中国东北和华北地区紫花苜蓿根瘤菌遗传多样性研究

【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。

Genetic Diversity Analysis of Rhizobia Associated with Medicago sativa Cultivated in Northeast and North China

【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.