基于毒性表型和基因型的主要冬繁区小麦条锈菌群体遗传分析

doi:10.3864/j.issn.0578-1752.2023.14.001

摘要/Abstract

摘要：

【目的】明确中国主要冬繁区小麦条锈菌群体毒性结构和遗传多样性，为冬繁区及黄淮海麦区小麦条锈病的防控及小麦抗性基因的合理布局提供参考依据。【方法】从四川盆地、湖北和河南南部等主要冬繁区采集并分离得到148个小麦条锈菌菌株，利用中国鉴别寄主和单基因系鉴别寄主进行毒性表型鉴定；并利用17对KASP-SNP引物对菌株进行标记，完成基因型分析。【结果】基于中国鉴别寄主共鉴定出14个已知小种和63个未知致病类型，其中CYR34（16.2%）、G22-14（12.2%）、CYR32（6.8%）、CYR33（5.4%）为优势小种（致病类型）；基于单基因系鉴别寄主鉴定得到113个小种（致病类型），其中race1（7.4%）、race2（3.4%）、race3（3.4%）为优势小种（致病类型）。贵农22类群是中国冬繁区小麦条锈菌群体的最大流行类群，供试条锈菌均不侵染携带Yr5和Yr15的单基因系品种。单基因系毒性鉴定及分子标记均显示CYR34和G22-14的毒性表型及基因型呈现多样化，表明这两个优势小种内部存在高度分化。基于两套鉴别寄主的毒性数据聚类显示，四川盆地与湖北南部条锈菌群体相似，而湖北西北部与河南南部条锈菌群体相似；基于KASP-SNP分子数据的遗传聚类显示，四川盆地、湖北南部条锈菌群体与湖北西北部、河南南部条锈菌群体基因型存在分化；Structure分析显示四川盆地、湖北南部群体主要有2种遗传背景，湖北西北部、河南南部群体主要有一种遗传背景；群体遗传分化分析显示四川盆地条锈菌群体与河南南部条锈菌群体二者Fst值最大，为0.118，遗传差异最大且遗传分化明显；湖北西北部群体与河南南部群体遗传分化程度最小，Fst值为0.010；基因流分析得到湖北西北部群体与河南南部群体之间的Nm值为25.236，Nm>4，二者存在较大的基因流，湖北西北部和河南南部群体与四川盆地群体之间的Nm值分别为2.923和1.864，均存在较小的基因流；遗传多样性分析结果表明，四川盆地、湖北南部地区条锈菌群体遗传多样性水平均较高，湖北西北部、河南南部条锈菌群体遗传多样性水平较低。上述结论均支持四川盆地、湖北南部群体同湖北西北部、河南南部群体存在遗传分化。【结论】单基因系鉴别寄主能够精准地进行中国小麦条锈菌小种鉴定；中国主要冬繁区的小麦条锈菌群体存在不同来源。

关键词: 小麦条锈菌, 冬繁区, 毒性鉴定, KASP-SNP

Abstract:

【Objective】To clarify the virulence structure and genetic diversity of Pst populations in the major winter-increasing areas of China, and to provide reference for the prevention and control of Pst and the rational layout of wheat resistance genes in the winter-increasing areas and the wheat production in Huang-huai-hai. 【Method】A total of 148 Pst isolates were collected and isolated from the major winter-increasing areas such as Sichuan Basin, Hubei and southern Henan, and the virulence phenotype was identified by using Chinese differentials and single-gene lines, and 17 pairs of KASP-SNP primers were used to mark the isolates and complete the genotype analysis. 【Result】Based on the Chinese differentials, 14 known races and 63 unknown pathotypes were identified, among which CYR34 (16.2%), G22-14 (12.2%), CYR32 (6.8%), CYR33 (5.4%) were the dominant races (pathotypes); based on the single-gene lines, 113 races (pathotypes) were identified, among which race1 (7.4%), race2 (3.4%), race3 (3.4%) were the dominant races (pathotypes). The Guinong 22 group was the largest epidemic group of Pst population in China’s winter-increasing area, and all tested Pst isolates did not infect single-gene lines varieties carrying Yr5 and Yr15. The virulence phenotype and genotype of CYR34 and G22-14 showed diversification by single-gene lines virulence identification and molecular marker, indicating that there was high differentiation within these two dominant races. The clustering based on the virulence data of two sets of differentials showed that the Pst populations in Sichuan Basin and southern Hubei were similar, while the Pst populations in northwestern Hubei and southern Henan were similar; the genetic clustering based on KASP-SNP molecular data showed that there was genotype differentiation between the Pst populations in Sichuan Basin, southern Hubei and northwestern Hubei, southern Henan; Structure analysis showed that Sichuan Basin, southern Hubei population mainly had two genetic backgrounds, northwestern Hubei, southern Henan population mainly had one genetic background; population genetic differentiation analysis showed that Sichuan Basin Pst population and southern Henan Pst population had the largest Fst value, which was 0.118, with the largest genetic difference and obvious genetic differentiation; northwestern Hubei population and southern Henan population had the smallest degree of genetic differentiation, Fst value was 0.010; gene flow analysis obtained Nm value between northwestern Hubei population and southern Henan population was 25.236, Nm>4, there was a high-level gene flow between them, northwestern Hubei and southern Henan population and Sichuan Basin population had Nm values of 2.923 and 1.864 respectively, both had a low-level gene flow; genetic diversity analysis results showed that Sichuan Basin, southern Hubei region Pst population had a high-level of genetic diversity, northwestern Hubei, southern Henan Pst population had a low-level of genetic diversity. The above conclusions all support that Sichuan Basin, southern Hubei population has genetic differentiation with northwestern Hubei, southern Henan population. 【Conclusion】Single-gene lines can accurately identify Chinese Pst races; Pst populations in China’s major winter-increasing areas have different sources.

Key words: wheat stripe rust, winter-increasing area, virulence identification, KASP-SNP

高新培, 赵鋆, 刘博凡, 郭一, 康振生, 詹刚明. 基于毒性表型和基因型的主要冬繁区小麦条锈菌群体遗传分析[J]. 中国农业科学, 2023, 56(14): 2629-2642.

GAO XinPei, ZHAO Jun, LIU BoFan, GUO Yi, KANG ZhenSheng, ZHAN GangMing. Population Genetic Analysis of Puccinia striiformis tritici in Main Winter-Increasing Areas Based on Virulent Phenotypes and Genotypes[J]. Scientia Agricultura Sinica, 2023, 56(14): 2629-2642.

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地点 Location	经纬度 Latitude and longitude	海拔 Elevation (m)	样本数 No. of isolate
四川省绵阳市安州区黄土村 Huangtu Village, Anzhou District, Mianyang City, Sichuan Province	104°87′37.72″E，31°78′74.90″N	500	11
四川省绵阳市梓潼县柴坝村 Chaiba Village, Zitong County, Mianyang City, Sichuan Province	105°14′87.27″E，31°58′72.00″N	480	11
四川省绵阳市游仙区新桥镇 Xinqiao Town, Youxian District, Mianyang City, Sichuan Province	104°81′22.71″E，31°55′41.80″N	482	40
四川省泸州市古蔺县丹桂镇 Dangui Town, Gulin County, Luzhou City, Sichuan Province	106°24′11.42″E，27°93′17.21″N	716	19
湖北省荆州市江陵县普济镇 Puji Town, Jiangling County, Jingzhou City, Hubei Province	112°54′15.12″E，29°97′78.52″N	29	19
湖北省随州市曾都区何店镇 Hedian Town, Zengdu District, Suizhou City, Hubei Province	113°33′22.60″E，31°59′22.01″N	77	4
湖北省随州市曾都区万店镇 Wandian Town, Zengdu District, Suizhou City, Hubei Province	113°50′17.24″E，31°83′14.21″N	93	3
湖北省襄阳市枣阳市兴隆镇 Xinglong Town, Zaoyang City, Xiangyang City, Hubei Province	112°90′71.50″E，32°04′67.11″N	157	2
湖北省襄阳市宜城市板桥店镇 Banqiao Dian Town, Yicheng City, Xiangyang City, Hubei Province	112°51′66.40″E，32°31′74.71″N	59	12
湖北省襄阳市襄州区黄集镇 Huangji Town, Xiangzhou District, Xiangyang City, Hubei Province	112°14′03.88″E，32°30′88.91″N	101	5
湖北省鄂州市华容区华容镇 Huarong Town, Huarong District, Ezhou City, Hubei Province	114°73′65.19″E，30°53′50.51″N	37	6
河南省南阳市淅川县厚坡镇 Houpo Town, Xichuan County, Nanyang City, Henan Province	111°76′79.90″E，32°60′77.39″N	147	16

SNP ID	CY32 Contig	CY32_PosIn	正向引物Forward primer (5′-3′)		反向引物 Reverse primer (5′-3′)
SNP ID	CY32 Contig	CY32_PosIn	正向引物-1 Forward primer-1	正向引物-2 Forward primer-2	反向引物 Reverse primer (5′-3′)
KASP1	1	75964	GTGGCTCCTGGTGTAAGTTCC	GTGGCTCCTGGTGTAAGTTCT	GTGGCCCAAACTACGCTAATTTA
KASP24	4	43862	GGCTTTTCCATTTAGGTCCTTT	GGCTTTTCCATTTAGGTCCTTC	TGAGGCTGATTGAATTGTGCTT
KASP93	13	397712	ATATCCTGAAAGAACTTGAGTGATTG	ATATCCTGAAAGAACTTGAGTGATTT	AGGCTTGGTTCGCTATACTCG
KASP150	23	3805	TGTTGATTTTATTTTACAGAGCGATT	TGTTGATTTTATTTTACAGAGCGATC	CACCTTAAGAAAATTGCGGGA
KASP153	23	3805	TGTTGATTTTATTTTACAGAGCGATT	TGTTGATTTTATTTTACAGAGCGATC	CACCTTAAGAAAATTGCGGGA
KASP157	24	223357	TGTACTAGTTGGCTTCTCAAGTTCTATT	TGTACTAGTTGGCTTCTCAAGTTCTATC	ATCACAGAACCTCCAGAGTGAAAT
KASP159	24	654444	CTTTGAATTTGAGGTGATGGCA	CTTTGAATTTGAGGTGATGGCG	CACGACTTCTGACTAGAACTGCTTG
KASP162	25	232930	AACCAGCCCTGCTCATTGAAATA	AACCAGCCCTGCTCATTGAAATC	CAGTGGATGTGATGCGTGAGGT
KASP167	26	419659	AAGGTAGGTCGGATATATTTGTCATCT	AAGGTAGGTCGGATATATTTGTCATCC	CCACTCATGGGCGAAGTTTT
KASP172	27	561290	ATCAAAACAAAGAACCCATCCCA	ATCAAAACAAAGAACCCATCCCG	CTGAATGTGATTGATTGCCAGGA
KASP177	28	702014	ACAAATTGTCCAACTGGAAAATCA	ACAAATTGTCCAACTGGAAAATCG	GCCAATCGGTGTCCTTTACG
KASP203	34	225219	CATTGTGACTATCAAGAAAGGATTGT	CATTGTGACTATCAAGAAAGGATTGC	GGAACCATCCGTCTAAACCC
KASP261	49	33389	TGAGGAGTACTTTCGATAGAATCAGG	TGAGGAGTACTTTCGATAGAATCAGA	GCCAGCATTCTCCCTCTTACAC
KASP285	56	96991	TAGTTCGTCGATTCCCAAGTTTG	TAGTTCGTCGATTCCCAAGTTTA	GATGCCGTCGTGATTGTTGC
KASP373	93	6570	CTGTTTGTCAATCGGACCAGC	CTGTTTGTCAATCGGACCAGT	TGAATAATTCGGTGAAGAGTTTAGTG
KASP392	108	154635	ATTCCACCCACATCAAGACCA	ATTCCACCCACATCAAGACCG	TACCCTCAGTCAAGTTCATCTATCTACC
KASP436	scaffold314	50233	TTCGTGGGTTACGGTGAGCTT	TTCGTGGGTTACGGTGAGCTC	AGTGGCACAGTCACCCCTCATA

毒性类群 Group	小种 Race	数量 Number	毒性频率 Frequency (%)	总毒性频率 Total frequency (%)
贵农22类群 Guinong 22 group	CYR34	24	16.2	40.0
	G22-14	18	12.2
	G22-13	2	1.4
	未知Unknown	15	10.1
杂种46类群 Hybrid 46 group	CYR32	10	6.8	29.7
	HY-21	3	2.0
	HY-8	2	1.4
	HY-30	2	1.4
	CYR31	1	0.7
	未知Unknown	26	17.6
水源11类群 Suwon 11 group	CYR33	8	5.4	18.9
	水11-41 Shui 11-41	1	0.7
	未知Unknown	19	12.8
洛夫林13类群 Lovrin 13 group	洛13-2 Luo 13-2	4	2.7	8.8
	洛13-8 Luo 13-8	3	2.0
	洛13-3 Luo 13-3	2	1.4
	未知Unknown	4	2.7
其他 Other	CYR24	2	1.4	2.7
其他 Other	未知Unknown	2	1.4	2.7

地区 Region	四川盆地 Sichuan Basin	湖北南部 Southern Hubei	湖北西北部 Northwestern Hubei	河南南部 Southern Henan
1	CYR34(9.9)	CYR34(28.1)	CYR34(20.0)	CYR34(18.8)
2	G22-14(9.9)	G22-14(12.5)	G22-14(15.6)	G22-14(18.8)
3	CYR32(4.9)	CYR32(12.5)	CYR33(11.1)	G22-13(12.5)

地区 Region	四川盆地 SCB	湖北南部 S-HB	湖北西北部 NW-HB	河南南部 S-HN
SCB		0.019	0.079	0.118
S-HB	13.123		0.036	0.036
NW-HB	2.923	6.631		0.010
S-HN	1.864	6.738	25.236