Pheno-morphological variation, genetic diversity and population structure of Tunisian Echinus Medic (Medicago ciliaris L.).

Medicago ciliaris L., considered as a valuable genetic resource, is a good candidate for the improvement of marginal or degraded lands with low fertility or high salinity. In this study, the pheno-morphological and genetic diversity were investigated in 14 Tunisian populations of M. ciliaris for the first time. Fourteen morphological traits showed significant differentiation between populations and high levels of diversity. Two amplified fragment length polymorphism primer combinations (E-AGC/M-CAA; E-AAG/M-CTG) were analyzed using an automated capillary electrophoresis system. A total of 528 loci were generated, of which 54% were polymorphic. Allelic polymorphism ranged from 0.02 to 0.5. Significant variation between populations was found for gene diversity, mean number of alleles per locus and Shannon index for which mean values were 0.17, 0.26, and 1.57, respectively. Analysis of molecular variance revealed a high rate of genetic variation within populations. Principal component analysis and genotypic clustering discriminated M. ciliaris populations according to their geographical origin. M. ciliaris clustered into three main groups. The first group was associated with high inland and cold areas, the second was defined by low areas with mild winters while the third described low coastal areas. Similarity of morphological and molecular results indicated that either markers could be used for the study of genetic diversity in this species.

Differential expression of TLP, ERF1, and R2R3MYB in annual Medicago species under salinity conditions.

The present study was conducted to evaluate the responses of three annual Medicago species (M. truncatula, M. laciniata, and M. polymorpha) to salinity. We analyzed publicly available microarray data in NCBI pertaining to salinity-response genes in M. truncatula. Our data search identified Tubby C2 (TLP) and ethylene responsive transcription factor 1 (ERF1) as genes that potentially respond to salinity. We evaluated morpho-physiological traits and the expression of the genes in three Medicago species that had been maintained under control and saline conditions. The analysis of morpho-physiological traits showed that M. polymorpha and M. laciniata were more tolerant to salinity than M. truncatula, as they had lower reductions in biomass and dry root weight and lower increases in anthocyanin concentration. The saline conditions caused a significant increase (P < 0.01) in the expression of TLP in all Medicago species, but caused a significant decrease in the expression of ERF1. Considerable variation in anthocyanin concentrations was found among the three Medicago species. To investigate the cause of this variation, we examined the expression of R2R3MYB, a gene involved in the biosynthesis of anthocyanins. Our analysis showed that saline conditions induced high over-expression of R2R3MYB in all three Medicago spp. The high efficiency of the primer pairs used in qRT-PCR enabled us to compare the expression levels of each gene in the three species. We concluded that the more salt tolerant species showed higher expression of TLP and R2R3MYB under both control and salinity conditions.

Sequence divergence of microsatellites for phylogeographic assessment of Moroccan Medicago species.

Six Medicago species were investigated to characterize and valorize plant genetic resources of pastoral interest in Morocco. Samples were obtained from the core collection of the South Australian Research and Development Institute (SARDI). The transferability of single sequence repeat markers of Medicago truncatula was successful with 97.6% efficiency across the five species. A total of 283 alleles and 243 genotypes were generated using seven SSR markers, confirming the high level of polymorphism that is characteristic of the Medicago genus, despite a heterozygosity deficit (HO = 0.378; HE = 0.705). In addition, a high level of gene flow was revealed among the species analyzed with significant intra-specific variation. The unweighted pair group method with arithmetic mean dendrogram generated by the dissimilarity matrix revealed that M. polymorpha and M. orbicularis are closely related, and that M. truncatula is likely the ancestral species. The Pearson correlation index revealed no significant correlations between the geographic distribution of the Moroccan species and genetic similarities, indicating local adaptation of these species to different ecological environments independent of their topographical proximities. The substantial genetic variation observed was likely due to the predominance of selfing species, the relative proximity of prospected sites, human impacts, and the nature of the SARDI core collections, which are selected for their high genetic diversity. The results of this first report on Moroccan Medicago species will be of great interest for establishing strategies aiming at reasonable management and selection programs for local and Mediterranean germplasm in the face of increasing environmental change.

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes.

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization.

An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes.

BACKGROUND: Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes Lotus and Medicago (both with substantially sequenced genomes). In contrast, peanut (Arachis hypogaea) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of Arachis with Lotus and Medicago and improve our understanding of the Arachis genome and legume genomes in general. To do this we placed on the Arachis map, comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed. RESULTS: The Arachis genetic map was substantially aligned with Lotus and Medicago with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of Arachis from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The Arachis and model legume genomes comparison made here, together with a previously published comparison of Lotus and Medicago allowed all possible Arachis-Lotus-Medicago species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in Medicago and possibly also in Lotus, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes than the more conserved regions, some harbor high densities of the fast evolving disease resistance genes. CONCLUSION: We suggest that gene space in Papilionoids may be divided into two broadly defined components: more conserved regions which tend to have low retrotransposon densities and are relatively stable during evolution; and variable regions that tend to have high retrotransposon densities, and whose frequent restructuring may fuel the evolution of some gene families.