MOLECULAR DIAGNOSTICS AND DNA TAXONOMY Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations DARINE TRABELSI,*† FRANCESCO PINI,† MARCO BAZZICALUPO,† EMANUELE G. BIONDI,† MOHAMMED E. AOUANI* and ALESSIO MENGONI† *Laboratoire des Interactions Le ´gumineuses-Microorganismes, Centre de Biotechnologie de Borj-Cedria, BP 901, Hammam-Lif 2050, Tunisia, †Department of Evolutionary Biology, University of Firenze, Via Romana 17, I-50125 Firenze, Italy Abstract The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmen- tal DNA is reported. The marker is based on terminal-restriction fragment length polymor- phism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymor- phism of S. meliloti populations. Keywords: bacterial population, IGS, Sinorhizobium meliloti, T-RFLP Received 26 August 2008; revision accepted 26 February 2009 Sinorhizobium meliloti is a nitrogen-fixing bacterial species which forms symbiotic nodules on the roots of legumi- nous plants. This species has been extensively used as model for bacterial population genetics (Carelli et al. 2000; Jebara et al. 2001; Bailly et al. 2006). However, due to the lack of efficient selective culture media, most of the population genetic studies on S. meliloti have been per- formed only on bacteria isolated from nodules with a few studies conducted on bacteria directly recovered from soil (Bromfield et al. 1995; Barran et al. 1997; Hartmann et al. 1998). These studies indicated that there is a frac- tion, possibly genetically differentiated, of the rhizobial population that cannot be detected on nodules and whose ecological role remains mostly unknown. To over- come the limitation due to isolation and cultivation of S. meliloti from soil, we developed a terminal-restriction fragment length polymorphism (T-RFLP; Marsh 1999) marker targeting the 16S-23S rDNA intergenic gene spacer (IGS) sequence to detect specifically S. meliloti DNA in soil samples and allow the analysis of its genetic polymorphism. IGS is known to be highly polymorphic among S. meliloti strains (Biondi et al. 2003). Marker spec- ificity and ability to detect the polymorphism were tested on DNA extracted from soil and on DNA from a collec- tion of S. meliloti isolates. Primer pairs for selective amplification of S. meliloti IGS region were developed based on comparison of nucleotide sequence of the IGS region of S. meliloti (and its closest phylogenetic relative, Sinorhizobium medicae. From the alignment (data not shown), a set of three prim- ers were designed based on the sequence of S. meliloti Rm1021 chromosome (accession number NC_003047): IGS-mel-57f, 5¢-CTCGAGTGCATGAAGTTGGA-3¢ (nt positions 83029–83048); IGS-mel-r, 5¢-CTGGCCTT- GCGAAGCCTTA-3¢ (nt positions 84586–84604); IGS-mel- 1392r, 5¢-AACCTATCCTCCTCGCTTGC-3¢ (nt positions 84345–84364). These primers were used for a semi- nested, two-step amplification reaction involving a first polymerase chain reaction (PCR) reaction with IGS- mel-57f ⁄ IGS-mel-r pair followed by a re-amplification of 1 lL of the first PCR product with primer pair IGS- mel-57f ⁄ IGS-mel-1392r. Due to the low abundance of S. meliloti cells in soil (usually estimated around 10 3 – 10 4 cells ⁄ g of soil out of 10 8 –10 9 total bacteria ⁄ g of soil as reported in Trabelsi et al. 2009), one-step amplification reaction produced faint or no amplification products on soil DNA, while the semi-nested reaction with Correspondence: Alessio Mengoni, Fax: +39 055 2288250; E-mail: alessio.mengoni@unifi.it Ó 2009 Blackwell Publishing Ltd Molecular Ecology Resources (2010) 10, 170–172 doi: 10.1111/j.1755-0998.2009.02697.x