Genetic Diversity and Differentiation of Eleven Medicago Species from Campania Region Revealed by Nuclear and Chloroplast Microsatellites Markers.

The species belonging to the genus Medicago are considered a very important genetic resource at global level both for planet's food security and for sustainable rangelands management. The checklist of the Italian flora (2021) includes a total number of 40 Medicago species for Italy, and 27 for Campania region, with a number of doubtful records or related to species no more found in the wild. In this study, 10 Medicago species native to Campania region, and one archaeophyte (M. sativa), identified by means of morphological diagnostic characters, were analyzed in a blind test to assay the efficacy of nine microsatellite markers (five cp-SSRs and four n-SSRs). A total number of 33 individuals from 6 locations were sampled and genotyped. All markers were polymorphic, 40 alleles were obtained with n-SSRs ranging from 8-12 alleles per locus with an average of 10 alleles per marker, PIC values ranged from 0.672 to 0.847, and the most polymorphic SSR was MTIC 564. The cp-SSRs markers were highly polymorphic too; PIC values ranged from 0.644 to 0.891 with an average of 0.776, the most polymorphic cp-SSR was CCMP10. 56 alleles were obtained with cp-SSRs ranging from 7 to 17 alleles per locus with an average of 11. AMOVA analysis with n-SSR markers highlighted a great level of genetic differentiation among the 11 species, with a statistically significant fixation index (FST). UPGMA clustering and Bayesian-based population structure analysis assigned these 11 species to two main clusters, but the distribution of species within clusters was not the same for the two analyses. In conclusion, our results demonstrated that the combination of the used SSRs well distinguished the 11 Medicago species. Moreover, our results demonstrated that the use of a limited number of SSRs might be considered for further genetic studies on other Medicago species.

Using Genomic Location and Coalescent Simulation to Investigate Gene Tree Discordance in Medicago L.

Several well-documented evolutionary processes are known to cause conflict between species-level phylogenies and gene-level phylogenies. Three of the most challenging processes for species tree inference are incomplete lineage sorting, hybridization and gene duplication, which may result in unwarranted comparisons of paralogous genes. Several existing methods have dealt with these processes but none has yet been able to untangle all three at once. Here, we propose a stepwise method by which these processes can be discerned using information on genomic location coupled with coalescent simulations. In the first step, highly discordant genes within genomic blocks (putative paralogs) are identified and excluded from the data set and, in the second step, blocks of linked genes are grouped according to their hybrid history. Existing multispecies coalescent software can then be applied to recover the principal tree(s) that make up the species tree/network without violating the underlying model. The potential of the approach is evaluated on simulated data derived from a species network composed of nine species, of which one is of hybrid origin, and displaying a single-gene duplication that leads to paralogous comparisons. We apply our method to an empirical set of 12 genes from 7 species sampled in the plant genus Medicago that display phylogenetic discordance. We identify the causes of the discordance and demonstrate that the Medicago orbicularis lineage experienced an episode of ancient hybridization. Our results show promise as a new way to explore phylogenetic sequence data that can significantly improve species tree inference in presence of hybridization and undetected paralogy or other causes leading to extremely discordant gene trees. [Coalescent simulation; gene tree; genomic location; hybridization; incomplete lineage sorting; paralogy; phylogenetic incongruence; principal tree; species tree.].

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.

The single evolutionary origin of chlorinated auxin provides a phylogenetically informative trait in the Fabaceae.

Chlorinated auxin (4-chloroindole-3-acetic acid, 4-Cl-IAA), a highly potent plant hormone, was once thought to be restricted to species of the tribe Fabeae within the Fabaceae, until we recently detected this hormone in the seeds of Medicago, Melilotus and Trifolium species. The absence of 4-Cl-IAA in the seeds of the cultivated species Cicer aeritinum from the Cicerae tribe, immediately basal to the Fabeae and Trifolieae tribes, suggested a single evolutionary origin of 4-Cl-IAA. Here, we provide a more robust phylogenetic placement of the ability to produce chlorinated auxin by screening key species spanning this evolutionary transition. We report no detectable level of 4-Cl-IAA in Cicer echinospermum (a wild relative of C. aeritinum) and 4 species (Galega officinalis, Parochetus communis, Astragalus propinquus and A. sinicus) from tribes or clades more basal or sister to the Cicerae tribe. We did detect 4-Cl-IAA in the dry seeds of 4 species from the genus Ononis that are either basal to the genera Medicago, Melilotus and Trigonella or basal to, but still within, the Fabeae and Trifolieae (ex. Parochetus) clades. We conclude that the single evolutionary origin of this hormone in seeds can be used as a phylogenetically informative trait within the Fabaceae.

Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific symbiotic phenotype.

In order to effectively manipulate rhizobium-legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.

Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

Molecular characterization of five betacryptoviruses infecting four clover species and dill.

The family Partitiviridae includes plant (Alphacryptovirus and Betacryptovirus), fungal (Partitivirus) and protozoan (Cryspovirus) viruses with bisegmented dsRNA genomes and isometric virions. Cryptic viruses commonly occur in different plant species without causing any symptoms. So far, numerous sequences have been determined for viruses of the genus Alphacryptovirus, but no sequence is available for any assigned member of the genus Betacryptovirus. Following extraction, cloning and sequence analysis of double-stranded RNA in this study, we report the molecular properties of three assigned members of the genus Betacryptovirus, white clover cryptic virus 2, red clover cryptic virus 2 and hop trefoil cryptic virus 2, and two new putative betacryptoviruses found in crimson clover and dill. Betacryptoviruses share sequence motifs with members of the genus Partitivirus. In phylogenetic analyses, members of the genus Betacryptovirus formed a new sub-cluster within the clusters containing members of the genus Partitivirus. Our results provide evidence for a distinct evolutionary lineage of dsRNA viruses of plants and fungi.

Contrasted patterns of selective pressure in three recent paralogous gene pairs in the Medicago genus (L.).

BACKGROUND: Gene duplications are a molecular mechanism potentially mediating generation of functional novelty. However, the probabilities of maintenance and functional divergence of duplicated genes are shaped by selective pressures acting on gene copies immediately after the duplication event. The ratio of non-synonymous to synonymous substitution rates in protein-coding sequences provides a means to investigate selective pressures based on genic sequences. Three molecular signatures can reveal early stages of functional divergence between gene copies: change in the level of purifying selection between paralogous genes, occurrence of positive selection, and transient relaxed purifying selection following gene duplication. We studied three pairs of genes that are known to be involved in an interaction with symbiotic bacteria and were recently duplicated in the history of the Medicago genus (Fabaceae). We sequenced two pairs of polygalacturonase genes (Pg11-Pg3 and Pg11a-Pg11c) and one pair of auxine transporter-like genes (Lax2-Lax4) in 17 species belonging to the Medicago genus, and sought for molecular signatures of differentiation between copies. RESULTS: Selective histories revealed by these three signatures of molecular differentiation were found to be markedly different between each pair of paralogs. We found sites under positive selection in the Pg11 paralogs while Pg3 has mainly evolved under purifying selection. The most recent paralogs examined Pg11a and Pg11c, are both undergoing positive selection and might be acquiring new functions. Lax2 and Lax4 paralogs are both under strong purifying selection, but still underwent a temporary relaxation of purifying selection immediately after duplication. CONCLUSIONS: This study illustrates the variety of selective pressures undergone by duplicated genes and the effect of age of the duplication. We found that relaxation of selective constraints immediately after duplication might promote adaptive divergence.

The reticulate history of Medicago (Fabaceae).

The phylogenetic history of Medicago was examined for 60 accessions from 56 species using two nuclear genes (CNGC5 and beta-cop) and one mitochondrial region (rpS14-cob). The results of several analyses revealed that extensive robustly supported incongruence exists among the nuclear genes, the cause of which we seek to explain. After rejecting several processes, hybridization and lineage sorting of ancestral polymorphisms remained as the most likely factors promoting incongruence. Using coalescence simulations, we rejected lineage sorting alone as an explanation of the differences among gene trees. The results indicate that hybridization has been common and ongoing among lineages since the origin of Medicago. Coalescence provides a good framework to test the causes of incongruence commonly seen among gene trees but requires knowledge of effective population sizes and generation times. We estimated the effective population size at 240,000 individuals and assumed a generation time of 1 year in Medicago (many are annual plants). A sensitivity analysis showed that our conclusions remain unchanged using a larger effective population size and/or longer generation time.

Relationships of the woody Medicago species (section Dendrotelis) assessed by molecular cytogenetic analyses.

BACKGROUND AND AIMS: The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy. METHODS: Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis. KEY RESULTS: The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions. CONCLUSIONS: FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular cytogenetic data do not suggest the hypothesis that M. arborea and M. strasseri were involved in the origin of M. citrina. FISH mapping can be used as an efficient tool to determine the genomic contribution of M. citrina in somatic hybrids with other medic species.

Lotus japonicus: legume research in the fast lane.

Legumes are of immense importance to humanity and a key to sustainable agriculture. Two model species, Lotus japonicus and Medicago truncatula, are the focus of genome sequencing and functional genomics programmes, but most researchers focus exclusively on one or the other. In spite of this, legume researchers now have a unique opportunity to integrate work on these and other legume species, including soybean, common bean and pea to create a platform for comparative genomics second to none of any other plant family. The question is: do we have the scientific fortitude and political will to achieve this?

Metabolomics spectral formatting, alignment and conversion tools (MSFACTs).

MOTIVATION: The amplified interest in metabolic profiling has generated the need for additional tools to assist in the rapid analysis of complex data sets. RESULTS: A new program; metabolomics spectral formatting, alignment and conversion tools, (MSFACTs) is described here for the automated import, reformatting, alignment, and export of large chromatographic data sets to allow more rapid visualization and interrogation of metabolomic data. MSFACTs incorporates two tools: one for the alignment of integrated chromatographic peak lists and another for extracting information from raw chromatographic ASCII formatted data files. MSFACTs is illustrated in the processing of GC/MS metabolomic data from different tissues of the model legume plant, Medicago truncatula. The results document that various tissues such as roots, stems, and leaves from the same plant can be easily differentiated based on metabolite profiles. Further, similar types of tissues within the same plant, such as the first to eleventh internodes of stems, could also be differentiated based on metabolite profiles. AVAILABILITY: Freely available upon request for academic and non-commercial use. Commercial use is available through licensing agreement http://www.noble.org/PlantBio/MS/MSFACTs/MSFACTs.html.

Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti.

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis.

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.