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

Andronov, E.E.

Dammann-Kalinowski, T.

Keller, M.

Roumiantseva, M.L.

Sharypova, L.A.

Simarov, B.V.

Young, J.P.W.

English

White Rose Research Online

This is a repository copy of Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/295/Article:Roumiantseva, M.L., Andronov, E.E., Sharypova, L.A. et al. (4 more authors) (2002) Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center. Applied and Environmental Microbiology. pp. 4694-4697. ISSN 0099-2240 https://doi.org/10.1128/AEM.68.9.4694–4697.2002eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2002, p. 4694–4697 Vol. 68, No. 90099-2240/02/$04.000 DOI: 10.1128/AEM.68.9.4694–4697.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.Diversity of Sinorhizobium meliloti from the Central AsianAlfalfa Gene CenterMarina L. Roumiantseva,1* Evgeny E. Andronov,1 Larissa A. Sharypova,1Tatjana Dammann-Kalinowski,2 Mathias Keller,2 J. Peter W. Young,3and Boris V. Simarov1Research Institute for Agricultural Microbiology, St.-Petersburg-Pushkin 8, 196608, Russia1; Lehrstuhl für Genetik,Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany2; and Department of Biology,University of York, York YO10 5YW, United Kingdom3Received 29 October 2001/Accepted 10 June 2002Sinorhizobium meliloti was isolated from nodules and soil from western Tajikistan, a center of diversity of thehost plants (Medicago, Melilotus, and Trigonella species). There was evidence of recombination, but significantdisequilibrium, between and within the chromosome and megaplasmids. The most frequent alleles matchedthose in the published genome sequence.Bacteria that nodulate alfalfa form effective symbioses withthree related genera: Medicago (alfalfa and perennial and an-nual medics), Melilotus (sweet clover), and Trigonella (fenu-greek). The bacteria fall into two closely related species, Sino-rhizobium meliloti and Sinorhizobium medicae (16, 17), thelatter mainly associated with annual medics around the Med-iterranean. The alfalfa-Sinorhizobium symbiosis is one of thebest-studied plant-microbe associations, and the complete ge-nome sequence of S. meliloti strain 1021 has been determined(10). Central Asia was recognized by N. I. Vavilov (19), thepioneer of plant biogeography, as a gene center of alfalfadiversity, where it is believed that Medicago sativa plants werefirst cultivated by humans and the tetraploid alfalfa formsarose. We examined rhizobia from this region in the expecta-tion that they might also be very diverse and shed light on thenatural gene pool of S. meliloti.Rhizobia were obtained from nodules and soil collectedduring 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) of10 single-copy loci and four insertion sequence (IS) elements(Table 1). All 27 isolates were identified by ribosomal DNARFLP as S. meliloti rather than S. medicae, and all formedeffective nodules on M. sativa cv. Europe. Isolates trappedfrom soil differed significantly from those from field nodules ingenotype frequencies at the recA, exo, and exp loci; the pres-ence of a 200-kb plasmid; and distribution of three of the fourIS elements.If Central Asia is the center of origin of alfalfa rhizobia aswell as of their host plants, a wider range of genetic variantswould be expected there than elsewhere. In fact, though, thelevel of polymorphism among these isolates was surprisinglymodest, despite the variety of hosts and sites sampled. Brom-field et al. (7) studied the diversity in strains isolated fromalfalfa in Canada, using RFLP at a different but comparable setof loci. They found 22 chromosomal types, 33 pSymA types,and 18 pSymB types, which contrasts with 7, 9, and 3, respec-tively, in our Central Asian isolates. Admittedly, they examinedmany more isolates, but this comparison certainly does notsupport the idea that Central Asia is a repository of alfalfasymbionts with many divergent endemic genotypes.At each locus that we examined (four chromosomal, four onpSymA, and two on pSymB), the most common variant amongthe isolates was the “a” type, which by our definition was thetype found in the standard laboratory strain 2011. Neverthe-less, 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 representa-tive of the species for genome sequencing (10) was a remark-ably fortunate one. It has the most typical genotype, withalleles that are common not just in agricultural inoculants butalso in a population that would be expected to include thebreadth 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 megaplasmidgenotype that differs at all four loci examined. In particular, itis 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 allthe other strains. This deserves further investigation, as it mayhave significant functional differences in its symbiosis genesfrom those of the well-studied alfalfa-nodulating rhizobia. Cu-riously, the chromosomal and pSymB markers of this isolateare indistinguishable from those of Rm2011, so it seems thatthe “exotic” pSymA has been transferred into a very typicalgenetic background.There is evidence of recombination between loci, bothwithin replicons and between replicons. In this context, “re-combination” between plasmid and chromosomal loci can beinterpreted as transfer of plasmids from one chromosomalbackground to another, whereas recombination between mark-ers linked on the same replicon implies physical breakage andreunion of the DNA. If there are at least two different alleles* Corresponding author. Mailing address: Research Institute forAgricultural 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@yandex.ru.4694at each of two loci, and they occur in all combinations, thismust indicate either recombination or independent parallelmutation to the same allelic state in different lineages. Forexample, at the two loci exoP to exoZ and expA10 to expE8 onmegaplasmid 2, all four combinations, “aa,” “ab,” “ba,” and“bb,” were found. There are many pairs of loci for which thisis true (“” in Table 2), which implies that recombination hasbeen frequent, since so much parallel evolution is implausible.TABLE 1. Origin and features of S. meliloti isolates native to the Central Asian center of diversityIsolate Sourcea SitebPlasmid size(kb)cRFLP type(s)d IS elementeChr pSymA pSymB i ii iii ivCA67 Trap 1 — aa aaaa aa 8 (a) 4 (a) 0 2CA69 Trap 3/d — ab afab aa 0 0 2 (a) 0CA72 Trap 4/a 240 aa agaa ab 12 (a) 1 (a) 6 5 (a)CA82 Trap 4/a 200 aa hbdc aa 0 5 9 9CA52 Trap 5/a 100 ba faba aa 8 (b) 0 0 0CA85 Trap 5/a — ba idab aa 4 (a) 1 (b) 2 (b) 0CA79 Trap 5/b — cc caaa aa 7 (a) 0 1 (a) 0CA96 Trap 5/b — cb dcaa bb 4 (b) 1 (c) 5 (a) 0CA97 Trap 6 — cb dcaa bb 4 (b) 1 (c) 5 (a) 0CA76 Trap 7 — aa bebb ba 3 1 (d) 2 (c) 0CA107 Trap 7 270 ac aaaa aa 12 (b) 1 (e) 2 (d) 0CA101 Trap 8 — cb dcaa bb 4 (b) 1 (c) 5 (a) 0CA81 M. sativa 3/a 200 ac aaaa ac 12 (c) 4 (b) 1 (b) 1 (a)CA104 M. sativa 3/b 200 ac aaaa aa 16 2 1 (c) 0CA110 M. sativa 3/b 200 ac ahaa aa 11 0 5 (b) 5 (b)CA112 M. sativa 3/c 600, 200, 80 ba gaba ca 9 0 0 0CA114 M. sativa 3/d 200 ac ahaa aa 11 0 0 1 (b)CA56 M. sativa 5/c — ba caaa aa 21 0 0 0CA80 M. sativa 5/d — bc ifab aa 7 (b) 2 4 (a) 3CA78 M. sativa 5/b 360 aa faca aa 15 (a) 6 8 8CA99 Medicago lupulina 5/b 300, 200, 80 ac egaa aa 13 1 (f) 0 1 (c)CA98 Medicago lupulina 5/b — ba gaba ca 9 4 (c) 0 0CA116 Melilotus officinalis 4/b — aa egaa aa 15 (b) 6 4 (b) 4CA83 Melilotus officinalis 4/b — ac aiaa aa 10 0 5 (c) 0CA105 Trigonella germiniflora 4/a 200 ac ahaa aa 11 0 5 (d) 0CA102 Trigonella popovi 3/a 200 ac ahaa aa 11 0 0 0CA108 Trigonella verae 2 — ba gaba aa 8 (c) 0 0 02011f M. sativa — aa aaaa aa 14 8 0 0a Trap, M. sativa cv. Europe inoculated with soil; others are from field nodules.b Sites are indicated in Fig. 1; letters indicate different locations at one site.c All isolates contained two megaplasmids (1,000 kb), except that CA69, CA76, and CA85 had a single megaplasmid band, like strain MVII (12); only smallerplasmids are listed. —, none were detected.d Letters indicate distinct EcoRI RFLP patterns at each of eight loci, determined with cloned or PCR-amplified probes: leuB (SMc04405) (1,2), recA (SMc00760)(8) on the chromosome, nodD1 (SMa0870) (14, 17), nodD2 (SMa0757) (17), nodH (SMa0850) (9), nifKDH (SMa0829, SMa0827, and SMa0825) (3, 15) on pSymA, exoPto exoZ (SMb20943 to SMb20961) (4, 5), and expA10 to expE8 (SMb21307 to SMb21327) (6) on pSymB. Accession numbers of genes used as hybridization probes arecited according to sequence data (10). The additional chromosomal locus mucR (SMc00058) (13) was invariant. Chr, chromosomal.e Number of copies of ISRm2011-2 (i), ISRm2011-1 (ii), ISRm220-12-3 (iii), and ISRm102F34-1 (iv) (18). Letters distinguish different fingerprints with the same copynumber.f 2011 is a standard laboratory strain of S. meliloti for comparison; it is the parent of 1021, the source of the complete genome sequence (10).TABLE 2. Pairwise tests of association and recombination between genetic markersaGeneticmarkerResult for genetic marker:leu recA nodD1 nodD2 nodH nifHDK exo exp 200-kb plasmidb IS2011-2 IS2011-1 IS220-12-3 IS102F34-1leu 0.003 0.000 0.030 NS NS 0.002 0.012 NS 0.002 NS NS NSrecA  0.008 0.001 0.021 NS 0.004 0.005 0.020 0.004 0.044 0.012 NSnodD1  0.001 0.000 0.010 0.000 NS NS 0.000 NS NS NSnodD2   NS 0.000 0.017 0.038 0.045 0.000 0.025 0.004 NSnodH  NS NS NS NS NS NS NS NSnifHDK    NS NS NS NS NS 0.039 NSexo     0.018 NS 0.033 0.028 NS NSexp    NS NS 0.003 0.037 NS200-kb plasmid       NS NS NS NSIS2011-2  0.001 0.009 NSIS2011-1     0.009 0.016IS220-12-3 NSIS102F34-1a The upper half shows probabilities based on the null hypothesis of no association (exact contingency test); NS, P  0.05, i.e., no significant association. The lowerhalf shows evidence () for recombination or parallel evolution of loci (see text).b Presence or absence of a 200-kb plasmid.VOL. 68, 2002 DIVERSITY OF CENTRAL ASIAN SINORHIZOBIUM MELILOTI 4695At the same time, recombination has not been so frequent asto eliminate linkage disequilibrium, which was significant be-tween many pairs of loci (values of P  0.05 in Table 2).Linkage disequilibrium was analyzed by an extension of Fish-er’s exact test, suitable for small samples and implemented inArlequin 2.0 (S. Schneider, D. Roessli, and L. Excoffier, Ge-netics and Biometry Laboratory, University of Geneva, Ge-neva, Switzerland). The estimation was based on sampling thespace of possible contingency tables via a random Markovchain of 2 million steps, following 10,000 dememorizationsteps. All significant P values were within 0.002 in duplicateruns. In part, this linkage disequilibrium may reflect the com-plex sampling structure, but in any case such disequilibrium isnot surprising in a bacterial population, because it can begenerated rapidly by “epidemic” reproduction of individualclones. There is, however, no indication of a predominantlyepidemic population structure in this case, since almost everyisolate was genetically different. The only “clonal” isolateswere the three trap isolates CA96, CA97, and CA101. Thesehad a distinctive allele combination on each of the three rep-licons and clearly represent a well-established clone since theywere isolated from three different locations. They form part ofthe rich genetic structure of the population, which must reflectthe joint effects of mutation, recombination, and selection.We thank B. Winterholler (Botanical Garden, Alma-Ata, Kazakh-stan) 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) fordiscussion, and I.-M. Pretorius-Guth for help during the preparation ofthe manuscript.This study was supported by the German and Russian Ministries ofAgriculture and by INTAS 694.REFERENCES1. Andronov, E. E., M. L. Roumiantseva, and B. V. Simarov. 2001. Geneticdiversity of a natural population of Sinorhizobium meliloti revealed in anal-ysis of cryptic plasmids and ISRm2011–2 fingerprints. Russ. J. Genet. 37:494–499.2. Aronshtam, A. A., B. R. Umarov, V. N. Yerko, E. E. Andronov, and B. V.Simarov. 1993. The use of the cosmid gene bank of Rhizobium meliloti forthe molecular cloning of leucine biosynthesis gene involved in genetic con-trol of nitrogen fixing symbiosis with alfalfa. Genetika 29:235–245. (In Rus-sian.)3. Banfalvi, Z., E. Kondorosi, and A. 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Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center

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Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center

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