuss an effective conservation strategy for this endangered species in China. High genetic variations were detected in R. rugosa populations in China. The mean percentage of polymorphic loci （P%） within four local populations was 57.99%, with the P% of the total population being 75.30%. Mean Shannon＇s information index （H0） was 0.2826, whereas total Ho was 0.3513. The genetic differentiation among populations was 0.1878, which indicates that most genetic diversity occurs within populations. Population Tumenjiang （TMJ） showed the highest genetic diversity （P% = 66.27%; H0 = 0.3117） and contained two exclusive bands. Population Changshandao （CSD） showed higher genetic diversity （P% =59.04%; H0 = 0.3065）. Populations TMJ and CSD contained 95.33% and 99.33%, respectively, of loci with moderate to high frequency （P〉0.05） of the total population. These results indicate that populations TMJ and CSD should be given priority for in situ conservation and regarded as seed or propagule sources for ex situ conservation. The results of the present study also suggest that R. rugosa in China has become endangered as a result of human actions rather than genetic depression of populations; thus, human interference should be absolutely forbidden in R. rugosa habitats.     

Genetic diversity analysis of Hepatacodium miconioides at different altitude in Tiantai Mountain, Zhejiang Province, China and its relationship with environmental factors

The genetic diversity within and among populations of Hepatacodium miconioides collected at three different altitudes in Tiantai Mountain, Zhejiang Province and its relationships to environmental factors were analyzed by random amplified polymorphic DNA (RAPD) technique. Amplification using 12 random primers of 60 plants and 122 repetitive loci were produced. The percentage of polymorphic loci of three populations ranged from 18.85% to 23.77% with an average of 21.86%, indicating the relatively low genetic diversity of H. miconioides. The average Shannon index of phenotypic diversity (0.1329) and Nei index (0.0925) within populations were relatively low. A distinct genetic differentiation existed among populations of H. miconioides in spite of the relatively small geographical distribution. The average genetic diversity within populations of H. miconioides accounted for 33.58% of the total genetic diversity while the genetic diversity among populations accounted for 66.42% as estimated by the Shannon index of phenotypic diversity, The genetic differentiation among populations of H. miconioides was 0.6546, as estimated by Nei index. The gene flow estimated from G _ST was only 0.2656 and it indicated that gene flow among populations of H. miconioides was relatively low. The mean value of the genetic identity among populations of H. miconioides was 0.7126 and the average of genetic distance of H. miconioides was 0.3412. The genetic identity between populations at the elevation of 990 m and at the elevation of 780 m was the highest. The genetic identity between population at the elevation 500 m and other two populations was relatively low. The correlation analysis showed that the genetic diversity within populations was significantly related with the soil total nitrogen. __________ Translated from Journal of Zhejiang University (science Edition) 2005, 32 (4)[译81EA;: 浙江大学学报(理学版), 2005,32(4)]     

Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA

Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly ifferentiated from T. mentagrophytes by using primer 5-GAAGGCTCCC-3 (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genetic diversity analysis of Hepatacodium miconioides at different altitude in Tiantal Mountain,Zhejiang Province,China and its relationship with environmental factors

The genetic diversity within and among populations of Hepatacodium miconioides collected at three different altitudes in Tiantai Mountain,Zhejiang Province and its relationships to environmental factors were analyzed by random amplified polymorphic DNA(RAPD)technique.Amplification using 12 random primers of 60 plants and 122 repetitive loci were produced.The percentage of polymorphic loci of three populations ranged from 18.85% to 23.77% with an average of 21.86%,indicating the relatively low genetic diversity of H.miconioides.The average Shannon index of phenotypic diversity(0.1329)and Nei index(0.0925)within populations were relatively low.A distinct genetic differentiation existed among populations Of H miconioides in spite of the relatively small geographical distribufion.The average genetic diversity within populations of H.miconioides accounted for 33.58% of the total genetic diversity while the genetic diversity among populations accounted for 66.42% as estimated by the Shannon index of phenotypic diversity,The genetic differentiation among populations of H.miconioides was 0.6546,as estimated by Nei index.The gene flow estimated from Gsr was only 0.2656 and it indicated that gene flow among populations of H.miconioides was relatively low.The mean value of the genetic identity among populations of H.miconioides was 0.7126 and the average of genetic distance of H.miconioides was 0.3412.The genetic identity between populations at the elevation of 990m and at the elevation of 780 m was the highest.The genetic identity between population at the elevation 500 m and other two populations was relatively low.The correlation analysis showed that the genetic diversity within populations was significantly related with the soil total nitrogen.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

湖南四种尾矿环境下的狗牙根遗传多样性的RAPD分析

选择湖南4种有代表性的有色金属尾矿地所生长的野生狗牙根作为研究对象,并以正常生境下的野生狗牙根作为对照,采用RAPD技术分析了这些特别环境下的狗牙根的遗传多样性。17条10个核苷酸长的引物用于PCR扩增,共得到432条DNA带。检测位点160个,其中多态位点134个,占83.75%。各个类群间的Jaccard相似系数平均为0.4475±0.0806,遗传距离平均为0.3825±0.0712。结果显示生长于这些不同尾矿环境下的狗牙根种群在遗传本质上均产生了明显的分化,完全可以根据其尾矿基质的不同而划分为不同的生态型或品种,甚至可能处理为新的变种。狗牙根种群具有丰富的遗传多样性,作为先锋植物可为尾矿的复垦提供更多的品种选择。     

条形柄锈菌33号生理小种的分子标记

采用RAPD-SCAR分子标记技术,从300条RAPD随机引物中筛选到了对条形柄锈菌Puccinia striiformis f. sp. tritici 33号生理小种特异的2条引物,将特异性片段回收、克隆和测序后（GenBank注册号为AB914691和AB914692）,依据其序列设计出了2对引物S261F33/S261R33和S300F33/S300R33,能够特异性地从33号生理小种基因组DNA及发病小麦叶片总DNA中分别扩增出247bp和763bp的片段,其结果与采用常规的鉴别寄主法鉴定的结果一致。因此,这2对引物都可用于条形柄锈菌33号生理小种的快速鉴定与监测。     

Genetic Diversity and Chemical Polymorphism of Some Thymus Species

To ascertain whether there are chemical and genetic relationships among some Thymus species and also to determine correlation between these two sets of data, the essential‐oil composition and genetic variability of six populations of Thymus including: T. daenensis ?elak. (two populations), T. fallax Fisch . & C.A.Mey ., T. fedtschenkoi Ronniger , T. migricus Klokov & Des .‐Shost ., and T. vulgaris L. were analyzed by GC and GC/MS, and also by randomly amplified polymorphic DNA (RAPD). Thus, 27 individuals were analyzed using 16 RAPD primers, which generated 264 polymorphic scorable bands and volatiles isolated by distillation extraction were subjected to GC and GC/MS analyses. The yields of oils ranged from 2.1 to 3.8% (v/w), and 34 components were identified, amounting to a total percentage of 97.8–99.9%. RAPD Markers allowed a perfect distinction between the different species based on their distinctive genetic background. However, they did not show identical clustering with the volatile‐oil profiles.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.