Genetic Diversity Of Rhizobia Isolated From Different Varieties Alfalfa

Based on the numerical taxonomy, the genetic diversity of rhizobial strains, isolated from nodules of different varieties alfalfa, was analyzed by methods of BOX-PCR, SSCP-PCR, IGS-RFLP, and 16S–23S rDNA internal spacer (IGS) sequences of 4 strains were sequenced.Box-PCR fingerprinting was a method of amplifying anti-repeat sequence, which can effectively reflect genetic diversity of bacterial isolates. Results of BOX-PCR fingerprinting showed abundant genetic patterns of all tested rhizobial strains were obtained, and showed large variety. According to the results of dendrogram, all strains were grouped into 6 genetic groups at 74% similarity level. GroupⅠandⅡwas capable of 13 tested strains respectively; groupⅣwas the largest group and had 18 tested strains. Except NWMSH072 100% similarity to NWMSH073, certain genetic diversity was existed in tested strains.SSCP (single strand conformational polymorphysim) is a high sensitive genetic measure showed one or several bases changed and is widely used in gene differentia and mutation. SSCP is a good method to identify genetic dib\versity of different isolates belonged to one species. In this test, 44 rhizobial strains V2-V3 and V4-V5 regions of 16S rDNA were amplified by PCR and denatured into single strand by SSCP. The results showed there were 12 different genotypes in V2-V3 and 13 genotypes in V4-V5 region. The SSCP electrophoresis lanes of all strains were incongruence, and some strains had several conformation. Besides, different genotype was into the same variety host plants and same genotype in different varieties host plants.IGS is capable of large variety, which fingerprinting is easier to reflect genetic diversity in genus, species or even different isolates from one species. IGS-RFLP analysis suggested 65 tested strains and 2 reference strains were well grouped together at 64% similarity level. With the results of IGS ARDRA, all tested strains were formed 2 genetic groups. GroupⅠwas the bigger one and had 51 strains, 47 strains of them were 100% similarity. GroupⅡhad 11 tested strains and 1 reference strain. There were 2 subgroups of 100% similarity within groupⅡ.IGS sequences of four type strains based on the results of IGS-RFLP analysis were sequenced and analyzed their phylogeny with the IGS of rhizobial reference strains. IGS phylogenetic analysis suggested all reference strains were formed five phylogenetic branches: Sinorhizobium, Rhizobium, Mesorhizobium, Bradyrhizobium, Agrobacterium. The types strains NWMSH063 and NWMSH001 were closed to S. medicae USDA1037, their similarity distance was 89.7% and 83.2% respectively. NWMSH050 and NWMSH035 were far with reference strains. NWMSH050 and NWMSH035 from two distinct fields, NWMSH050 and NWMSH035 from the same field had higher similarities level, was 92.5%, 97% respectively. The strains isolated from same field, the NWMSH001 genetic distance with NWMSH050 and NWMSH035 was very low, just 39.8%. The result of IGS phylogentic analysis was good conformed to IGS-RFLP and rhizobial phylogeny was mainly determined by host plant, less in influence with region and climate.