BackgroundAs our world becomes warmer, agriculture is increasingly impacted by rising soil salinity and understanding plant adaptation to salt stress can help enable effective crop breeding. Salt tolerance is a complex plant phenotype and we know little about the pathways utilized by naturally tolerant plants. Legumes are important species in agricultural and natural ecosystems, since they engage in symbiotic nitrogen-fixation, but are especially vulnerable to salinity stress.ResultsOur studies of the model legume Medicago truncatula in field and greenhouse settings demonstrate that Tunisian populations are locally adapted to saline soils at the metapopulation level and that saline origin genotypes are less impacted by salt than non-saline origin genotypes; these populations thus likely contain adaptively diverged alleles. Whole genome resequencing of 39 wild accessions reveals ongoing migration and candidate genomic regions that assort non-randomly with soil salinity. Consistent with natural selection acting at these sites, saline alleles are typically rare in the range-wide species' gene pool and are also typically derived relative to the sister species M. littoralis. Candidate regions for adaptation contain genes that regulate physiological acclimation to salt stress, such as abscisic acid and jasmonic acid signaling, including a novel salt-tolerance candidate orthologous to the uncharacterized gene AtCIPK21. Unexpectedly, these regions also contain biotic stress genes and flowering time pathway genes. We show that flowering time is differentiated between saline and non-saline populations and may allow salt stress escape.ConclusionsThis work nominates multiple potential pathways of adaptation to naturally stressful environments in a model legume. These candidates point to the importance of both tolerance and avoidance in natural legume populations. We have uncovered several promising targets that could be used to breed for enhanced salt tolerance in crop legumes to enhance food security in an era of increasing soil salinization.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1160) contains supplementary material, which is available to authorized users.

Multilocus sequence typing (MLST) is a sequence-based method used to characterize bacterial genomes. This method was used to examine the genetic structure of Medicago-nodulating rhizobia at the Amra site, which is located in an arid region of Tunisia. Here the annual medics Medicago laciniata and M. truncatula are part of the natural flora. The goal of this study was to identify whether distinct chromosomal groups of rhizobia nodulate M. laciniata because of its restricted requirement for specific rhizobia. The MLST analysis involved determination of sequence variation in 10 chromosomal loci of 74 isolates each of M. laciniata and M. truncatula. M. truncatula was used as a control trap host, because unlike M. laciniata, it has relatively unrestricted rhizobial requirements. Allelic diversity among the plasmid nodC alleles in the isolates was also determined. The 148 isolates were placed into 26 chromosomal sequence types (STs), only 3 of which had been identified previously. The rhizobia of M. laciniata were shown to be part of the general Medicago-nodulating population in the soil because 99.95% of the isolates had chromosomal genotypes similar to those recovered from M. truncatula. However, the isolates recovered from M. laciniata were less diverse than those recovered from M. truncatula, and they also harbored an unusual nodC allele. This could perhaps be best explained by horizontal transfer of the different nodC alleles among members of the Medicago-nodulating rhizobial population at the field site. Evidence indicating a history of lateral transfer of rhizobial symbiotic genes across distinct chromosomal backgrounds is provided.Traditionally, legumes have been important in a practice referred to as low-input sustainable agriculture. The recognition of their agricultural importance led to the demonstration that legumes bearing nodules inhabited by bacteria assimilated dinitrogen in a process termed biological nitrogen fixation. The bacteria, commonly referred to as rhizobia, harbor the genetic information necessary for establishing the nitrogen fixation process when they reside symbiotically inside the nodules. Plants of the genus Medicago are legumes that benefit from such a symbiosis with rhizobia that are currently divided into two species, Sinorhizobium meliloti (11) and S. medicae (25).The plant genus Medicago L., as currently defined, consists of about 85 species (28), including Medicago sativa (alfalfa), the world's most important cultivated forage crop. Heyn (16) reported that this genus is native to Western Asia and the Mediterranean countries, although many annual species have invaded wide areas in both the Old and New Worlds.Among the 85 species, M. laciniata has symbiotic properties that are unusual. While most Medicago species nodulated and formed nitrogen-fixing symbioses when they were grown in Australian soils with naturalized rhizobial populations, Ballard and Charman (3) observed that there was a lower incidence of nodulation with M. laciniata. They attributed this observation to the spec...

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Variation in growth of Medicago ciliaris was recorded across soils from 5 different regions in Tunisia that represented different soil types and climatic zones. In 4 of these soils (Mateur, Enfidha, Rhayet, and Soliman) this variation appeared to be related to the nodule number on the roots of the plants. With the exception of one isolate the rhizobia isolated from these nodules had 16S rRNA PCR-RFLP fingerprint patterns that were characteristic of Sinorhizobium medicae. Plant growth in the fifth soil (Jelma) was the poorest; plants had few nodules that yielded exclusively rhizobia with 16S rRNA fingerprint patterns characteristic of S. meliloti. In subsequent plant tests, S. medicae isolates formed effective nitrogen fixation symbioses with M. ciliaris, while S. meliloti formed small, white, ineffective nodules. Therefore, plant growth in Jelma soil was poor because only S. meliloti are present and this species is ineffective with M. ciliaris. In a co-inoculation experiment with M. ciliaris, S. medicae was more competitive for nodulation than S. meliloti, perhaps explaining why the majority of the isolates from Enfidha and Rhayet were S. medicae, since S. meliloti is present in these soils. However, it is not clear how the host influences rhizobia for nodulation by S. medicae in preference to S. meliloti when present.

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.