Discovery of single-nucleotide mutations in acetolactate synthase genes by Ecotilling

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to discover induced point mutations in genes of interest. The use of the TILLING technique to survey natural variation in genes is called Ecotilling. We report an adaptation of Ecotilling for rapid detection of single-nucleotide mutations in the acetolactate synthase (ALS) genes of sulfonylurea (SU)-resistant (R) Monochoria vaginalis (Pontederiaceae), a paddy weed, in Japan. Genomic DNA of a SU-R plant (target DNA) was mixed with the DNA of a SU-susceptible (S) plant (reference DNA). Ecotilling detected two nucleotide mutations in the ALS gene of SU-R M. vaginalis. These 2 mutations were confirmed by DNA sequencing. A single nucleotide mutation (C to A), in the codon CCT to CAT and another mutation (C to T), in the codon CCT to TCT were identified by sequencing. Both mutations result in the disruption of a Pro codon in the conserved Domain A region with the consequent substitution of a His residue in the first mutation and a Ser residue in the second. Substitution of the Pro residue in Domain A of the ALS gene has been reported to result in insensitivity to SUs in many weed biotypes. This study demonstrates that Ecotilling is a fast, reliable, economical method for detecting single-nucleotide mutations in genes arising from herbicide selection.     

MircoRNA156家族在小麦非生物胁迫中的表达分析

MircoRNA (miRNA)作为一类20碱基左右的非编码RNA,它在转录水平调控基因的表达,在植物抗逆境胁迫的调节网络中发挥了重要作用.通过对小麦miRNAs的研究,可以扩展小麦抵抗自然灾害的途径,减少损失.有报道表明miR156作为保守的miRNA在多种植物中参与到抗逆反应[1],Xin等[2]发现不同小麦品种的miR156在不同时间点分别受到白粉菌和热胁迫的诱导,尽管对miR156家族个别成员的研究已经有了相关报道,但对小麦miR156的整个家族的报道较少,对它们在小麦受到非生物胁迫网络中发挥的作用还不清楚.本研究拟明确小麦中miR156家族成员,并选取机械伤害、冷害和对植物损害较大的UV-B三种处理方法,探索miR156家族成员在不同的非生物胁迫中的作用.     

Plant somatic mutations in nature conferring insect and herbicide resistance

Because of the role of the meristem in plant growth and reproduction, somatic mutations in plants have long been suspected of conferring herbivore and pathogen resistance on individual plants and, in the case of trees, individual branches within single plants. A few instances of resistance to phytophagous insects owing to somatic mutations have been reported in the literature. More recently, a striking example has demonstrated how somatic mutations confer resistance to an herbicide on an invasive plant, Hydrilla verticillata. The array of new methods for manipulating genomes (e.g., gene‐editing) plus existing examples of somatic mutation‐associated resistance suggest that such mutations might be useful in silviculture, agriculture, and horticulture. Answering several general questions about somatic mutations in plants would facilitate such applications: Why are so few examples reported? Do other cases exist but go undetected for want of adequate attention or methods? Under what circumstances do somatic mutations enter gametophytes? © 2018 Society of Chemical Industry     

Proposal for a unified nomenclature for target‐site mutations associated with resistance to fungicides

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild‐type amino acid, the codon number and the new amino acid, using the one‐letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well‐studied ‘archetype’ species. Orthologous amino acids in all species are then assigned numerical ‘labels’ based on the position of the amino acid in the archetype protein. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Target site insensitivity mutations in the AChE and LdVssc1 confer resistance to pyrethroids and carbamates in Leptinotarsa decemlineata in northern Xinjiang Uygur autonomous region

A survey of resistance to five conventional insecticides was conducted in 2009 and 2010 for the first generation 4th-instar larvae of Leptinotarsa decemlineata from Urumqi, Changji, Qitai and Qapqal. Compared with the Tekes population, a reference susceptible population, the Changji and Qapqal populations exhibited very high to moderate levels of resistance to cyhalothrin and deltamethrin, moderate to high levels of resistance to carbosulfan and carbofuran, and low levels of resistance to azinphosmethyl. Moreover, the Urumqi and the Qitai populations reached a high and a moderate level of resistance to carbosulfan, respectively. Synergistic effects of triphenyl phosphate, diethylmeleate, and piperonyl butoxide on cyhalothrin and carbosulfan in Changji population revealed that cytochrome P450s were involved in the resistance to cyhalothrin but not carbosulfan. A modified bi-PASA was developed to simultaneously detect point mutations of S291G in the AChE and L1014F in the LdVssc1 genes. The former mutation resulted in the resistance to carbamates and the latter in the resistance to pyrethroids. The rates of homozygous and heterozygous resistant individuals to carbamates (S291G mutation) were 17.6% and 14.7%, 50.6% and 42.2%, 49.9% and 41.7%, 51.3% and 41.4%, and 44.8% and 47.4%; to pyrethroids (L1014F mutation) were 5.8% and 8.7%, 36.1% and 27.0%, 41.8% and 24.8%, 12.2% and 9.7%, and 7.9% and 10.6%, respectively, in samples from Tekes, Changji, Qapqal, Urumqi and Qitai. I392T point mutation in the AChE was detected by RT-PCR among 18 individuals from Changji, Qapqal, Urumqi and Qitai. These results demonstrated that point mutations of S291G in the AChE and L1014F in the LdVscc1 are responsible for, at least partially, the resistance to carbamates and pyrethroids in L. decemlineata in some field populations in northern Xinjiang Uygur autonomous region.     

安徽省草地贪夜蛾对杀虫剂的敏感性和靶标突变检测

采用饲料混毒法测定了安徽省6个地区的草地贪夜蛾3龄幼虫对4种常用杀虫剂的敏感性,并对其靶标的抗性突变频率进行检测,以明确2020年迁入安徽省的草地贪夜蛾对杀虫剂的敏感性现状,为后续制定科学防控措施提供依据。结果显示,马鞍山和宿州草地贪夜蛾种群对氟苯虫酰胺分别产生了11.3倍和10.1倍的抗性;而对甲氨基阿维菌素苯甲酸盐、茚虫威和氯虫苯甲酰胺尚未产生抗性。且这4种杀虫剂的靶标基因均未检测到抗性位点突变情况。以上结果表明,6个地区的草地贪夜蛾对甲维盐、氯虫苯甲酰胺和茚虫威均较敏感,在田间防治中仍可轮换使用该类型杀虫剂。部分地区的草地贪夜蛾对氟苯虫酰胺产生了中等水平抗性,因此以后应该减少该药剂的使用频次。     

PWL基因家族在黑龙江省水稻稻瘟病菌中的分布与变异

根据已公布的PWL家族基因序列设计4对特异性引物, 对329个采自2016年及2017年黑龙江省各稻区的水稻稻瘟病菌单孢菌株DNA进行PCR扩增与序列分析, 研究了水稻稻瘟病菌中PWL家族基因的组成及变异特征?结果显示, PWL2与PWL4在黑龙江省各稻区稻瘟菌中均有分布且稳定存在, 出现频率分别为73.86%与73.25%; PWL3出现频率为40.73%; PWL1在329个菌株DNA中均未扩增出目的条带, 再次验证了PWL1在水稻的稻瘟病菌中显示为完全缺失?对部分菌株的PCR产物进行测序分析发现, PWL2?PWL3和PWL4基因编码区在黑龙江省水稻稻瘟病菌株中的主要变异为点突变, 且引起氨基酸的突变?     

短管赤眼蜂Argonaute蛋白基因家族鉴定及表达分析

动物和植物Argonaute蛋白在RNA诱导的基因沉默中发挥重要作用。本研究采用生物信息学方法在全基因组水平鉴定短管赤眼蜂Argonaute蛋白基因家族,鉴定并命名6个TpAGO蛋白。蛋白结构及系统进化分析将TpAGO家族蛋白分为PIWI和Ago两个亚家族。TpAGO蛋白二级结构的数量比例差异不大,分布却存在一定差异,其中α-螺旋分布差异尤为显著。TpAGO蛋白基因结构表现明显的多样性,基因外显子数量差异明显,从1到20个数量不等;Ka/Ks分析表明TpAGO2和TpAGO5,TpAGO3和TpAGO4两对基因间进化动力为正选择作用,其他组合间表现为纯化选择。EST表达分析发现TpAGO基因在昆虫的卵期、幼虫期、蛹期和成虫期均有不同程度的表达,而且靶向EST在同一物种中具有单一性别(雌性或雄性)表达特性,表明短管赤眼蜂TpAGO蛋白基因可参与赤眼蜂性别及发育调控。该研究不仅有助于揭示赤眼蜂产雌孤雌生殖的表观遗传机理,也有助于推动赤眼蜂等天敌的生防应用。     

卵菌与真菌Argonaute家族基因的生物信息学分析

 Argonaute蛋白广泛存在于真核生物与原核生物中,可在非编码小RNA或DNA的引导下,对完全匹配或部分匹配的靶标进行切割、翻译抑制或染色体修饰。本研究利用生物信息学对127种卵菌与真菌的基因组进行分析,旨在了解各个物种中AGO家族基因的数量、蛋白结构域、进化关系及转录模式等。结果发现,大部分卵菌与真菌的基因组中(51%)含有2个AGO基因,而疫霉菌和壶菌等平均含有4个以上。卵菌与真菌的AGO基因在进化上相互独立,多拷贝AGO基因可能是通过基因复制形成;大部分AGO基因具有6个可预测的功能域(即:N端、Linker 1、PAZ、Linker 2、MID和PIWI),并且在PAZ和PIWI功能域上,与核酸5'和3'端结合及与催化活性相关的氨基酸位点整体相对保守,仅个别位点存在一定的差异。侵染大豆过程中,大豆疫霉和终极腐霉的两对同源AGO基因具有保守的表达模式,且基因表达水平相对较高,可能具有相似的生物学功能。上述结果将为深入解析AGO介导的RNA干扰机制及生物学功能奠定基础。     

卵菌与真菌Argonaute家族基因的生物信息学分析

Argonaute蛋白广泛存在于真核生物与原核生物中,可在非编码小RNA或DNA的引导下,对完全匹配或部分匹配的靶标进行切割、翻译抑制或染色体修饰。本研究利用生物信息学对127种卵菌与真菌的基因组进行分析,旨在了解各个物种中AGO家族基因的数量、蛋白结构域、进化关系及转录模式等。结果发现,大部分卵菌与真菌的基因组中(51%)含有2个AGO基因,而疫霉菌和壶菌等平均含有4个以上。卵菌与真菌的AGO基因在进化上相互独立,多拷贝AGO基因可能是通过基因复制形成;大部分AGO基因具有6个可预测的功能域(即:N端、Linker 1、PAZ、Linker 2、MID和PIWI),并且在PAZ和PIWI功能域上,与核酸5’和3’端结合及与催化活性相关的氨基酸位点整体相对保守,仅个别位点存在一定的差异。侵染大豆过程中,大豆疫霉和终极腐霉的两对同源AGO基因具有保守的表达模式,且基因表达水平相对较高,可能具有相似的生物学功能。上述结果将为深入解析AGO介导的RNA干扰机制及生物学功能奠定基础。     

防御相关激素与纹枯病菌处理下水稻VQ基因家族的转录分析

VQ蛋白与WRKY转录因子互作,调节植物防御反应.本研究分析了水稻VQ基因家族在水杨酸(SA)、茉莉酸(JA)、乙烯(ET)3种防御相关激素和纹枯病菌(Rhizoctonia solani)处理下的转录表达谱.结果表明,水稻VQ基因家族有1/3以上成员至少响应一种处理,OsVQ2、-11和-35的表达在3种激素处理下均...     

A strategy to identify sources of quantitative resistance in pathosystems involving disease escape and physiological resistance: the case study of rice sheath blight

An epidemiology‐based strategy using rice sheath blight (ShB) as a biological model was developed that enables identification of sources of resistance. A set of 163 cultivated rice genotypes, including genotypes which had been reported to express partial resistance to ShB, and a few genotypes reported as very susceptible, were assessed using two complementary methods. First, microfield experiments allowed measurement of disease intensification at, and spread from, inoculated sources, along with morphological traits of each genotype. Secondly, detached tiller tests allowed measurement of the physiological resistance to the disease under conditions where morphology does not come into play. Multivariate analysis involving hierarchical cluster analysis, followed by multiple correspondence analysis, indicated that levels of physiological resistance, groups of plant morphology and disease levels in microfields were associated. Results from logistic regressions further indicated that a decreased number of lesions measured on detached tillers increased the odds of a rice genotype belonging to the group with low disease intensity in microfields. The combined results from microfield and detached tiller tests allowed identification of 23 genotypes with low level of disease intensity, which may be used as sources of resistance to ShB in breeding programmes. The results suggest that this strategy, which combines the crop stand and the individual tiller scales, may be applied to the identification of sources of resistance to a range of diseases with similar life cycle traits.     

Mutants in wheat showing multipathogen resistance to biotrophic fungal pathogens

Five fast-neutron-derived mutants were isolated from the wheat line Hobbit 'sib' that show enhanced field resistance towards Puccinia striiformis f.sp. tritici , the causal agent of yellow rust. Subsequent testing showed the yellow rust resistance phenotypes to differ between mutants, to be expressed at different growth stages and, in some cases, to show an isolate interaction. Three mutants, I3-48, I3-49 and I3-54, exhibited an enhanced yellow rust resistance phenotype from the third seedling leaf onwards, while mutants I3-27 and I3-30 did not show an altered yellow rust phenotype until later growth stages. Additional resistance for brown rust (causal agent Puccinia triticina ) was identified in mutants I3-27, I3-30, I3-48 and I3-49, and for powdery mildew caused by Blumeria graminis f.sp. tritici in mutants I3-27, I3-30, I3-48 and I3-54, although in some cases the resistance was isolate-specific.