中国不同地理来源谷瘟病菌rDNA-IGS序列分析

由谷瘟病菌引起的谷瘟病是我国谷子的主要病害之一,谷瘟病发生可造成谷子大量减产。为了研究谷瘟病菌群体遗传结构和进化关系,本研究对64个谷瘟病菌的r DNA-IGS序列进行扩增检测和测序分析,结果表明,谷瘟病菌之间IGS序列的长度存在着明显的多态性。进一步序列分析发现,IGS序列中重复单元(GGGGGTGCAGGGTA和CATTTTT)的重复次数的不同是造成序列长度差异的主要原因,并且发现IGS基因序列具有寄主特异性。采用最大似然法对所获得的IGS序列进行系统发育树分析,将谷瘟病菌划分为3个谱系。谷瘟病菌遗传分化明显,具有丰富的多样性,分析表明影响谷瘟病菌IGS基因遗传分化的环境因素主要为寄主因素。研究结果为今后深入研究谷瘟病菌群体遗传结构提供理论支持。     

山东省稻瘟病病菌无毒基因鉴定及分析

为了明确山东省稻瘟病病菌无毒基因类型及在不同地区分布情况,根据已经公布的AVR1-CO39、AVR-Pita、ACE1、AvrPia、AvrPii、AvrPik、AvrPiz-t、AvrPi9 8个无毒基因引物,通过PCR扩增的方法 ,对2018年在山东水稻主产区收集分离的123个稻瘟病菌单孢菌株进行了无毒基因类型鉴定和分析。试验结果表明,无毒基因ACE1、AvrPiz-t、AvrPi9出现频率都为100%;无毒基因AVR1-CO39、AVR-Pita、AvrPia出现频率分别为87.80%、90.24%、10.57%;无毒基因AvrPii、AvrPik出现频率都为0。根据基因对基因假说,可以在山东稻区选育和推广携带抗病基因Pi-33、Piz-t、Pi-9、Pi-CO39、Pi-ta的水稻品种来减轻稻瘟病为害,而抗病基因Pi-a、Pi-i、Pi-k不适合在山东稻区使用。     

油茶暹罗刺盘孢菌群体遗传结构分析

暹罗刺盘孢菌是油茶炭疽病病原之一,在我国多个油茶产区均有分布。研究油茶暹罗刺盘孢菌群体遗传结构可为全面、有效防治油茶炭疽病害提供理论依据。本研究对分离自海南、江西、湖南、广西4省(自治区)6个地区暹罗刺盘孢菌菌株的ITS、CAL和GAPDH 3个基因的序列进行群体遗传结构分析。根据拼接的上述3个基因的序列,57个暹罗刺盘孢菌菌株可定义为13个单倍型,其中单倍型H7为主要单倍型,分布于本研究所涉及的所有地区。病菌不同地理种群间的遗传分化较大,AMOVA分析显示,遗传变异主要发生在种群内,病菌未经历过大规模的种群扩张。研究结果表明油茶炭疽病原暹罗刺盘孢菌种群具有丰富的遗传多样性。     

海南橡胶树白粉菌的群体遗传结构研究

白粉病是海南橡胶树上发生最严重的叶部病害,为了明确海南橡胶树白粉菌群体的遗传结构,本研究对海南7个市(县)的橡胶树白粉菌的ITS序列进行分析。结果表明,95个病菌样品可推导出5种单倍型,其中单倍型H1包含样品数为88个,且在7个市(县)均有分布;三亚种群的单倍型多样性和核苷酸多样性最高。总体和各地理群体的中性检验结果显示,群体扩张遵循中性进化,群体大小保持相对稳定。遗传分化指数(Fst)表明三亚种群与白沙、儋州、海口和琼中等四市(县)种群的遗传分化较大。AMOVA分析显示,遗传变异主要发生在种群内,占总变异的89.66%。因此海南橡胶树白粉菌菌株间虽存在一定的遗传分化,但整体遗传多样性偏低。     

海南橡胶树暹罗刺盘孢菌群体遗传结构分析

暹罗刺盘孢菌Colletotrichum siamense是引起海南橡胶树炭疽病的重要病原之一,为了明确海南橡胶树暹罗刺盘孢菌群体的遗传结构,本研究对海南5个市(县)的橡胶树暹罗刺盘孢菌的ITS序列进行分析。结果表明,58个病菌样品可推导出11种单倍型,其中单倍型H11包含样品数最多且在5个市(县)均有分布,其次为单倍型H4,在4个市(县)有分布。遗传分化指数(Fst)表明保亭种群与其他种群的遗传分化较大。AMOVA分析显示,遗传变异主要发生在种群内,占总变异的93.9%。对所有地理种群病原菌ITS序列的核苷酸不配对进行分析,表明病菌未经历过大规模的种群扩张。系统发育分析表明,来自相同地区的病菌单倍型并没有聚在一起。研究结果表明海南橡胶树暹罗刺盘孢菌种群具有丰富的遗传多样性。     

我国烟草赤星病菌遗传多样性的ISSR分析

为明确我国烟草赤星病的2种主要致病菌链格孢菌Alternaria alternata和长柄链格孢菌A.longipes的地理差异与遗传结构,采用ISSR标记对分离自9个省市的135株烟草赤星病菌进行遗传多样性分析。结果显示,通过正交优化试验建立的烟草赤星病菌ISSR-PCR最佳反应体系稳定性较好,筛选出17条多态性高且稳定的引物,共扩增出192条谱带,其中有177条具有多态性,多态率为92.19%。UPGMA聚类分析结果显示,链格孢菌和长柄链格孢菌的遗传相似性系数分别在0.67~1.00和0.66~1.00之间,遗传相似性系数为0.83时可使链格孢菌和长柄链格孢菌分别划分为5个和6个亚群,其中前者不同地理种群间表现出地理相关性,后者不同菌株随机分组。烟草赤星病菌种群的基因多态性和遗传多样性丰富,链格孢菌和长柄链格孢菌的群体间遗传分化系数分别为0.36和0.37,均存在遗传分化;群居每代迁移数分别为0.89和0.85,不同地理种群间存在基因交流;2种烟草赤星病菌的遗传分化结构表现出相似性。表明我国烟草赤星病菌中的链格孢菌和长柄链格孢菌均存在丰富的遗传多样性,且二者进化方向相似,ISSR标记能较好地揭示烟草赤星病菌种群间的亲缘关系和遗传差异性,可用于其遗传多样性分析。     

新疆蟾蜍的遗传变异与地理分化

分布在新疆境内的蟾蜍在长期适应性进化中,其表型差异较大。为了解新疆蟾蜍的遗传多样性和系统发育关系,采集了新疆26个地区254只蟾蜍,对其线粒体Cytb基因进行了扩增和测序,得到长度为880 bp的片段序列,群体单倍型多样度为0.900,核苷酸多样性为0.005 5,遗传分化指数Fst为0.695 61,基因流Nm为0.35,254条序列共定义了55种单倍型,其中H23、H15是群体共享最多的单倍型,分子变异分析(AMOVA)发现,种群间遗传变异为68.55%,种群内遗传变异为31.45%。26个地理种群中乌苏群体和尉犁群体发生过种群扩张事件,扩张时间约为10~22万年前。群体间遗传距离较小,与地理距离呈低度正相关,系统发育树显示55种单倍型分为2个支系。新疆蟾蜍具有丰富的遗传多样性,群体间的遗传分化很大,各地种群间的基因流较小,遗传变异主要来自于种群间,而非种群内,单倍型H23、H15可认为是新疆蟾蜍的原始单倍型,天山在各地理群体之间不但起屏障作用,在冰期还提供了避难场所。     

基于mt COI片段广西橘小实蝇种群遗传分化研究

橘小实蝇Bactroceradorsalis (Hendel)是一种重要的世界性入侵害虫,寄主范围广,适应性强,除两极地区外均有分布。本文对17个地理种群177头橘小实蝇线粒体DNA COI基因片段序列(667 bp)进行测序和分析,利用MEGA7.0,DnaSP 6.1和Arlequin 3.5等软件对橘小实蝇不同地理种群的碱基组成、遗传多样性、遗传分化程度和种群结构等进行分析,结合Network10.0构建单倍型网络图。在177个序列样本中,获得了82个单倍型,其中单倍型Hap1为12个地理种群所共享,总发生频率达30.49%。橘小实蝇总群体遗传多样性高,单倍型多样性(Hd)为0.957 6,核苷酸多样性(Pi)为0.00855,核苷酸平均差异数(K)为5.702 17;总群体出现极度遗传分化(F_(ST)=0.452 61),根据海岛模式换算出N_m=0.302 35,说明种群之间的遗传分化由遗传漂变造成;分子方差分析(AMOVA)结果表明,遗传变异来源为群体内;总群体Tajima's D检验值为极显著负值,说明橘小实蝇在较近历史时期经历了明显的群体扩张。Mantel检测结果说明种群间的遗传距离和遗传分化系数与地理距离没有相关性。邻接法(Neighbor-Joiningmethod,NJ)系统发育进化树与单倍型网络图显示,橘小实蝇各地理种群中的单倍型散布在不同的分布群中,分布格局较为混杂,未形成明显的系统地理结构。研究结果显示,广西区橘小实蝇各种群间已产生分化,并且基因交流并未受到地理距离的影响,推测橘小实蝇种群遗传多样性受当地果蔬种植面积及种类影响显著。     

基于线粒体COI基因序列的大豆食心虫不同地理种群遗传分化

为揭示大豆食心虫Leguminivora glycinivorella不同地理种群间的遗传分化情况,通过测定19个地理种群214头老熟幼虫的线粒体COI基因序列,利用MEGA 6.0和Dna SP 5.0软件对不同地理种群间序列变异和遗传分化进行分析。结果表明,在214条408 bp的COI基因序列中发现46个变异位点,获得49种单倍型。总群体单倍型多样度为0.8277,固定系数为0.59,基因流为0.17。总群体Tajima’s D检验结果不显著,说明在较近时间内大豆食心虫未经历群体扩张。大豆食心虫不同地理种群间基因交流较少,贵阳种群、都安种群与其它地理种群分化明显,整体的遗传多样性和遗传分化程度较高,地理距离是影响不同地理种群间遗传距离的重要因素。     

中国4省猕猴桃细菌性溃疡病菌的生物型检测和遗传多样性分析

由丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae (Psa)侵染引起的猕猴桃细菌性溃疡病(kiwifruit bacterial canker)是全球猕猴桃生产上最具毁灭性的细菌病害。为探明福建、安徽、四川和陕西4省Psa菌株的生物型和遗传多样性,用5对PCR特异性引物PsaJ-F/-R、PsaK-F/-R、Tac-F/-R、Con002-F/-R和avrRps4-F1/-R2检测Psa菌株的生物型;用4对PCR引物27F/1492R、PsaF1/PsaR2、gapA-Fps/Rps和rpoD+364s/-1222ps分别扩增16S rRNA、ITS、gapA和rpoD基因,进行多基因联合分析Psa菌株的遗传多样性。结果表明,特异性引物Tac-F/-R从47株Psa菌株中均能扩增出一条545 bp的特异条带,其他4对引物未扩增出任何条带,说明供试Psa菌株的生物型均为biovar 3。多基因联合分析表明,4省Psa存在丰富的遗传多样性,4个群体共检测出27个单倍型,单倍型多样性为0.955。安徽、福建、四川和陕西群体的单倍型数差异较大,分别为1、8、12个和12个。4个群体的多态性位点数、核苷酸多样性和平均核苷酸差异数差异极显著(P<0.01),其中福建群体的多态性最丰富,而安徽群体的多态性最低。AMOVA分析表明,3.6%的遗传变异来源于种群间,而96.4%的遗传变异来源于种群内,说明种群内变异是遗传变异的主要来源。遗传分化分析表明,安徽省Psa群体与其他3个群体间的遗传分化极高(Fst>0.175),福建、四川和陕西群体间的遗传分化水平较低(Fst<0.017)。研究结果有利于了解福建省Psa的来源,为阻断Psa的传播和猕猴桃细菌性溃疡病的长期可持续控制提供了理论参考。     

Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea

A previous study of the diversity and population structure of the rice blast fungus, Magnaporthe oryzae, over a 20-year period in Korea, found novel fingerprint haplotypes each year, and the authors hypothesized that populations might experience annual bottlenecks. Based on this model, we predicted that M. oryzae populations would have little or no genetic differentiation among geographic regions because rice blast is commonly found throughout Korea each year and M. oryzae would have to disperse from small populations surviving annually between rice crops. To test this hypothesis, we sampled M. oryzae from rice fields in eight provinces in Korea in a single year (1999). In four provinces, we sampled from a set of rice cultivars commonly grown in commercial fields (group I); because of low disease incidence in four other provinces, we could not sample from commercial fields and instead sampled from scouting plots of different cultivars set up for detecting new pathotypes of M. oryzae (group II). All isolates were genotyped with DNA fingerprint probes MGR586 and MAGGY, a telomere-linked gene family member TLH1, the PWL2 host specificity gene and mating type. Fingerprint haplotypes clustered into two distinct lineages corresponding to the two sets of cultivars (groups I and II), with haplotype similarities of 71% between lineages and >76% within lineages. Isolates from the same cultivar within group I were genetically differentiated among locations, and isolates within the same location were differentiated among cultivars. Differentiation for TLH1 and PWL2 was significant (P < 0.03), but not as strong as for fingerprint markers. Similar analyses were not possible among group II isolates because too few isolates were available from any one cultivar. All isolates were in the same mating type, Mat1-1, ruling out sexual reproduction as a source of novel haplotypes. When the 1999 samples were compared with the historical samples from the previous study, haplotypes of group I formed a separate cluster, while those of group II clustered with haplotypes from the historical sample. Altogether, geographic subdivision, monomorphism of mating type, and correlation of haplotypes to sets of cultivars are not consistent with the hypothesis of repeated turnover of haplotypes. Instead, the previous correlations of haplotypes to year might have been caused by inadequate sampling of haplotypes each year, highlighting the need for studies of population genetics to be conducted with systematic samples collected to address specific questions.     

Identification of a novel locus Rmo2 conditioning resistance in barley to host-specific subgroups of Magnaporthe oryzae

Barley cultivars show various patterns of resistance against isolates of Magnaporthe oryzae and M. grisea. Genetic mechanisms of the resistance of five representative barley cultivars were examined using a highly susceptible barley cultivar, 'Nigrate', as a common parent of genetic crosses. The resistance of the five cultivars against Setaria, Oryza, Eleusine, and Triticum isolates of M. oryzae was all attributed to a single locus, designated as Rmo2. Nevertheless, the Rmo2 locus in each cultivar was effective against a different range of isolates. Genetic analyses of pathogenicity suggested that each cultivar carries an allele at the Rmo2 locus that recognizes a different range of avirulence genes. One allele, Rmo2.a, corresponded to PWT1, which conditioned the avirulence of Setaria and Oryza isolates on wheat, in a gene-for-gene manner. The other alleles, Rmo2.b, Rmo2.c, and Rmo2.d, corresponded to more than one avirulence gene. On the other hand, the resistance of those cultivars to another species, M. grisea, was conditioned by another locus, designated as Rmo3. These results suggest that Rmo2 is effective against a broad range of blast isolates but is specific to M. oryzae. Molecular mapping revealed that Rmo2 is located on the 7H chromosome.     

New Avirulence Genes in the Phytopathogenic Fungus Leptosphaeria maculans

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (Brassica napus), develops gene-for-gene interactions with oilseed rape, and four L. maculans avirulence (AVR) genes (AvrLm1, AvrLm2, AvrLm4, and alm1) were previously genetically characterized. Based on the analysis of progeny of numerous in vitro crosses between L. maculans isolates showing either already characterized or new differential interactions, this work aims to provide an overview of the AVR genes that may specify incompatibility toward B. napus and the related species B. juncea and B. rapa. Two novel differential interactions were thus identified between L. maculans and B. napus genotypes, one of them corresponding to a complete resistance to European races of L. maculans. In both cases, a single gene control of avirulence was established (genes AvrLm3 and AvrLm7). Similarly, a single gene control of avirulence toward a B. rapa genotype, also resistant to European L. maculans isolates, was demonstrated (gene AvrLm8). Finally, a digenic control of avirulence toward B. juncea was established (genes AvrLm5 and AvrLm6). Linkage analyses demonstrated that at least four unlinked L. maculans genomic regions, including at least one AVR gene cluster (AvrLm1-AvrLm2-AvrLm6), are involved in host specificity. The AvrLm3-AvrLm4-AvrLm7 region may correspond either to a second AVR gene cluster or to a multiallelic AVR gene.     

Selection and mutation of the avirulence gene AVR-Pii of the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae avirulence (AVR) genes are predicted to be involved in pathogen invasion and their virulence functions are restricted by the presence of the cognate resistance (R) genes. In this study, the distribution and variation of the avirulence (AVR-Pii) gene of M. oryzae in Yunnan province, China were analysed to understand haplotype diversity of AVR-Pii under field conditions. The presence of AVR-Pii in 454 field isolates of M. oryzae collected in Yunnan province was examined using gene-specific PCR markers. The results showed that 82 M. oryzae isolates carried AVR-Pii. Among them, 39 (35.5%), 5 (15.2%), 4 (14.3%), 2 (13.3%), 25 (12.8%) and 7 (9.7%) of the M. oryzae isolates carried AVR-Pii from central, southeastern, southwestern, northwestern, western and northeastern Yunnan province, respectively. Of these isolates, 55 were sequenced, while the remaining 27 isolates were not suitable for PCR-based sequencing and were not used for further analysis. Moreover, three AVR-Pii haplotypes were identified among the 55 isolates, in which H1 was identical with a previous sequence in GenBank (accession no. AB498874 ) and H2 and H3 were novel variants. All DNA sequence variations were found to occur in the protein-coding region resulting in amino acid substitutions. One virulent haplotype of AVR-Pii to Pii was identified among 55 field isolates, suggesting that the AVR-Pii gene has lost avirulence function through base substitution. These findings suggest that AVR-Pii is under positive selection and AVR-Pii mutations are responsible for overcoming race-specific resistance in nature.     

菲律宾稻瘟病菌生理小种中AVR-Pita及其同源基因的序列与功能分析

根据已克隆的AVR-Pita及其同源基因序列设计4对引物,对来自菲律宾的74个稻瘟病单孢菌株DNA进行PCR扩增和序列分析,研究了与水稻抗病基因Pi-ta互作的稻瘟病菌无毒基因AVR-Pita及其同源基因序列多态性与进化关系。结果显示,在42个菌株中扩增出AVR-Pita1基因目的条带,31个菌株中扩增出AVR-Pita3基因目的条带,55个菌株中扩增出AVR-Pita4基因目的条带,但所有供试菌株均未扩增出AVR-Pita2基因目的条带。对PCR产物进行测序分析,发现AVR-Pita1基因在菲律宾的生理小种中存在序列多态性,可划分为5个单倍型,共有10个多态性位点;AVR-Pita3和AVR-Pita4基因序列较保守,序列比对后没有发现多态性。对不同单基因系接种鉴定,发现除第3种AVR-Pita1单倍型外,含有AVR-Pita1单倍型1、2、4及5的生理小种均不能与Pi-ta基因互作,从而使Pi-ta单基因系IRBLta-CT2在接种后表现为感病。以上试验结果说明AVR-Pita1基因变异性较强,且序列改变后会导致基因功能的变化。     

福建省稻瘟病菌致病性及其无毒基因分析

利用41个已知抗性基因水稻品种测定2003—2006年从福建省闽东、闽南、闽西、闽北和闽中5个主要稻区采集分离的87个稻瘟病单孢菌株的致病性。结果表明,福建省稻瘟病菌群体含有与所有测试抗病基因相应的无毒基因,其中66.67%的稻瘟病菌株表现较强致病力。病菌群体对水稻抗病基因Pi-d2、Pi-k(1)、Pi-km、Pi-kh、Pi-1(1)、Pi-z5(1)、Pi-z5(2)和Pi-1(2)的毒力频率均低于10%,提示这些抗病基因在福建省可作抗源使用。2003—2006年福建省稻瘟病菌群体中分别出现了40、37、36和38个无毒基因,其中有34个无毒基因在各年份均有分布,有30个无毒基因在5个主要稻区均有分布,Avr-a(2)、Avr-3(2)、Avr-ks、Avr-4b、Avr-b、Avr-kp(C)、Avr-km(C)、Avr-ta(C)、Avr-11(C)、Avr-19(t)、Avr-t和Avr-a(1)无毒基因的出现频率均低于30%,提示与之相对应的抗病基因在福建省水稻品种抗稻瘟病育种中应慎用。含有17、14、23、18和16个无毒基因组合的病菌较多,其组合频率分别为13.79%、10.34%、9.20%、8.05%和8.05%。     

Analysis of Host Species Specificity of Magnaporthe grisea Toward Foxtail Millet Using a Genetic Cross Between Isolates from Wheat and Foxtail Millet

ABSTRACT Host species specificity of Magnaporthe grisea toward foxtail millet was analyzed using F(1) cultures derived from a cross between a Triticum isolate (pathogenic on wheat) and a Setaria isolate (pathogenic on foxtail millet). On foxtail millet cvs. Beni-awa and Oke-awa, avirulent and virulent cultures segregated in a 1:1 ratio, suggesting that a single locus is involved in the specificity. This locus was designated as Pfm1. On cv. Ki-awa, two loci were involved and one of them was Pfm1. The other locus was designated as Pfm2. Interestingly, Pfm1 was not involved in the pathogenic specificity on cv. Kariwano-zairai. These results suggest that there is no "master gene" that determines the pathogenic specificity on all foxtail millet cultivars and that the species specificity of M. grisea toward foxtail millet is governed by cultivar-dependent genetic mechanisms that are similar to gene-for-gene interactions controlling race-cultivar specificity.     

Taxonomic characterization of Pyricularia isolates from green foxtail and giant foxtail, wild foxtails in Japan

Twenty-eight Pyricularia isolates from two wild foxtails—green foxtail (Setaria viridis) and giant foxtail (S. faberii)—in Japan were taxonomically characterized by DNA analyses, mating tests, and pathogenicity assays. Although most of the isolates failed to produce perithecia in mating tests with Magnaporthe oryzae, a diagnostic polymerase chain reaction-restriction fragment length polymorphism phenotype of M. oryzae was detected in the beta-tubulin genomic region in all isolates. The pathogenicity assays revealed that host ranges of the isolates were similar to those of isolates from foxtail millet (S. italica), which were exclusively pathogenic on foxtail millet. In addition to the 28 isolates from wild foxtails, 22 Pyricularia isolates from 11 other grasses were analyzed by RFLP using single-copy sequences as probes. In a dendrogram constructed from the RFLP data, isolates that were previously identified as M. oryzae formed a single cluster. All the wild foxtail isolates formed a subcluster with foxtail millet isolates within the M. oryzae cluster. From these results, we conclude that Pyricularia isolates from the wild foxtails are closely related to isolates from foxtail millet and should be classified into the Setaria pathotype of M. oryzae.     

Identification and genetic diversity of Mycosphaerella species on banana and plantain in Nigeria

Ribosomal coding DNA was sequenced and compared in 95 isolates of Mycosphaerella spp. collected in Nigeria and single nucleotide polymorphism (SNP) was used to identify the species and to determine the genetic structure of the sampled geographical populations. Using reference GenBank accessions with intercontinental distributions as controls, and shared species-specific SNPs in these control accessions, 84 (88·4%) isolates that grouped into 14 SNP haplotypes were identified as M. fijiensis , while 11 (11·6%) isolates represented by seven SNP haplotypes were characterized as M. eumusae . None of the isolates were either M. musicola or M. musae . The presence of M. fijiensis and M. eumusae in the collection was further confirmed using previously published species-specific probes designed on actin and β-tubulin gene sequences. A pairwise comparison of the population genetic distances revealed significant genetic differentiation between most populations ( P  < 0·001), with an average F _ST of 0·126, and a population structure corresponding to the four sampled geographical zones. The intraspecific dissimilarity of M. eumusae was 4·6%, compared with 2% for M. fijiensis . Compared to all the GenBank reference accessions, three sequence variations were unique to some Nigerian M. fijiensis haplotypes. Twenty-one sequence haplotypes were identified, geographically mapped and registered in GenBank. The results indicate that M. musicola has been replaced by more frequently occurring M. fijiensis and M. eumusae , against which disease management and resistance breeding efforts should be directed in Nigeria.