黑龙江省和海南省稻瘟病菌中AVR-Pita家族的分布及变异分析

doi:10.3864/j.issn.0578-1752.2023.03.006

摘要/Abstract

摘要：

【目的】通过检测黑龙江省和海南省不同稻瘟病菌（Magnaporthe oryzae）菌株群体中AVR-Pita家族的分布情况和变异特征，了解其变异类型的致病表型，为区域内抗病种质的筛选与选育提供参考。【方法】通过参考NCBI中公布的AVR-Pita家族序列分别对启动子区和CDS区设计特异性引物共6对，对2020年采自黑龙江省和海南省不同地区的397个稻瘟病菌单孢菌株提取DNA，进行PCR扩增。通过电泳检测结果，分别选取囊括不同地区的代表菌株对扩增片段进行测序。测序结果与NCBI中相应的碱基与氨基酸序列进行比较分析，并利用水稻抗性单基因系，对不同变异类型的稻瘟病菌菌株进行无毒功能验证。【结果】在PCR电泳检测结果中，黑龙江省稻瘟病菌菌株携带所有待测基因，分布广泛且检出频率较高；而海南省稻瘟病菌菌株中仅携带AVR-Pita1和AVR-Pita2，并以低频率集中存在。无毒基因组成分析结果显示，黑龙江稻区的稻瘟病菌菌株复杂多样，携带的基因型种类较海南菌株丰富。PCR产物测序检测结果显示，AVR-Pita家族以点突变、插入和缺失为主要变异类型划分为19种,且不同稻瘟病菌群体来源的菌株，其变异类型具有特异性。AVR-Pita1检测出10种变异类型，其中AVR-Pita1-（1—5）为黑龙江省稻瘟病菌菌株独有的变异类型，AVR-Pita1-（6—10）为海南省稻瘟病菌菌株独有。对这10种变异类型经功能验证后发现，无毒功能均已丧失。AVR-Pita2检测出8种变异类型，其中AVR-Pita2-（1—4）为黑龙江省稻瘟病菌菌株独有，AVR-Pita2-（5—8）为海南省稻瘟病菌菌株独有。经功能验证后发现，无毒功能均已丧失。AVR-Pita3在黑龙江省仅检测出1种变异类型AVR-Pita3-1。【结论】黑龙江和海南稻瘟病菌群体中AVR-Pita家族均为突变后的等位基因型存在，经致病性鉴定后，检测出的所有突变类型均不能被相对应抗性基因Pi-ta和Pi-ta²所识别，表现为感病。因此，抗性基因Pi-ta和Pi-ta²在黑龙江省和海南省对稻瘟病的抗病育种与利用过程中可聚合其他抗性基因应用来保证品种抗病性。同时，不同地理来源的稻瘟病菌菌株群体中AVR-Pita家族的分布和变异类型具有特异性。

关键词: 稻瘟病菌, AVR-Pita家族, 变异, 无毒基因, 黑龙江省, 海南省

Abstract:

【Objective】By detecting the distribution and variation characteristics of the AVR-Pita family in different Magnaporthe oryzae strains from Heilongjiang Province and Hainan Province, the pathogenic phenotypes of the variation types were understood, which provided a reference for the screening and breeding of disease-resistant germplasm in the region.【Method】Six pairs of specific primers were designed for the promoter region and CDS region by referring to the AVR-Pita family sequence published in NCBI. DNA was extracted from 397 single spore strains of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020, and PCR amplification was performed. By electrophoresis test results, representative strains from different regions were selected to sequence the amplified fragments. The sequencing results were compared with the corresponding base and amino acid sequences in NCBI, and the avirulence function of different variation types of M. oryzae strains was verified by using rice resistant single gene lines.【Result】In the PCR electrophoresis results, the M. oryzae strains in Heilongjiang Province carried all the genes to be tested, which were widely distributed and had a high detection frequency. However, only AVR-Pita1 and AVR-Pita2 were carried by M. oryzae strains in Hainan Province, and concentrated in low frequency. The results of avirulence gene composition analysis showed that the strains of M. oryzae in Heilongjiang were complex and diverse, and the genotypes were more abundant than those in Hainan. The sequencing results of PCR products showed that the AVR-Pita family was divided into 19 types with point mutation, insertion and deletion as the main variation types, and the variation types of strains from different M. oryzae populations were specific. Ten variation types were detected in AVR-Pita1, among which AVR-Pita1- (1-5) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita1- (6-10) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification of these ten variation types, it was found that the avirulence functions were lost. Eight variation types were detected in AVR-Pita2, among which AVR-Pita2- (1-4) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita2- (5-8) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification, it was found that the avirulence functions were lost. Only one variation type (AVR-Pita3-1) was detected in AVR-Pita3 in Heilongjiang Province.【Conclusion】The AVR-Pita family in Heilongjiang and Hainan populations were mutated alleles. After pathogenicity identification, all mutation types could not be identified by the corresponding resistance genes Pi-ta and Pi-ta². Therefore, the resistance genes Pi-ta and Pi-ta² can be used to polymerize other resistance genes to ensure the disease resistance of varieties in the process of disease resistance breeding and utilization of rice blast in Heilongjiang Province and Hainan Province. At the same time, the distribution and variation types of AVR-Pita family in M. oryzae strains from different geographical sources are specific.

Key words: Magnaporthe oryzae, AVR-Pita family, mutation, avirulence gene, Heilongjiang Province, Hainan Province

刘瑞, 赵羽涵, 顾欣怡, 王艳霞, 靳学慧, 吴伟怀, 张亚玲. 黑龙江省和海南省稻瘟病菌中AVR-Pita家族的分布及变异分析[J]. 中国农业科学, 2023, 56(3): 466-480.

LIU RUI, ZHAO YuHan, GU XinYi, WANG YanXia, JIN XueHui, WU WeiHuai, ZHANG YaLing. Distribution and Variation Analysis of AVR-Pita Family in Magnaporthe oryzae from Heilongjiang Province and Hainan Province[J]. Scientia Agricultura Sinica, 2023, 56(3): 466-480.

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省份 Province	采集地点 Sampling location	菌株编号 Strain number	菌株数 Number of strains
黑龙江省（215^a） Heilongjiang Province（215^a）	绥化北林区Beilin District, Suihua	BL20001-BL20006	6
	绥化庆安Qing’an, Suihua	QA20007-QA20009	3
	绥化青冈Qinggang, Suihua	QG20010-QG20012	3
	伊春铁力Tieli, Yichun	TL20013-TL20018	6
	哈尔滨方正Fangzheng, Harbin	FZ20019-FZ20044	26
	哈尔滨通河Tonghe, Harbin	TH20045-TH20069	25
	哈尔滨木兰Mulan, Harbin	ML20070-ML20081	12
	哈尔滨依兰Yilan, Harbin	YL20082-YL20085	4
	鹤岗绥滨Suibin, Hegang	SB20086-SB20102	17
	佳木斯富锦Fujin, Jiamusi	FJ20103-FJ20106	4
	佳木斯东风区Dongfeng District, Jiamusi	DF20199-DF20215	17
	鸡西虎林Hulin, Jixi	HL20107-HL20110	4
	鸡西密山Mishan, Jixi	MS20111-MS20169	59
	牡丹江东宁Dongning, Mudanjiang	DN20170-DN20198	29
海南省（182^b） Hainan Province（182^b）	东方感城Gancheng, Dongfang	DF20001-DF20004	4
	文昌铺前Puqian, Wenchang	WC20005-WC20038	34
	定安定城Dingcheng, Dingan	DC20039-DC20065	27
	定安雷鸣Leiming, Dingan	LM20066-LM20084	19
	陵水Lingshui	LS20085-LS20094	10
	澄迈金江Jinjiang, Chengmai	CM20095-CM20182	88

无毒基因 Avr-gene	引物序列 Primer sequence (5°-3°	片段大小 Length of targeted fragments (bp)	登录号GenBank accession number
AVR-Pita1	F1：GCCGAGTCGTTCTGA	475	AF207841
	R1：TGTTAATTGTGCAGAAGTTTTT	475
	F2：TGCCAATAGACTAGCTTCCG	1957
	R2：ATTCCCTCCATTCCAACACT	1957
AVR-Pita2	F1：CTTAGAGTAGAATCTTCGTCG	481	AB607344
	R1：GTATGAAAAGGTGCCAATG	481
	F2：TTGGCACCTTTTCATACCCAGTTT	687
	R2：CAACTTACTTGTGAATCCCATCCC	687
AVR-Pita3	F1：AAATATTACCTGCCAGCTGG	218	DQ855958
	R1：TTAAAGCCCCGTGGATATCA	218
	F2：AAATATTACCTGCCAGCTGG	692
	R2：GCGCTAGTTCATGCATAATC	692

基因型 Genotype	黑龙江Heilongjiang		海南Hainan
基因型 Genotype	菌株数 Number of strains	频率Frequency (%)	菌株数 Number of strains	频率Frequency (%)
AVR-Pita(f)	10	4.65	92	50.55
AVR-Pita2	3	1.40	0	0
AVR-Pita3	22	10.23	0	0
AVR-Pita1+AVR-Pita2	19	8.84	90	49.45
AVR-Pita1+AVR-Pita3	3	1.40	0	0
AVR-Pita2+AVR-Pita3	3	1.40	0	0
AVR-Pita1+AVR-Pita2+AVR-Pita3	155	72.09	0	0
AVR-Pita(f)：不含待测基因的基因型，其中，f表示不存在 Genotypes that do not contain the gene to be tested, where, f stands for free

等位基因型 Allele genotype	碱基突变位点Base mutation site (bp)
等位基因型 Allele genotype	-413	-376	-246	-116	-52	18—20	207	245	247	263	312	407	520	575	580	583
AVR-Pita1	TGA	TTA	CAA	TAA	AAC	***	TGT	AAT	GAC	AGA	AAG	GGA	GTT	TAC	TGG	GAT
AVR-Pita1-2	-A-	--G	T--	-C-	--A	GTT	--C	-G-	A--	-A-	--T	---	A--	-G-	---	---
AVR-Pita1-3	-A-	--G	---	-C-	--A	GTT	--C	-G-	A--	-A-	--T	---	A--	-G-	---	---
AVR-Pita1-4	-A-	--G	T--	-C-	--A	GTT	--C	-G-	A--	-A-	--T	-A-	A--	-G-	---	---
AVR-Pita1-5	-A-	--G	T--	-C-	--A	GTT	--C	-G-	A--	-A-	--T	-A-	A--	---	---	---
AVR-Pita1-6	---	---	---	-C-	--A	GTT	--C	-G-	A--	-A-	--T	-A-	A--	---	C--	---
AVR-Pita1-7	---	--G	---	-C-	--A	GTT	--C	-G-	A--	-A-	--T	-A-	A--	---	C--	---
AVR-Pita1-8	---	---	T--	-C-	--A	GTT	--C	-G-	A--	-A-	--T	-A-	A--	---	C--	---
AVR-Pita1-9	---	---	T--	---	---	GTT	---	---	---	---	---	---	---	-T-	---	C--
AVR-Pita1-10	-A-	--G	T--	-C-	--A	GTT	---	---	---	---	---	---	---	-T-	---	C--

等位基因型 Allele genotype	碱基突变位点Base mutation site (bp)
等位基因型 Allele genotype	55	103	122	135	148	183	267	307	315	319—321	333	479	484	508
AVR-Pita3	ATA	TCA	GCT	CGG	AAA	GAA	TTC	GAA	AAC	AAG	GAA	CAA	GAG	ACC
AVR-Pita3-1	G--	C--	-T-	--A	C--	--C	--A	A--	--T	CGT	--C	-G-	A--	G--