马铃薯晚疫病抗性基因分子标记检测及抗性评价

马铃薯是重庆优势特色作物之一,但其安全生产受到晚疫病的严重威胁。马铃薯生产中种植抗病品种是防控马铃薯晚疫病最为经济有效和环境友好的途径。为了了解来自国内外不同种质的晚疫病抗性基因组成以及确定在重庆市具有晚疫病抗性的马铃薯品种(系),本研究以218份来自国内外的品种(系)为材料,进行了6个晚疫病抗性(R)基因分子标记检测,同时进行了田间晚疫病抗性评价及室内接种鉴定和筛选。研究结果显示,6个R基因的分子标记在供试材料中均有分布,但分子标记的组成不尽相同,主要分为4大类。第Ⅰ类含有具有广谱抗性基因的RB,晚疫病抗性评价表现为中抗以上;第Ⅲ类缺失R2 family基因标记,绝大部分表现为晚疫病敏感型;第Ⅱ类和第Ⅳ类中分别主要含有3个R基因(R2 Family+R3a+R3b)标记类型和4个R基因(R1+R2 Family+R3a+R3b)标记类型,这两类材料中表现出一定比例的晚疫病抗性水平,但第Ⅳ类中出现抗性表现的比例高于第Ⅱ类。结果说明含有RB基因标记贡献了较高的晚疫病抗性,缺失R2 famlily基因标记的材料可能不利于晚疫病抗性,利用这些基因标记辅助筛选有助于提高重庆地区晚疫病抗性育种效率。本研究评价了218份马铃薯材料6个重要R基因组成,并筛选出重庆地区表现抗性的多个材料,为新品种(系)的推广应用以及抗病育种选育提供了科学依据,同时为发掘新的抗病基因提供了遗传资源。     

萝卜种质资源对黑腐病的抗性鉴定与筛选

对40份初选萝卜种质分别接种Xcc8004和XccBJ两个菌株,进行黑腐病苗期抗性鉴定,对其中8份代表性萝卜种质肉质根切片接种Xcc8004进行抗性鉴定和27份萝卜种质幼苗接种8个效应物基因进行过敏反应鉴定。结果表明:不同萝卜种质苗期对黑腐病的抗性存在显著差异,筛选出高抗Xcc8004的材料3份、抗病1份、中抗4份,高抗XccBJ的材料1份、抗病2份、中抗5份。萝卜苗期对Xcc8004和XccBJ的抗病性极显著相关,幼苗和肉质根对Xcc8004的抗病性极显著相关。筛选出17份对不同效应物表现过敏反应的萝卜种质。对效应物XC₀241表现过敏反应的种质数最多,对XC₀542和XC₀541表现过敏反应的种质数次之。不同抗源对不同效应物的过敏反应程度有所不同。稳定可靠抗病资源的获得为萝卜抗病育种和抗病机理的深入研究提供了基础材料。     

玉米种质资源对六种重要病虫害的抗性鉴定与评价

在2003－2005年间,对604份玉米种质进行了抗弯孢菌叶斑病和玉米螟鉴定,筛选出抗弯孢菌叶斑病的材料93份,抗玉米螟材料22份。2006－2009年间,对836份玉米种质进行了抗大斑病、茎腐病、穗腐病和瘤黑粉病的鉴定与评价,筛选出一批高抗和多抗的资源。在836份资源中,对大斑病1、2和N号3个生理小种具有抗性的材料均为50％左右;抗茎腐病材料为41.3％,高抗和抗性种质分别为264和81份;穗腐病高抗和抗性种质分别为5和171份,占比为21.1％;瘤黑粉病高抗和抗性种质各261和14份,占总鉴定材料的32.9％。上述结果表明抗大斑病、茎腐病和瘤黑粉病的种质资源较为丰富。通过对抗性结果进行对比分析,发现不同生态区玉米种质的抗性强弱以及抗性多样性存在明显差异,黑龙江和内蒙的种质对病虫害的抗性强弱及多样性程度明显高于四川种质。此外,玉米自交系对病虫害的抗性强弱以及多抗性程度高于农家种。     

玉米种质资源对六种重要病虫害的抗性鉴定与评价

在2003-2005年间,对604份玉米种质进行了抗弯孢菌叶斑病和玉米螟鉴定,筛选出抗弯孢菌叶斑病的材料93份,抗玉米螟材料22份。2006-2009年间,对836份玉米种质进行了抗大斑病、茎腐病、穗腐病和瘤黑粉病的鉴定与评价,筛选出一批高抗和多抗的资源。在836份资源中,对大斑病1、2和N号3个生理小种具有抗性的材料均为50%左右;抗茎腐病材料为41.3%,高抗和抗性种质分别为264和81份;穗腐病高抗和抗性种质分别为5和171份,占比为21.1%;瘤黑粉病高抗和抗性种质各261和14份,占总鉴定材料的32.9%。上述结果表明抗大斑病、茎腐病和瘤黑粉病的种质资源较为丰富。通过对抗性结果进行对比分析,发现不同生态区玉米种质的抗性强弱以及抗性多样性存在明显差异,黑龙江和内蒙古的种质对病虫害的抗性强弱及多样性程度明显高于四川种质。此外,玉米自交系对病虫害的抗性强弱以及多抗性程度高于农家种。     

绿豆种质资源枯萎病抗性鉴定及抗性资源筛选北大核心CSCD

绿豆枯萎病是影响绿豆产量最严重的病害之一。筛选苗期抗性资源,培育抗病品种对枯萎病防治具有重要意义。本研究采用剪根浸根接种法,对来自全国18个省市及国外的215份绿豆核心种质资源和85份绿豆新品系进行了苗期枯萎病抗性鉴定。结果显示,不同地区种质间的枯萎病抗性水平存在差异。国内产区中,东北、华东、华中地区约50%的种质具有枯萎病抗性;华北地区抗枯萎病种质占比40.4%;西北、西南和华南地区种质抗病水平较高;国外材料抗病种质占比40.0%。本研究共筛选出17份高抗(HR)枯萎病种质资源,并利用部分材料建立了枯萎病抗性研究RIL群体;6份高抗高代品系材料,在田间全生育期表现高抗且农艺性状优异。本研究期望为今后抗枯萎病绿豆新品种选育及抗性遗传相关研究提供优异资源和理论依据。     

辣椒种质资源抗青枯病的鉴定与评价

采用青枯菌FJC100301菌株对田间辣椒（Capsicum annuum）抗病品种76a和感病品种TW-1分别作了不同温度、不同接种量和不同接种方法的接种试验。结果表明,辣椒青枯病抗性的室内鉴定以接种温度28℃、浸根20 min和3×10^8cfu/mL接种浓度为宜;辣椒种质田间抗青枯病接种鉴定宜选择5月上旬进行,浸根20 min,接种浓度为3×10^8cfu/mL。采用田间抗性接种鉴定的方法,用青枯菌FJC100301菌株对106份辣椒材料进行了抗性鉴定。田间接种后每隔10 d统计病情指数,划分辣椒抗青枯病鉴定分级标准,获得了高抗材料14份、抗病材料8份、中抗材料23份、中感材料23份、感病材料20份、高感材料18份;采用离体叶片接种法对田间筛选得到的高抗和高感纯度较高品种进行抗性分析,结果与田间鉴定一致。     

不结球白菜种质资源对TuMV的抗性鉴定与评价

分别利用苗期人工接种鉴定及ELISA检测方法,对127份不结球白菜种质资源进行芜菁花叶病毒(TuMV)的抗性鉴定。结果显示,苗期人工接种鉴定的病情指数(DI)分布在3.55~95.68之间,不同种质间表现出较大的抗性差异。不同抗性级别的次数分布图基本符合正态分布,略向感病区域偏离。基于病情指数的聚类分析结果与抗病性分级基本一致。通过苗期鉴定共筛选获得高抗TuMV的不结球白菜种质6份,抗病种质13份,其主要农艺性状表现出一定的多样性。其中叶面皱缩和具有刺毛的抗病材料所占比例较高,可能与TuMV抗性存在一定的相关性。ELISA检测结果显示,117份供试种质的P/N值分布在3.10~25.37之间,不同种质间表现出病毒含量的差异,但与DI值未呈现明显相关性,可作为抗病材料筛选的辅助指标。     

不同薄皮甜瓜种质白粉病抗性研究

本研究采用田间自然鉴定和离体叶片接种鉴定的方法,研究了7个薄皮甜瓜种质对白粉病的抗性,并分析了气孔大小、气孔密度和叶绿素含量与白粉病抗性的关系。结果表明,离体叶片接种鉴定和田间自然鉴定的白粉病抗性结果一致,共鉴定出5个抗病种质,2个感病种质,四倍体的抗病性优于二倍体;气孔大小和叶绿素含量与病情指数呈负相关,气孔密度与病情指数呈正相关,除气孔大小与离体鉴定病情指数呈显著性相关外,其它指标与病情指数相关性不显著,抗病种质和感病种质在气孔大小、气孔密度和叶绿素含量上差异均不显著,因此,气孔大小、气孔密度和叶绿素含量与薄皮甜瓜白粉病抗性不相关。     

小白菜种质资源对小菜蛾的抗性评价

本文就小白菜种质资源对小菜蛾的抗性进行室内鉴定,结果表明268份材料的虫害指数分布在18.10～100.00之间,种质资源间抗虫性差异达到显著水平.聚类分析将268份种质资源分为6大类,与高抗、抗、中抗、中感、感、高感相对应,其中筛选出高抗和抗性材料分别为6和11份.对268份材料的主要形态性状与虫害指数和抗性级别的相关分析表明,小白菜种质资源叶面的皱缩度与抗虫性存在一定的相关性,认为从叶面平滑型的种质资源中筛选抗虫性材料的可能性更大.     

湖南地区新收集烟草种质资源的鉴定与遗传多样性分析

"第三次全国农作物种质资源普查与收集行动"湖南项目组经过系统调查共收集到烟草种质资源32份。本研究从收集到的烟草种质资源中筛选出23份特色种质材料,对其22个农艺性状进行鉴定和遗传多样性分析,结果表明供试种质材料遗传多样性丰富,其中数量性状的多样性指数普遍较高,多样性指数最高的3个数量性状分别是节距、叶数和叶宽,质量性状以叶形的多样性指数最高;性状变异系数最大的是叶柄,其次是株高和叶宽。对新收集烟草种质资源进行聚类分析,可以将其分为3个类群,其中第Ⅱ类群的种质叶片数多,第Ⅲ类群的植株较高,可用于高产育种实践或作为中间材料使用。对特色烟草种质进行主要病害的抗性鉴定,发现13份种质材料对黑胫病、TMV或CMV表现出抗性,其中2份种质兼抗黑胫病和TMV,可用于抗病育种。由此可见,开展烟草种质资源的普查收集工作意义重大,可以为新品种的选育提供更丰富的亲本材料或中间材料。     

Genetic and physical mapping of homologues of the virus resistance gene <Emphasis Type="Italic">Rx1</Emphasis> and the cyst nematode resistance gene <Emphasis Type="Italic">Gpa2</Emphasis> in potato

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.     

马铃薯StAP1基因的分子克隆与表达分析

采用RT-PCR方法从马铃薯品种‘Désirée’中克隆了StAP1cDNA序列（GenBank登录号GU220568）。该基因cDNA开放阅读框长度为735bp,编码一个由244个氨基酸残基组成的蛋白,该蛋白分子量为28.57kDa,理论等电点为8.32。StAP1蛋白含有1个高度保守的MADS结构域,与烟草NAP1具有较高一致性。组织表达分析显示,StAP1在马铃薯植株的顶芽、花和叶中有较高水平的转录表达,在块茎中有微量表达。利用反义StCOL转基因马铃薯植株进一步分析表明,StAP1的表达受StCOL的调控。     

Identification of RFLP markers linked to the H1 gene conferring resistance to the potato cyst nematode Globodera rostochiensis.

The H1 gene from Solanum tuberosum ssp. andigena confers high levels of resistance to the potato cyst nematode Globodera rostochiensis and is used extensively in potato breeding. Using a dihaploid segregating population, a search was conducted for linkage between this gene and markers on the potato/tomato RFLP map. A total of 60 RFLP markers covering the entire genome were screened on bulk resistant and susceptible segregants. Linkage was indicated for eight markers on chromosome 5. Individual plant analysis placed the closest marker, CD78, at a maximum map distance of 2.7 cM from H1. A molecular marker for the H1 should be useful both as a correlative screening tool for incorporation of resistance into new cultivars and as starting point for map-based cloning of this important gene.     

Quantitative Resistance to Phytophthora Infestans in Potato: A Case Study for Qtl Mapping in an Allogamous Plant Species

Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance-the latter being more important for potato breeding- are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F(1) progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.     

Phytochrome B mediates the photoperiodic control of tuber formation in potato

To determine whether phytochrome B is involved in the response of potato plants to photoperiod, a potato PHYB cDNA fragment was inserted in the antisense orientation behind the 35S CaMV promoter in Bin19 and this construct was transformed into Solanum tuberosum ssp. andigena plants which normally require short days for tuberization. Two independent transformants were obtained that had much lower levels of PHYB mRNA and protein, and which exhibited phenotypes characteristic of phyB mutants, for example, elongated stems and decreased chlorophyll content. The level of phyA, and of several phytochrome A-controlled responses, was unaffected in these plants. The photoperiodic control of tuberization in these antisense PHYB plants was abolished, the plants tuberizing in short day, long day, or short day plus night break conditions. This result shows that phytochrome B is required for the photoperiodic control of tuberization in potato ( Solanum tuberosum ssp. andigena ) and that it regulates this developmental process by preventing tuber formation in non-inductive photoperiods rather than by promoting tuberization in inductive photoperiods.     

Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo

S. tuberosum ssp. andigena potato plants require short days (SD) for tuberization. We have isolated PHOR1 (photoperiod-responsive 1), which shows upregulated expression in induced leaves (SD). PHOR1 encodes an arm repeat protein with homology to the Drosophila segment polarity protein armadillo. Antisense inhibition of PHOR1 produces a semidwarf phenotype similar to that of GA-deficient plants, and the antisense lines show reduced GA responsiveness combined with a higher endogenous GA content than wild-type plants. Feedback regulation of GA biosynthetic genes is also altered in these lines. Conversely, transgenic lines overexpressing PHOR1 show an enhanced response to GA. GA application induces rapid migration of PHOR1-GFP protein to the nucleus. Thus, PHOR1 appears to be a general component of GA signaling pathways that relocalizes to the nucleus in the presence of GA.     

Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.     

Potato late blight in Morocco: characterization of Phytophthora infestans populations (virulence and mating type)

Late blight caused by Phytophthora infestans, is the most important disease of potato in Morocco. Use of partially resistant cultivars should be an essential component of a sustainable management strategy of potato late blight, provided the durability of this form of resistance. It is therefore important to determine the nature of P. infestans Moroccan populations. Mating types were determined for 91 strains of P. infestans collected in the northern (Larache-northern plain), north western (Kénitra) and north eastern (Méknès, Middle Atlas) potato cropping areas of Morocco in 1999-2000, 2000-2001 and 2003-2004. They showed a clear regional structure of these populations, with the presence of both mating types (A1 and A2). Of all isolates collected since 1999, A2 mating type constituted 56% (54 isolates), following by A1 mating type (40.7%, 31 isolates) and A1-A2 (self-fertile) mating type (3.30%, 3 isolates). Populations from Méknès and Kénitra consisted mainly of A2 mating type, whereas populations from Larache predominantly included A1 mating type. Physiological race study revealed the presence of 19 races of P. infestans in the first collection of 25 isolates tested between 1999 and 2001. All known virulence genes were detected in western and northern Moroccan isolates, except virulence for resistance genes R2, R5, and R6 which were absent. All isolates were able to overcome two or more R genes except one isolate (5-1) corresponding to race 1.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.