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Applied and Environmental Microbiology, September 2002, p. 4694-4697, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4694-4697.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center

Marina L. Roumiantseva,^1* Evgeny E. Andronov,¹ Larissa A. Sharypova,¹ Tatjana Dammann-Kalinowski,² Mathias Keller,² J. Peter W. Young,³ and Boris V. Simarov¹

Research Institute for Agricultural Microbiology, St.-Petersburg-Pushkin 8, 196608, Russia,¹ Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany,² Department of Biology, University of York, York YO10 5YW, United Kingdom³

Received 29 October 2001/ Accepted 10 June 2002

Top Abstract Introduction References

 
Sinorhizobium meliloti was isolated from nodules and soil from western Tajikistan, a center of diversity of the host plants (Medicago, Melilotus, and Trigonella species). There was evidence of recombination, but significant disequilibrium, between and within the chromosome and megaplasmids. The most frequent alleles matched those in the published genome sequence.

Top Abstract Introduction References

 
Bacteria that nodulate alfalfa form effective symbioses with three related genera: Medicago (alfalfa and perennial and annual medics), Melilotus (sweet clover), and Trigonella (fenugreek). The bacteria fall into two closely related species, Sinorhizobium meliloti and Sinorhizobium medicae (16, 17), the latter mainly associated with annual medics around the Mediterranean. The alfalfa-Sinorhizobium symbiosis is one of the best-studied plant-microbe associations, and the complete genome sequence of S. meliloti strain 1021 has been determined (10). Central Asia was recognized by N. I. Vavilov (19), the pioneer of plant biogeography, as a gene center of alfalfa diversity, where it is believed that Medicago sativa plants were first cultivated by humans and the tetraploid alfalfa forms arose. We examined rhizobia from this region in the expectation that they might also be very diverse and shed light on the natural gene pool of S. meliloti.

Rhizobia were obtained from nodules and soil collected during an expedition to Tajikistan (Fig. 1) in early summer. Their genetic diversity was characterized by plasmid profiling (11), RsaI digestion of amplified 16S ribosomal DNA (1, 17, 20), and restriction fragment length polymorphism (RFLP) of 10 single-copy loci and four insertion sequence (IS) elements (Table 1). All 27 isolates were identified by ribosomal DNA RFLP as S. meliloti rather than S. medicae, and all formed effective nodules on M. sativa cv. Europe. Isolates trapped from soil differed significantly from those from field nodules in genotype frequencies at the recA, exo, and exp loci; the presence of a 200-kb plasmid; and distribution of three of the four IS elements.

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FIG. 1. Map of Central Asia showing the Tajikistan region where nodules from Medicago, Melilotus, and Trigonella species and soil samples were collected. The black line indicates the route of the expedition; numbers from 1 to 8 represent the collection sites.

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TABLE 1. Origin and features of S. meliloti isolates native to the Central Asian center of diversity

If Central Asia is the center of origin of alfalfa rhizobia as well as of their host plants, a wider range of genetic variants would be expected there than elsewhere. In fact, though, the level of polymorphism among these isolates was surprisingly modest, despite the variety of hosts and sites sampled. Bromfield et al. (7) studied the diversity in strains isolated from alfalfa in Canada, using RFLP at a different but comparable set of loci. They found 22 chromosomal types, 33 pSymA types, and 18 pSymB types, which contrasts with 7, 9, and 3, respectively, in our Central Asian isolates. Admittedly, they examined many more isolates, but this comparison certainly does not support the idea that Central Asia is a repository of alfalfa symbionts with many divergent endemic genotypes.

At each locus that we examined (four chromosomal, four on pSymA, and two on pSymB), the most common variant among the isolates was the "a" type, which by our definition was the type found in the standard laboratory strain 2011. Nevertheless, there was sufficient polymorphism that only one isolate, CA67, had the "a" genotype at every locus. It seems, therefore, that the choice of 1021 (a derivative of 2011) as a representative of the species for genome sequencing (10) was a remarkably fortunate one. It has the most typical genotype, with alleles that are common not just in agricultural inoculants but also in a population that would be expected to include the breadth of the genetic variation in the species.

Although all the isolates share some RFLP alleles with 2011, there is one strain (CA82) that has a SymA megaplasmid genotype that differs at all four loci examined. In particular, it is the only one that has two novel fragments in place of the 3.9-, 1.8-, and 0.6-kb bands that hybridize to the nifKDH probe in all the other strains. This deserves further investigation, as it may have significant functional differences in its symbiosis genes from those of the well-studied alfalfa-nodulating rhizobia. Curiously, the chromosomal and pSymB markers of this isolate are indistinguishable from those of Rm2011, so it seems that the "exotic" pSymA has been transferred into a very typical genetic background.

There is evidence of recombination between loci, both within replicons and between replicons. In this context, "recombination" between plasmid and chromosomal loci can be interpreted as transfer of plasmids from one chromosomal background to another, whereas recombination between markers linked on the same replicon implies physical breakage and reunion of the DNA. If there are at least two different alleles at each of two loci, and they occur in all combinations, this must indicate either recombination or independent parallel mutation to the same allelic state in different lineages. For example, at the two loci exoP to exoZ and expA10 to expE8 on megaplasmid 2, all four combinations, "aa," "ab," "ba," and "bb," were found. There are many pairs of loci for which this is true ("+" in Table 2), which implies that recombination has been frequent, since so much parallel evolution is implausible.

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TABLE 2. Pairwise tests of association and recombination between genetic markersa

At the same time, recombination has not been so frequent as to eliminate linkage disequilibrium, which was significant between many pairs of loci (values of P < 0.05 in Table 2). Linkage disequilibrium was analyzed by an extension of Fisher's exact test, suitable for small samples and implemented in Arlequin 2.0 (S. Schneider, D. Roessli, and L. Excoffier, Genetics and Biometry Laboratory, University of Geneva, Geneva, Switzerland). The estimation was based on sampling the space of possible contingency tables via a random Markov chain of 2 million steps, following 10,000 dememorization steps. All significant P values were within 0.002 in duplicate runs. In part, this linkage disequilibrium may reflect the complex sampling structure, but in any case such disequilibrium is not surprising in a bacterial population, because it can be generated rapidly by "epidemic" reproduction of individual clones. There is, however, no indication of a predominantly epidemic population structure in this case, since almost every isolate was genetically different. The only "clonal" isolates were the three trap isolates CA96, CA97, and CA101. These had a distinctive allele combination on each of the three replicons and clearly represent a well-established clone since they were isolated from three different locations. They form part of the rich genetic structure of the population, which must reflect the joint effects of mutation, recombination, and selection.

 
We thank B. Winterholler (Botanical Garden, Alma-Ata, Kazakhstan) for plant identification, A. Rasulov (University of Tashkent, Tashkent, Uzbekistan) for technical help during the expedition, R. Bahro (Bielefeld, Germany) for providing nodD primers, A. Pühler (Bielefeld, Germany) and E. Bromfield (Ottawa, Ontario, Canada) for discussion, and I.-M. Pretorius-Guth for help during the preparation of the manuscript.

This study was supported by the German and Russian Ministries of Agriculture and by INTAS 694.

 
* Corresponding author. Mailing address: Research Institute for Agricultural Microbiology, Sh. Podbelsky 3, St.-Petersburg-Pushkin 8, 196608, Russia. Phone: 7 812 470 28 02. Fax: 7 812 470 43 62. E-mail: genet{at}yandex.ru.

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Applied and Environmental Microbiology, September 2002, p. 4694-4697, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4694-4697.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roumiantseva, M. L. Articles by Simarov, B. V. Search for Related Content PubMed PubMed Citation Articles by Roumiantseva, M. L. Articles by Simarov, B. V. Agricola Articles by Roumiantseva, M. L. Articles by Simarov, B. V.