海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证

在对黄萎病菌胁迫处理的海岛棉Pima 90-53根组织全长c DNA文库分析中,筛选到一个与黄萎病胁迫相关的杂合富含脯氨酸蛋白(hybrid proline-rich protein)基因,将其命名为Gb Hy PRP1。该基因c DNA序列全长1747 bp,开放阅读框945 bp,编码一个由314个氨基酸残基组成的蛋白,包含信号肽、N端富含脯氨酸域及C端Pollen Ole e I域。同源序列分析显示,Gb Hy PRP1与来自雷蒙德氏棉、陆地棉和亚洲棉的Hy PRP1蛋白序列相似性最高,分别为95.95%、93.87%和91.34%。q RT-PCR分析结果显示,受黄萎病菌胁迫后海岛棉根部Gb Hy PRP1表达显著下调。将Gb Hy PRP1基因克隆至植物超表达载体,农杆菌介导转化拟南芥获得转基因植株。病指统计分析表明Gb Hy PRP1过量表达显著降低了拟南芥对黄萎病的抗性。据此推测Gb Hy PRP1参与棉花抗黄萎病,可能是一个重要的负调控因子。     

The Gossypium hirsutum TIR-NBS-LRR gene GhDSC1 mediates resistance against Verticillium wilt

Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome-wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR-NBS-LRR receptor-like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA-signalling-related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P-loop motif encoded by GhDSC1. This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana, and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

黄萎病菌诱导的海岛棉抗病反应的SSH文库构建及分析

在Pima90幼苗接种黄萎病病原菌后2、4、8、12、24、48、72和96h分批取幼根以抽提棉花总RNA。以未接种病原的棉花cDNA为驱动方,利用SSH法对接种病原后的棉花cDNA进行差减杂交。对杂交后的cDNA进行T／A克隆并转化到大肠杆菌中完成文库构建,共获得了534个克隆。以M13引物对扩增插入片段进行PCR扩增,电泳结果显示插入片段大小从0．2—1．2kb不等,平均大小为0．5kb。将克隆中的插入片段点种于尼龙膜上,分别以病原诱导前后的总cDNA为探针进行反向Northern杂交。对78个在海岛棉的抗病反应中呈上升表达的克隆进行了测序并对测序结果去载体后在NCBI上利用Blastn和Blastx进行序列相似性分析。结果表明,大部分阳性克隆与拟南芥等多个物种不同逆境条件下诱导的相关基因或表达序列相同或同源,如棉花病程相关蛋白家族10,拟南芥抗病反应家族蛋白等。SSH文库的构建和分析将有助于了解棉花抗黄萎病反应的分子机制。     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.     

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl-ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2-A, GhSSI2-B, and GhSSI2-C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia-lyase-mediated salicylic acid (SA) accumulation and activated a SA-independent defence response in GhSSI2s-silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2-dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance-related genes and nitric oxide (NO) signal were activated in GhSSI2s-silenced cotton. Taken together, these results indicate that SA-independent defence response, multiple resistance-related proteins, and elevated NO level play an important role in GhSSI2s-regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance.     

甘肃省甜瓜黄萎病的病原鉴定

【背景】2013年11月在甘肃省兰州市皋兰县的日光温室秋冬茬甜瓜种植棚发现黄萎症状的甜瓜植株,病株率约为1%。【目的】明确甜瓜黄萎病的病原。【方法】采用组织分离法进行病原菌分离;通过科赫氏法则(Koch’s法则)明确分出病菌的致病性;采用形态学和分子生物学方法对病原菌进行种类鉴定。【结果】分离得到轮枝菌属真菌8株,轮枝菌属真菌的病株分出率达100%;2个代表性菌株GLTG-2和GLTG-5(显微特征相似但菌落形态和生长速率不同),在温度18-24℃及昼/夜光周期为11.5 h/12.5 h的试验条件下,人工接种可引起甜瓜苗矮化、枯萎;接种后40 d,枯死株率分别为70%和40%;BLASTn分析结果显示,菌株GLTG-2的rDNA-ITS序列与Verticillium dahliae菌株MRHf7的序列相似性达99.78%,菌株GLTG-5的rDNA-ITS序列与V.dahliae菌株MRHf7和Vd414的序列相似性达100.00%。【结论】引起甜瓜黄萎病的病原菌被鉴定为大丽轮枝菌(V. dahliae),这是大丽轮枝菌引起甜瓜黄萎病在我国和亚洲地区的首次报道。     

海岛棉品种抗黄萎病基因SSR标记的验证及克隆

以陆地棉与海岛棉杂交组合中棉所8号×Pima90-53 F2群体的100个单株和邯208×Pima90-53 F2的131个单株为材料,在条件可控的培养室鉴定抗病性,进行海岛棉黄萎病抗性基因连锁SSR标记(BNL3255-208)分析,进一步检验该标记的应用价值,并对该标记进行克隆、测序。结果表明,2个组合的F2群体中抗病、感病植株比例经x2c适合性测验均符合3∶1分离比例;中棉所8号×Pima90-53 F2的79个抗病单株中有70株检测出BNL3255-208标记,抗病单株中有该标记的百分率为88.6%,邯208×Pima90-53 F2的100个抗病单株中有85株检测出BNL3255-208标记,抗病单株中有该标记的百分率为85.0%。通过回收BNL3255-208片段,并进行克隆和测序,表明该标记是一条长211bp、含10次TG重复的片段,该标记为辅助选育抗病品种奠定了基础。     

Soil inoculum density of Verticillium dahliae and Verticillium wilt of olive in Lebanon

In the Mediterranean basin, Verticillium Wilt of Olive (VWO) is diffused throughout its range of cultivation, causing severe yield losses and tree mortality. The disease was reported in almost all the Mediterranean and Middle East countries, and in Lebanon it is of increasing significance also on many valuable crops. The disease has already been reported on potato, peach and almond in the Bekaa valley; however, to date no information is available about the incidence of VWO and the inoculum density of Verticillium dahliae microsclerotia in soil of the main agricultural areas of Lebanon. Results from the present investigations demonstrate a high V. dahliae frequency in soils (75.3%), coupled with a mean soil inoculum density of 17.0 MS g^?1, clearly indicating a great impact on the production of susceptible hosts in Lebanon, mainly in Bekaa region. Molecular method to assess the microsclerotia inoculum density in soil allowed the detection of a higher frequency of infested soils, as compared with the traditional plating, thus confirming its higher sensitivity. The overall Verticillium wilt prevalence in the inspected olive orchards was 46.2%, and the frequency of V. dahliae‐infected trees was 25.7%. The widespread presence of V. dahliae in all olive growing areas of Lebanon enforces the adoption of measures aimed at reducing the soil inoculum density before any new olive plantation, and the use of strong phytosanitary regulations to improve the certification schemes of propagating material.     

Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

Molecular mapping of Verticillium wilt resistance QTL clustered on chromosomes D7 and D9 in upland cotton

Verticillium wilt is a destructive disease with international consequences for cotton production. Breeding broad-spectrum resistant cultivars is considered to be one of the most effective means for reducing crop losses. A resistant cotton cultivar, 60182, was crossed with a susceptible cultivar, Jun-mian 1, to identify markers for Verticillium resistance genes and validate the mode of its inheritance. Genetic segregation analysis for Verticillium wilt resistance was evaluated based upon infected leaf percentage in the seedling stage using major gene-polygene mixed inheritance models and joint analysis of P₁, P₂, F₁, B₁, B₂ and F₂ populations obtained from the cultivar cross. We found that resis-tance of upland cotton cultivar 60182 to isolates BP2, VD8 and T9, and their isoconcentration mixture was controlled by two major genes with additive-dominance-epistatic effects, and the inheritance of the major gene was dominant. Furthermore, a genetic linkage map was constructed using F₂ segregating population and resistance phenotypic data were obtained using F_2︰3 families inoculated with different isolates and detected in different developmental stages. The genetic linkage map with 139 loci was comprised of 31 linkage groups covering 1165 cM, with an average distance of 8.38 cM between two markers, or 25.89% of the cotton genome length. From 60182, we found 4 QTL on chromosome D7 and 4 QTL on D9 for BP2, 5 QTL on D7 and 9 QTL on D9 for VD8, 4 QTL on D7 and 5 QTL on D9 for T9 and 3 QTL on D7 and 7 QTL on D7 for mixed pathogens. The QTL mapping results revealed that QTL clusters with high contribution rates were screened simultaneously on chromosomes D9 and D7 by multiple interval mapping (CIM), whether from resistance phenotypic data from different developmental stages or for different isolates. The result is consistent with the genetic model of two major genes in 60182 and suggests broad-spectrum resistance to both defoliating isolates of V. dahliae and nondefoliating iso-lates. The markers associated with resistance QTL may facilitate the use of Verticillium wilt resistance genes in improving breeding programs for cotton.