基于SSR序列的江西省不同生态地区稻瘟病菌遗传多样性分析

为明确江西省稻瘟病菌Magnaporthe oryzae群体遗传结构及其多样性水平,选用13对SSR引物对分离自5个不同生态地理县(市)水稻穗颈瘟标样的稻瘟病菌单孢菌株的全基因组进行PCR扩增,利用最长距离法和POPGENE 32生物学软件对其进行聚类分析和群体遗传多样性分析。结果显示,共分离获得189株稻瘟病菌菌株,13对SSR引物对其均能扩增出1条大小相同且清晰的条带,多态性位点百分率高达100.00%。供试189株稻瘟病菌菌株在相似系数为0.74时可划分为15个遗传宗谱,其中宗谱JXL01包含71株菌株,占总菌株数的37.57%,为优势宗谱;宗谱JXL02、JXL14为亚优势宗谱,分别包含31、26株菌株,占总菌株数的16.40%和13.76%;宗谱JXL03、JXL08、JXL10为次要宗谱,包含10～17株菌株;其它9个宗谱为小宗谱,包含菌株都在5株以下。在群体水平上,来源于不同生态型地区的5个稻瘟病菌群体的Nei’s基因多样性指数为0.375,Shannon信息指数为0.558,具有丰富的遗传多样性,且群体间差异较大;这5个种群基于非加权配对平均法大多聚为一类,种群遗传谱系与地理区域分布呈一定相关性,群体遗传多样性均值为0.373,存在一定的遗传分化,且群体内多样性大于群体间多样性,总遗传变异的64.56%存在于群体内。表明江西省稻瘟病菌群体结构既包含明显的优势宗谱,又存在复杂多变的特异性小宗谱,遗传多样性丰富,且与地理分布有一定的相关性。     

基于毒性相关基因序列的稻瘟病菌群体遗传多样性分析

 选用16对毒性相关基因特异性引物对四川和重庆9个县(市)分离到的200个稻瘟病菌单孢菌株进行PCR扩增,并采用最长距离法进行聚类分析,结果显示各引物均能扩增出其目的条带,多态位点百分率(P)高达93.75%,扩增频率差异较大;200个菌株可归为70个不同的单元型,其中单元型SCH13为优势单元型;在0.86遗传相似水平上,200个菌株可划分为27个遗传宗谱,包括1个优势宗谱,3个亚优势宗谱,14个次要宗谱,9个小宗谱,层次丰富;在群体平均水平上,病菌群体具有丰富的遗传多样性(H=0.324 4,I=0.484 2),且群体间差异较大;9个种群在遗传距离为0.05水平上可分为4个类群,种群遗传谱系与地理区域分布呈一定相关性。同时,该地区的群体存在一定的遗传分化(HT=0.320 0),群体内多样性大于群体间多样性(Hs=0.179 6,Dst=0.140 4),总遗传变异的56.13%存在于群体内(Gst=0.438 7),群体间基因流动性较小(Nm=0.639 6)。本研究揭示了四川和重庆部分区域稻瘟病菌群体遗传结构、遗传多样性及其与地理分布之间的关系,为抗病育种和品种布局奠定了基础。     

四川籼稻区稻瘟病菌群体遗传结构

应用rep-PCR分子指纹技术对2000～2002年采自四川6个籼稻自然生态区的137个稻瘟病菌菌株进行了DNA分子指纹扩增和聚类分析,共获得73个不同的DNA指纹图谱(单元型)和62条分子量不等的DNA带型.结果显示,无论以何种遗传相似水平划分,四川稻瘟病菌的群体结构都表现很突出的优势宗谱,又存在着具有较多遗传多样性的次要小宗谱和特异性宗谱,蕴含着极其丰富的遗传信息;在0.19遗传相似水平,所有供试菌株可以划分成37个遗传宗谱,层次较为丰富.四川稻瘟病菌群体结构具有明显的时空特点,不同年度间稻瘟病菌群体存在一定的亲缘关系,又各自拥有当年的特异性宗谱;在空间上,不同稻作区表现出从复杂到简单的病菌群体变化特点.稻瘟病菌的遗传宗谱与生理小种致病型不存在一一对应的关系,作者认为将二者横向比较没有可比性.     

江西省稻区稻瘟病菌遗传宗谱与致病型的关系

为了探寻稻瘟病菌无性世代DNA水平的变异,明确江西省稻区稻瘟病菌遗传宗谱与致病型之间的对应关系,利用rep-PCR(repetitive element-based PCR)分子指纹分析技术,对稻区稻瘟病菌的群体结构和遗传多样性进行分析,并用41株代表性菌株对35个水稻品种进行了致病性测定。结果表明,以相似度75%为界,可以将不同稻区采集的99个菌株划分为14个遗传宗谱,其中,宗谱4、1和10为优势宗谱,分别包含37、18和12个菌株,占总数的37.37%、18.18%和12.12%;稻瘟病菌遗传宗谱与致病型间存在复杂的关系,同一宗谱的菌株对应多个致病型,而同一致病型的菌株,分属于不同的遗传宗谱,两者之间不存在简单的对应关系。     

云南元阳哈尼梯田稻瘟病菌遗传多样性分析

 利用12对SSR引物,对来自云南元阳哈尼梯田不同海拔高度、不同水稻品种上的稻瘟病菌菌株进行遗传多样性分析,并运用日本清泽建立的鉴别品种对其中62个菌株进行生理小种鉴定。结果显示:稻瘟病菌总体遗传多样性水平较高(He=0.57±0.27,I=1.17±0.60)。聚类分析表明,在80%相似水平下菌株可划分为9个多样性群体。供试菌株划分为16个生理小种,其中002、000和006为优势小种。分离的不同海拔稻瘟病菌生理小种和遗传多样性存在差异。海拔1600~1700m生理小种最丰富,达9个生理小种;海拔1500~1600m、1400~1500m和1700~1800m,生理小种分别为6、5和4个;遗传多样性分析表明,从海拔1400~1500m分离的菌株遗传多样性水平最高(He=0.57±0.24),并划分为6个群体。综合分析表明,在元阳哈尼梯田系统中,栽培传统品种遗传多样性越丰富的区域,其稻瘟病菌群体多样性和生理小种也越丰富。     

安徽省水稻条斑病菌群体遗传结构分析

 水稻条斑病菌是近年来影响安徽水稻生产的主要有害生物。本研究利用rep-PCR指纹技术分析了来自安徽11个不同县市的水稻条斑病菌群体遗传结构。用引物BOX、REP和ERIC分别对94个菌株的基因组DNA进行了PCR扩增,结果表明3组引物共扩增出了49条指纹条带,且所扩增出的DNA条带均为多态带。在群体平均水平上,安徽省水稻条斑病菌群体Nei’s基因多样性指数(H)为 0.32,Shannon 信息指数(I)为 0.49,表明安徽省水稻条斑病菌的遗传多样性丰富,但病菌的遗传多样性在地区间存在差异。UPGMA聚类分析表明,来自毗邻地区的水稻条斑病菌种群大都聚为一类,水稻条斑病菌种群遗传谱系与地理区域分布呈现一定相关性。同时,安徽省水稻条斑病菌群体存在一定的遗传分化,遗传变异主要来源于群体内部。     

广东省稻瘟病菌DNA指纹分析及谱型结构

 利用散布性重复序列(dispersed repetitive sequence) MGR 586探针与EcoRI组合,分析了采自广东省4个自然生态稻作区的112个猪瘟病菌株的限制性片断长度多态性(RFLPs),根据彼此间RFLPs单型(haplotype)的带型位置相似率达80%为度,把这些菌株划分为15个遗传宗谱(genetic lineage),其中宗谱1及宗谱2占总数的78.58%,遍布全省各地,是优势宗谱。供测菌株的RFLPs单型的多样性显示出我省稻瘟病菌的群体结构呈现多样性。参试菌株的群体遗传多样性值为0.64。谱型分析表明,我省多数病原型是杂合群体。在分子水平上划分的病菌宗谱与寄生品种的遗传背景有密切关系。研究结果也初步建立了病菌宗谱与用以鉴别寄主反应划分的生理小种致病谱型的相互关系。     

拟轮枝镰孢菌EST-SSR信息分析与标记开发

为开发可用于拟轮枝镰孢菌Fusarium verticillioides及其近缘种遗传多样性分析的SSR引物,利用生物信息学方法和PCR技术,通过对从NCBI下载的87 086条拟轮枝镰孢菌的EST序列信息进行分析,设计EST-SSR引物,检测其在拟轮枝镰孢菌及其近缘种中的扩增情况,并用筛选出的多态性引物对15株拟轮枝镰孢菌进行SSR遗传多样性分析。结果表明,在EST序列中,共查找到11 952个SSR位点,592种重复基元,SSR出现频率为1.09%,重复基元出现数量最多的为三核苷酸(54.00%),其中(CAA/TTG)n基元出现频率最高。设计的25对EST-SSR引物在拟轮枝镰孢菌种内的有效扩增率和多态率分别为80.00%与32.00%,对5种近缘镰孢菌种的通用率和多态率分别为40.00%和8.00%。遗传多样性分析结果表明,在相似系数为0.664水平下,供试菌株可划分为4个SSR类群,但类群的划分与菌株的地理来源无关;不同菌株间存在明显的遗传分化。表明基于拟轮枝镰孢菌EST序列开发的SSR引物可用于拟轮枝镰孢菌及其近缘种的遗传多样性分析。     

甘肃中部及周边地区小麦条锈菌种群的遗传结构分析

为明确甘肃中部与周边地区小麦条锈菌种群的遗传结构及关系,利用SSR分子标记技术对采自甘肃、陕南、青海及新疆等7个地区共369份小麦条锈病菌标样的群体遗传结构进行了分析。结果表明,小麦条锈菌群体Nei’s基因多样性指数为0.39、Shannon信息指数为0.57,各地区条锈菌群体遗传多样性较为丰富,且在不同地区之间存在明显差异,7个条锈菌群体中以天水种群的遗传多样性相对较高,其Nei’s基因多样性指数为0.42、Shannon信息指数为0.61。该地区小麦条锈菌群体间和群体内都存在着一定的遗传分化,群体间遗传变异占总变异的2.24%,群体内遗传变异占总变异的97.76%。表明甘肃中部及周边地区小麦条锈菌群体存在一定的遗传分化,但遗传变异主要发生在群体内部;甘肃中部、陇南及陕南3地的小麦条锈菌种群遗传相似度较高,菌源交流密切。     

青藏高原地区青稞茎基腐病病原菌的群体遗传多样性、毒素化学型及其地理分布

为明确青藏高原沿线甘肃省甘南藏族自治州和青海省青稞茎基腐病燕麦镰孢菌Fusarium avenaceum的群体遗传多样性、毒素化学型及其地理分布,采用SSR分子标记法对6个地理种群91株燕麦镰孢菌菌株的遗传多样性进行分析。结果表明,6对SSR引物在91株燕麦镰孢菌中共检测到等位位点数14个,多态性位点数13个,多态性条带百分率为92.86%。6个地理种群的平均等位基因数为1.82,有效等位基因数为1.55,Nei’s基因多样性指数为0.32,Shannon信息指数为0.47,多态性位点数为11.50,多态位点百分率为82.15%。6个地理种群的Nei’s遗传相似度为0.83～0.99,遗传距离为0.01～0.18。种群间的遗传距离和地理距离、遗传相似度与海拔差距无显著相关性。燕麦镰孢菌地理种群聚为3个大类群,Group Ⅰ由甘肃省临潭县、合作市和卓尼县种群组成,Group Ⅱ由青海省互助土族自治县和刚察县种群组成,Group Ⅲ由青海省海晏县种群组成。燕麦镰孢菌种群的遗传变异主要来自种群内部,占总变异的93.63%。燕麦镰孢菌毒素化学型分为NIV、DON、3-AcDON三大类,没有15-AcDON毒素化学型,其中DON毒素化学型在6个地理种群中均有分布。表明燕麦镰孢菌地理种群的遗传多样性高。     

Genetic structure of Iranian <Emphasis Type="Italic">Pyricularia grisea</Emphasis> populations based on rep-PCR fingerprinting

The population structure of the rice blast fungus Pyricularia grisea was analyzed in two major rice-growing provinces of Iran using rep-PCR DNA fingerprinting. A total of 221 monoconidial isolates of the fungus was collected from 12 cultivars at ten regions during 1997–2000. Long-PCR conditions were used to amplify sequences lying between adjacent Pot2 elements. The frequencies of Pot2 lineages (isolates with 70% amplicon similarity) and haplotypes within lineages were determined. Phenetic analysis differentiated five Pot2 fingerprint lineages, designated A, B, C, D and E. The most common fingerprint group, Lineage E, was recovered from all rice cultivars sampled and was distributed throughout the region. Haplotype E6, the most common haplotype within lineage E, was recovered from almost all regions. Lineage A, the second most common lineage, was found mainly in the western part of the sampled region. Haplotype A1 was found in most sites in the western province. Lineage A occurred at relatively high frequency on the susceptible local cultivar Binam, suggesting that lineage A is specifically adapted to Binam. To test this hypothesis, 193 additional isolates were recovered from four fields at two sites separated by approximately 100 km. This second, field-specific collection of isolates contained lineages A, C, D, and E. Approximately 64% and 29% of the isolates recovered from Binam (the shared cv. at two sites) grouped into lineages A and E, respectively. The other two susceptible cultivars at these sites were infected by lineage E at frequencies of 100% and 71%. Overall, these data indicated a low level of genetic diversity in the Iranian P. grisea population similar to that reported in other countries.     

我国北方部分稻区稻瘟病菌群体遗传结构研究

 本文利用rep-PCR指纹分析方法研究了我国北方部分稻区稻瘟病菌的单元型(haplotype)和谱系(lineage)。根据单元型和谱系组成以及优势单元型和优势谱系的变化,分析了稻瘟病菌的群体结构和遗传变异性,证实了稻瘟病菌在DNA水平上是高度异质和极易变异的。对病原菌群体的时空变化分析发现,稻瘟病菌群体的单元型和谱系因年度和地区而不同。不同年度、不同地区的稻瘟病菌亚群体间几乎无共同的单元型和谱系。对138个供试菌株的致病型分析发现,单元型和谱系与致病型之间不存在规律性的对应关系,这说明用rep-PCR方法揭示的稻瘟病菌的遗传变异很可能与致病性变异不存在必然的联系。     

DNA fingerprinting of Pyricularia grisea by rep-PCR using a single primer based on the terminal inverted repeat from either of the transposable elements Pot2 and MGR586

We investigated the use of single primers complementary to sequences in the terminal inverted repeat (TIR) of either Pot2 or MGR586, transposable elements found in Pyricularia grisea, for DNA fingerprinting by repetitive-element-based polymerase chain reaction (rep-PCR). Under standard amplification conditions, rep-PCR with each single primer generated distinct fingerprint patterns among rice-infecting P. grisea isolates collected in Japan. With the Pot2-TIR primer, bands ranging in size from 0.2 to 8 kb and in number from 8 to 13 per isolate were amplified. Although fewer bands were amplified with the MGR586-TIR primer, this molecular technique should be more reliable to identify and classify P. grisea isolates by combining the data of fingerprint patterns from each TIR primer. In a cluster analysis based on DNA fingerprints from this rep-PCR with the Pot2-TIR primer, 10 reference isolates and 12 field isolates from Saga Prefecture in 2002 were separated into six clonal lineages. We also demonstrated that the 12 field isolates belonged to one clonal lineage. Thus, this rep-PCR method using the single primer Pot2-TIR will be useful for the analysis of the population structure of rice blast pathogens.     

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。     

内蒙古亚洲小车蝗种群遗传多样性和遗传分化的ISSR分析

亚洲小车蝗Oedaleus asiaticus Bei-Bienko是我国北方草原和农牧交错区的主要害虫。为评价内蒙古地区亚洲小车蝗种群的遗传多样性和遗传分化,应用ISSR标记方法对内蒙古15个亚洲小车蝗种群遗传多样性及遗传分化进行了分析。结果表明,7条引物扩增出85条ISSR条带,均为多态性条带。多态性比例（P）、Nei''s遗传多样性指数（H）和香农多样性指数（I）分别为82.59%、0.2319和0.3421,表明亚洲小车蝗种群具有较高的遗传多样性。基因流（Nm）和基因分化系数（Gst）分别为1.2298和0.3352,表明亚洲小车蝗不同地理种群具有明显的遗传分化。遗传距离与地理距离呈极显著正相关关系。表明地理距离和地形差异可能是形成亚洲小车蝗种群遗传分化的主要原因。     

Suppression of rice blast by phylloplane fungi isolated from rice plants

Rice phylloplane fungi were evaluated for their potential as biocontrol agents for rice blast disease caused by Magnaporthe grisea. A total of 1923 fungal isolates were obtained from rice plants in fields at Ishigaki and Iwama and from potted plants placed in a cedar woods in Iwama as bait. Although 82.9% of isolated fungi could not be identified, species of Epicoccum were the most prevalent among identified isolates. Of the 1923 isolates, 967 were randomly selected for screening against rice leaf blast. Nine isolates (MKP5111B, MKP5112, J2JMR3-2, K2J131-2, I5R3-1, NOP541, K1KM134-1, NOP5112, MKP33222) suppressed the disease when a conidial or hyphal suspension of both the phylloplane fungus and pathogen were simultaneously used to inoculate rice plants cultured in pots in a growth chamber. Five of the isolates originated from potted plants in the woods and four from Ishigaki, a subtropical island. Five (MKP5111B, MKP5112, NOP541, NOP5112, MKP33222) of the nine isolates strongly suppressed conidial germination of M. grisea (0.7%) and formed inhibition zones (3–5mm width) in dual cultures with the pathogen. Methanol extracts from the isolates also inhibited mycelial growth of the pathogen. These results suggest that the five isolates produced antibiotic(s). These five isolates are likely identical or closely related fungal species because the sequence of their ITS regions were 100% similar. ITS sequence analysis also suggested that J2JMR3-2 was associated with a species of Fusarium. Under field conditions, J2JMR3-2 reduced both leaf and panicle blast severity, and three other isolates (MKP5111B, K1KM134-1, K2J131-2) suppressed leaf blast in one of the three experiments.