中国东北和华北地区紫花苜蓿根瘤菌遗传多样性研究

doi:10.3864/j.issn.0578-1752.2021.16.003

摘要/Abstract

摘要：

【目的】紫花苜蓿（Medicago sativa L.）被誉为牧草之王,近年来,中国东北和华北地区种植面积不断扩大,但紫花苜蓿质量与产量仍不足以满足中国畜牧业发展的需求。本研究旨在分析中国东北和华北地区紫花苜蓿根瘤菌遗传多样性,为紫花苜蓿高效固氮根瘤菌的筛选与应用提供参考。【方法】采用表面消毒和平板划线法从根瘤中分离纯化根瘤菌单菌落;使用BOX-PCR方法对供试菌株进行基因型划分;选取代表菌株进行持家基因（atpD、glnII和rpoB）和共生基因（nifH和nodC）的系统发育分析。【结果】从中国东北和华北19个采样地共分离纯化了499株根瘤菌,BOX-PCR可将供试菌株分为37种BOX型,BOX型存在显著的地理分布现象,同时寄主品种对根瘤菌基因型具有一定的选择作用。97.60%（487/499）的根瘤菌为Sinorhizobium meliloti。其余12株分别为S. adhaerens、Mesorhizobium huakuii、Rhizobium loessense、R. mesosinicum、R. vignae、Mesorhizobium sp.和Phyllobacterium sp.,这12株根瘤菌仅在中国东北地区发现,华北地区根瘤菌均为S. meliloti。3个持家基因在紫花苜蓿根瘤菌种间系统发育分析结果一致,但其揭示优势种S. meliloti的种内多样性存在差异。共生基因系统发育结果显示,在根瘤菌属间和属内发生了共生基因的水平转移现象。nifH在S.meliloti种内显示出比nodC更丰富多样性。【结论】中国东北和华北地区紫花苜蓿根瘤菌具有较丰富的遗传多样性,且根瘤菌存在显著的地理分布特征和寄主选择现象。

关键词: 紫花苜蓿, 根瘤菌, 遗传多样性, 系统发育, 水平转移

Abstract:

【Objective】Medicago sativa is reputed to be the “Queen of Forages”, its cultivated area has increased in Northeast and North China in recent years. However, the quality and yield of M. sativa still do not meet the needs of livestock husbandry development in China. The objective of this study was to analyze the genetic diversity of rhizobia associated with M. sativa from Northeast and North China, which will provide valuable reference for screening and applying high efficient nitrogen fixing rhizobia from M. sativa.【Method】Surface sterilization and plate streaking method were used to recover and purify the rhizobia from M. sativa root nodules; BOX-PCR was applied to identify the genotypes of the tested rhizobia; Three housekeeping (atpD, glnII and rpoB) and two symbiotic genes (nodC and nifH) were selected for phylogenetic analysis of the representative genotypes. 【Result】A total of 499 rhizobia strains were obtained from 19 sampling regions located in Northeast and North China. All strains were classified into 37 BOX types. The BOX types showed a strong geographic distribution, and host cultivars imposed a certain selection pressure to the genotypes of rhizobia. 97.60% (487/499) of the strains was identified as Sinorhizobium meliloti, while the rest 12 strains were identified as S. adhaerens, Mesorhizobium huakuii, Rhizobium loessense, R. mesosinicum, R. vignae, Mesorhizobium sp. and Phyllobacterium sp.. All the 12 strains were collected from Northeast China, while all strains from North China were S. meliloti. The housekeeping genes revealed similar phylogenetic trends when they were used for inter-species analysis, but revealed differently for intra-species analysis. Phylogeny of symbiotic genes revealed that horizontal gene transfer happened between genera and species within genus. nifH showed high genetic diversity than nodC in S. meliloti strains.【Conclusion】 Rhizobia associated with M. sativa from Northeast and North China showed high genetic diversity, moreover, they had a strong characteristics of geographic distribution and host selection.

Key words: Medicago sativa, Sinorhizobium meliloti, genetic diversity, phylogeny, horizontal transfer

李昕芫, 娄金秀, 刘清源, 胡健, 张英俊. 中国东北和华北地区紫花苜蓿根瘤菌遗传多样性研究[J]. 中国农业科学, 2021, 54(16): 3393-3405.

LI XinYuan, LOU JinXiu, LIU QingYuan, HU Jian, ZHANG YingJun. Genetic Diversity Analysis of Rhizobia Associated with Medicago sativa Cultivated in Northeast and North China[J]. Scientia Agricultura Sinica, 2021, 54(16): 3393-3405.

图/表 5

表1

表2

紫花苜蓿根瘤菌BOX型种类及其分布"

BOX型 BOX type	菌株数 No. of strains	占比 Percentage (%)	比对序列号 Accession No.	菌种 Species	采样地^b Sampling site	区域 Region
1	38	7.62	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、凤城、黑龙江（5）^b、建平（6）、辽阳（21）、前郭、沈阳、五河（2） Baicheng, Fengcheng, Heilongjiang (5), Jianping (6), Liaoyang (21), Qianguo, Shenyang, Wuhe (2)	NE、N
2	2	0.40	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、五河 Baicheng, Wuhe	NE、N
3	1	0.20	EF549450 (91.34) ^a	中慢生根瘤菌属 Mesorhizobium sp.	白城 Baicheng	NE
4	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	辽阳 Liaoyang	NE
5	6	1.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、前郭、特尼河、通榆（2）、五河 Baicheng, Qianguo, Turney river, Tongyu (2), Wuhe	NE、N
6	1	0.20	KF206625 (100)	黄土根瘤菌 Rhizobium loessense	白城 Baicheng	NE
7	93	18.64	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城（2）、沧州、衡水（28）、新乡（23）、荥阳、郑州（13）、辽阳（10）、前郭（2）、聊城（3）、沈阳（7）、五河（3） Baicheng (2), Cangzhou, Hengshui (28), Xinxiang (23), Xingyang, Zhengzhou (13), Liaoyang (10), Qianguo (2), Liaocheng (3), Shenyang (7), Wuhe (3)	NE、N
8	2	0.40	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、沈阳 Baicheng, Shenyang	NE
9	3	0.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、郑州、齐齐哈尔 Baicheng, Zhengzhou, Qiqihar	NE、N
10	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城 Baicheng	NE
11	1	0.20	KF206625 (100)	黄土根瘤菌 Rhizobium loessense	白城 Baicheng	NE
12	4	0.80	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、聊城（3） Baicheng, Liaocheng (3)	NE、N
13	15	3.00	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城（5）、建平、通榆（4）、谢尔塔拉（4）、延边 Baicheng (5), Jianping, Tongyu (4), Cher tara (4), Yanbian	NE
14	8	1.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城、前郭、通榆（3）、五河（2）、延边 Baicheng, Qianguo, Tongyu (3), Wuhe (2), Yanbian	NE、N
15	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城 Baicheng	NE
16	2	0.40	KP251033 (100)	华癸根瘤菌 Mesorhizobium huakuii	通榆 Tongyu	NE
17	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	前郭 Qianguo	NE
18	15	3.00	-	草木樨中华根瘤菌 Sinorhizobium meliloti	齐齐哈尔（3）、沈阳、特尼河（3）、五河（6）、谢尔塔拉（2） Qiqihar (3), Shenyang, Turney river (3), Wuhe (6), Cher tara (2)	NE、N
19	36	7.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	沧州、衡水（5）、新乡（2）、荥阳（2）、郑州（5）、前郭、胶州（12）、通榆（2）、五河（4）、延边（2） Cangzhou, Hengshui (5), Xinxiang (2), Xingyang (2), Zhengzhou (5), Qianguo, Jiaozhou (12), Tongyu (2), Wuhe (4), Yanbian (2)	NE、N
20	13	2.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	齐齐哈尔、沈阳（8）、通榆、五河（3） Qiqihar, Shenyang (8), Tongyu, Wuhe (3)	NE、N
21	1	0.20	AM418747 (99.67)	附着剑菌 Sinorhizobium adhaerens	沈阳 Shenyang	NE
OX型 BOX type	菌株数 No. of strains	占比 Percentage (%)	比对序列号 Accession No.	菌种 Species	采样地^b Sampling site	区域 Region
22	58	11.62	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城（35）、凤城（2）、齐齐哈尔、建平（4）、前郭、沈阳（8）、五河（5）、谢尔塔拉（2） Baicheng (35), Fengcheng (2), Qiqihar, Jianping (4), Qianguo, Shenyang (8), Wuhe (5), Cher tara (2)	NE、N
23	141	28.26	-	草木樨中华根瘤菌 Sinorhizobium meliloti	凤城（3）、沧州（25）、衡水（14）、新乡（20）、荥阳（14）、郑州（12）、辽阳（2）、胶州（12）、聊城（20）、沈阳、通榆（2）、五河（15）、谢尔塔拉 Fengcheng (3), Cangzhou (25), Hengshui (14), Xinxiang (20), Xingyang (14), Zhengzhou (12), Liaoyang (2), Jiaozhou (12), Liaocheng (20), Shenyang, Tongyu (2), Wuhe (15), Cher tara	NE、N
24	8	1.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	五河 Wuhe	N
25	3	0.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	荥阳（2）、前郭 Xingyang (2), Qianguo	NE、N
26	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	五河 Wuhe	N
27	4	0.80	-	草木樨中华根瘤菌 Sinorhizobium meliloti	特尼河（3）、五河 Turney river (3), Wuhe	NE、N
28	6	1.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	荥阳、建平、五河（4） Xingyang, Jianping, Wuhe (4)	NE、N
29	3	0.60	-	草木樨中华根瘤菌 Sinorhizobium meliloti	白城（2）、谢尔塔拉 Baicheng (2), Cher tara	NE
30	1	0.20	EU120727 (98.01)	华中根瘤菌 Rhizobium mesosinicum	沈阳 Shenyang	NE
31	2	0.40	AM418747 (99.67)	附着剑菌 Sinorhizobium adhaerens	沈阳 Shenyang	NE
32	1	0.20	KF580979 (92.72)	芸苔叶杆菌 Phyllobacterium brassicacearum	沈阳 Shenyang	NE
33	19	3.81	-	草木樨中华根瘤菌 Sinorhizobium meliloti	沧州（4）、新乡（11）、郑州、聊城（2） Cangzhou (4), Xinxiang (11), Zhengzhou, Liaocheng (2)	N
34	2	0.40	-	草木樨中华根瘤菌 Sinorhizobium meliloti	新乡 Xinxiang	N
35	1	0.20	-	草木樨中华根瘤菌 Sinorhizobium meliloti	沧州 Cangzhou	N
36	1	0.20	KF206603 (99.33)	绿豆根瘤菌 Rhizobium vignae	白城 Baicheng	NE
37	2	0.40	-	草木樨中华根瘤菌 Sinorhizobium meliloti	五河 Wuhe	N

表2

图1

图2

表3

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采样地 Sampling site	省份 Province	区域 Region	采样时间 Sampling date (Y.M)	寄主品种 Host cultivar	菌株数量 No. of strains
五河Wuhe	安徽Anhui	华北North	2016.1	未知Unknown	58
沧州Cangzhou	河北Hebei	华北North	2017.5	中苜1号、中苜3号、甘农3号 Zhongmu1, Zhongmu3, Gannong3	32
衡水Hengshui	河北Hebei	华北North	2017.5	中苜1号、WL363 Zhongmu1, WL363	48
新乡Xinxiang	河南Henan	华北North	2017.5	M343、M354	58
荥阳Xingyang	河南Henan	华北North	2017.5	三得利 Sandeli	20
郑州Zhengzhou	河南Henan	华北North	2017.5	劲能5010 Jinneng5010	32
齐齐哈尔Qiqihar	黑龙江Heilongjiang	东北Northeast	2015.9	806	11
白城Baicheng	吉林Jilin	东北Northeast	2017.5	公农5号 Gongnong5	59
前郭Qianguo	吉林Jilin	东北Northeast	2015.9	敖汉 Aohan	9
通榆Tongyu	吉林Jilin	东北Northeast	2015.9	东苜1号 Dongmu1	16
延边Yanbian	吉林Jilin	东北Northeast	2015.9	公农1号 Gongnong1	4
凤城Fengcheng	辽宁Liaoling	东北Northeast	2016.6	未知 Unknown	6
建平Jianping	辽宁Liaoning	东北Northeast	2016.6	未知 Unknown	12
辽阳Liaoyang	辽宁Liaoning	东北Northeast	2016.6	龙牧、WL319 Longmu, WL319	34
沈阳Shenyang	辽宁Liaoning	东北Northeast	2016.6	公农1号 Gonglong1	32
特尼河Turney river	内蒙古Inner Mongolia	东北Northeast	2016.1	未知 Unknown	7
谢尔塔拉Cher tara	内蒙古Inner Mongolia	东北Northeast	2015.9	未知 Unknown	9
胶州Jiaozhou	山东Shandong	华北North	2017.6	三得利、中苜3号 Sandeli, Zhongmu3	24
聊城Liaocheng	山东Shandong	华北North	2017.6	三得利、WL363 Sandeli, WL363	28
总计 Total					499

采样地 Sampling site	品种^a Cultivar	菌株数 No. of strains	BOX型^b BOX type
新乡 Xinxiang	M343	37	BOX7(4)^b、BOX19(2)、BOX23(23)、BOX33(9)、BOX34(2)
新乡 Xinxiang	M354	21	BOX7(19)、BOX33(2)
沧州 Cangzhou	中苜1号Zhongmu1	12	BOX7、BOX23(9)、BOX33、BOX35
	中苜3号Zhongmu3	16	BOX19、BOX23(12)、BOX33(3)
	甘农3号Gannong3	4	BOX23(4)
胶州 Jiaozhou	三得利Sandeli	12	BOX19(8)、BOX23(4)
胶州 Jiaozhou	中苜3号Zhongmu3	12	BOX19(4)、BOX23(8)
衡水 Hengshui	中苜1号Zhongmu1	31	BOX7(28)、BOX23(3)
衡水 Hengshui	WL363	17	BOX 19(5)、BOX23(11)、BOX33
辽阳 Liaoyang	龙牧Nongmu	13	BOX1(11)、BOX7、BOX23
辽阳 Liaoyang	WL319	21	BOX1(10)、BOX7(9)、BOX4、BOX23