【作者】 赵飞；

【导师】 董莎萌； 钱扬文；

【作者基本信息】 南京农业大学 ， 农业硕士（专业学位）， 2022， 硕士

【摘要】 马铃薯(Solanum tuberosum)是全球最重要的主粮作物之一,而由致病疫霉(Phytophthora infestans)引起的马铃薯晚疫病是马铃薯生产上的最重大威胁。19世纪40年代,马铃薯晚疫病在爱尔兰地区爆发成灾并引发了粮食供给危机,所引起的“爱尔兰大饥馑”(Great Irish Famine)对西方产生了重要而深远的影响。现代农业生产中应对马铃薯晚疫病的主要手段是应用化学农药进行防治,不但增加农民经济负担、提高环境负荷,而且化学农药的不当使用易引发抗药性并进一步推高经济成本和生态成本。选育抗晚疫病马铃薯品种是一种经济绿色且高效的替代手段,但我国马铃薯抗病育种开展相对迟缓,主要限制因素在于晚疫病抗性资源少、抗病基因挖掘手段欠缺。因此,亟待开展高效的抗晚疫病资源挖掘工作。本研究以抗晚疫病基因的预测和功能验证为目标,以对晚疫病高抗的二倍体马铃薯野生近缘种Solanum hintonii为研究材料,取得了如下结果:马铃薯野生近缘种S.hintonii具有良好的晚疫病抗性并且其中至少存在2个抗晚疫病基因。采用离体叶片接种游动孢子的方法测定S.hintonii对致病疫霉的抗性,结果显示叶片仅接种点附近呈现小范围坏死斑,未观察到扩展病斑和产孢菌丝,而感病马铃薯栽培品系Desiree上则出现明显扩展病斑,说明S.hintonii具有良好的晚疫病抗性。利用PVX介导的农杆菌穿刺接种法,将致病疫霉131个效应子依次在S.hintonii叶片上进行瞬时表达,结果显示含有效应子PITG＿04085和效应子Avrblb1的农杆菌可产生过敏性坏死反应,提示S.hintonii可能存在一个未知的抗晚疫病基因和一个抗晚疫病基因RB的等位基因。对S.hintonii进行基因组测序组装并预测到593个候选NLR基因。为进一步解析S.hintonii的晚疫病抗性遗传基础并挖掘其携带的抗晚疫病基因,采用Pac Bio Hi Fi测序技术对其进行测序并进行基因组的从头组装。完成组装的单倍型基因组大小为932 Mb,contig N50为14.1 Mb,BUSCO评估基因组完整度为97.2%。利用抗病基因预测软件NLR-Annotator对该基因组中可能编码Nucleotide-binding and leucine-rich repeat immune receptors(NLR)的基因位点进行预测,随后结合转录组数据将没有表达量的位点剔除,并利用算法不同的另一种抗病基因预测软件NLRtracker对转录的NLR基因编码位点进行了二次NLR基因预测,共获得593个高可信度的NLR候选基因,为后续开展S.hintonii的抗晚疫病基因遗传挖掘奠定了工作基础。比对到56个RB等位基因并选取部分克隆进行功能验证。采用BLASTN进行序列比对分析,从预测得到的593个NLR中比对得到56个与已知持久抗病基因RB的序列相似度大于80%的NLR基因。本研究最终选取了9个与RB亲缘关系最近的基因,认为其最有可能是RB的等位基因并进行抗病功能验证。烟草瞬时表达结果显示本研究所克隆的9个候选基因与效应子Avrblb1共注射均不能产生过敏性坏死反应,同时将这9个候选基因在烟草叶片中瞬时表达后离体接种携带Avrblb1基因的致病疫霉游动孢子,结果显示表达了这9个候选基因的叶片均能被晚疫病侵染,说明候选NLR基因中并没有能够识别效应蛋白AVRblb1的抗病基因,推测S.hintonii中可能存其他NLR基因介导了对Avrblb1的特异性识别。更多还原

【Abstract】 Potato(Solanum tuberosum)is one of the most important staple crops in the world,and potato late blight caused by Phytophthora infestans is the most serious threat to potato production.The "Great Irish Famine" caused by the outbreak of potato late blight in the1840 s had an important and far-reaching impact on western history.In modern agricultural production,the main means to control potato late blight is the application of chemical fungicides,which not only increases farmers’ economic burden and environmental burden,but also leads to drug resistance and further increase economic and ecological costs.Selection of potato varieties resistant to late blight is an economical,green and efficient means of control.However,the progress of potato resistance breeding in China is relatively slow,and the main limiting factors are the lack of resources for resistance to late blight and the lack of technical means for mining resistant genes.Therefore,it is urgent to carry out the mining of resources for resistance to late blight.Aiming at the prediction and functional verification of resistance genes to late blight,this study took Solanum hintonii,a wild relative of potato with high resistance to late blight,obtained the following results:The wild potato species S.hintonii had good resistance to late blight and had at least two resistance genes.The resistance of S.hintonii was tested by inoculating zoospores on detached leafs.The results showed that there were necrotic ring spots near the inoculation site of Phytophthora infestans,and no spreading spots or even sporogenous mycelia were observed.In contrast,there were obvious spreading spots and sporangia on the susceptible potato cultivar Desiree,indicating that S.hintonii has good resistance to potato late blight.By PVX-mediated agrobacterium puncture method,131 effector of Phytophthora infestans were expressed in S.hintonii leaves.The results showed that effector PITG＿04085 and effector Avrblb1 could induce hypersensitive reaction in the leaves of S.hintonii,suggesting that there may be an unknown potato late blight resistance gene and an allele or homolog of resistance gene RB.S.hintonii was sequenced to assemble the genome and 593 NLR sequences were predicted.In order to further explore the source of S.hintonii resistance to late blight,and lay a foundation for excavating the resistance gene of late blight.We sequenced and de novo assembled the genome using Pac Bio Hi Fi technology,the third generation sequencing technology.The size of the assembled haplotype genome was 932 Mb,the contig N50 was14.1Mb,and the genome integrity assessed by BUSCO was 97.2%.To further identify potential Nucleotide-binding and leucine-rich repeat immune receptors(NLR)genes,we used NLR-Annotator to directly predict NLR loci in the genome,and 1397 NLR gene loci were obtained.After removing the loci with no expression combined with transcriptome data,a total of 593 high confidence candidate NLR sequences were obtained by using NLR-Tracker,another disease-resistance gene prediction software with different algorithms,for secondary prediction near the transcribed NLR sites.It laid a foundation for the subsequent excavation of S.hintonii resistance genes to late blight56 RB alleles sequences were identified and some of them were cloned for functional verification.In order to find sequences with high similarity to RB from predicted NLR sequences,sequence alignment software BLASTN was used for sequence alignment analysis,56 NLR genes with sequence similarity greater than 80% to the known durable resistance gene RB were compared from the 593 predicted NLR genes to construct an evolutionary tree.The 9 sequences most similar to RB protein sequences were selected for cloning.Transient expression in Nicotiana benthamiana showed that none of the 9candidate sequences could produce hypersensitive reaction with Avrblb1,that is,effector Avrblb1 could not be recognized by NLR receptor.In addition,after transient expression of9 candidate sequences in Nicotiana benthamiana,detached leaf infection was carried out.After inoculation with zoospores of Phytophthora infestans containing Avrblb1 gene showed that none of the 9 sequences could provide resistance to late blight in Nicotiana benthamiana,suggesting that the candidate genes could not provide resistance to late blight by recognizing effector Avrblb1.It is speculated that there may be other NLR genes in S.hintonii that mediate the specific recognition of AVRblb1.更多还原