【作者】 邢俊杰；

【导师】 袁隆平； 曹孟良； 贾育林；

【作者基本信息】 中南大学 ， 植物学， 2013， 博士

【摘要】 在美国南部含有稻瘟病抗性基因Pi-ta的水稻品种被用于控制稻瘟病已经超过了20年。根据基因对基因假说,Pi-ta基因能够有效阻止含有无毒基因AVR-Pita的稻瘟病菌侵染。从含有或不含Pi-ta基因的水稻品种上分离稻瘟病菌株,通过检测他们的遗传组成,以判断Pi-ta基因在美国南部的功能性。1996年及2009年共收集了169个稻瘟病菌株,通过国际稻瘟病鉴别体系,致病性检测,重复性PCR,稻瘟病菌无毒基因AVR-Pital的DNA序列测定等方法对这些菌株进行了分析。结果表明,所有菌株可分为5类生理小种：IA1, IB1, IB17, IC1以及IC17；15种重复性PCR带型,且每一类生理小种都含有一种主要带型。IB17, IC17,以及大多数的IA1, IB1都不能侵染含有Pi-ta基因的水稻品种Katy,这表明了Pi-ta基因仍然能有效的防止稻瘟病发生。利用AVR-Pital基因的特异性引物,在78个无毒菌株中扩增到了一个预期的AVR-Pital等位基因,然而,在另外12个无毒菌株中扩增出2-3个AVR-Pital等位基因,这再次验证了Pi-ta基因的有效性。通过对扩增到的AVR-Pital等位基因进行测序、序列比对后,共发现了10种不同的基因型。其中6种基因型与已发现的基因型一致,4种基因型为新发现的基因型。将所有菌株按照宿主是否含有Pi-ta基因分为两类,并对这两类菌株中有毒菌株出现的频率进行了独立性检测统计分析,结果表明从含有Pi-ta基因水稻品种中分离出来的菌株中毒性菌株出现的概率远大于从不含有Pi-ta基因的水稻品种中分离出来的菌株,这也说明了稻瘟病宿主中是否含有Pi-ta基因与有毒菌株出现频率有密切关系。尽管如此,毒性IC1生理小种能够侵染含有Pi-ta基因的抗性品种Katy,说明一些克服了Pi-ta抗性基因的有毒菌株已经发生了变异,并适应了美国南部的田间环境。利用稻瘟病部分抗性亲本Lemont与抗性亲本Jasmine85构建的重组自交系群体227个单株,结合199个多态性的SSR分子标记,针对10个新分离到的分别属于生理小种IA1, IB1, IB17以及IC1等的稻瘟病菌株,在染色体8、10、12上共定位到6个稻瘟病抗性QTL:qBLR8, qBLR10-1, qBLR10-2, qBLR10-3, qBLR12-1以及qBLR12-2,其中位于10号染色体的qBLR10-3为新定位的QTL。qBLR8,qBLR12-1以及qBLR12-2来自于父本Jasmine85,而qBLR10-1,qBLR10-2以及qBLR10-3来自于母本Lemont.所有QTL的表型差异贡献率在5.37%～39.18%之间。qBLR12-2是在本研究中鉴定到的对稻瘟病菌抗谱最广的QTL,并且表现出很高的效应值,尤其对于新鉴定到生理小种IA1,以及地区优势生理小种都具有明显的抗性效应,但对于毒性生理小种IC1菌株没有表现出抗性效应,而qBLR8,qBLR10-1,qBLR10-2等却对IC1菌株表现出一定的抗性效应。本研究中,定位到的稻瘟病抗性QTL,来自于商业化推广的水稻品种Lemont以及Jasmine85,所以这些抗性QTL,可直接通过杂交转育而应用于今后的稻瘟病抗性育种。更多还原

【Abstract】 The rice blast resistance gene Pi-ta has been effectively deployed in the Southern US rice germplasm for over two decades. Pi-ta is effective in preventing infection of blast fungus strains carry the corresponding avirulence gene AVR-Pital in a gene-for-gene fashion. In the present study, we examined genetic makeup of the field isolates of rice blast fungus from rice cultivars with or without Pi-ta to predict the functionality of the Pi-ta gene in the southern US. A total of169field isolates from1996and2009collections were analyzed using an international differential system, pathogenicity assay, rep-PCR and DNA sequencing of the avirulence gene AVR-Pital. These isolates were determined to belong to the races IA1, IB1, IB17, IC1, and IC17of M. oryzae, and were classified as15distinct groups within a major rep-PCR pattern in each group. Most of the fungal isolates of the races IA1, IB1, IB17, and IC17were unable to infect the Pi-ta-containing rice cultivar Katy suggesting that Pi-ta was effective. One AVR-Pital allele was consistently amplified by AVR-Pital-specific primers from78avirulent fungal isolates as expected; however, two to three AVR-Pital alleles were amplified in each of other12avirulent fungal isolates verifying again the effectiveness of the Pi-ta gene. Through sequencing and comparing the AVR-Pital gene that amplified, we found10different AVR-Pital alleles, in which6of them were same with those found before, and4of them were new.We separated all the blast isolates into two types according if there was Pi-ta gene and did the Chi square independent statistics test to the relation between the advent of virulent isolate and rice with or without Pi-ta gene. The test demonstrated that there was a significant difference of the occurrence of the virulent isolates between isolates from Pi-ta and non-Pi-ta cultivars, and Pi-ta gene was related to the occurrence of the virulent isolates. However, most isolates from the race IC1were virulent to Katy suggesting that some virulent isolates overcoming Pi-ta resistance have been adapted to the Southern US environment.10blast isolates that belong to IA1, IB1, IB17and IC1respectively were used to examine the blast resistance QTL. Finally,6blast resistant Quantitative trait loci (QTLs):qBLR8, qBLR10-1, qBLR10-2, qBLR10-3, qBLR12-1and qBLR12-2were identified on the chromosome8,10and12respectively with the recombinant inbred lines population that inherited from the hybridization between Lemont (partial resistant) and Jasmine85(resistant), which included total227lines. qBLR10-3was the new QTL that identified in this study, and the other5QTLs have been reported in the previous research. qBLR8、qBLR12-1and qBLR12-2were from Jasmine85, but qBLR10-1, qBLR10-2and qBLR10-3were from Lemont。The phenotype variance contribution of QTL was from5.37%～39.18%. qBLR12-2was the most widely resistance to the blast isolates in this study, especially to the new isolates belong to IA1and the dominant races IB land IB17that was just identified recently, but for another one, IC1, there was no resistance. However, qBLR10-1, qBLR10-2and qBLR10-3show part resistance to IC1. The blast resistance QTLs that identified from commercial rice Lemont and Jasmine85in this study can provide reference to rice breeding for blast resistance.更多还原