The use of neutral and non-neutral SSRs to analyse the genetic structure of a Tunisian collection of Medicago truncatula lines and to reveal associations with eco-environmental variables.

In this study, we investigated the genetic diversity of a collection of 136 Medicago truncatula lines from 10 Tunisian natural populations collected in well-defined locations and in various ecological conditions of soil, salinity and water availability. The genetic diversity was evaluated using a set of 18 microsatellites (SSRs), representing the 8 chromosomes of M. truncatula. A neutrality test showed that 7 SSRs were non-neutral with evidence of balancing selection. The 11 neutral SSRs revealed a geographical pooling with the Tunisian Dorsale axis restricting migration of alleles. The 7 non-neutral alleles demonstrate a correlation with rainfall, altitude and salinity environmental variables suggesting that these SSRs are linked to genes involved in water use efficiency, resistance to salinity or adaptation to altitude, and that there is local adaptation of M. truncatula to these variables. This demonstrates that the choice of so-called neutral markers should be carefully evaluated in population genetic studies. This study illustrates the genetic diversity occurring in natural Tunisian populations of M. truncatula and describes the first collection of this species dedicated to natural variation involved in adaptation to the environment.

Development of real-time PCR assay for detection and quantification of Sinorhizobium meliloti in soil and plant tissue.

AIMS: Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most-probable-number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real-time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species-specific fashion, the chromosomal gene rpoE1 and the pSymA gene nodC. METHODS AND RESULTS: rpoE1 and nodC primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti. Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples. CONCLUSIONS: The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation. SIGNIFICANCE AND IMPACT OF THE STUDY: These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil.

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

Genetic diversity of Sinorhizobium populations recovered from different medicago varieties cultivated in Tunisian soils.

A collection of 468 rhizobial isolates was obtained from different ecological areas of Tunisia by trapping them on Medicago sativa cv. Gabes, Medicago scutelleta cv. Kelson, Medicago truncatula, and Medicago ciliaris. A subsample of 134 rhizobia was chosen to determine their plasmid profile, and 89 isolates were subjected to multilocus enzyme electrophoresis (MLEE) and PCR/RFLP analysis using 16S, IGS (inter genic spacer), and nifKD probes. Twenty-five representatives from these isolates were evaluated for their nodulation and nitrogen fixation capacities. MLEE studies revealed two groups with highly heterogeneous host specificity and geographical origin. The discriminatory power was found to be slightly better with the amplified ribosomal intergenic region, than the nifKD genes. Divisions detected by nifKD amplified DNA analysis matched those established by ribosomal PCR- RFLPs. The comparison between different analyses revealed that MLEE illustrated better phenotypic properties of isolates than PCR-RFLP or plasmid content analysis. Clear distinction between Sinorhizobium meliloti and Sinorhizobium medicae were observed by analysis of the IGS symbiotic regions between nifD and nifK genes. Were able to distinguish three inoculation groups; isolates trapped from M. sativa cv. Gabes and M. scutelleta cv. Kelson formed one inoculation group which was more closely related to isolates trapped from M. truncatula than those trapped from M. ciliaris.