Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays

BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

大白菜果胶甲酯酶基因BrPME1的克隆及特征分析

 【目的】克隆大白菜果胶甲酯酶基因,为进一步探讨果胶代谢在大白菜育性调控中的分子机制提供帮助。【方法】利用cDNA-AFLP技术分析大白菜核雄性不育两用系‘AB02’可育株(msms)和不育株(Msms)花蕾的基因表达谱,在可育株混合花蕾cDNA中扩增出1条特异条带TDF-24,通过RACE技术扩增该基因的cDNA全长序列,采用生物信息学软件分析所克隆基因的编码蛋白特性,利用荧光定量PCR技术分析基因时空表达模式。【结果】该基因编码大白菜果胶甲酯酶(EC 3.1.1.11),被命名为BrPME1(GenBank登录号：HM185497)。BrPME1全长cDNA序列为1 290 bp,编码1个包含363个氨基酸的前体蛋白。BrPME1蛋白N末端1—23位为信号肽,成熟蛋白含有保守的果胶甲酯酶酶活结构域,而不含酶活抑制位点。预测BrPME1蛋白含有10个磷酸化位点、1个酰胺化位点和6个N端豆蔻酰基化位点。BrPME1在两用系不育株花蕾中表达量很低,在可育株的大花蕾以及成熟花药中高水平表达。【结论】BrPME1是一个受大白菜细胞核雄性不育基因抑制的果胶甲酯酶基因家族成员。     

Development of a CAPS marker for the badh2.7 allele in Sri Lankan fragrant rice (Oryza sativa)

Fragrance in rice is caused by mutations in the badh2 (betaine aldehyde dehydrogenase) gene. It was previously reported that exons 1, 2, 7, 10, 13 and 14 of badh2 are hot spots for various mutations leading to fragrance in most aromatic rice. This study was carried out to sequence the 14th exon of badh2 gene of Sri Lankan aromatic rice varieties that lack the badh2.1 allele. The aims of the study were to predict the aberrant protein structure and to develop a functional DNA marker. In view of this, we sequenced the 14th exon of four traditional aromatic accessions and compared with a published sequence. Four accessions contained a nucleotide ‘G’ insertion in the 14th exon. This novel mutation can be classified as the badh2.7 allele. The predicted three‐dimensional protein structure of the mutant shows loss of part of the oligomerization and coenzyme binding domains, a change that is predicted to result in fragrance. A CAPS‐based novel marker, Bad2.7CAPS, was developed to identify varieties possessing this badh2.7 allele, and it can be utilized in rice breeding programmes.     

微卫星位点AciG-35在中华鲟野生个体中不同等位基因的序列变异研究

本研究利用PCR扩增和序列测定得到了1个特殊4碱基重复微卫星位点AciG-35在中华鲟野生个体中的34个等位基因序列。根据序列测定的结果,初步研究了该位点的核心重复区序列以及两端侧翼序列的变异情况。该位点不同等位基因的核心序列和侧翼序列都表现出一定程度的变异,其中核心重复序列主要表现为点突变,侧翼序列主要表现为各种碱基的替换、插入和缺失以及片段的插入缺失。此外,还发现了AciG-35引物在中华鲟中的多位点扩增现象。研究结果对于准确解释微卫星分子标记用于群体遗传分析时的数据结果具有重要意义,对在微卫星标记的应用过程中可能存在的一些问题一并进行了探讨。     

四川地方小麦品种产量与品质相关性状SSR标记位点的优异等位变异遗传解析

为发掘控制小麦产量及品质相关性状的优异等位变异和携带优异等位变异的基因资源,本研究采用与小麦产量及品质相关性状显著关联的13个SSR标记,利用Breseghello提出的无效等位变异(null allele)方法对64份四川地方小麦品种构成的自然群体的等位变异进行遗传解析。共鉴定出38个控制产量相关性状、18个控制品质相关性状的等位变异。其中,7份四川地方小麦品种携带有较多的等位变异(50个)。优异等位变异分析显示,等位变异产生的表型效应值在方向和大小上均有所不同,在与产量性状关联的5个优异等位变异中,2个具有较大增效效应值(效应值3.00),其余3个则具有较大的减效效应值(效应值3.00);与品质性状关联的12个优异等位变异中,9个具有较大的增效效应值,3个具有较大的减效效应值。SSR标记Xgwm372的4个等位变异与产量和品质相关性状均显著关联。基于上述研究结果,这些与小麦产量与品质相关性状显著关联的SSR等位变异可为小麦育种杂交亲本的选择和分子辅助选择育种提供依据。     

南方红豆杉4个微卫星位点等位基因变异分析

以南方红豆杉（Taxus chinensis var. mairei）的4个微卫星位点为例,通过等位基因长度和序列变异分析,对各位点等位基因的变异特点、变异来源和进化关系进行研究。结果表明：这4个位点在南方红豆杉种内表现出丰富而复杂的变异特点,既有SSR重复区突变形成的等位基因,也有因侧翼序列插入/缺失（Indel）形成的等位基因,还存在同一长度形态的同形异源等位基因（allelic homoplasy）,同时,在1个mtSSR位点中检测到了线粒体基因的异质性（heteroplasmy）。SSR重复序列和侧翼序列突变的共同作用是引起等位基因长度异常的主要原因。等位基因分布及其进化关系研究表明,4个位点的序列突变更符合逐步突变模型（SMM）,常见等位基因在进化上具有原始性。     

中国柑橘大实蝇遗传多样性分析

利用4对微卫星引物对采自中国7省市的18个柑橘大实蝇地理种群进行标记,并进行遗传多样性分析。结果表明,从供试柑橘大实蝇地理种群检测到的等位基因数为19~30个,平均为23.06个,且4个微卫星位点(Bp125、BcuF 1.6、Bcac 5.10、Bcac 6.10)的等位基因数分别为7、14、9、8个。对18个柑橘大实蝇种群的哈迪—温伯格平衡测试结果显示,其卡方值的变化范围为5.582~∞,各种群间差异显著(变化范围为0.000~0.694)。表明中国柑橘大实蝇种群的遗传多样性较高,且不同种群间存在较大的遗传分化。     

牦牛和黄牛三个微卫星基因座种间特异性等位基因分析

采用PCR产物直接测序和克隆测序相结合的方法分析了牦牛、普通牛和瘤牛在ILSTS013、ILSTS050和SPS115 3个微卫星基因座上存在的特异性等位基因座位的特异性.结果表明:牦牛、普通牛和瘤牛在这3个基因座上等位基因的DNA序列存在种间特异性差异,依据这些特点可以将牦牛与普通牛和瘤牛在基因型或DNA序列水平上进行区分,这将成为评价牛种间的基因交流水平和建立牛肉及其产品分子追溯系统的可靠分子标记.