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。     

玉米大斑病菌ISSR反应体系的优化和遗传多样性分析

以玉米大斑病菌基因组DNA为模板,采用单因素水平优化的方法对DNA聚合酶的来源及浓度、引物浓度、dNTPs浓度、DNA模板浓度、Tm(退火温度)、PCR反应循环数等重要参数进行摸索和优化,建立了玉米大斑病菌ISSR-PCR优化反应体系,并从40条ISSR引物中筛选出9条多态性较好的ISSR引物。对来自河北、河南、辽宁等玉米主产区的44个菌株进行ISSR分析表明,ISSR标记在我国玉米大斑病菌中存在较高的多态性,多态性条带占40.3%。聚类分析显示,在阈值为0.8时菌株被分为7个类群。对ISSR揭示的玉米大斑病菌的遗传多样性与菌株交配型、地理来源之间的关系进行分析,结果显示菌株的遗传多样性与交配型间的关系密切,而与其地理来源无明显相关性。     

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。     

应用UP-PCR进行玉米丝黑穗病菌遗传多样性研究

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250～2 000 bp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。     

福建省丙环唑不同敏感性玉米小斑病菌的遗传多样性和致病性

 利用菌丝生长法、ISSR分子标记和分生孢子悬浮液喷雾接种法,探讨了福建省玉米小斑病菌对丙环唑的敏感性以及不同敏感性病菌群体的遗传多样性和致病性。敏感性测定结果表明,福建省玉米小斑病菌对丙环唑产生了抗药性,抗药性菌株的抗性倍数达到2.1~9.4倍。筛选获得的10条ISSR引物对55个菌株共检测出153个位点,其中多态性位点百分比高达93.46%。在敏感型、中间型和抗药型群体中多态性位点百分比分别为77.12%、69.93%和81.70%,在抗药型群体中,等位基因观测值、等位基因有效值、Nei’s遗传多样性指数和Shannon’s信息指数均高于敏感型群体,表明玉米小斑病菌抗药型群体的遗传多样性最丰富。聚类分析结果表明,病菌群体遗传多样性与抗药性水平和地理来源均有较高的相关性。致病性测定表明,丙环唑不同敏感性病菌群体对11个鲜食玉米品种均具有较强的致病性,但是,在9个玉米品种上,敏感型群体中强致病力菌株出现频率明显低于抗药型群体。研究结果为深入研究玉米小斑病菌群体遗传结构及其田间抗药性监测提供了理论基础。     

陕西省苹果树腐烂病菌基因多态性的ISSR分析

为了从分子水平上揭示苹果树腐烂病菌的群体遗传多样性,采用正交设计对ISSR-PCR体系进行了4因素3水平的筛选,并从47条ISSR引物中筛选出11条多态性较好的引物。对供试的87个分离株进行扩增的结果显示,11条引物在129个位点扩增出稳定的条带,其中多态性位点119个,多态性位点率为92.25%。POPGENE分析显示,病菌种群的遗传多样性和基因多态性丰富,群体间的遗传分化系数(Gst)为0.109,群体内为0.891,群体内多样性大于群体间多样性。两个地理种群间的居群每代迁移数(Nm)为2.046,两者之间存在广泛的基因交流。在遗传相似系数为0.88时,可将21个自然种群划分为9个不同的类群,表明陕西省苹果树腐烂病菌的各个自然种群之间的遗传亲缘关系与其地理来源之间无明显的相关性。     

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%。     

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。     

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。     

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。     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

Population genetic structure of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1IA from rice field in North India

Rhizoctonia solani AG1IA is an important fungal pathogen causing significant yield and quality losses in rice production. However, little is known about the levels of genetic diversity and structure of this pathogen in North India. Out of 240 samples collected from different rice-growing regions of North India, 112 isolates were identified as R. solani AG1IA subgroups using species-specific primers. All 112 isolates were organized into four groups on the basis of percent disease index (PDI). The majority of the isolates were weakly virulent. Population genetic analysis was performed within and between populations using inter simple sequence repeat (ISSR) markers. A total of 8249 alleles were identified from the 112 isolates of R. solani AG1IA through analysis of the ten inter simple sequence repeat markers. All the ten ISSR markers were polymorphic. The average number of bands per primer was 7.3 which ranged in size from 250 to 1500 bp. Genetic structure of the isolates using inter simple sequence repeat primers showed high degree of polymorphism (PIC ≥0.81). The analysis of molecular variance (AMOVA) indicated that most of the genetic diversity occurred within populations (60%), while the variability among populations and among regions contributed 25 and 15%, respectively. Overall, the present study reveals that a large variation exists among rice-infecting isolates of R. solani AG1IA in North India. Fingerprinting of the isolates using ISSRs along with phenotypic characterization and virulence analysis will help epidemiological studies that can provide new insights into pathogen biology and disease spread.     

甘肃省大麦条纹病病原菌致病力分化、rDNA-ITS及遗传多样性分析

为明确甘肃省大麦条纹病病原菌麦类核腔菌Pyrenophora garminea的致病力分化、r DNA-ITS序列特征及变异,采用"三明治"法测定甘肃省大麦条纹菌的致病力,通过r DNA-ITS序列分析菌株间变异类型,并对菌株进行ISSR遗传多样性分析。结果表明,共筛选到43株大麦条纹病菌,生长7 d后的菌落直径为2.60~7.93 cm,其中菌株TB生长速度最快,菌株JT生长速度最慢,强、中等和弱致病力菌株分别为2、21和20株;43株菌的核糖体DNA-ITS序列与麦类核腔菌菌株SMCD2015同源性在99%以上;在9个菌株中检测到15个变异位点,共17种变异类型;8个ISSR标记从43个菌株中扩增出41条带,平均每个标记5.13条带,90.24%片段具有多态性,遗传相似系数为0.44~1.00,平均值为0.71,当遗传相似系数为0.68时,可将供试菌株划分为4个类群。表明甘肃省大麦条纹病病原菌存在致病力分化,菌株核糖体DNA-ITS序列变异丰富,且菌株间遗传结构复杂。     

玉米圆斑病病原的快速检测

玉米生平脐蠕孢菌(Bipolaris zeicola)是引起玉米圆斑病的病原菌。本研究通过对玉米生平脐蠕孢菌及其近似种的EF-1α基因(elongation factor 1α)部分序列进行比对,设计出玉米生平脐蠕孢菌的特异性引物Y-EF-F和Y-EF-R,利用该引物可以从B.zeicola中扩增出137 bp的特异片段,而其余的17个参试菌株扩增结果为阴性。灵敏度实验表明该对引物可以检测到目标DNA的浓度为1 pg·μL~(-1)。用B.zeicola接种玉米叶片、苞叶以及玉米粒,然后以接种发病的病组织DNA为模板,利用引物Y-EF-F和Y-EF-R进行PCR扩增,可以扩增出137 bp的特异性条带,而健康玉米组织DNA中未能扩增出任何条带。用B.zeicola孢子悬浮液接种大田玉米叶片,接种第3 d可以检测到未发病组织中有B.zeicola病原菌,第5 d可以看到明显的病斑。研究结果表明该方法可用于快速、准确和灵敏地检测玉米组织中的潜伏期玉米生平脐蠕孢菌,为玉米圆斑病的快速检测,进而及早采取防治措施提供积极的指导。     

Characterization of a Fusarium poae world-wide collection by using molecular markers

Fusarium poae has been considered as a minor species among those that cause Fusarium Head Blight (FHB) disease but in recent years several researchers have documented a high frequency of occurrence of this species. In this study, a total of 173 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated by using inter simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) to evaluate genetic variability within F. poae and to amplify MAT idiomorphs as a possible mechanism that could explain part of the variability found in this species. The molecular analysis obtained from both molecular markers showed a high intraspecific variability. However, a partial clustering between F. poae isolates and their geographic origin was obtained by ISSR markers while AFLP showed isolates from different geographic locations distributed throughout the dendrogram. Moreover, ISSR grouped all the F. poae isolates into a different cluster from the F. langsethiae and F. sporotrichioides isolates used as outgroups compared with the dendrogram obtained using AFLP markers. Analysis of molecular variance (AMOVA) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between, American and European populations by using both molecular markers. Regarding MAT idiomorphs, for most F. poae isolates both MAT-1 and MAT-2 were present from each isolate.     

Genetic diversity and structure of the Fusarium oxysporum f.sp. lini populations on linseed (Linum usitatissimum) in China

The genetic diversity of specific Fusarium oxysporum f.sp. lini from six provinces in China was investigated using molecular markers, inter-simple sequence repeats (ISSR). Based on the morphological features and the internal transcribed spacer (ITS) sequences, 96 isolates were identified as Fusarium oxysporum. The 96 isolates were amplified by PCR with 12 ISSR primers. The number of bands amplified by each primer ranged from 43 to 142, with sizes ranging from 250 to 4,500 bp. A total of 800 bands were observed, out of which 797 were polymorphic (99.62%). The percentage of polymorphic loci varied from 17.25% in Gansu and Inner Mongolia to 33.75% in Sinkiang. Nei’s gene diversity index (h) ranged from 0.0428 in Gansu to 0.0666 in Sinkiang, and Shannon’s information index (I) ranged from 0.0675 in Gansu to 0.1117 in Sinkiang. The genetic identity using the Nei’s genetic identity varied from 0.9643 between the populations from Hebei and Gansu to 0.9844 between the populations from Sinkiang and Shanxi. Unweighted pair group mean analysis (UPGMA) cluster analysis, as indicated by the Nei’s genetic distance, showed the distances ranging from 0.0158 between the populations from Sinkiang and Shanxi to 0.0364 between the populations from Hebei and Gansu. The six populations were clustered into three subgroups. The Gansu population was clustered into one subgroup, the same as the Inner Mongolia population. The four other populations were clustered into the third subgroup. The Nei’s GST (0.2972) and gene flow among populations (Nm =1.1825) revealed large gene exchanges among populations.     

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.     

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.