甜椒育种材料N1345的疫病抗性遗传分析

阐明了以甜椒N1345为抗原的疫病抗性遗传机制,为甜辣椒抗疫病新品种选育提供依据。通过稳定高抗疫病甜椒育种材料N1345,与高感疫病辣椒材料N1308构建的P1、P2、F1、B1、B2、F2六个联合世代,应用植物数量性状主基因+多基因联合分离分析方法,进行了疫病抗性遗传分析。结果显示,以甜椒N1345为抗原的疫病抗性由2对加性-显性-上位性主基因控制（B-1-1）,两主基因加性效应、显性效应均相等,主基因遗传率在B1、B2和F2世代分别为63.43%、82.32%和83.46%。     

两个葡萄杂交后代根系抗葡萄根瘤蚜及抗寒性鉴定

【目的】为了筛选抗葡萄根瘤蚜Daktulosphaira vitifoliae Fitch且抗寒的葡萄砧木以适应我国葡萄生产需求。【方法】以山葡萄Vitis amurensis Rupr.左山1号×SO4杂种F1代的45个株系(A系列)和左山1号×101-1杂种F1代27个株系(B系列)为试材,采用离体根接种鉴定法进行抗葡萄根瘤蚜鉴定及抗性分级;采用差热分析系统(differential thermal analysis,DTA)进行各株系根系的低温放热(low temperature exotherms,LTE)分析,建立各株系根系韧皮部及木质部的温度-伤害度(LT-I)回归方程,评估各株系根系的抗寒性。【结果】葡萄根瘤蚜在杂交株系根系上的产卵量均显著低于敏感品种巨峰,筛选出被葡萄根瘤蚜侵染后不能形成根瘤,抗葡萄根瘤蚜级别为0级的A系列杂交株系18个和B系列株系11个。被葡萄根瘤蚜侵染后形成根瘤比例低于10%的抗葡萄根瘤蚜级别为1级的A系列杂交株系9个和B系列株系4个;筛选出A系列综合低温放热温度隶属度函数、韧皮部和木质部低温放热温度隶属度函数3个指标均低于贝达的株系27个,B系列各指标均低于贝达的株系3个。【结论】本研究筛选出抗寒性强且对葡萄根瘤蚜抗性强的A系列株系15个和B系列株系2个。其中,A14,A16,A18,A22,A23,A28,A34,A35,A38,A44,A50,B24和B26对葡萄根瘤蚜抗性级别为0;A11,A15,A17和A27对葡萄根瘤蚜抗性级别为1。     

辣椒上CMV株系鉴别寄主的筛选与应用

根据“基因对基因”理论和日本小室与都凡按寄主的科属关系及被害症状划分株系的方法,研究了辣椒CMV的“基因型株系”和“致病型株系”。从373个甜、辣椒品种(系)中,筛选出一套抗性不同的差别品种,编号为:LS-8501(HR)、LS-8502(R)、LS-8503(T)、LS-8504(S)、LS-8505(HS)。用这套差别品种做“基因型”株系鉴别寄主,将59个CMV分离物划分为5个株系,命名为:CMV-P0,CMV-P1,CMV-P2,CMV-P3,CMV-P4。又从7科39种不同科属寄主值物中,筛选出一套“致病型”株系的鉴别寄主谱7种,用这套鉴别寄主将59个CMV分离物划分为5个株系群,即十字花科株系群,藜科株系群,茄科、葫芦科株系群,豆科株系群,普通黄色花叶株系群。文中比较了两种方法划分的株系致病性与辣椒病症表现型之间的关系,以及各株系的分布。还讨论了“基因型”鉴别寄主谱及“基因型”株系划分方法盼学术价值和实用性,比较了5个株系与国内外已分化的CMV株系的异同点。     

黄瓜花叶病毒山东株(CMV\|SD)RNA2全长cDNA克隆的构建及全序列分析

分别利用5’RACE和3’RACE确定了CMV\|SD RNA2的5’和 3’末端序列,在此基础上,利用RTPCR得到了RNA2的5’端一半的cDNA克隆pC25和3’端一 半的cDNA克隆pC23,并通过拼接构建了RNA2全长cDNA克隆pC2F。通过对pC25和pC23进行序列 测定,得到了RNA2的全序列。序列分析结果表明CMVSD RNA2由3048nt组成,其中存在2个 部分重叠的阅读框ORF1(79～2652nt)和ORF2(2414～2746nt),分别编码858aa的2a蛋白和111 aa的2b蛋白,并在2a蛋白的序列中发现了动植物病毒复制酶所特有的两个保守序列。该株系 RNA2核苷酸序列与分属CMV I亚组的Fny株系和II亚组的Q株系RNA2的核苷酸序列同源性分别 为917%和756%;2a蛋白的氨基酸序列同源性分别为938%和677%,2b蛋白的氨基酸序 列同源性分别为830%和513%。同源性比较的结果表明SD株系属于CMV I亚组。     

抗茎腐病转基因小麦新种质的筛选

为拓宽小麦茎腐病(又称茎基腐病)抗源种类,筛选抗茎腐病小麦新种质,对43份转TaPIMP1、AtNPR1和Gastrodianin基因小麦纯合株系,进行目的基因表达分析,以及茎腐病、纹枯病和赤霉病抗性鉴定。结果表明,转基因株系的目的基因均能正常表达;转基因株系间茎腐病抗性差异明显,24份转基因株系茎腐病抗性,比受体对照扬麦12显著提高;转基因株系茎腐病抗性与纹枯病抗性相关性显著,与赤霉病相关性不显著。结合农艺性状鉴定,筛选出5份抗茎腐病转基因株系,其中2份兼抗纹枯病和赤霉病,1份兼抗纹枯病,可作为长江中下游麦区茎腐病备用抗源。     

分泌抗芜菁花叶病毒株系特异性单克隆抗体杂交瘤细胞株的建立及抗体理化性质测定

用经芜菁花叶病毒(TuMV)免疫的BALB／c小鼠脾细胞与骨髓瘤细胞(Sp2／0－Agl4)融合,经3次克隆化培养和ELISA筛选,建立了5类分泌抗TuMV的单克隆抗体(McAb)的杂交瘤细胞株。其中4类分别对TuMV－CI株系、TuMV—C3株系、TuMV—C4株系和TuMV－c5株系具有特异性反应,第5类对TuMV 5个株系均有反应。以双抗体夹心ELISA和间接ELISA检测,上述McAb同CaMV、CMV、TMV、PVX和PVY等均不产生交叉反应,小鼠腹水McAb的滴度在1：256000--2048000之间,多为1：1024000,比常规多抗血清高400倍左右。对上述杂交瘤细胞系和McAb的生物学特性及理化性质进行了鉴定。用SDS—PAGE、Western—blotting对TuMV外壳蛋白亚基、McAb识别位点进行了分析,并就McAb区分TuMV株系进行了讨。     

转反义trxs基因小麦株系01TY18遗传分析及抗穗发芽特性

以豫麦18为受体导入反义trxs基因获得的株系01TY18(T0)为材料,运用PCR、实时荧光RT-PCR以及离体整穗发芽的方法,对反义trxs基因在转基因小麦中的遗传稳定性、基因表达和抗穗发芽特性进行了研究.结果表明,8个T1代转基因株系中有6个株系目的基因检测呈阳性,且在以后的世代中能够稳定遗传并呈典型的孟德尔单基因3∶1分离规律;反义trxs基因在6个转基因株系中能够正常表达且表现出显著的抗穗发芽特性.与非转基因对照相比,转基因株系穗粒发芽率和穗发芽度平均分别降低62%(P<0.01)和50.8%(P<0.01).     

表达黄瓜花叶病毒外壳蛋白的转基因番茄抗黄瓜花叶病毒侵染

通过土壤农杆菌(Agrobacterium tumefaciens)介导将黄瓜花叶病毒外壳蛋白(CMV CP)的cDNA成功地引入番茄(Lycopersicon esculentum)植株中,并得到转基因植株。用强致病力CMV株系接种后,表达CMV外壳蛋白的转基因植株表现出对CMV侵染的抗性。转基因植株RI代的抗性基因以接近3:1比例分离。对R_1代接种CMV后,表达CMV CP的植株病症减轻,发病率、病情指数及病毒积累量明显低于对照。病症出现推迟1个多月。     

通过农杆菌介导将番茄铁转运蛋白基因导入八棱海棠

用农杆菌介导法,成功地将番茄铁转运蛋白基因导入了苹果砧木八棱海棠.获得了19个卡那霉素抗性株系,其中有11个株系经PCR鉴定为阳性.Southern杂交结果显示:有9个转基因株系基因组中整合了完整的目的基因.选择其中含有单拷贝和3个拷贝目的基因的各一个株系进行水培试验,结果表明整合了单拷贝目的基因的转基因株系表现出较强的抗缺铁胁迫能力,5周后其植株的鲜重比对照高21%～34%.     

水稻品种矮梅早3号抗稻瘟病的遗传

水稻杂交组合(矮梅早3号×华矮837)的F_3株系接种菌株78-189(ZD_3)和83-182(ZD_1),应用累积分布曲线法进行了抗稻瘟遗传分析,结果表明:水稻品种矮梅早3号含有4个主效抗性基因P_i-A_1、P_i-A_2、P_i-A_3和P_i-A_4,其中P_i-a_4为隐性基因。P_i-A_1和P_i-A_2控制对菌株78-189(ZD_3)的高抗性,同时,P_i-A_2兼控M类型对菌株78-189(ZD_3)的抗性。P_i-A_3,控制对菌株83-182(ZD_1)的抗性。P_i-A_4控制M类型对菌株83-182(ZD_1)的抗性。水稻品种华矮837对以上两菌株不存在抗性基因。     

Involvement of Suppressor-Glucans and Plant Epidermal Cells in Host-Selective Pathogenesis of Phytophthora capsici

Water-soluble glucans (WSG) from a virulent isolate of Phytophthora capsici (PCAP-3) which were released during germination of cystospore markedly suppressed the elicitor-induced death of suspension-cultured cells of susceptible sweet pepper and tomato but not that of resistant pepper and tobacco. PCAP-3, its polygalacturonase (PGase)-deficient mutant (PCAP3-M16), and galacturonic acid non-utilizable mutant carrying the PGase (PCAP-1) activity could penetrate in epidermal cells of host leaves, but similarly caused a hypersensitive response (HR) on non-injured leaves of resistant host (sweet pepper). In the case of inoculation on press-injured leaves, however, both of the resistant and nonhost plant leaves became quite susceptible to PCAP-3similar to susceptible hosts, but not to PCAP3-M16 and PCAP-1. The results suggested that host-selectivity of P. capsici may be determined in the leaf epidermal cells where the suppressor glucans released during infection effectively suppressed the occurrence of hypersensitive reaction. Furthermore, during growth of the fungus in intercellular spaces of leaf tissues, PGase may contribute not only to the virulence experession but also the supply of initial nutrition for fungal growth in the intercellular space of host tissues.     

疫霉侵染下辣椒幼苗消减文库的构建与初步分析

为了分离和鉴定辣椒中疫霉诱导基因,以高抗疫霉病辣椒品种L11为材料,以接种辣椒疫霉菌的幼嫩叶片为处理（tester）,以未接种自然生长的幼嫩叶片为对照(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了疫霉侵染下辣椒幼苗的消减文库。从消减文库中随机挑取30个阳性克隆,提取质粒进行PCR鉴定,显示插入片段大小大部分集中在200～1 000 bp之间,文库质量良好。随机挑取40个克隆进行测序,共获得35个有效EST序列。经Blastx分析表明：有30个EST与GenBank中其他序列有同源性,5个EST为未知功能序列。已知功能的EST序列分别编码NAC转录因子、丝氨酸/苏氨酸蛋白激酶、P450单加氧酶、叶绿素a/b结合蛋白、谷胱甘肽转移酶、几丁质酶等,这些蛋白涉及抗病信号传递、抗氧化作用、转录调控及光合作用等多种生理过程。本研究为抗病基因克隆和系统研究疫霉侵染下辣椒基因的表达奠定了重要的理论基础。     

辣椒疫霉菌侵染模型和侵染条件定量研究

在生长箱内控制条件下分析测定了土壤温度、水分含量对辣椒疫病死苗率的影响.结果表明:土壤温度和水分状况是决定辣椒疫病菌侵染的重要因子,病菌侵染的最适土壤温度为22 ℃～28 ℃,最适土壤含水量为40%,土壤过于干燥和过饱和都不利于病菌侵染发病;辣椒疫病死苗率与土壤温度、水分含量及其互作可用数学模式描述.田间调查发现,辣椒疫病田间流行趋势可用Gompertz模型描述,发病率与初始发病率、土壤温度、水分含量以及空气温度密切相关.建立了田间辣椒疫病发病率预测模型.     

Characterisation of Phytophthora capsici isolates from black pepper in Vietnam

Phytophthora foot rot of black pepper caused by Phytophthora capsici is a major disease of black pepper (Piper nigrum) throughout Vietnam. To understand the population structure of P. capsici, a large collection of P. capsici isolates from black pepper was studied on the basis of mating type, random amplified microsatellites (RAMS) and repetitive extragenic palindromic (REP) fingerprinting. Two mating types A1 and A2 were detected in four provinces in two climatic regions, with A1:A2 ratios ranging from 1:3 to 1:5. In several instances A1 and A2 mating types were found to co-exist in the same farm or black pepper pole, suggesting the potential for sexual reproduction of P. capsici in the field in Vietnam although its contribution to disease epidemics is uncertain. RAMS and REP DNA fingerprinting analysis of 118 isolates of P. capsici from black pepper showed that the population was genetically more diverse where two mating types were found, although the overall genetic diversity was low with most of the isolates belonging to one clonal group. The implication of these findings is discussed. The low diversity among isolates suggests that the P. capsici population may have originated from a single source. There was no genetic differentiation of isolates from different climatic regions. In addition to the large clonal group, several isolates with unique RAMS/REP phenotypes were also detected. Most of these unique phenotypes belonged to the minority A1 mating type. This may have significant implications for a gradual increase in overall genetic diversity.     

辣椒疫病拮抗菌株筛选、鉴定及其防效

从疫病发病严重田块的健康辣椒植株根际分离到98株拮抗菌,从中筛选出两株具有广谱抗性并可在贫营养条件下生长良好的高效拮抗菌株HL-3和LZ-8.通过形态观察、生理生化特性和16S rDNA序列分析,确定HL-3为多粘类芽孢杆菌,LZ-8为短小芽孢杆菌.HL-3和LZ-8对辣椒疫霉菌丝生长抑制率分别为72%和68%.HL-3和LZ-8还对黄瓜枯萎病菌、辣椒枯萎病菌、棉花黄萎病菌、黄瓜立枯病菌、烟草黑胫病菌和番茄青枯病菌具有显著的抑制作用.盆栽试验表明,HL-3和LZ-8对辣椒苗期疫病防治效果分别为72%和83%,且对辣椒生长表现出明显的促生作用.     

辣椒疫毒致病毒素

辣椒疫霉（PhytophthoracapsiciLeonian）在培养液中振荡培养时可分泌毒素,这种毒素能引起辣椒叶片形成水渍状褐腐斑,类似病原菌侵染后形成的症状。Plich’s和V8C培养液适于P.capsici的生长和产毒;生长适宜温度和pH范围分别为20～30℃与pH6～7,在25～30℃、pH5～8的条件下培养滤液毒性最强;培养15天产毒达到最高值。滤液先后经85％硫酸铰沉淀、DE52、CM32和SephadexG－75柱层析将毒素纯化。经鉴定该纯毒素为39．8kD酸性糖蛋白,并对80℃以上高温或蛋白酶敏感,而β-D-葡萄糖苷酶处理不影响其毒性,蛋白亚基为毒素的活性中心。毒素处理寄主叶片引致病理性坏死,这一作用与辣椒品种抗病性有关。     

QTLs for a component of partial resistance to cucumber mosaic virus in pepper: restriction of virus installation in host-cells

 Ninety four doubled-haploid (DH) lines obtained from the F₁ between Perennial, a cucumber mosaic virus (CMV)-partially resistant Capsicum annuum line, and Yolo Wonder, a CMV-susceptible C. annuum line, were analysed with 138 markers including mostly RFLPs and RAPDs. Clustering of RAPD markers was observed on five linkage groups of the intraspecific linkage map. These clusters could correspond to the centromeric regions of pepper chromosomes. The same progenies were evaluated for restriction of CMV installation in pepper cells in order to map quantitative trait loci (QTLs) controlling CMV resistance. This component of partial resistance to CMV was quantitatively assessed using a CMV strain that induced necrotic local lesions on the inoculated leaves. The number of local lesions gave an estimation of the density of the virus-infection sites. Genotypic variance among the DH lines was highly significant for the number of local lesions, and heritability was estimated to be 0.94. Using both analysis of variance and non-parametric tests, three genomic regions significantly affecting CMV resistance were detected on chromosomes Noir, Pourpre and linkage group 3, together explaining 57% of the phenotypic variation. A digenic epistasis between one locus that controlled significant trait variation and a second locus that by itself had no demonstrable effect on the trait was found to have an effect on CMV resistance. For each QTL, the allele from Perennial was associated with an increased resistance. Implications of QTL mapping in marker-based breeding for CMV resistance are discussed. Received: 16 September 1996     

Construction of 2 intraspecific linkage maps and identification of resistance QTLs for Phytophthora capsici root-rot and foliar-blight diseases of pepper (Capsicum annuum L.).

Two linkage maps of pepper were constructed and used to identify quantitative trait loci (QTLs) conferring resistance to Phytophthora capsici. Inoculations were done with 7 isolates: 3 from Taiwan, 3 from California, and 1 from New Mexico. The first map was constructed from a set of recombinant inbred lines (RILs) of the PSP-11 (susceptible) x PI201234 (resistant) cross; and the second map was from a set of F(2) lines of the Joe E. Parker' (susceptible) x 'Criollo de Morelos 334' (resistant) cross. The RIL map covered 1466.1 cM of the pepper genome, and it consisted of 144 markers -- 91 amplified fragment length polymorphisms (AFLPs), 34 random amplified polymorphic DNA (RAPDs), 15 simple sequence repeats (SSRs), 1 sequence characterized amplified region (SCAR), and 3 morphological markers -- distributed over 17 linkage groups. The morphological markers mapped on this population were erect fruit habit (up), elongated fruit shape (fs(e)), and fasciculate fruit clusters (fa). The F(2) map consisted of 113 markers (51 AFLPs, 45 RAPDs, 14 SSRs, and 3 SCARs) distributed in 16 linkage groups, covering a total of 1089.2 cM of the pepper genome. Resistance to both root rot and foliar blight were evaluated in the RIL population using the 3 Taiwan isolates; the remaining isolates were used for the root-rot test only. Sixteen chromosomal regions of the RIL map contained single QTLs or clusters of resistance QTLs that had an effect on root rot and (or) foliar blight, revealing a complex set of genetics involved in resistance to P. capsici. Five QTLs were detected in the F(2) map that had an effect on resistance to root rot.     

Genetic transformation of the plant pathogens Phytophthora capsici and Phytophthora parasitica.

Phytophthora capsici and P.parasitica were transformed to hygromycin B resistance using plasmids pCM54 and pHL1, which contain the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Ustilago maydis heat shock hsp70 gene. Enzymes Driselase and Novozyme 234 were used to generate protoplasts which were then transformed following exposure to plasmid DNA and polyethylene glycol 6000. Transformation frequencies of over 500 transformants per micrograms of DNA per 1 x 10(6) protoplasts were obtained. Plasmid pCM54 appears to be transmitted in Phytophthora spp. as an extra-chromosomal element through replication, as shown by Southern blot hybridization and by the loss of plasmid methylation. In addition, transformed strains retained their capacity of infecting Serrano pepper seedlings and Mc. Intosh apple fruits, the host plants for P.capsici and P.parasitica, respectively.