落叶松-杨栅锈菌遗传分化的RAPD分析

用随机扩增多态性ＤＮＡ（Ｒａｎｄｏｍ ａｍｐｌｉｆｉｅｄ ｐｏｌｙｍｏｒｐｈｉｃ ＤＮＡ,ＲＡＰＤ）技术对来自陕西及青海的７个地区的１３个落叶松－杨栅锈菌（Ｍｅｌａｍｐｓｏｒａ ｌａｒｉｃｉ－ｐｏｐｕｌｉｎａａ Ｋｌｅｂ．）的分离物进行了基因组ＤＮＡ多态性分析。１３个１０－核苷酸随机引物（Ｏｐｅｒｏｎ公司）对１３个菌株共扩增出８１条ＲＡＰＤ带,其中６９个ＤＮＡ片断呈现多态性,占总扩增片断的８５．２％。供试菌株的相似系数在０．６０８～１．０００之间,各菌株之间的差异在０～３３．１％之间,并建立了聚类树状图。１３个菌株在相似性７６．１％时被分为４个类群：Ⅰ组包括秦岭宁陕火地塘Ｃ的２个分离物,Ⅱ组为火地塘Ｂ的１个分离物;Ⅲ组为青海西宁、互助,陕西太白宝太路、宝鸡天台山、周至厚畛子（ＨＺａ）等５个地区的８个分离物;第Ⅳ     

林木遗传多样性研究中的DNA标记

遗传多样性是物种长期存活和进化的基础。应用ＤＮＡ标记的研究可以提供大量新的多样性信息。本文介绍和描述了限制性片段长度多态性（ＲｅｓｔｒｉｃｔｉｏｎＦｒａｇｍｅｎｔＬｅｎｇｔｈＰｏｌｇｍｏｒｐｈｉｓｍｓ，ＲＦＬＰｓ）和随机扩增多型性ＤＮＡ（ＲａｎｄｏｍＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃＤＮＡ，ＲＡＰＤ）方法，以及它们作为ＤＮＡ标记的分子基础。就这两种ＤＮＡ标记的遗传特性和人们已熟悉的同工酶标记进行了比较。简要地评述了ＤＮＡ标记近年在林木群体遗传学、系统分类学中的应用。     

马尾松随机扩增多态性DNA标记的分离研究

马尾松随机扩增多态性ＤＮＡ标记的分离研究魏令波，唐谦，郑先武，顾万春（中国林业科学研究院林业研究所北京１０００９１）李小兵（中国科学院遗传研究所植物生物技术开放实验室北京１０００１２）关键词马尾松，ＲＡＰＤ，Ｍｅｎｄｅｌｉａｎ分离随机扩增多态性ＤＮＡ...     

美洲黑杨(Populus deltoides Marsh)×青杨(P.cathayana Rehd.)分子连锁图谱的构建

利用随机扩增ＤＮＡ多态性ＲＡＰＤ方法，在美洲黑杨（Ｐ．ｄｅｌｔｏｉｄｅｓＭａｒｓｈ）×青杨（Ｐ．ｃａｔｈａｙａｎａＲｅｈｄ．）３代谱系中分析分子标记，构建出第１张美洲黑杨×青杨分子连锁图谱。共从３００个１０ｍｅｒ随机引物中筛选出７９个适合引物，检测出可供构图的分离标记１８０个。该图谱由２０个连锁群，１１０个ＲＡＰＤ标记组成。总图距为覆盖基因组总长度的７０３５％，标记间的平均间距为１７２７ｃＭ．连锁群长度在３７１～１８９８，相应标记分别在３～１０。本图谱为杨树抗病、虫和其它性状基因定位提供了框架结构，为实现杨树分子遗传育种迈进了最重要的一步。     

RAPD标记在杉木种源遗传变异上的应用

本实验首先建立了适合杉木ＲＡＰＤ分析的ＰＣＲ扩增实验体系。在此基础上从全国不同杉木种源区选择７个代表性的种源,然后从这７个种源中采样并提取ＤＮＡ,利用２３个不同随机引物对７个种源进行了ＤＮＡ序列多态性分析。实验结果表明杉木种源间遗传多态性水平较高,在被检测的１１４个ＲＡＰＤ位点中,多态位点占到７９８％;７个种源彼此间的遗传距离在０２２５～０４４０７范围内变动,平均为０３２２。根据种源彼此之间的遗传相似度构建了它们之间的聚类图,从聚类图上显示的杉木种源间亲缘关系可知,分布在南岭山脉西部周围的４个种源相对聚在一起,由此可推测南岭山脉中西部为杉木中心产区之一,并从此处向四周扩散。     

用RAPD分析麻核桃起源与分类地位

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）方法对麻核桃、核桃楸、核桃、奇异核桃和核桃×核桃楸的人工杂种进行了基因组多态性分析。选用２８个１０ｂｐ随机引物扩增出３３２个ＤＮＡ片断,片断大小在２５９ｂｐ～３０５４ｂｐ之间,其中２４３个ＤＮＡ片断呈现多态性,占总扩增片断的７３２％。依据扩增结果进行相似性系数和遗传距离分析,据此构建聚类树状图。研究结果表明：核桃楸×核桃的天然杂交是麻核桃形成的主要机制;在麻核桃形成过程中,核桃楸的遗传贡献率大于核桃;在胡桃属分类中,麻核桃应归为核桃楸组,这与传统分类学的结果一致。     

利用RAPD分析大青杨天然群体的遗传结构

本文利用ＲＡＰＤ分子标记技术从ＤＮＡ分子水平上探测了大青场（ｐｌｐｕｌｕｓｕｓｓｕｒｉｅｎｓｉｓＫｏｍ．）天然群体的遗传结构和分化程度。结果得出：用１４个随机寡核苷酸引物共产生１８０个扩增片段，扩增片断在２１１ｂｐ至１６３６ｂｐ之间。Ｓｈａｎｎｏｎ表型多样度（ＨＯ）估测值在群体间变动范围为０．２７１至０．３９２，平均为０．３１０。对分子水平变异分为群体间和群体内两部分进行分析，群体间分量占总变异的６２．３％，群体内只占３７．７％。不同引物在群体内探测能力也各不相同，ＣＨｌ－ｌ引物探测多样度（ＨＯ）最高（０．５４０），而２１１６引物最低（０．１５１）。     

筛选与杨树抗云斑天牛基因连锁的RAPD标记

筛选与杨树抗云斑天牛基因连锁的ＲＡＰＤ标记王京兆，王斌，卞学瑜，韩一凡（中国科学院遗传研究所北京１００１０１）（中国林业科学研究院林业研究所北京１０００９１）关键词杨树，云斑天牛，ＲＡＰＤ，多态性产物植物抗虫基因工程是当前比较活跃的研究领域，但真正能...     

大青杨及其近缘种的遗传变异和系统关系研究

对杨属青杨派主要树种大青杨、甜杨、香杨和马氏杨种间及种内遗传变异进行了ＲＡＰＤ检测，结果表明：７个随机引物对４个树种ＤＮＡ扩增产物绝大部分呈现为单型性，而有３个引物扩增产物显示出丰富的种间多型性；所有引物均在４个树种内扩增出不同程度的多态性。统计分析建立的系统树还说明了４树种可能的亲缘关系及系统发育史，同时，各树种内也均存在着遗传多样性。这对今后进行这些树种的遗传改良具有重要意义。此外还得出：分子     

中国3种松干锈菌在随机扩增DNA多态性水平上的遗传分化

用ＲＡＰＤ手段分析了我国３种松干锈菌Ｃｒｏｎａｒｔｉｕｍｒｉｂｉｃｏｌａ、Ｃ．ｆｌａｃｃｉｄｕｍ及Ｃ．ｑｕｅｒｃｕｕｍ在ＤＮＡ水平上的遗传分化。用５个随机引物从１０个菌株（含１个不同属参照菌株）中检测出５１个多态ＤＮＡ片段进行聚类分析,３种锈菌清晰地显示为不同类群,此结果为传统分类的成立提供了分子遗传学证据。３种锈菌中Ｃ．ｒｉｂｉｃｏｌａ和Ｃ．ｆｌａｃｉｄｕｍ的亲缘关系较近,这同由症状和冬孢子寄主反映出的相似关系一致。种内菌株间存在遗传差异,但其程度小于种间差异。锈孢子及冬孢子寄主都不同的Ｃ．ｆｌａｃｉｄｕｍ菌株间差异明显,可能反映着专化型分化的遗传学基础。不同松树寄主的Ｃ．ｑｕｅｒｃｕｕｍ菌株间也存在差异,暗示我国的Ｃ．ｑｕｅｒｃｕｕｍ同北美一样存在对松类的寄生专化性分化。研究结果还提示松干锈菌的遗传分化主要同寄主有关,而与地理分布关系不大。     

RAPD analysis of genetic relationships among Sphaeropsis sapinea isolates

Genetic relationships were studied among 23 isolates of Sphaeropsis sapinea collected from China, the United States, England, South Africa and Chile by using a random amplification of a polymorphic DNA (RAPD) analytical method. One hundred and 35 DNA fragments were amplified with 12 random primers by a polymerase chain reaction PCR technique and 96.3% were polymorphic. The genetic dendrogram based on RAPD analysis showed that the S. sapinea isolates could be divided into three types. Isolate CWS41 from Chile was separated genetically as the first type that was different from other isolates and isolates F2 and J2 from China comprised the second group. The third RAPD group accommodated other isolates including the B morphotype isolate CWS43 from the United States. __________ Translated from Journal of Nanjing Forestry University, 2006, 30(1): 13–16 [译自: 南京林业大学学报]     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

Isozyme and RAPD polymorphisms in Heterobasidion annosum in Italy

Isozyme and random amplified polymorphic DNA (RAPD) polymorphisms were used to study variability in a group of 41 isolates from the Italian population of Heterobasidion annosum. The isolates belonged to the intersterility groups P and S, and particularly to the group that is most widely distributed in Italy, group F. Isozyme analysis was effective in identifying the three intersterility groups and revealed a high degree of genetic divergence within the P group isolates; the mannose phosphate isomerase (MPI-2) locus was diagnostic in the attribution of isolates to the more correlated F and S groups. RAPDs were detected following amplification by the polymerase chain reaction (PCR). 74 RAPD fragments, obtained through amplifications with eight primers, were scored. Isolates from the 3 intersterility groups were clearly divergent based on analysis of RAPD markers. However, a similarity index calculated for the isolates within the F population indicated a high uniformity of the isolates collected throughout the Italian peninsula.     

撑篙竹遗传变异的RAPD分析

采用随机扩增多态DNA(RAPD)方法对6个群体30丛撑篙竹个体进行了遗传变异的研究。28个随机引物共检测到173个位点,其中85个是多态位点,平均每个引物提供6．18个RAPD信息量,扩增出的DNA片段大小一般在200～2000bp范围之间;用POPGENE1．31版软件进行遗传多样性分析：平均Nei’s基因多样性为0．2114,Shannon’s信息指数为0．3277,基因分化系数0．1853,表明群体间有一定的分化;各群体平均遗传距离0．0350,表明群体亲缘关系较近;试用UPGMA方法对不同产地的撑篙竹群体作聚类分析,初步可将6个群体聚为3类。     

黄心夜合的遗传多样性研究

运用随机扩增多态DNA（RAPD）技术,对黄心夜合[Michelia martinii（Levl.）Levl.]自然分布区的6个自然种群遗传多样性进行了研究。从100个随机引物中筛选出能产生清晰、稳定的多态性标记引物18个,共检测出110个位点,其中多态性位点为96个,占87.27%;在物种水平上,有效等位基因数目（Ne）,Nei’s基因多样性系数（He）和Shannon表型多样性指数（I）分别为1.735 2,0.416 3和0.597 1;总的遗传变异量（Ht）为0.339 8,其中居群内遗传变异量（Hs）为0.258 7,各居群间的遗传分化系数（Gst）达到0.323 1.应用UPGMA法对遗传距离进行聚类分析并构建树系图,结果表明：黄心夜合自然种群具有较高的遗传多样性,其种群间的遗传差异与其地理分布有关。     

白术不同居群亲缘关系RAPD分析

用随机引物扩增多态性DNA(RAPD)技术对白术的3个居群进行基因组DNA多态性分析,从100个随机引物中筛选出20个10bp随机引物,共扩增出187个DNA片段,片段大小在200bp～2800bp之间,其中多态性谱带为135条,表现出了丰富的RAPD多态性(占72.2%),根据遗传距离,利用UPGMA构建了居群亲缘关系柱状图。结果表明:於术86和於术76的亲缘较近,而於术86和白术天台之间的亲缘关系较远。就单个样本来看,於术86的15个样都聚在了一起,表明了於术86较强的同源性,於术76的14个样也聚到了一起,也表明了於术76的同源性。而於术76第14个样与白术天台聚到了一起,表明了於术76与白术天台有一定的同源关系。     

四种竹子的RAPD指纹图谱的初步研究

本实验以RAPD技术对考顺竹、凤尾竹、绿竹、白绿竹四个品种用20种已知序列的10nt引物进行PCR反应扩增。其中有17种可扩增出DNA条带,构成RAPD指纹图谱。各种引物PCR扩增的DNA条带数目在0—6条之间,大小在0．3—2．0kb之间。各种竹子的17种引物扩增的DNA条带总数在23—44条之间。四种竹子两两之间的相似系数在36—73％之间。     

Characterization of Cryphonectria parasitica strains by random amplified polymorphic DNA (RAPD) technique and conventional methods

In recent years damage to Austrian Castanea sativa populations caused by Cryphonectria parasitica has increased. A total of 34 isolates out of 13 observation plots of this phytopathogen in the south-east of Austria were examined for laccase activity, virulence, number of vegetative compatibility groups and for the existence of hypovirulence associated dsRNA. Furthermore, the applicability of the random amplified polymorphic DNA (RAPD) technique for investigating population structure was tested. Small dsRNA fragments were found in two Austrian strains. The same two strains exhibited reduced virulence. Seven vegetative compatibility groups were found in the areas examined. RAPD analysis proved to be an efficient method for distinguishing between different C. parasitica genotypes.     

应用RAPD分析快速鉴定外生菌根蘑菇分离物的真伪

采用DNA指纹比较技术对部分外生菌根蘑菇分离菌株的真伪进行了鉴定，结果表明：不同来源的点柄乳牛肝菌Suillus granulatus子实体之间的DNA相似系数为0.939。但各菌丝体分离菌株与其来源子实体的DNA相似系数为1.000，每个供试引物获得的RAPD指纹图谱全部对应相同，证实分离物为真正的牛肝菌分离菌株。试验还鉴定与子实体DNA具极高同源性的细裂硬皮马勃Sclerodema areolatum的组织分离菌丝体是真正的分离菌株。供试大红菇Russula rubra分离培养物与供试子实体之间存在很大的DNA异质性，因此判定该分离物为杂菌或其它生物体分离物，并非大红菇的分离菌株。试验结果显示：RAPD指纹技术是外生菌根蘑菇分离物真伪鉴定的一种快速可靠的方法。文章就RAPD鉴定共生真菌分离菌株的可靠性与技术范围进行了讨论。