小菜蛾抗药性分子遗传机理的探讨与分析

采用核型分析和代表性差异分析方法,探讨了小菜蛾抗药性的分子遗传机理.结果表明,敏感品系和抗性品系小菜蛾的染色体数目均为2n=30条,两者的核型无明显差异;以敏感品系作为驱赶扩增子,抗溴氰菊酯、抗杀虫双、抗杀螟丹近等基因系分别作为检测扩增子进行消减杂交和差异片段的富集.最后随机选取部分差异片段作探针,Southern blot验证,分别在抗杀虫双、抗溴氰菊酯和抗杀螟丹近等基因系中筛选出2、2和3个特异片段.从研究结果可以看出,与敏感品系相比,三种抗性品系小菜蛾的核型没有发生明显的变化,但在DNA水平上存在明显的差异,说明小菜蛾的抗药性与基因组DNA的变化有关.     

小菜蛾溴氰菊酯抗性和敏感种群遗传差异的cDNARDA分析

寻找小菜蛾抗溴氰菊酯品系中差异表达基因,初步分析小菜蛾敏感品系与抗溴氰菊酯品系间的遗传差异.采用改进的cDNA代表性差异分析法,以小菜蛾抗溴氰菊酯品系的cDNA为Driver扩增子、敏感品系的cDNA为Tester扩增子,通过四轮消减杂交,获得1条200bp左右的差异扩增带.将此差异扩增带克隆于pMD18-T载体,随机挑选10个克隆测序.将所得的10个序列与GenBank等数据库作同源比较,发现仅有1个序列与已知泛素基因有较高同源性,但它与小菜蛾抗药性的关系尚未查明.     

Mariner转座子与小菜蛾抗性关系的研究

以抗溴氰菊酯、杀虫双、杀螟丹小菜蛾种群及其敏感品系为研究对象,借助聚合酶链式反应(PCR)、载体筛选、琼脂糖凝胶电泳等实验技术,检测了小菜蛾4个品系的Mariner转座子的存在及其与抗性的关系。结果表明,在抗溴氰菊酯、杀虫双、杀螟丹以及敏感品系的小菜蛾中都存在两种大约500 bp的Mariner转座子片断,初次证明在小菜蛾4个品系中均含有两种Mariner转座子,并发现一种新的转座子基因片断,但是没有发现Mariner转座子与抗性之间存在直接的联系。研究结果将为利用Mariner转座子标签法在小菜蛾中分离定位基因、转化外源基因、转基因昆虫防治害虫等研究提供理论基础。     

小菜蛾对阿维菌素的抗性遗传分析及交互抗性研究

利用室内选育的抗阿维菌素小菜蛾品系和敏感品系分析了小菜蛾对阿维菌素的抗性遗传。结果表明,小菜蛾对阿维菌素的抗性为常染色体、不完全隐性遗传,而且可能是由多基因控制的抗性遗传。阿维菌素抗性品系对4 种杀虫剂的抗性谱测定结果表明,对马拉硫磷、溴氰菊酯、灭多威和农梦特无交互抗性。     

小菜蛾对杀虫双的抗性遗传研究

利用室内选育的敏感品系和抗杀虫双品系为亲本,采用剂量对数—死亡机率值回归线(LD-P线)分析法,研究了小菜蛾对杀虫双的抗性遗传方式。结果表明,小菜蛾对杀虫双的抗性为多基因、常染色体遗传,正、反交F_1的显性度(D)值分别为0.39、0.28,即其主效基因为不完全显性。小菜蛾对杀虫双的抗性现实遗传力较低,h~2=0.052,产生抗性的速率较慢,室内选育119代,抗性仅达122.8倍。抗杀虫双品系和遗传杂交后代(F_1、F_2、BC)对拟除虫菊酯类、氨基甲酸酯类、有机磷类的代表杀虫剂溴氰菊酯、灭多威、敌敌畏等的交互抗性测定结果表明,它们对3种杀虫剂无交互抗性;亲本和杂交后代的多功能氧化酶环氧化活性与杀虫双的抗性水平呈正相关性;乙酰胆碱酯酶活性要比敏感品系低;羧酸酯酶活性与敏感品系无明显差异。     

小菜蛾对溴氰菊酯的抗性消退与抗性恢复动态

利用选育的抗溴氰菊酯小菜蛾 Plutella xylostella L. 品系第100代为虫源,在不接触任何杀虫剂条件下继代繁殖50代,观察了小菜蛾对溴氰菊酯的抗性消退动态;同时对消退过程中的小菜蛾施加溴氰菊酯选择压力和利用消退过程中的小菜蛾与抗性品系、非抗性品系杂交,观察了小菜蛾对溴氰菊酯的抗性恢复动态.结果表明:①小菜蛾对溴氰菊酯的抗性消退表现为1～15代较慢,15～25代较快,25代后又缓慢的趋势,抗性消退也使氯菊酯、氯氰菊酯交互抗性水平下降;②溴氰菊酯抗性消退过程中,当连续受到一定程度的溴氰菊酯选择压力时,抗性会在几代之内很快恢复;③与其它品系杂交的后代受到一定剂量溴氰菊酯连续处理后,3～4代就能达到或接近抗溴氰菊酯品系第100代的抗性水平,对氯菊酯、氯氰菊酯的交互抗性水平也随之迅速上升.     

小菜蛾对阿维菌素的抗性机制及交互抗性研究

用叶片药膜法研究了阿维菌素抗性小菜蛾 Plutella xylostella （L.）品系 对常用药剂的交互抗性谱以及增效醚（PB）和磷酸三苯酯（TPP）的增效作用。小菜蛾对阿 维菌素与高效氯氰菊酯、溴氰菊酯、氰戊菊酯和联苯菊酯等菊酯类药剂间具有比较低的交互 抗性，对后者抗性为3～20倍，对阿维菌素的抗性为575.6倍；对氟虫脲和氟啶脲没有交互抗 性。PB和TPP对阿维菌素分别增效8.2和5.5倍，说明小菜蛾对阿维菌素的抗性可能与多功能 氧化酶（MFO）和羧酸酯酶有关。通过差光谱技术测定了阿维菌素抗性和敏感小菜蛾细胞色 素P450的含量，抗性品系是敏感品系的1.38倍。     

武汉地区小菜蛾对溴氰菊酯的抗性回复及交互抗性

与小菜蛾敏感品系相比,武汉市张家湾小菜蛾田间种群的F₁、F₄和F₇,代对溴氰菊酯的抗性指数分别为362.79、30.78和10.91倍,抗性回复显著。用浸叶法测定Bt、抑太保和杀虫双制剂对两个小菜蛾种群的毒力,证明小菜蛾对这3类药剂与溴氰菊酯间无交互抗性。研究结果可为小菜蛾抗药性治理方案的制订提供依据。     

小麦新品系YW243抗条锈性鉴定和遗传分析

 YW243是最近培育出的兼抗条锈病、白粉病和黄矮病的小麦新品系,本文对其条锈病抗性进行研究。小麦条锈菌苗期接种鉴定表明,YW243高抗CY29、CY30、CY31、CY32、CYSu-11等我国条锈菌流行小种。用26个来自世界各地的菌系接种进行基因推导,结果表明,YW243具有较宽的抗谱,试验中21个已知基因系,仅有Yr5的抗性与YW243相似,但YW243的系谱表明不含有Yr5基因,因此YW243很可能含有一个新的抗条锈病基因。用YW243和京771分别作为抗感亲本进行杂交,后代分离结果表明,YW243对条锈菌CY31的抗性由1个显性基因控制。分子标记初步研究表明,该基因与RAPD引物OPY08扩增出的1条特异DNA片段连锁。     

小菜蛾对阿维菌素B1抗药性选育及交互抗性

用阿维菌素B1（abamectin)对小菜蛾敏感种群在室内进行抗性品系选育。经过25代连续汰选，获得抗性种群Laba-R，与选育前比较，抗性提高100倍，La-ba-R种群在不接触任何药剂条件下饲养20代，抗性逐渐下降，很难恢复到选育前的敏感状态。抗性汰选前后分别测定10种药剂的剂量-死亡率毒力回归线，发现Laba-R抗性种群对乙酰甲胺磷、锐劲特、灭多威、敌敌畏不存在交互抗性；对溴氰菊酯、氯氰菊酯、杀虫双、巴丹和Bt的第三性略有下降，但无明显交互抗性。活性增效剂试验表明，增效醚（PBO）和磷酸三苯酯（TPP）对阿维菌素B1均有明显的增效作用，其中PBO的增效活性尤为显著，它能使对阿维菌素B1产生100多倍抗药性的小菜蛾完全恢复其敏感性，说明多功能氧化酶解毒代谢增强可能是小菜蛾对阿维菌素B1产生抗性的主导因素之一。     

苜蓿抗褐斑病基因ISSR标记的筛选及验证

运用ISSR分子标记技术和BSA法,对四倍体紫花苜蓿(Medicago sativaL.)F1代进行抗褐斑病基因连锁的分子标记筛选。结果表明,在93个随机引物中,有32个引物能够产生清晰稳定的扩增条带,其中6个引物在中抗杂交F1代、高抗、高感各12单株所组成的抗、感DNA池间出现差异性条带。随后,对此6个标记进行单株检验,结合均值差检验法,结果显示20-R750与苜蓿褐斑病的抗病基因紧密连锁,11-S750与苜蓿褐斑病感病的基因紧密连锁,研究结果为紫花苜蓿抗褐斑病的鉴定和分子标记辅助抗病育种提供了依据。     

Molecular mapping of the crown rust resistance gene rpc1 in barley

ABSTRACT Crown rust of barley, caused by Puccinia coronata var. hordei, occurs sporadically and sometimes may cause yield and quality reductions in the Great Plains region of the United States and Canada. The incompletely dominant resistance allele Rpc1 confers resistance to P. coronata in barley. Two generations, F(2) and F(2:3), developed from a cross between the resistant line Hor2596 (CIho 1243) and the susceptible line Bowman (PI 483237), were used in this study. Bulked segregant analysis combined with random amplified polymorphic DNA (RAPD) primers were used to identify molecular markers linked to Rpc1. DNA genotypes produced by 500 RAPD primers, 200 microsatellites (SSRs), and 71 restriction fragment length polymorphism (RFLP) probes were applied to map Rpc1. Of these, 15 RAPD primers identified polymorphisms between resistant and susceptible bulks, and 62 SSR markers and 32 RFLP markers identified polymorphisms between the resistant and susceptible parents. The polymorphic markers were applied to 97 F(2) individuals and F(2:3) families. These markers identified 112 polymorphisms and were used for primary linkage mapping to Rpc1 using Map Manager QT. Two RFLP and five SSR markers spanning the centromere on chromosome 3H and one RAPD marker (OPO08-700) were linked with Rpc1 and, thus, used to construct a 30-centimorgan (cM) linkage map containing the Rpc1 locus. The genetic distance between Rpc1 and the closest marker, RAPD OPO08-700, was 2.5 cM. The linked markers will be useful for incorporating this crown rust resistance gene into barley breeding lines.     

Mapping of clubroot (Plasmodiophora brassicae) resistance in canola (Brassica napus)

Clubroot disease, caused by Plasmodiophora brassicae, has become a major problem in the production of cruciferous crops worldwide. In this study, a population of 121 doubled haploid (DH) lines derived from a crossing between a resistant and a susceptible canola (Brassica napus) genotype was subjected to phenotypic and genotypic studies to determine the inheritance and location of the resistance gene(s). After inoculation with pathotype 3 of P. brassicae, the lines showed a 1:1 segregation ratio for resistance, indicating that resistance in this population is controlled by a single gene. Fifteen PCR‐based markers that were known to be linked to clubroot resistance (CR) genes were screened against genomic DNA from parents and resistant and susceptible bulks. Marker GC1680, linked to the CR gene CRa, exhibited polymorphism between the parents and between the resistant and susceptible bulks. CRa target primers were used to amplify fragments from the two parents and the resultant sequences were compared. A high degree of sequence similarity was found between the parents in the nucleotide binding site domain of CRa. In contrast, sequence polymorphisms were detected in the leucine‐rich repeat (LRR) domain. One pair of primers that amplify a band from the LRR region of the resistant parent but not the susceptible parent was used to screen the DH population. Amplicons were obtained from 60 of the 61 resistant lines and two of the 60 susceptible lines; thus, three recombinants were found. Based on these results, a resistance locus linked to CRa was found.     

不同猕猴桃品种RAPD分析及其与抗溃疡病的关系

对不同猕猴桃品种的分子生物学试验表明：猕猴桃的DNA浓度在920 μg/mL符合RAPD分析的要求。通过60个随机引物的PCR扩增,报道了6个不同品种和类型猕猴桃种质资源的RAPD多态性,计算了它们之间的遗传距离,构建了聚类图,并讨论了其亲缘关系。聚类分析图反映出来源于安徽省主要猕猴桃产区的6个样品可以分为3组,其中抗病与感病的相对较为集中,由此可推断出现这种聚类的原因可能是由于它们基因组中有相同的DNA片段。抗病品系都有一条1 458 bp DNA片段,而感病品系均没有该带。故该片段可能与猕猴桃植株抗溃疡病相关。RAPD多态性从分子水平上反映出了猕猴桃种质资源不同品种及不同类型间复杂的遗传背景,为抗病育种的亲本选配提供了依据,也为合成猕猴桃抗溃疡病探针并用于检测猕猴桃抗溃疡病种质和分子标记辅助育种奠定了基础。     

韩国栗疫病抗病品种的遗传变异分析

为了分析韩国栗疫病的抗病品种和感病品种的遗传变异和抗病性的筛选,利用抗病性的快速检测法和RAPD(random amplified polymorphic DNA)方法对13个栗树品种进行了抗病性检测和RAPD标记分析。抗病性的快速检测选出了5个抗病品种、5个感病品种和3个中度抗病(或中度感病)品种,并且这一结果与该品种的田间表现相一致。利用筛选的12个随机引物,扩增了100个多态性RAPD片段,但未发现与抗病性或感病性相关的特异RAPD片段。聚类分析结果表明,12个品种大致分为抗病、感病和中度抗病(或中度感病)等3个大组,并与抗病性的快速检测结果基本一致。抗病品种“MANSEKI”表现出了相对于12个品种较远的亲缘关系。     

玉米抗粗缩病遗传及分子标记研究

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is transmitted by planthopper in China. Identification and development of resistant hybrids are complicated because of the inconsistencies in viral disease pressure every year. Marker-assisted selection can provide means for main-taining virus resistance alleles even in the absence of disease. In this paper a F₂ segregation population was constructed to identity the molecular markers linked to the resistance gene using a cross between a resistant and a susceptible parents (Qi319×Ye107). Fifteen-day-old seedlings of F₂ population were exposed to small brown planthoppers carrying RBSDV for 3 days in specific inoculation chamber. The inoculated plants were transplanted to screenhouse after removing the insects completely. In plant maturity stage the disease resistance of all the individuals were visually assessed. The results showed that 17, 8, 11, 51 and 122 plants were scaled from 0-4 respectively, in which 0 means no symptoms and 4 represents highly susceptible. Chi-square test demonstrated that the segregation ratio of phenotype was 1∶15 (resistant: susceptible) or 1∶6∶9 (resistant∶moderate∶susceptible) in the F₂ population, indicating RBSDV resistance of maize was controlled by two recessive genes. The F₂ individuals DNA were extracted and 261 SSR (simple sequence repeat) primers derived from maize genome ten chromosomes were selected from maize GDB database to construct genetic linkage map. The linkage map consisted of 71 polymorphic SSR markers, spanning a genetic distance of 996.6 cM with an average interval of 14.0 cM between adjacent markers. The resistant and susceptible gene pools were set up for BSA (bulked segregant analysis) and 6 polymorphism markers were obtained with BSA-SSR method between the two pools. The F₂individuals were further analyzed with 6 polymorphism markers. Chi-square test showed that phi 051, umc1407 and umc1432, mapped on chromosome 7 and 10, exhibited segregation distortion significantly and very significantly in susceptible individuals. These three SSR markers were identified as potential markers linked to the resistant loci.     

Quantifying Aphanomyces euteiches in Alfalfa with a Fluorescent Polymerase Chain Reaction Assay

ABSTRACT A polymerase chain reaction (PCR) assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial cultivars based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.     

Assessment of latent infection with <Emphasis Type="Italic">Verticillium longisporum</Emphasis> in field-grown oilseed rape by qPCR

Improvement of cultivar resistance is the key strategy to control the host-specialized pathogen Verticillium longisporum in oilseed rape (OSR). A special feature of this pathogen is its systemic, non-homogenous and delayed colonization of the plant xylem resulting in an extended symptomless period of latency. As a result, severity of infection in the field is difficult to score as it becomes apparent only at crop maturity stages when it may be confused with natural senescence. Assessment of Verticillium disease severity in OSR by visual scoring of microsclerotia on harvested stubbles unsatisfactorily reflects genotypic resistance as it is strongly affected by the ripening stage of the plant. To overcome these limitations, we developed a qPCR method, which unambiguously differentiates levels of quantitative resistance to V. longisporum in OSR genotypes under field conditions. The specificity and sensitivity of two primer pairs targeting ITS or tubulin loci in the V. longisporum genome were tested. While tubulin primers showed a high specificity to V. longisporum isolates, ITS primers exhibited a significantly higher sensitivity in detecting fungal DNA in stem tissue (limit of quantification =0.56 fg DNA) of field-grown pre-symptomatic plants. The best discrimination of resistant and susceptible OSR cultivars based on fungal DNA analysis in stem tissue was achieved at growth stage 80, at the transition of fungal vascular growth in viable plants to saprotrophic colonization of senescent stem tissues. Field screening data obtained with qPCR at growth stage 80 confirmed results from greenhouse testing thus corroborating the relevance and reliability of seedling assays for determining cultivar responses to V. longisporum in the field, as a useful tool for breeders in first selection of elite OSR genotypes with improved resistance to Verticillium.     

一个与马铃薯青枯病抗性连锁的RAPD标记

 用原始栽培种Solanum phureja作为抗源构建了一个二倍体马铃薯作图群体,并且用于进行群分法(bulked segregant analysis,BSA)分析,以筛选检定马铃薯青枯病抗性的RAPD连锁标记。使用300个随机引物进行RAPD检测,发现引物OPG09可在抗感性DNA池之间产生960 bp的稳定的相斥型多态性产物。进一步分析分离群体并得到与抗性相连锁的标记OPG09₉₆₀。该标记已有效地应用于检测其它具相近遗传背景的二倍体群体的抗性。