水稻褐飞虱抗性基因的遗传定位及近等基因系的构建和抗性评价

发布时间：2018-07-14 09:11

【摘要】：水稻(Oryza sativa L.)是全球超过一半人口的主食。褐飞虱(Nilaparvata lugens St?l,BPH)是水稻最为严重的虫害之一。控制褐飞虱最为经济环保的策略是利用水稻抗性基因培育抗虫品种。目前,水稻中已经报道了30个抗性基因,其中7个已被克隆。然而,真正应用于育种的基因比较少,且大多数基因是在不同遗传背景下鉴定的,其效应和育种前景难以准确评价。因而,在本研究中,我们利用图位克隆的方法发掘了新的抗性基因,并通过构建分子标记辅助选择(marker-assisted selection,MAS)体系和回交育种,构建了前人已定位或克隆的13个抗性基因在9311背景下的近等基因系(near-isogenic line,NIL),并结合基因型和抗性表型,准确区分和评价了这些抗性基因的效应。主要结果如下:利用分子标记遗传连锁分析,我们在感虫品种9311和抗虫品种AUS69杂交衍生的F2:3群体中,定位到两个主效QTL,并分别暂时命名为Bph30和Bph31。它们分别位于第4号染色体长臂标记J63和RM252之间和第11染色体短臂标记RM536和RM1355之间,加性效应分别为1.56和1.14,LOD值分别为9.46和6.18,解释的表型变异分别为11.65%和5.95%。通过重组单株的筛选及其基因型和表型分析,我们最终把Bph31定位在标记11-165和In88之间(400 kb)。通过参考序列日本晴在此区段的基因注释,我们找到了两个卷曲螺旋(coiled-coil,CC),核苷酸结合位点(nucleotide-bingding site,NBS),亮氨酸重复(leucine-rich repeat,LRR)(CC-NBS-LRR)类基因,分别命名为LRR1和LRR2。我们测序并比较两个亲本9311和AUS69在这两个基因上的差异,但只测通亲本AUS69的LRR1的前部分和LRR2的全长基因。我们对比了LRR1和LRR2分别在AUS69和日本晴中的氨基酸序列,发现这两个基因在两品种间都有大量变异,结果初步表明,LRR1和LRR2可能在AUS69的抗性中起到一定的作用,但还有待遗传转化的验证。利用MAS和回交育种技术,我们将前人报道的来源于9个供体的13个褐飞虱抗性基因如RH(Bph3和Bph17)、B5(Bph14和Bph15)、IR54751-1-2-44(QBph3和QBph4)、IR65482-4-136(Bph10)、IR71033-121(Bph20和Bph21)、IR65482-7-216(Bph18)、Swarnalata(Bph6)、Pokkali(Bph9)和BR96(Bph24)等,分别导入到9311中,通过正向选择、反向选择和RICE6K芯片的背景筛选,我们最终得到了13个9311背景的NILs。我们考查了这13个NILs的苗期抗性,褐飞虱在NILs上的蜜露分泌量和存活率。结果显示,抗性基因的导入显著地提高了褐飞虱抗性,减少了褐飞虱的蜜露分泌量和存活率。在苗期抗性方面,Bph24-NIL和Bph6-NIL这两个家系表现为高抗水平,抗性等级分别为1.31和1.96;其次是表现为抗的家系Bph3-NIL、QBph3-NIL、Bph15-NIL、Bph9-NIL和QBph4-NIL,抗性等级分别为2.34、2.09、2.38、2.42和2.49;再次是表现为中抗的家系Bph17-NIL、Bph20-NIL和Bph14-NIL,抗性等级分别为2.74、3.16和3.37;最后是表现为中感的家系Bph10-NIL、Bph21-NIL和Bph18-NIL,抗性等级分别为4.73、5.69和6.25。综合来看除Bph9外,第4号染色体上的抗性基因(QBph4、Bph17、Bph15、Bph20、Bph24和Bph6)普遍比第12号染色体上的抗性基因(Bph10、Bph18和Bph21)在9311背景下表现出更强的抗性。褐飞虱取食后蜜露的分泌量和存活率的趋势基本上与苗期抗性一致。本研究所用的13个抗性基因中存在三个基因簇内,其中已克隆的基因有Bph14、Bph17、Bph18和Bph26。根据序列比较Bph18和Bph26是的不同等位基因。Bph10和Bph21编码的氨基酸序列与Bph26完全一样。Bph9和Bph26在第一个和第二个外显子上有一些氨基酸的不同。QBph3编码的氨基酸序列跟Bph14的有明显的差异,它们在LRR区域有很多氨基酸的替换。Bph15编码的氨基酸序列和Bph17完全一致,而QBph4、Bph20和Bph24编码的氨基酸序列与Bph17相比有一些氨基酸的替换。基于上述基因簇内不同基因NILs的表型和氨基酸序列比较,我们推测Bph10、Bph21和Bph26,Bph15和Bph17可能是同一个基因;QBph3和Bph14,QBph4和Bph20可能是不同的等位基因;Bph9、Bph18和Bph26可能是复等位基因;而Bph24和Bph17可能是不同的基因。
[Abstract]:Rice (Oryza sativa L.) is the staple food for more than half of the world's population. Nilaparvata lugens St? L (BPH) is one of the most serious insect pests in rice. The most economical and environmentally friendly strategy for controlling the brown planthopper is to use rice resistance gene to breed the insect resistant varieties. At present, 30 resistant genes have been reported in rice, of which 7 have been cloned. However, there are fewer genes used in breeding, and most of the genes are identified in different genetic backgrounds, and their effects and breeding prospects are difficult to be accurately evaluated. Therefore, in this study, we have exploited the new resistance gene by using the method of map cloning and constructed the marker-assisted selection, MAS body by molecular marker. The near isogenic line (near-isogenic line, NIL) of 13 resistant genes which have been located or cloned by predecessors in the 9311 background were constructed, and the effects of these resistance genes were accurately distinguished and evaluated by combining with the genotypes and resistant phenotype. The main results are as follows: the molecular linkage analysis of the use of molecular markers, we are in the susceptible variety 9311. In the F2:3 population derived from AUS69 hybrid, two main effects QTL were located, and they were temporarily named Bph30 and Bph31., respectively, between J63 and RM252 on chromosome fourth and eleventh chromosome short arm markers RM536 and RM1355 respectively, the additive effects were 1.56 and 1.14 respectively, LOD values were 9.46 and 6.18, respectively. The variant was 11.65% and 5.95%. through the screening of the recombinant single plant and its genotype and phenotype analysis. We finally located Bph31 between marker 11-165 and In88 (400 KB). We found two curl helix (coiled-coil, CC), nucleotide binding site (nucleotide-bingdin) through the reference sequence of the gene annotation in this section of Japan. G site, NBS), leucine repeat (leucine-rich repeat, LRR) (CC-NBS-LRR) genes, named LRR1 and LRR2., respectively, we sequenced and compared the difference between the two parents 9311 and AUS69 in these two genes, but only measured the front part of the LRR1 and the full length gene. The sequence of amino acids found that the two genes had a large variation between two varieties. The results showed that LRR1 and LRR2 might play a role in the resistance of AUS69, but there was still to be verified by genetic transformation. Using MAS and backcross breeding techniques, we originated 13 brown planthopper resistance genes, such as RH (Bph3), from 9 donors. And Bph17), B5 (Bph14 and Bph15), IR54751-1-2-44 (QBph3 and QBph4), IR65482-4-136 (Bph10), IR71033-121 (Bph20 and Bph21), respectively, through forward selection, reverse selection and background screening of the chip, we finally get 13 9311 backgrounds. NILs. we examined the nectar secretion and survival rate of the 13 NILs seedlings on the NILs, and the results showed that the resistance gene was significantly increased by the resistance gene, and the honeydew secretion and survival rate of the brown planthopper were reduced. In the seedling resistance, the two families of Bph24-NIL and Bph6-NIL were highly resistant and resistant to high resistance. The sex levels were 1.31 and 1.96, followed by the resistant families Bph3-NIL, QBph3-NIL, Bph15-NIL, Bph9-NIL and QBph4-NIL, and the resistance grades were 2.34,2.09,2.38,2.42 and 2.49, and the secondary resistant families were Bph17-NIL, Bph20-NIL and Bph14-NIL, the resistance grade was divided into 2.74,3.16 and 3.37, and the last was the median sense of family Bph10. The resistance genes on chromosome fourth (QBph4, Bph17, Bph15, Bph20, Bph24 and Bph6) were generally more resistant than the resistance genes on chromosome twelfth (QBph4, Bph17, Bph15, Bph20, Bph24 and Bph6) on chromosome fourth. The resistance genes on chromosome twelfth were more resistant to the 9311 background. The secretion of honeydew from the brown planthopper after feeding. The trend of the quantity and survival rate is basically consistent with the resistance of the seedling stage. There are three gene clusters in the 13 resistant genes used in this study, of which the cloned genes are Bph14, Bph17, Bph18, and Bph26., according to the sequence, the.Bph10 and Bph21 encoded amino acid sequences of the different alleles of Bph18 and Bph26 are exactly the same as Bph26.Bph9 and Bph26. The amino acid sequences of different.QBph3 encoded amino acids with some amino acids in the first and second exons are distinctly different from that of Bph14. They have a lot of amino acids in the LRR region and the amino acid sequences encoded by a lot of amino acids are exactly the same as Bph17, while the amino acid sequences encoded by QBph4, Bph20 and Bph24 have some amino acids compared to Bph17. Based on the comparison of the phenotypic and amino acid sequences of different genes NILs in the above gene cluster, we speculate that Bph10, Bph21 and Bph26, Bph15 and Bph17 may be the same genes; QBph3 and Bph14, QBph4 and Bph20 may be different alleles; Bph9, Bph18 and may be complex alleles, and may be different genes.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.112.3

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