应用UP-PCR进行玉米丝黑穗病菌遗传多样陛研究

本研究探讨了UP-PCR技术在玉米丝黑穗病菌遗传多样性分析中的利用可行性。在13个通用引物中，筛选出9个扩增多态性好且稳定的通用引物，共扩增出113条DNA条带，大小分布于250~2000bp之间，其中多态性条带为95条，为总条带数的84．07％。遗传距离为0．76处时，所有丝黑穗病菌菌株被聚为6个组。利用UP-PCR技术，可以充分展现玉米丝黑穗病菌菌株间的亲缘关系及差异性，可用于玉米丝黑穗病菌遗传多样性研究，为有效地开展玉米丝黑穗病菌的遗传进化甚至探讨病菌致病性的生理分化提供了一个新的技术方法。     

利用UP-PCR、ISSR和AFLP标记分析玉米丝黑穗病菌遗传多样性

利用UP-PCR、ISSR和AFLP分子标记方法研究了我国主要玉米产区34株玉米丝黑穗病菌的遗传多样性。从供试引物中筛选获得具多态性的UP-PCR引物9个、ISSR引物11个和AFLP引物组合22对,分别扩增出113、72和293条谱带,多态性条带比率分别为91.15%、84.7%和83.27%。聚类分析表明,玉米丝黑穗病菌存在丰富的遗传变异,与地理来源无明显相关性。3种分子标记的遗传相似系数矩阵相关性分析表明,UP-PCR与AFLP具有较高的相关性,相关系数为0.698;UP-PCR与ISSR、ISSR与AFLP的相关系数分别为0.659和0.633。从多态性水平、稳定性和可操作性可以看出,UP-PCR技术更适于分析玉米丝黑穗病菌遗传多样性。此外,UP-PCR、ISSR和AFLP标记划分的类群与鉴别寄主划分的致病类型之间存在一定的相关性,吻合率分别为50.0%、60.0%和47.6%。     

我国玉米灰斑病菌遗传多样性的ISSR分析

为明确我国发生的玉米灰斑病菌地理差异及遗传结构,利用简单序列重复区间(ISSR)对玉米灰斑病菌遗传多样性进行了分析,并利用尾孢菌特异引物对分离自四川、云南、湖北、贵州等西南地区的16个玉米灰斑病菌菌株进行了分子鉴定。结果显示,通过ISSR标记筛选出10个扩增多态性好且稳定的通用引物,共扩增出81条DNA条带,均为多态性条带,扩增片段大小在200~2 000 bp之间,菌株遗传相似系数为0.19~1.00。在遗传相似系数为0.19时,供试菌株被聚为2大类群,来自西南地区和东北地区的菌株各自聚为一组,在DNA水平上表现出明显差异,认为是2类不同的致病类群。分子鉴定结果显示引起西南各地区玉米灰斑病的主要致病菌均为玉米尾孢菌Cercospora zeina。表明我国玉米灰斑病菌存在丰富的遗传多样性,ISSR标记可揭示出玉米灰斑病菌株间的亲缘关系及遗传差异性,可用于其遗传多样性研究。     

玉米圆斑病菌(Bipolaris zeicola)遗传多样性ISSR分析

利用简单序列重复间隔区(inter-simple sequence repeats,ISSR)标记对玉米圆斑病菌(Bipolaris zeicola)的遗传多样性进行了分析。筛选出9个扩增多态性好且稳定的通用引物,共扩增出47条DNA条带,大小分布于250~2 000bp之间,其中多态性条带为33条,为总条带数的70.21%。遗传距离为0.91处时,所有菌株被聚为6个组。ISSR标记可以揭示菌株间的亲缘关系及差异性,可用于玉米圆斑病菌遗传多样性研究。此外,通过分子标记划分的类群与利用寄主反应型之间存在一定相关性,但其关系并不密切。     

玉米根际球孢白僵菌群体遗传多样性的ISSR分析

为了明确玉米根际球孢白僵菌的遗传分化情况及亲缘关系,通过ISSR-PCR分子标记技术对分离自玉米根际的球孢白僵菌的遗传多样性进行了研究。从40个引物中共筛选出11个多态性高、稳定性好的引物用于正式的扩增分析,在37个菌株中共扩增出83条谱带,其中多态性条带占69条,多态性百分率为83.13%。平均每引物扩增条带在7.5条。群体的多态位点百分率(PPL)为83.13%,Nei基因多样性指数(H)为0.316 9,Shannon信息指数(I)为0.465 7。结果表明,分离自安徽省涡阳、萧县、蒙城三个地区的球孢白僵菌具有较高的遗传多样性。研究结果对进一步探讨玉米根际球孢白僵菌不同菌株的生防效果具有重要意义。     

棉花黄萎病菌ISSR反应体系优化及其遗传多样性分析

为给棉花黄萎病菌分子变异及遗传多样性研究提供可靠的检测方法,以棉花黄萎病菌基因组DNA为模板,采用正交优化方法对PCR体系中DNA聚合酶、引物、dNTPs、DNA模板、Mg2+及10×Buffer等重要参数进行6因素4水平优化,建立了棉花黄萎病菌ISSR-PCR优化反应体系,并从20条ISSR通用引物中筛选出多态性较好的10条引物。采用该优化反应体系和10条ISSR引物对采自陕西棉花主产区的21个棉花黄萎病菌菌株和3个参照菌株进行ISSR分析。结果显示,10条ISSR引物共扩增出87条谱带,条带分子量均在250~2 000 bp之间,平均每条引物扩增出8.7个条带,其中58条为多态性条带,占65.2%。聚类分析结果显示,在相似系数0.59处,供试菌株分为2个遗传类型。表明棉花黄萎病菌菌株间的亲缘关系与地理来源存在一定的相关性,而与其病害症状类型无相关性。     

产纳他霉素生防菌A01和A02与已知产生菌的RAPD比较

为了明确自主分离的天然抗真菌活性产物——纳他霉素产生菌A01和A02与已知纳他霉素产生菌的遗传相似性,采用RAPD技术对这2株菌和3个产纳他霉素的标准菌株进行了比较分析。利用筛选出的7个随机引物对5个菌株的PCR扩增共得到154条清晰稳定的DNA条带,其中同源性条带86条,多态性条带68条,分别占总条带数的55.8%和44.2%,平均每个引物扩增出9.7个多态性条带,得到了丰富的DNA指纹图谱。所得数据经NTSYS pc 2.10e软件聚类分析,表明5个菌株间的遗传距离为0.0991~0.4738,其中A02与其它菌株间的遗传距离均较远。结合前期的分类研究结果,确证了菌株A02为新的纳他霉素产生菌。     

西瓜枯萎病菌遗传多样性的相关序列扩增多态性分析

对30个西瓜枯萎病菌Fusarium oxysporum f.sp.niveum菌株基因组DNA进行相关序列扩增多态性(SRAP)分子标记分析,以探究其遗传多样性与地理来源的关系。采用尖孢镰刀菌西瓜专化型Fusarium oxysporumf.sp.niveum0、1、2号生理小种的基因组DNA为模板,对225对SRAP引物进行筛选,筛选出20对多态性、重复性较好且条带清晰的引物,对30个菌株进行PCR扩增,共扩增出386条带,其中多态性条带有371条,多态性比率为96.11%,平均每对引物扩增出19.3个位点和18.55个多态性位点。UPGMA法聚类分析结果显示,供试菌株两两之间的遗传相似系数范围为0.69~0.90,平均为0.79,说明尖孢镰刀菌西瓜专化型的遗传多样性较为丰富。基于SRAP标记聚类分析表明,30个菌株在遗传相似系数为0.70处被划分为3个类群,I类群包含24个菌株,其中18个来自湖南省,Ⅱ类群只包含1个来自黑龙江省哈尔滨市的菌株,它和另一个来自黑龙江地区的菌株被划分到不同的类群,且遗传距离相对较远;Ⅲ类群包含了5个菌株,其中3个来自海南三亚,其余两个来自湖南省。根据菌株的分布情况来看,菌株的聚集与地理来源没有明显的相关性。     

尖镰孢菌EST-SSR遗传多样性分析及通用性评价

为了解38株尖镰孢菌的遗传多样性并开发可在近缘镰孢菌种中通用的尖镰孢菌EST-SSR标记,利用设计和筛选的18对多态性EST-SSR引物对38株尖镰孢菌和5种近缘镰孢菌进行SSRPCR扩增,经6%非变性聚丙烯酰胺凝胶分离扩增产物,并用NTSYS软件分析供试尖镰孢菌的PCR扩增结果。结果表明,18对EST-SSR标记引物在38株尖镰孢菌中检测到75条多态性条带,多态性比率达92.6%,平均每对引物可扩增4.2条;各菌株间的遗传相似系数介于0.565~0.946之间,平均为0.721;来源于同科寄主植物群体的菌株间的平均遗传相似系数以葫芦科最大,锦葵科最小,依次为葫芦科兰科豆科亚麻科茄科锦葵科。在相似系数为0.756时,供试38株菌有35株按照不同科寄主植物聚为不同的类群,说明尖镰孢菌SSR类群的划分与其寄主来源具有一定的相关性。18对EST-SSR引物在近缘镰孢菌种中均能有效扩增的引物数及通用性比率为10对和55.6%,均显示多态性的引物有2对,占供试引物总数的11.1%。表明尖镰孢菌ESTSSR区域遗传多样性丰富,基于尖镰孢菌EST序列开发镰孢菌通用SSR标记是可行的。     

黑龙江省水稻纹枯病菌的致病力分化与AFLP分析

为了明确黑龙江省水稻纹枯病菌遗传多样性，为水稻抗病育种和水稻纹枯病的综合防治提供依据。本文对采自13个水稻种植地区的29个水稻纹枯病菌菌株进行了致病力测定和AFLP分析。结果表明9对AFLP引物对供试菌株扩增出396条带，其中多态性带187条，占总扩增带数的47.22%。黑龙江省水稻纹枯病菌的遗传距离变化在0.50~0.92之间，平均为0.71，群体遗传多样性较为丰富。UPGMA法可以将供试菌株分成4个AFLP聚类组群(Ⅰ、Ⅱ、Ⅲ和Ⅳ)，相同地理来源的菌株基本上聚集在同一组群内，表明AFLP类群划分与菌株的地理来源有较强的相关性。黑龙江省水稻纹枯病菌致病性分化较为明显，并且AFLP类群划分与菌株的致病性鉴定之间存在一定相关性。     

蔬菜保护地木霉菌rDNA-ITS序列和UP-PCR遗传多样性分析

采用传统形态学分类和ITS序列比对的方法,研究蔬菜保护地土壤中木霉菌种群分布和遗传多样性。木霉菌分离培养结果显示,共获得397株木霉菌,鉴定出11个种,分别为:长枝木霉Trichoderma longibrachiatum、深绿木霉T.atroviride、哈茨木霉T.harzianum、粘绿木霉T.viren、微孢木霉T.minutisporum、拟康木霉T.pseudokoningii、黄绿木霉T.aureoviride、非钩木霉T.inhamatum、棘孢木霉T.asperellum、长孢木霉T.longipile和螺旋木霉T.helicum。经ITS序列建立系统发育树后,将木霉菌分为5个组。用5条通用引物经UP-PCR扩增后,扩增出46条谱带,其中多态性条带43条,占总条带数的93.5%。遗传多样性分析表明,当相似系数为0.80时,可将24个菌株划分为9个组。UP-PCR与ITS序列相比,更能体现木霉菌种间和种内的亲缘关系及遗传差异性,可以作为木霉菌分类的辅助方法。     

Genetic and virulence diversity, and mating type distribution of Togninia minima causing grapevine trunk diseases in Spain

Fifty eight single-spore Togninia minima (anamorph Phaeoacremonium aleophilum) isolates were recovered from grape rootstock wood of plants that showed symptoms of Petri disease and esca from 2001 to 2008 in Spain. These isolates were studied by means of mating type distribution, UP-PCR analysis, and virulence assays. Analysis of clone-corrected data sets showed equal frequencies of both mating types in the entire Spanish population, in the Ciudad Real region, at inter-vineyard and intra-vine spatial scales; while unequal mating type distribution was detected in Valencia and Zaragoza regions, at intra-vineyard and intra-vine spatial scales. This is the first study on distribution of T. minima mating types on spatial scales varying from vineyards to regions. A total of 49 polymorphic UP-PCR markers were obtained using seven UP-PCR primers. Four optimal clusters were inferred with Bayesian structure and multivariate analyses from the UP-PCR data. The high number of unique genotypes observed within the Spanish population, combined with a near-equal distribution of mating types, suggested that sexual reproduction probably does occur. However, based on allele distribution and frequency, each of the three subpopulations appeared to be evolving independently. Gene and genotype diversities across the subpopulations were similar and ranged from 0.24 to 0.27 and from 0.27 to 0.37, respectively. The detection of genetically identical isolates within and among subpopulations indicates that an asexual reproductive component should not be excluded. Contrast analysis among groups defined by UP-PCR analyses showed no significant differences in the virulence of T. minima isolates.     

烟草疫霉SSR分子标记的开发与初步应用

为开发烟草疫霉的SSR分子标记,利用MISA软件搜索烟草疫霉基因组序列中的SSR位点,共发现1311个SSR位点,优势SSR位点为二核苷酸和三核苷酸,分别占总SSR位点的56.45%和39.36%;根据分析到的SSR位点使用Primer 5.0软件设计48对SSR引物,以7株烟草疫霉的DNA为模板对这些引物进行筛选,共获得扩增条带清晰且具有多态性的SSR引物20对,然后使用其中的6对SSR对32株烟草疫霉进行UPGMA聚类分析,遗传相似系数在0.60~1.00之间,在相似系数0.70水平上,可将其划分为3个类群,类群Ⅰ包含29个菌株,类群Ⅱ包含1个菌株,类群Ⅲ包含2个菌株,显示出较低的遗传分化水平。这些SSR引物的开发将为研究我国烟草疫霉的遗传多样性、群体遗传结构以及遗传图谱构建等奠定基础。     

中国部分地区稻曲病菌培养特性及其遗传多样性分析

采用生物学方法和RAPD-PCR技术,对来自11个省(市)84个菌株的菌丝生长速率、分生孢子数量、孢子萌发率及其遗传多样性进行分析,以明确中国部分地区稻曲病菌株的培养特性和遗传多样性。依据菌丝生长速率,菌株可被划分为快和慢2种类型,分别占58.33%和41.67%。依据产孢力和分生孢子萌发力,菌株可划分为强、中和弱3种类型。采用12条RAPD引物共扩增出323条带,多态性条带比率为98.14%,遗传距离变化范围为0.02～1.00。在遗传距离0.725水平上,所有菌株被划分成7个遗传聚类组,聚类组R4和R5为优势聚类组,并存在一些亚组。不同地区之间和同一地区内的菌株表现出不同程度的变异,内陆地区的菌株群体变异程度明显高于沿海地区。从采用相同接种体接种的水稻品种上分离的稻曲病菌具有紧密的亲缘关系。     

水稻上三种条斑病细菌DNA的多态性初析

 试用40个引物对我国水稻条斑病菌、"稻短条斑病菌"和李氏禾条斑病菌等14个代表菌株进行RAPD分析,其中11个引物的扩增产物表现明显的多态性,共扩增出158条谱带,多态性为89.74%。聚类分析结果显示14个菌株可区分为3个类群,第1群包括LLS2、LLS3、LLS4、RS05、R1008、TAS和TAX;来自不同稻区水稻条斑病菌的群体结构差异明显,分属于第2群(如RS-Hai等)和第3群(如RS105等)。菌株DNA-RAPD指纹分析和致病性测定结果证明:李氏禾条斑病菌、"稻短条斑病菌"在水稻和李氏禾上不仅可相互侵染,而且遗传背景的相似性较高,确认是同一种病菌,其与小麦黑颖病菌亲缘关系较近,但与水稻细菌性条斑病菌具有明显差异。     

山东小麦纹枯病菌致病性与遗传分化关系的研究

 对山东麦区被鉴定为双核丝核菌的35个菌株进行致病性测定的基础上,选用15个随机引物对上述菌株进行了RAPD分析,共标记出171条DNA片段,其中多态性片段161个,多态率为94.15%。用UPGMA法构建系统树,以遗传距离0.33为阈值,被鉴定为AG-D融合群的33个菌株隶属于同一个RAPD组,而3个未定融合群菌株(WK-6、WK-37和WK-13)均为独立的RAPD组。以遗传距离0.25为阈值,将属于同一个融合群的33个AG-D群菌株划分为7个亚组,说明受试小麦纹枯病菌株间存在丰富的遗传多样性。综合分析受试菌株的致病力测定结果与RAPD分析发现,供试菌株的RAPD组与菌株致病力的强弱无明显的相关性,但少数菌株的致病力强弱与亚RAPD组有一定的相关性。     

Molecular variability among isolates of <Emphasis Type="Italic">Mycosphaerella graminicola</Emphasis>, the causal agent of septoria tritici blotch,in Argentina

The genetic structure and diversity of Mycosphaerella graminicola population were studied with ISSR molecular markers, using isolates from several locations of the Argentinean wheat region: subregion IV (SE of Buenos Aires Province) and II South (central part of Buenos Aires Province). Samples were taken from different bread wheat (Triticum aestivum) cultivars. A total of 126 isolates were subjected to molecular analysis to compare the genetic structure of the isolates from both wheat subregions. Ten ISSR primers were used: (GACA)₄; (AAC)₇; (ATC)₇; (AC)₉; (AAG)₇; (AG)₉; (AGC)₅; (CAG)₅, (GTG)₅ and (GACAC)₃. Eighty-four bands ranging from 200 bp to 8.000 were amplified. Eighty-one distinct haplotypes were identified and 43 isolates did not generate any amplification products. The highest number of polymorphic DNA fragments were produced using ISSR primers (ATC)₇ and (GTG)₅, which detected bands in 38 isolates. The molecular analysis revealed the existence of 81 different haplotypes among the 126 isolates studied. These results revealed a high degree of genetic diversity in the M. graminicola population in Argentina.     

中国北方番茄细菌性溃疡病菌的rep-PCR基因指纹图谱分析

对来自全国若干省市的番茄细菌性溃疡病菌(Clavibacter michiganensis subsp.michiganensis)的33个菌株进行rep-PCR分析。结果表明,用引物BOX分别扩增出6～15条多态性条带,条带大多数集中在500～2800bp之间;用引物ERIC扩增的条带不清晰,可能是反应条件不适合,也可能是其不适合对番茄细菌性溃疡病菌进行多态性分析;BOX-PCR表明番茄细菌性溃疡病菌菌株具有丰富的遗传多态性和较大的遗传变异,对产生的指纹图谱进行分析:在遗传距离为0.18时,测试的33个菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ等7个遗传相似组群,其中Ⅵ组群包含的菌株最多。研究还表明番茄细菌性溃疡病菌的遗传分群与菌株的地理来源关系密切,但与该病的发病年份上没有必然联系,这也从另一侧面说明了该病害是土壤带菌引起的。     

Molecular diversity of binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-A in China

AG-A belongs to the binucleate Rhizoctonia (BNR) anastomosis group (AG) of the Ceratobasidium teleomorph, which parasitizes the roots of many plant species. Ninety nine isolate species of AG-A were obtained from Tibet, Sichuan, and Yunnan Province in China. All isolates were divided into three types based on their cultural characteristics. Type I: abundant aerial mycelia, dense hyphae, loose sclerotia; Type II: abundant aerial mycelia, no sclerotia. Type III: sparse aerial mycelium and no sclerotia. All of the isolates infected the seedlings of Chinese mustard and Chinese cabbage, causing the formation of lesions on the stem and a brown discoloration of the roots. Sequence analysis of the 5.8S rDNA-ITS showed a similarity of 98–100% among the isolates. Inter Simple Sequence Repeat (ISSR) was used to detect genetic variation in binucleate Rhizoctonia spp. Forty two AG-A isolates were amplified using 15 random primers. From a total of 164 bands, 144 bands (87.8%) were polymorphic in the 42 tested isolates. A dendrogram showing genetic relationships between the isolates was constructed using unweighted pair-group averages based on genetic distances. According to the dendrogram, the 42 tested isolates could be aligned into three clusters with a genetic similarity coefficient of 0.29, the first clusters including 27 isolates with III of culture characteristics on PDA; the second clusters included eight isolates with I of cultural characteristics on PDA; the third cluster included seven isolates with II of cultural characteristics on PDA. The results of ISSR analysis showed an association between the hosts of these isolates. Our results showed that ISSR analysis can reveal more molecular variation among isolates of AG-A than sequence analysis using the 5.8S rDNA-ITS.