松杨栅锈菌无毒基因型性状分离及AvrL567同源序列分析

doi:10.11707/j.1001-7488.20170511

摘要/Abstract

摘要： [目的] 分析松杨栅锈菌小种无毒基因型性状分离规律和其无毒基因序列与欧美小种无毒基因序列的差异和系统学关系。[方法] 以秦岭厚畛子落叶松上松杨栅锈菌2号小种HZ3542的性子器为雄性亲本，与秦岭火地塘落叶松上2号小种HF2369性子器为雌性亲本进行人工有性杂交，初步获得F₁代锈孢子堆。通过对太白杨离体叶片接种反应型统计，分析松杨栅锈菌2号生理小种无毒基因型，并根据亚麻锈菌无毒基因AvrL567（accession：AAS66952）设计和筛选引物，利用同源扩增技术，对松杨栅锈菌无毒基因片段进行PCR扩增，经测序、比对后构建最大似然系统树。[结果] 2号生理小种无毒基因表现型符合孟德尔分离定律，来自秦岭厚畛子太白杨不亲和性亲本为Aa无毒基因型，来自秦岭火地塘的亲和性亲本为aa基因型。在F₁代锈孢子堆群体（P=0.01）和F₁代夏孢子堆群体（P=0.05）中，抗∶感表现型均按1∶1分离。无毒基因型以坏死斑有无和褪绿斑大小为重要标准，潜育期长短和夏孢子堆密度、直径大小等数量性状为辅助标准进行判断，并在PCR扩增中得到检验和证实。筛选引物对AvrPr1（5’-TAATCCTCGTTGACATCAGTC-3’，5’-AAGCTTGAGAGCTCCGCTC-3’，T_m=53.5℃）可以稳定扩增出800~900 bp的产物。ML系统树表明，真菌无毒基因序列总体上同源性较低，但本研究所供试的17条序列可分为2个群，栅锈菌无毒基因序列聚在一个分支上，其中5条中国松杨栅锈菌无毒基因序列同加拿大2条MLP无毒基因序列聚在同一个亚分枝上，另一条序列与法国松杨栅锈菌、美国亚麻锈菌等菌聚在另一个亚分支上。[结论] 中国松杨栅锈菌2号小种无毒基因为单显性遗传，其无毒基因序列同加拿大的小种无毒基因序列同源性高。

关键词: 松杨栅锈菌, 无毒基因, 遗传分离, 系统进化树

Abstract: Rust fungus of Melampsora larici-populina (abbr. MLP) is a great enemy of poplar industry in China, and it threatened short rotation forest seriously, including poplars of Sect. Tacamachacae, and Sect. Aigerios, and their hybrids. [Objective]To understand the genetics of avirulence genes in their family, and to detect the phylogenic relationship of avirulence gene sequences between Chinese MLP and the other races in European and USA.[Method] Population of F₁ aecia of MLP was harvested by cross-fertilization with spermogonia of physiologic race 2 (MLP₂) collected from Houzhengzi (in which, isolate HZ3542 as a male maternal,incompatible with Populus purdomii) and Huoditang (in which, isolate HF2369 as a female maternal, compatible with purdomii poplar) in Qinling Mountain, and segregation pattern of avirulence genes in MLP₂ family as detected and inoculating aciospores and urediospores on P.purdomii leaves in vitro. Primers were designed and selected by referring to AvrL5671-8;9-16;17-26;27-37;38-45;46-56;57-67;68-74;75-81;82-88;89-94;95-105;106-112;113-123;124-130;131-137;138-144;145-152;153-183;160-167;168-176;177-183;184-191;192-197;198-203;204-211;212-224(accession: AAS66952) in Melampsora lini. Avirulence genes were amplified by PCR homogenized technology and then sequenced. All putative sequences were revised and aligned with other avirulence genes in the related public molecular information data, and a maximum likehood (abbr.,ML) phylogeny tree was then constructed. [Result] Segregation of avirulence genes in MLP₂ was coincident to the Mendel's law, and showed a single dominant heterozygous phenotype (Aa) in the incompatible isolate, and homozygote phenotype (aa) in the compatible isolate. Their F₁ population in both aeciospores (P=0.01) and urediospores (P=0.05) segregated by following 1∶1 with dominant to recessive phenotype. The certification for dominant avr phenotype is revised and confirmed by combination with quality traits of necrosis and chlorosis, quantitative trait loci (QTLs) including latent days, both diameter size and density of uredinia. PCR productions were also used to test and confirm avirulence phenotype under annealing by selected primer pair AvrPr1 (5'-TAATCCTCGTTGACATCAGTC-3',5'-AAGCTTGAGAGCTCCGCTC-3'). ML phylogeny tree differentiated two groups from all tested sequences at a low bootstrap data. Among them, all Melampsora sequences were grouped together with a relatively high homology, and five of six AvrL567 sequences from China MLP₂ isolates grouped into a sub-clad together with two Canada MLP sequences, and another one was grouped into another sub-clad with French MLP, USA M. lini, etc.[Conclusion] Incompatible race 2 of MLP in China was a phenotype of Aa, and its avirulence gene sequences showed a high homology with that of tested Canada isolates.

Key words: Melampsora larici-populina, avirulence gene, segregation law, phylogeny tree

中图分类号:

S718.81

余仲东, 陈祖静, 曹支敏, 任争争, 冯世强, 张瑶琦. 松杨栅锈菌无毒基因型性状分离及AvrL567同源序列分析[J]. 林业科学, 2017, 53(5): 88-96.

Yu Zhongdong, Chen Zujing, Cao Zhimin, Ren Zhengzheng, Feng Shiqiang, Zhang Yaoqi. Segregation Patterns and Phylogeny Analysis of AvrL567 Gene in Melampsora larici-populina[J]. Scientia Silvae Sinicae, 2017, 53(5): 88-96.

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