【作者】 黄利兴；

【导师】 梁康迳；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2009， 博士

【摘要】 稻瘟病是广泛发生在世界各稻区的一种最具毁灭性的真菌性病害。培育持久、广谱抗稻瘟病水稻系列不育系,并揭示其抗性遗传机理是杂交稻抗病育种的理论与实践基础。本文在评述水稻抗稻瘟病遗传和育种研究现状、阐述水稻品种与稻瘟菌互作机制、植物抗病基因同源序列的克隆与测序的基础上,以一个历时22年成功育成的一个水稻不育系抗稻瘟病系谱为材料,进行其抗谱、与稻瘟菌互作、抗稻瘟病基因遗传及抗病基因同源序列相似性等方面研究,初步揭示水稻系列不育系对稻瘟病的抗性遗传规律,为进一步有效利用和克隆其抗稻瘟病基因提供科学依据。主要结果如下:1.采用苗期室内喷雾接菌鉴定和稻瘟病重发区田间自然诱发鉴定相结合的方法,以10个水稻系列不育系和6个恢复系,按NCII设计配制一套包括16个亲本和60个杂种一代为研究对象,利用20个来自福建省主要稻瘟病区有代表性的致病力强的菌株,并结合2个包括福建省龙岩市茶地乡和湖北省恩施州白果乡稻瘟病重发区田间自然诱发点的数据,探讨了其抗谱特征。发现从1995年成功育成福伊A,到2001年育成的夏丰A、谷丰A、昌丰A,再到2004年育成的安丰A、全丰A、长丰A、乐丰A、富丰A等9个抗稻瘟病不育系及其配制的54个杂种一代,对近年福建省流行的稻瘟病菌生理小种及茶地和恩施的田间稻瘟病菌的群体毒力都具有很强的抗病反应,抗性频率均达100%,抗谱广,持续时间久,均含有显性主效抗瘟基因,对杂交稻均具有很强的显性抗瘟遗传效应;连丰A不育系及其配制的6个杂种一代,平均抗性频率为21.93%。在供试的6个水稻恢复系中,蜀恢527的抗性频率为72.3%,抗谱较广;明恢77、晚3、福恢13、明恢86、福恢5138等5个恢复系的抗性频率幅度为4.5%～13.6%,抗谱较窄。根据供试亲本抗瘟性系统聚类结果,可将16个亲本分成两大类型。第1大类为抗病类型,包括9个抗稻瘟病水稻不育系福伊A、夏丰A、谷丰A、昌丰A、安丰A、全丰A、长丰A、乐丰A、富丰A和1个中抗稻瘟病水稻恢复系蜀恢527。但福伊A、夏丰A等9个抗稻瘟病水稻不育系之间的抗病性表现差异不大,而与蜀恢527之间存在一定的差异。第2大类为感病类型,包括1个感病水稻不育系连丰A及5个水稻恢复系明恢77、晚3、福恢13、明恢86、福恢5138。2.在抗谱分析的基础上,同样以上述10个水稻系列不育系为母本,6个恢复系为父本,按NCII设计配制一套包括16个亲本(P)、60个F1和60个F2的遗传材料,采用数量性状的加性?显性?上位性及与环境互作的遗传模型和统计分析方法,以22个稻瘟病菌株和稻瘟病重发区田间自然诱发点作为环境,深入探讨了水稻系列不育系与稻瘟菌互作的遗传基础。发现供试水稻品种抗瘟性的表现是由供试水稻品种中的抗瘟基因与稻瘟病菌株互作的结果。对供试水稻品种抗瘟性的遗传控制作用从大到小依次为基因加性效应、显性×菌株互作效应、显性效应、加性×菌株互作效应、加性×加性上位性效应,而不存在加性×加性上位性×菌株互作效应。夏丰A、昌丰A、安丰A、福伊A、乐丰A、长丰A、全丰A、富丰A、谷丰A等9个水稻不育系及蜀恢527的抗瘟性可以稳定地传递给后代,都能极显著地提高其后代的抗瘟性,在抗瘟性方面有很高的育种利用价值。供试水稻品种抗瘟性的加性基因遗传概率是非加性基因遗传概率的2.2倍,加性基因产生的抗瘟性可以稳定地传递给后代;普通狭义遗传力是互作狭义遗传力的6.4倍,显示在不同的稻瘟病菌株胁迫的环境条件进行水稻抗瘟性的选择是有效的。3.在抗稻瘟病水稻不育系系谱中,选用抗源谷农13、天谷B、福伊B、谷丰B、全丰B为材料,在一套以CO39为轮回亲本的6个近等基因系和1个感病对照丽江新团黑谷(LTH)的遗传背景下,人工分别组配了谷农13、天谷B、福伊B、谷丰B、全丰B与CO39近等基因系和感病对照的包括亲本(P)、F1、F2 3个世代的遗传材料,以3个稻瘟病菌株进行苗期室内喷雾接菌鉴定,应用经典遗传学分析方法研究该不育系系谱抗稻瘟病基因的遗传及其等位性关系。发现谷农13、天谷B、福伊B、谷丰B和全丰B等5个亲本的抗瘟性都由显性基因控制。谷农13、福伊B、谷丰B和全丰B中都含有2对与CO39近等基因系中的Pi-1、Pi-2、Pi-3、Pi-4a、Pi-4b不等位的显性抗稻瘟病基因。天谷B中含有3对显性抗稻瘟病基因,它们都与Pi-1、Pi-2、Pi-4a、Pi-4b不等位,其中有1对与Pi-3等位。4.根据已知NBS-LRR类抗病基因结构中氨基酸的保守区域设计简并引物PR2和ER4,采用PCR方法对福伊A、谷丰A等10个水稻系列不育系,谷农13、V41B等11个亲本,及感稻瘟病对照丽江新团黑谷(LTH)的NBS-LRR类抗病基因同源序列(RGAs)进行克隆、测序,分析抗病基因同源序列相似性关系。在供试材料中,共获得33个NBS-LRR类抗病基因的同源片段。其中,从谷农13、福伊A、地谷B、昌丰A、夏丰A、连丰A、乐丰A、V41B、龙特甫B、博白B、金23B等11亲本中各获得2个同源片段,从天谷B、谷丰A、安丰A、全丰A、长丰A、富丰A、LTH、R931022、Y20、Y27、Y12等11亲本中各获得1个同源片段。运用Clustal W方法和DNAstar软件对这些氨基酸序列进行聚类分析,可分为12类NBS-LRR类抗病基因同源序列。用DNAsis软件对这12类抗病基因同源片段再次聚类分析,可分为9类NBS-LRR类抗病基因同源片段。其中,氨基酸片段间同源性最高达98.8%,最低仅为19.8%。更多还原

【Abstract】 Rice blast is one of the most devastating fungal diseases in rice region of world. To breed durable and broad-spectrum blast resistance of rice series sterile lines, and to reveal the genetic mechanism of resistance, it is hybrid rice breeding for disease resistance based on the theory and practice. This dissertation expatiated on the rice blast resistance breeding, the interaction between rice variety and Magnaporthe grisea and cloning and sequencing the RGA of plant. For breeding the male sterile lines resistance to rice blast during the 22 years as an examples. The study on the spectrum of this rice variety, interaction between rice variety and Magnaporthe grisea, rice blast resistence gene and homologous resistance gene analogues and revealed an male sterile line of rice blast resistance of inheritance, in order to use and clone of rice blast resistance gene effectively. Main research results are as follows:1.The use of indoor spraying seedling identification and blast strains then re-issued identification of areas of field-induced combination of natural methods for the 10 series male sterile line of rice and 6 restorer lines. According to the design of NCII, 2 generations genetic research materials including 16 parents(P), 60 hybrid rice varieties(F1 generations). We discuss its resistance spectrum using 20 strong blast strains which come from fields that susceptible to blast strains of FuJian province and 2 datas of serious blast areas of LongYan ChaDi country of FuJian province and EnShiZhou BaiGuo Country of Hubei Province. Fuyi A was cultivated in 1998. Xiafeng A, Gufeng A and Changfeng A were cultivated in 2001. Anfeng A, Quanfeng A, Changfeng A, Lefeng A, Fufeng A were cultivated in 2004. The results showed 9 sterile lines resistaance to blast fungus and 54 hybrid varieties represented high resistance to Fujian popular blast races and blast strain in ChaDi and Enshi, and resistance frequencies were 100%, have broad resistance spectrum. These varities not only contain the main effect dominant blast resistance gene, but also have a strong dominant genetic effect of blast resistance for the hybrid rice combinations. The average of resistance frequencies of 6 sterile resistance to blast fungus and 6 hybrid varities were 100%. Among 6 restoring lines, Shuhui 527 have broad resistance spectrum with resistance frequency of 72.3%. 5 restoring lines (Minghui 77, Wan 3, Fuhui 13, Minghui 86 Fuhui 5138) and Lianfeng A range have narrow resistance spectrum with resistance frequency of 4.5% to 13.6%.According to the results of blast resistance cluster analysis, we divided the 16 parents into two types. One type is blast resistance, including nine sterile lines of resistance to blast fungus(Fuyi A, Xiafeng A, Gufeng A, Changfeng A, Quanfeng A, Changfeng A, Lefeng A, Fufeng A, respectively.) and one sterile lines of moderate resistance, Shuhui 527. However, there were significant differences in resistance between Shuhui 527 and the other 9 sterile lines, while there were no significant differences in resistance between the other 9 sterile lines. The other type is susceptible to blast resistance, including one sterile lines of susceptible to blast fungus (LianfengA), and five restoring lines (Minghui 77, Wan 3, Fuhui 13, Minghui 86 Fuhui 5138).2. The 10 series male sterile line of rice for the female parent, 6 restorer lines of rice as male parent, similarly to the above-mentioned in the anti-spectrum analysis. According to the design of NCII, 3 generations genetic research materials including 16 parents(P), 60 F1 and 60 F2 were prepared. In order to reveal genetic mechanism of rice resistance to blast, genetic effect of interaction sterile line and blast fungus were analysised according to the interaction genetic model of additive-dominance-epistatic with the environment and statistical methods by 22 rice blast strains and in blast nursery nature achievement environment. The results showed the performance of blast resistance in rice were controlled by the blast resistance gene and environment interaction of the blast. The genetic control effects were followed in turn additive effect > interaction effect of dominant×blast stains > dominant effect > interaction effect of additive×blast stains > additive×additive epistatic effect. The interaction effect of additive×additive epistatic×blast stains was not existent. Blast resistance of 9 sterile lines (Xiafeng A, Changfeng A, Anfeng A, Fuyi A, Lefeng A, Changfeng A, Quanfeng A, Fufeng A, Gufeng A, respectively.) and Shuhui 527 can stably transmitted to offsprings, and can significantly improve blast resistance of offsprings. They have the very high breeding value in the anti-blast breeding. The heritability of additive genetic probability was 2.2 times of non-additive genetic probability in the tested rice varieties to blast resistance. The blast resistance causing from additive gene can stably transmitted to offsprings. The rice anti-blast ordinary narrow heritability was 6.4 times of the narrow mutual heritability. The results indicated it is effective for rice blast selection under environmental stress condition of the different rice blast strains.3. Male sterile line of rice resistance to blast in the pedigree, the selection of anti-agricultural source Gunong 13, TianguB, FuyiB, Gufeng B, Quanfung B for the whole material, in a set of recurrent parent CO39 for the six near-isogenic lines and a susceptible control Lijiangxintuanheigu (LTH) of the genetic background, the artificial group, respectively, with the Gumong 13, Tiangu B, Fuyi B, GufengB, QuanfengB and CO39-wide near-isogenic lines and a sense of disease control, including parental (P), F1, F2 3 generation of the genetic material to blast three strains spray indoor access to the seedling stage to identify bacteria. Using classical genetic analysis method, the maintainer line(Fuyi B, Gufeng B, Quanfeng B) of sterile line, the number and genetic mechanism of resistance gene to blast in resistance parents(Gunong 13, Tiangu B), and allelic relationship between known resistance gene to blast in near-isogenic lines(NILs) of CO39 were studied. The results indicated 5 parents have dominant resistance genes to blast. Gunong13, Fuyi B, Gufeng B and Quanfeng B contain 2 pairs of dominant resistance genes to blast, existing non-allelic relationship with Pi-1、Pi-2、Pi-3、Pi-4a、Pi-4b in NILs of CO39. Tiangu B contains 3 pairs of dominant resistance genes to blast, existing non-allelic relationship with Pi-1、Pi-2、Pi-4a、Pi-4b, while 1 pairs of dominant resistance genes existing allelic relationship with Pi-3.4. Based on conserved regions of the known NBS-LRR type R genes, a pair of degenerate primers PR2 and ER4 was designed. Using PCR method, the NBS-LRR type RGAs (resistance gene analogues) were isolated from 10 sterile lines( Fuyi A, Gufeng A etc.), 11 parents (Gunong13, V41B, etc.) and susceptible control LTH. The similarity and evolution relations of cloned RGAs were analysed. The reuslts indicated total 33 NBS-LRR type RGAs were obtained (2 RGAs from Gunong 13, Fuyi A, Digu B, Changfeng A, Xiafeng A, Lianfeng A, Lefeng A, V41B, Longtepu B, Bobai B, Jin 23B, respectively; 1 RGAs from Tiangu B, Gufeng A, Anfeng A, Quanfeng A, Changfeng A, Fufeng A, LTH, R931022, Y20, Y27 and Y12, respectively.). The deduced amino acids from 33 RGAs analysis showed 33 RGAs were divided into 12 classes by Clustal W and DNAstar. Further cluster analysis indicated 33 RGAs were divided into 9 classes by DNAsis software. Homology analysis showed sequence identity among the deduced amino acid sequences identity from 33 RGAs ranged from 19.8 % to 98.8%.更多还原