烟草抗黑胫病基因Ph的精细定位和克隆鉴定

Oomycete pathogens can cause highly devastating diseases, such as potato late blight caused by Phytophthora infestans and soybean stem and root rot caused by Phytophthora sojae, in many plants important to agriculture, forestry, and natural ecosystems. The tobacco black shank disease, caused by the soilborne oomycete Phytophthora parasitica var. nicotianae van Breda de Haan, is one of the most destructive diseases of cultivated tobacco (Nicotiana tabacum) in many tobacco producing areas. The wild diploid tobacco species, Nicotiana plumbaginifolia, is highly resistant to race 0 of P. nicotianae. The resistance is controlled by the dominant single gene Ph in N. plumbaginifolia. Although the resistance to race 0 of P. nicotianae coffered by fragment containing Ph gene has been introgressed into the flue-cured tobacco varieties, Ph gene has not been cloned. Based on the preliminary mapping results of Ph gene, the proposed project intends to fine mapping the Ph gene by developing new marker in the positioning region of Ph gene, and genotyping the recombinant plants. Furthermore, the candidate genes of Ph will be selected based on the information of the fine mapping, genome sequence of the parents, re-sequencing of near isogenic lines and transcriptome analysis. Finally, the candidate genes of Ph will be cloned and functional validated via overexpression and knocking down by RNAi. It is expected that the cloning and identification of black shank resistant gene Ph in tobacco will contribute to further understanding the molecular genetics mechanism of the tobacco resistance to black shank disease. The molecular markers tightly linked to Ph gene identified by fine mapping will be valuable for breeding new black shank disease-resistant tobacco varieties. The cloned Ph gene will provide new clues for the research on resistance to oomycetes in other solanaceous plant as well.

卵菌病原菌能造成许多植物的毁灭性病害如马铃薯晚疫病、大豆茎和根腐病等。由卵菌纲寄生疫霉烟草致病变种侵染引起的烟草黑胫病是为害烟草生产的主要病害之一。野生烟草Nicotiana plumbaginifolia高抗黑胫病0号小种，其抗性由显性单基因Ph控制、已转育至烤烟品种，但Ph基因尚未被克隆。本项目拟在Ph基因初步定位的基础上，经Ph基因定位区间的新标记开发和交换单株基因型分析，精细定位Ph基因；结合Ph基因精细定位结果、抗、感亲本基因组序列、抗黑胫病近等基因系重测序、黑胫病菌侵染前后的转录组测序等信息，筛选Ph候选基因；克隆Ph候选基因，通过过表达和RNAi等方法验证候选基因功能、鉴定Ph基因。项目预期将首次克隆获得烟草抗黑胫病基因Ph，这有助于阐明烟草抗黑胫病的分子遗传机理，精细定位结果可为选育抗黑胫病烟草新品种提供指导，也可为其他茄科植物抗卵菌研究提供参考。

由卵菌纲寄生疫霉烟草致病变种侵染引起的烟草黑胫病是为害烟草生产的主要病害之一。已往研究表明，野生烟草Nicotiana plumbaginifolia对黑胫病0号小种的高抗性由显性单基因Ph控制，其抗性已转育至烤烟品种，但Ph基因尚未被克隆。本项目在Ph基因初步定位在连锁群LG20的基础上，利用BC1大群体基因型分析、标记开发和交换单株分析，将Ph基因定位区间缩小到InDel标记D617和D1179之间。进一步利用BC7高世代回交群体，经标记开发和交换单株基因型分析，将Ph基因锁定在特异序列标记CX4-037和CX6-344之间；继续利用BC8和BC9群体进行交换单株分析，结果表明该定位区间很难再缩小。为了验证该结果，将两侧片段缩短的感病交换株系杂交，交换片段互补的杂交F1均表现抗病，表明Ph基因位点尽管从经典遗传学角度分析属于典型的显性单基因控制黑胫病抗性，但从分子角度看Ph基因位点决定的抗性由该区间的至少两个基因控制、或者1个基因及其相距较远的调控因子共同控制。利用Ph基因定位区间的标记序列比对抗病亲本RBST的基因组序列，推测大致物理距离约2.63Mb。结合抗黑胫病基因精细定位、抗病亲本RBST基因组序列、重测序数据、转录组数据等分析结果，初步确定了Ph基因的候选基因5个，获得了相应序列信息。通过候选基因过表达、RNAi抑制表达、基因编辑等研究表明，候选基因G55是控制Ph基因位点黑胫病抗性的重要基因，但该基因需与其他基因或调控因子协同作用才能控制黑胫病抗性（必要非充分）。本项目经精细定位获得的Ph基因紧密连锁标记可用于烟草品种的抗黑胫病分子标记辅助选择育种，并已选育出抗黑胫病红花大金元改良新品种云烟300。经典遗传学上属于典型显性单基因（Ph）控制的黑胫病抗性、从分子角度看并非单个基因控制，G55为Ph基因位点的抗黑胫病重要基因、但需与其他基因或调控因子协同作用才能控制黑胫病抗性等研究结果属本项目首次发现，为揭示Ph基因位点的抗黑胫病遗传机制开辟了新思路，也可为其它茄科作物抗疫霉研究提供借鉴。