Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (2): 264-274.doi: 10.3864/j.issn.0578-1752.2023.02.005

• PLANT PROTECTION • Previous Articles     Next Articles

Distribution and Variation of PWL Gene Family in Rice Magnaporthe oryzae from Heilongjiang Province and Hainan Province

LIU RUI1(),ZHAO YuHan1,FU ZhongJu1,GU XinYi1,WANG YanXia1,JIN XueHui1,YANG Ying1,WU WeiHuai2,ZHANG YaLing1()   

  1. 1College of Agronomy, Heilongjiang Bayi Agricultural University/Heilongjiang Plant Resistance Research Center, Daqing 163319, Heilongjiang
    2Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101
  • Received:2022-08-02 Accepted:2022-09-06 Online:2023-01-16 Published:2023-02-07

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Abstract:

【Objective】The objective of this study is to understand the distribution and variation characteristics of PWL gene family in different Magnaporthe oryzae strains, and to provide a basis for studying the genetic diversity and specificity of different M. oryzae strains. 【Method】A total of 8 pairs of specific primers were designed for the promoter region and CDS region of the PWL gene family by referring to the avirulence gene sequence published in NCBI. DNA was extracted from 397 single spore isolates of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020. PCR amplification of avirulence genes was conducted and the genes were detected by agarose gel electrophoresis. Representative strains from different regions were selected from the test results to sequence and analyze the amplified fragments. The sequencing results were compared with the base and amino acid sequences of the corresponding avirulence gene promoter region and CDS region in NCBI. 【Result】In the results of PCR electrophoresis, PWL1 was not detected in all strains. The specific fragments of PWL2, PWL3 and PWL4 were amplified in Heilongjiang Province and Hainan Province, indicating that these three genes were distributed in both provinces and had different distribution frequencies and variation types. Among them, PWL2 had the highest distribution frequency in Heilongjiang Province and Hainan Province, which were 98.14% and 100%, respectively. The distribution frequencies of PWL3 and PWL4 in the two provinces were significantly different. The frequencies of these two genes in Heilongjiang strains were 89.30% and 82.79%, respectively, while in Hainan strains were 5.49%. Through the analysis of avirulence gene combinations, the results showed that the combination types could be divided into six types, namely PWL(f), PWL2, PWL3, PWL2+PWL3, PWL2+PWL4, PWL2+PWL3+PWL4. Among them, Heilongjiang strains contained all combination types, and Hainan strains contained only two, indicating that Heilongjiang strains were more abundant in avirulent genotypes than Hainan strains. By sequencing the PCR products of the PWL gene family, it was found that the PWL gene family had abundant variation sites in the promoter region and CDS region, and was divided into nine types with point mutations and deletions as the main variation types, and the variation types of strains from different populations were specific and consistent. Among them, five variant types PWL2- (1, 2, 3, 4, 5) were detected in PWL2, base sequence changes led to amino acid sequence missense mutations; two mutation types were detected in PWL3 and PWL4, which were PWL3- (1, 2) and PWL4- (1, 2), respectively. Both of them had frameshift mutations that caused changes in the following amino acids. 【Conclusion】The distribution and variation types of PWL gene family in M. oryzae strains from different populations have regional differences, and the variation sites are abundant.

Key words: Magnaporthe oryzae, PWL gene family, mutation, avirulence gene, Heilongjiang Province, Hainan Province

Table 1

The strains of M. oryzae isolated from Heilongjiang Province and Hainan Province"

省份Province 采集地点Sampling location 菌株编号<BOLD>S</BOLD>train number 菌株数Number of strains
黑龙江省（215a）
Heilongjiang Province（215a）		 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（182b）
Hainan Province（182b）		 东方感城Gancheng, Dongfang DF20001-DF20004 4
陵水Lingshui LS20085-LS20094 10
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
澄迈金江Jinjiang, Chengmai CM20095-CM20182 88

Table 2

Primers used for Avr-gene amplification"

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
PWL1 F1：TTTCACGCCCATTAACGG 1262 AB480169.1
R1：ACGCACAACACGAGGT
F2：GTCGCAAATGGACTAACA 395
R2：AAACCCTATTCCAGCAGT
PWL2 F1：TTCGGGCACTCCGTTACT 521 U26313.1
R1：CCTCTCTTTCGCCTTTG
F2：ATGAAATGCAACAACATC 495
R2：CCTCACACTTAAGTTAACAC
PWL3 F1：TGCGAGTAAAAGCCTGAA 548 U36995.1
R1：GTGGGTGTTGTAATAGCGAT
F2：GCTGGGAAGCCTGTCACCT 1412
R2：GTTCACGCAACTGGAGGG
PWL4 F1：TCCTCTGGAACATGACAATAGT 385 U36996.1
R1：TCCGTGGGTGTTGTAGTAGC
F2：CGGCTCATTGTCTGTCGTAAAGG 292
R2：TTCGCTGGTCTTTACAAACTCCC

Fig. 1

Electrophoretic diagram of PCR amplification of PWL gene family"

Table 3

Detection results of avirulent genotypes of tested strains"

基因型
Genotype		 黑龙江Heilongjiang 海南Hainan
菌株数 Number of strains 频率Frequency (%) 菌株数 Number of strains 频率Frequency (%)
PWL(f) 1 0.47 0 0
PWL2 25 11.63 172 94.51
PWL3 1 0.47 0 0
PWL2+PWL3 14 6.51 0 0
PWL2+PWL4 3 1.40 0 0
PWL2+PWL3+PWL4 171 79.53 10 5.49

Table 4

PWL2 base sequence alignment results of some strains"

等位基因型
Allele genotype		 碱基突变位点Base mutation site (bp)
-307 -223 -169 -152 -129 -73 -51 268
PWL2 CGC TAT GGT CAT TGG TCG GAA GAT
PWL2-1 --T --- --- --- --- --- --- ---
PWL2-2 --- --- --- --- --- --- --- A--
PWL2-3 --- --- --- --- --- --- A-- A--
PWL2-4 --- --G --G -T- G-- --T A-- A--

Fig. 2

Deletion of PWL2-5 fragment"

Fig. 3

Comparative analysis of amino acid sequences of PWL2-2"

Table 5

PWL3 base sequence alignment results of some strains"

等位基因型
Allele genotype		 碱基突变位点Base mutation site (bp)
-187 -151 301 363 391 408 409，411
PWL3 AAA AGT TAT GGG AAG GAC GAC
PWL3-1 --- --C *-- --A C-- --A T-G
PWL3-2 --* --C *-- --A C-- --A T-G

Fig. 4

Comparative analysis of amino acid sequences of PWL3-1"

Fig. 5

PWL4 base sequence alignment results of some strains"

Fig. 6

Comparative analysis of amino acid sequences of PWL4-1"

Fig. 7

Proportion analysis of variation types of PWL gene family"

Fig. 8

Phylogenetic tree analysis of PWL gene family genotypes"

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