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Medicago ruthenica is an important perennial forage with multiple characteristics of resistance. In this study, we sequenced and characterized the complete chloroplast genome of M. ruthenica 'Taihang', which is 124, 254 bp in length. A total of 108 genes were identified, including 74 protein-coding, 30 tRNA, and four rRNA genes. Phylogenetic analysis based on 27 chloroplast genomes showed that M. ruthenica 'Taihang' has a close relationship with M. ruthenica from Qinghai Province, China. The data are useful in better understanding the genetic diversity and stress resistance of Medicago and contribute to the phylogenetic study of Trifolieae.

Seed morphology was described in detail for 12 species belonging to 5 genera of tribes Astragaleae and Trifolieae (Leguminosae; Papilionoideae) using scanning electron microscopy (SEM) to evaluate the taxonomic relevance of macromorphological and micromorphological seed characters. The study aims to search for diagnostic seed ultrastructural features that may help to elucidate species identification. For SEM analysis, seed morphological characters including seed form and shape, color and size, ornamentation, epidermal cell shape, and anticlinal wall pattern were investigated. As a result of the study, species-specific characters have been determined. Based on seed exomorphology, three characteristic cell patterns; irregular, round, and flat were observed. In majority of studied taxa, species may be further differentiated based on seed shape, size, and surface ornamentation. The inconsistency in testa cell pattern, shape, and distribution of papillae or protuberances may probably give further insight and significant morphological features at specific and generic level within the tribe. This study illustrated that considerable taxonomic knowledge can be obtained by examining the seed characters of Astragaleae and Trifolieae, particularly at the species level. The results demonstrated that the use of SEM in seed morphology could play a role in the identification of taxa particularly at genus and species level. Twelve species of Astragaleae and Trifolieae were studied in order to describe and investigate the seed morphology and to evaluate the diagnostic value of this character using a SEM. A broader taxon sampling is required for classification at generic and tribal level, besides molecular and phylogenetic studies.

Medicago archiducis-nicolai Sirj. is a well-known high-quality forage as its good palatability and strong tolerance to drought, cold and saline-alkali stress. Here, the complete chloroplast genome ...

The genus Ononis comprises 86 species worldwide distributed in temperate regions. It is represented in Egypt by ten species. During recent excursions to the mountains of southern Sinai Peninsula, a newly recorded species, namely O. viscosa subsp. breviflora, was collected. This new record was previously confused with O. sicula. These two species can be differentiated by corolla length and shape. This study provides a full taxonomic revision of the genus for the flora of Egypt, updated nomenclature for all taxa, amended descriptions, detailed hand drawings, and a diagnostic key to the species. The most valuable diagnositic characters in the taxonomy of the genus in Egypt are discussed. After critical examination and literature study, we found that two names (O. serrata and O. vaginalis) needed to be lectotypified.

The genus Medicago L. belongs to the family Fabaceae, subfamily Papilionoideae, tribe Trifolieae. Seed and seedling morphology of twenty-five taxa under two sections and three subsections of the genus Medicago have been studied based on fresh materials. Seeds were obtained from the Institute of Plant Genetics and Crop Plant Research (IPK) in Germany and the International Center for Agricultural Research in the Dry Areas (ICARDA) in Syria. External and internal morphological characteristics of seeds and seedlings were observed. The characters of seeds, hypocotyl, paracotyledons, epicotyl and first leaves of seedlings provide important information which has been used in construction of artificial keys for identification purpose. Significant characters like seed color, shape, radical length in relation to seed length, epicotyl color and hairiness, first foliage leaf shape and hairiness used as important morphological descriptors for species identification. Cluster analysis has been used to determine taxonomic interrelationships among the investigated taxa.

The genus Trifolium is the largest of the tribe Trifolieae in the subfamily Papilionoideae (Fabaceae). The paucity of mitochondrial genome (mitogenome) sequences has hindered comparative analyses among the three genomic compartments of the plant cell (nucleus, mitochondrion and plastid). We assembled four mitogenomes from the two subgenera (Chronosemium and Trifolium) of the genus. The four Trifolium mitogenomes were compact (294,911&ndash, 348,724 bp in length) and contained limited repetitive (6.6&ndash, 8.6%) DNA. Comparison of organelle repeat content highlighted the distinct evolutionary trajectory of plastid genomes in a subset of Trifolium species. Intracellular gene transfer (IGT) was analyzed among the three genomic compartments revealing functional transfer of mitochondrial rps1 to nuclear genome along with other IGT events. Phylogenetic analysis based on mitochondrial and nuclear rps1 sequences revealed that the functional transfer in Trifolieae was independent from the event that occurred in robinioid clade that includes genus Lotus. A novel, independent fission event of ccmFn in Trifolium was identified, caused by a 59 bp deletion. Fissions of this gene reported previously in land plants were reassessed and compared with Trifolium.

Medicago ruthenica is a well-known high-quality forage due to its good palatability and strong tolerance to drought, cold and saline-alkali stress. Here, the complete chloroplast genome sequence of...

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Two bacterial strains Ach-343 and Opo-235 were isolated, respectively from nodules of Miocene-Pliocene relict legumes Astragalus chorinensis Bunge and Oxytropis popoviana Peschkova originated from Buryatia (Baikal Lake region, Russia). For identification of these strains the sequencing of 16S rRNA (rrs) gene was used. Strain Opo-235 belonged to the species Mesorhizobium japonicum, while the strain Ach-343 was identified as M. kowhaii (100 and 99.9% rrs similarity with the type strains MAFF 303099T and ICMP 19512T, respectively). Symbiotic genes of these strains as well as some genes that promote plant growth (acdS, gibberellin- and auxin-synthesis related genes) were searched throughout the whole genome sequences. The sets of plant growth-promoting genes found were almost identical in both strains, whereas the sets of symbiotic genes were different and complemented each other with several nod, nif, and fix genes. Effects of mono- and co-inoculation of Astragalus sericeocanus, Oxytropis caespitosa, Glycyrrhiza uralensis, Medicago sativa, and Trifolium pratense plants with the strains M. kowhaii Ach-343 and M. japonicum Opo-235 expressing fluorescent proteins mCherry (red) and EGFP (green) were studied in the gnotobiotic plant nodulation assay. It was shown that both strains had a wide range of host specificity, including species of different legume genera from two tribes (Galegeae and Trifolieae). The effects of co-microsymbionts on plants depended on the plant species and varied from decrease, no effect, to increase in the number of nodules, nitrogen-fixing activity and plant biomass. One of the reasons for this phenomenon may be the discovered complementarity in co-microsymbionts of symbiotic genes responsible for the specific modification of Nod-factors and nitrogenase activity. Localization and co-localization of the strains in nodules was confirmed by the confocal microscopy. Analysis of histological and ultrastructural organization of A. chorinensis and O. popoviana root nodules was performed. It can be concluded that the strains M. kowhaii Ach-343 and M. japonicum Opo-235 demonstrate lack of high symbiotic specificity that is characteristic for primitive legume-rhizobia systems. Further study of the root nodule bacteria having complementary sets of symbiotic genes will contribute to clarify the evolutionary paths of legume-rhizobia relationships and the mechanisms of effective integration between partners.

Uromyces ciceris-arietini has been reported on Cicer arietinum (chickpea) and Medicago polyceratia. Plants of Medicago polymorpha in Riverside and San Diego, CA were collected with severe rust caused by U. ciceris-arietini. To confirm the identification and potential new host range, a monouredinial isolate of U. ciceris-arietini from M. polymorpha was inoculated on eight accessions each of C. arietinum and M. polyceratia. All plants showed symptoms of the disease. Consequently, a range of fabaceous hosts were evaluated for their reaction to U. ciceris-arietini. New hosts for U. ciceris-arietini included 29 species of Medicago, specifically M. arabica, M. blancheana, M. ciliaris, M. constricta, M. coronata, M. doliata, M. granadensis, M. intertexta, M. italica, M. laciniata, M. lanigera, M. lesinsii, M. lupulina, M. minima, M. murex, M. muricoleptis, M. orbicularis, M. praecox, M. radiata, M. rigidula, M. rotata, M. rugosa, M. sativa, M. sauvagei, M. scutellata, M. soleirolii, M. tenoreana, M. truncatula, and M. varia, and three species of Melilotus, specifically M. italicus, M. speciosus, and M. spicatus. This isolate of U. ciceris-arietini produced no symptoms on plants in the 33 accessions tested in the genera Anthyllis, Astragalus, Lotus, and Lupinus. DNA sequences are provided to aid in the identification of this pathogen.

Here, we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima with those of members of an Ensifer reference collection. Our goal was to determine if the T. suavissima rhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade within E. meliloti . The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere. IMPORTANCE In this study, we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study, we were unable to find persuasive evidence supporting this hypothesis. Instead, our results suggest either that the T. suavissima rhizobia are native to Australia or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.

Melilotus, an annual or biennial herb, belongs to the tribe Trifolieae (Leguminosae) and consists of 19 species. As an important green manure crop, diverse Melilotus species have different values as feed and medicine. To identify different Melilotus species, we examined the efficiency of five candidate regions as barcodes, including the internal transcribed spacer (ITS) and two chloroplast loci, rbcL and matK, and two non-coding loci, trnH-psbA and trnL-F. In total, 198 individuals from 98 accessions representing 18 Melilotus species were sequenced for these five potential barcodes. Based on inter-specific divergence, we analysed sequences and confirmed that each candidate barcode was able to identify some of the 18 species. The resolution of a single barcode and its combinations ranged from 33.33% to 88.89%. Analysis of pairwise distances showed that matK+rbcL+trnL-F+trnH-psbA+ITS (MRTPI) had the greatest value and rbcL the least. Barcode gap values and similarity value analyses confirmed these trends. The results indicated that an ITS region, successfully identifying 13 of 18 species, was the most appropriate single barcode and that the combination of all five potential barcodes identified 16 of the 18 species. We conclude that MRTPI is the most effective tool for Melilotus species identification. Taking full advantage of the barcode system, a clear taxonomic relationship can be applied to identify Melilotus species and enhance their practical production.

This is the first report describing isolates from root nodules of Ononis arvensis (field restharrow). The aim of this investigation was to describe the diversity, phylogeny, and plant growth promoting features of microsymbionts of O. arvensis, i.e., a legume plant growing in different places of the southern part of Poland. Twenty-nine bacterial isolates were characterized in terms of their phenotypic properties, genome fingerprinting, and comparative analysis of their 16S rRNA, nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies, the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of all the isolates tested exhibited the highest similarities to the corresponding gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in the genomes of rhizobia specific for O. arvensis implies that these bacteria may promote the growth and development of their host plant in stress conditions. The isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction, all of them (except three) exhibited DNA fingerprints specific only for them. Our studies showed that restharrow isolates formed effective symbiotic interactions with their native host (O. arvensis) and Ononis spinosa but not with Trifolium repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis species and not with Lotus corniculatus, representing the tribe Loteae.

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 Cicer 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 four 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.

The plastid genome retains several features from its cyanobacterial-like ancestor, one being the co-transcriptional organization of genes into operon-like structures. Some plastid operons have been identified but undoubtedly many more remain undiscovered. Here we utilize the highly variable plastome structure that exists within certain legumes of the inverted repeat lost clade (IRLC) to find conserved gene clusters. These plastomes exhibit an unusually high frequency of translocations and inversions. We analysed the plastomes of 23 legume species and identified 32 locally collinear blocks (LCBs), which are regions within the plastid genomes that occur in different orientation and/or order among the plastid genomes but are themselves free from internal rearrangements. Several represent gene clusters that have previously been recognized as plastid operons. It appears that the number of LCBs has reached saturation in our data set, suggesting that these LCBs are not random, but likely represent legume plastid operons protected from internal rearrangement by functional constraint. Some of the LCBs we identify, such as psbD/C/Z, are previously known plastid operons. Others, such as rpl32-ndhF-psbA-matK-rbcL-atpB-atpE, may represent novel polycistronic operons in legumes.

Efficiency of symbiotic nitrogen fixation in legumes depends on bringing together the processes of N2 fixation, assimilation of its products, supply of nitrogenase with energy, and development of nodule tissue and cellular structures. Coordination of these processes could arise from the evolutionary old functions of the nodules associated with deposition of the products of photosynthesis governed by systemic signals traveling between the above-ground organs and the roots. Further increase in symbiotic efficiency was associated with a pronounced ability to fix N2 by intracellular bacteroids that lost capability to propagate (as observed in galegoid legumes from the tribes Viciae, Trifolieae, and Galegae producing indeterminate nodules). However, efficiency of these symbioses is restricted by a slow removal from the nodules of the products of N2 fixation, which are assimilated along the same amide pathway as nitrogen compounds arriving from the soil. In legumes from the tribe Phaseoleae, such a restriction was overcome owing to a particular way of nitrogen assimilation via its incorporation into ureides (in determinate nodules). Development of symbioses where specialization of bacteroids in symbiotic fixation of atmospheric nitrogen is combined with its ureide assimilation will make it possible to produce new forms of plants highly efficient in symbiotic nitrogen fixation.

The aims of the present study were (i) to compare the anatomical structures of roots from legume species belonging to the tribes Aeschynomeneae, Fabeae (syn. Vicieae), and Trifolieae (Papilionoideae); (ii) to characterize those developmental stages of roots that are most useful for comparative studies; (iii) to characterize root developmental patterns during their growth from primary to adult structures; and (iv) to identify developmental patterns that are typical for particular tribes within this plant group. To this end, serial transverse cuts of the main roots of 20 species belonging to the above-mentioned tribes were made from the root tip to the root collar. Vessel density, vessel diameter, secondary cortex area, and xylem area were quantified using appropriate software. Based on root ontogeny, four stages giving evidence for important developmental events were chosen based on which a comparison of the species-specific structures became possible. The first stage corresponds to the primary structure of a root. The second stage corresponds to the onset of cambium and phellogen division, when their first division products become differentiated. The third and fourth stages correspond to adult roots with a well-developed secondary structure. These developmental stages that allowed between-species comparison turned out to be useful characterising those patterns that best typify a particular tribe within the subfamily. Vessel density, vessel diameter, and the xylem transverse area were found to differ significantly between the tribes examined. Some of the analyzed morphological variables had a tribe-specific consistency, so that, together with other characteristics, they should be included in approaches to clarify systematic problems in the legume family.

Nodules in the family Leguminosae come in many different shapes and forms, depending partly on the host that bears them. This review focuses on the peripheral tissues that surround the infected tissues of four anatomically distinct classes of nodules: nodules with indeterminate meristems (from the Trifolieae and Fabeae tribes); desmodioid nodules with determinate meristems (from the Phaseoleae and Loteae tribes); aeschynomenoid nodules ( Arachis hypogaea L., peanut); and lupinoid nodules ( Lupinus sp. L., lupine). I have especially stressed the importance of the three-dimensionality of the organ, because not all nodules display a radial symmetry as it is often assumed. The goal of this review is to provide a strong base of nodule structure so that forthcoming molecular studies can integrate this information into their approach.

A morphological study of Medicago sect. Dendrotelis was undertaken by using scanning electron microscope (SEM). Upper and lower surface of leaves, stomata, surface of wings, exine and number of apertures of pollen and seed testa, show remarkable characters for distinguishing among taxa. The western Mediterranean endemism M. citrina is characterized by unique features related to pollen (4-zonocolporate), surface of the upper basal and lower distal parts wing, and testa seed (reticules quite apparent). With regard to M. strasseri, the morphology of leaf cells, upper and lower distal part of wings and seed testa were useful for characterization. On the contrary, M. arborea shared most of characters with M. citrina (e.g. surface of leaves) or M. strasseri (e.g. pollen apertures), excepting the micromorphology of certain part of wings. Among all the studied characters, the surface of wings was found noticeable to characterize each taxa of Medicago sect. Dendrotelis. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The genus Trigonella (Fabaceae, tribe Trifolieae) includes potential new species for pasture legume production in phase farming systems of southern Australia. Performance under biotic stress is an important factor in selecting the most suitable species and genotypes for pasture legume breeding programs. Fifty‐nine accessions representing 26 Trigonella species and two Medicago species (formerly with Trigonella) were evaluated for resistance to two aphid species, bluegreen aphid (Acyrthosiphon kondoi Shinji), and spotted alfalfa aphid (Therioaphis trifolii (Monell) fm. maculata) in glasshouse experiments. An accession of Trigonella macrorrhyncha (SA 32223) was found to be highly resistant to both the aphids. Accessions of T. coerulescens, T. cylindracea, T. gladiata, T. mesopotamica, T. schlumbergeri and Medicago monspeliaca were found to be resistant to bluegreen aphid, but susceptible to spotted alfalfa aphid. The resistance mechanism in T. macrorrhyncha involved both antixenosis (non‐preference ...

The carbohydrate-binding sequences (CBS) in the lectin genes of Trifolium repens, T. pratense, and T. trichocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.

An analysis of the histone H1 subtype, H1-1, in eight legumes belonging to four genera of the tribe Vicieae (Pisum, Lathyrus, Lens, and Vicia), revealed an extended region consisting of the tandemly repeated AKPAAK motifs. We named this region the Regular zone (RZ). The AKPAAK motifs are organized into two blocks separated by a short (two or six amino acids) intervening sequence (IS). The distal block contains six AKPAAK motifs, while the number of repeats in the proximal block varies from six in V. faba to seven in the other species. In V. hirsuta, the first two repeated units of the proximal block are octapeptides AKAKPAAK. The apparent rate of synonymous substitutions in the blocks of RZ is much higher than in the rest of the gene. This can be explained by repeat shuffling within each block. In the C-domain of the orthologous H1 subtype from Medicago truncatula (tribe Trifolieae), a region corresponding to the RZ of Vicieae species was found. It also consists of two blocks of AKPAAK motifs (four and three repeats in the proximal and distal blocks, respectively). These blocks are separated by a 20-amino acid IS. The first 20 amino acids of the Medicago RZ are not part of AKPAAK repeats. We hypothesise that the RZ has most probably evolved as a result of an expansion of AKPAAK repeats from two separate sites in the C-domain. This process started tens of millions of years ago and was most likely directed by positive selection.

Trifolium kurdistanicum, a new species of Trifolium sect. Trifolium from Kurdistan Province, west of Iran is described. It grows at high altitude on subalpine regions with steep slopes and shallow soil. The new species is morphologically close to T. longidentatum and can be considered as a member of the subsect. Ochroleuca. Trifolium kurdistanicum is characterized by large, multiple and branched stems, less than half of the stipule length adnate to the petiole, inflorescences without bracts, calyx with 3-nerved lower and 1-nerved upper teeth and all slightly longer than the tube, calyx nearly half the length of corolla, flowers violet or rarely white and seeds reniform.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.

The first complete plastid genome (plastome) of the largest angiosperm genus, Astragalus, was sequenced for the Korean endangered endemic species A. mongholicus var. nakaianus. Its genome is relatively short (123,633 bp) because it lacks an Inverted Repeat (IR) region. It comprises 110 genes, including four unique rRNAs, 30 tRNAs, and 76 protein-coding genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. The putative pseudogene with abnormal stop codons is atpE. This plastome has no additional inversions when compared with highly variable plastomes from IRLC tribes Fabeae and Trifolieae. Our phylogenetic analysis confirms the non-monophyly of Galegeae.

We investigated the transferability of 31 soybean (Glycine max) simple sequence repeat (SSR) loci to wild congeners and to other legume genera. Up to 65% of the soybean primer pairs amplified SSRs within Glycine, but frequently, the SSRs were short and interrupted compared with those of soybeans. Nevertheless, 85% of the loci were polymorphic within G. clandestina. Cross-species amplification outside of the genus was much lower (3%-13%), with polymorphism restricted to one primer pair, AG81. AG81 amplified loci in Glycine, Kennedia, and Vigna (Phaseoleae), Vicia (Vicieae), Trifolium (Trifolieae), and Lupinus (Genisteae) within the Papilionoideae, and in Albizia within the Mimosoideae. The primer conservation at AG81 may be explained by its apparent proximity to the seryl-tRNA synthetase gene. Interspecific differences in allele size at AG81 loci reflected repeat length variation within the SSR region and indels in the flanking region. Alleles of identical size with different underlying sequences (size homoplasy) were observed. Our findings and the emerging patterns in other plant studies suggest that in contrast to animals, successful cross-species amplification of SSRs in plants is largely restricted to congeners or closely related genera. Because mutations in both the SSR region and the flanking region contribute to variation in allele size among species, knowledge of DNA sequence is essential before SSR loci can be meaningfully used to address applied and evolutionary questions.

Sinorhizobium meliloti is a well-known symbiont that fixes nitrogen with plants of three genera of the family Leguminosae, Tribe Trifolieae: Medicago, Melilotus, and Trigonella. We have found that S. meliloti strain RCR2011 forms ineffective nodules on a distantly related legume species, Sesbania macrocarpa. Nodules are indeterminate and lack leghemoglobin. Although S. meliloti deformed root-hairs of S. macrocarpa, infection threads were not detected in these cells. Instead, cells of S. meliloti were found in infection pockets between cortical cells and within infection threads that had penetrated into these cells. Bacteria eventually were released and gave rise to symbiosomes, which contained large quantities of an amorphous matrix with parallel arrays of electron-dense striations. Mutants of strain RCR2011 lacking nodA or nodH failed to nodulate S. macrocarpa, inactivation of nodG or nodE resulted in smaller nodules, while inactivation of region IIa, which contains nodIJ, had no apparent effect on nodulation.

The pollen morphology of 32 species of the genus Vicia was studied using scanning and transmission electron microscope (SEM and TEM). The interstitia of the pollen of the examined species were found to be either regular columellate, irregular columellate, or granular. The granular pattern was recognized to be apomorphy against the columellate patterns in the tribe Vicieae by comparison with the pollen in the tribes Vicieae, Cicereae, Galegeae, Hedysareae, and Trifolieae. The group of Vicia species having the apomorphy was congruent with that having a known apomorphy of the pistil character. This group with such synapomorphies may be monophyletic, though it is treated as polyphyletic in the present infrageneric system of Vicia.

The morphology and development of styles and stigmas ofVicia, one of the five genera of tribe Vicieae, were studied by scanning electron microscopy to contribute to a revision of infrageneric classification. Among the 22 species examined, variations were found as to: (1) whether a boundary between stigma and style is V-shaped when viewed from the adaxial side or not; (2) whether stylar hairs sprout on all sides of the style or only on the abaxial side; (3) whether a hairy area of the style connects with the stigma area first at lateral sides or at an adaxial side; (4) whether styles are tapered or bottle-neck-shaped; (5) whether styles are straight or bend abaxially. Based on these variations, five morphological types of styles and stigmas were provided with a diagnostic key. Comparisons with related tribes Cicereae, Coronilleae, Galegeae, Hedysareae, Loteae and Trifolieae as possible outgroups suggested that the V-shaped boundary between stigma and style is a synapomorphy of some species withinVicia, and that a dorsally compressed and abaxially tufted style, which was distributed in separate subgenera and sections sensu Kupicha but is now known to occur together with the V-shaped boundary, has evolved only one time in the genus, rather than more than once as supposed earlier.

Electrophoresis of seed proteins from the tribe Vicieae species was carried out on SDS-PAGE. On the basis of similarity matrix index, the homology of Cicer arietinum with the rest of the members of the tribe Vicieae is approximately the same as that of Trifolieae and Ononideae. This endorsed the removing of Cicer from Vicieae and assigned it to a separate tribe, the Cicerideae. The three dimensional model of the Cicer species grouped the species into five groups. One of these groups includes C. arietinum and C. reticulatum. These data support the suggestion that C. reticulatum is the wild progenitor of the cultivated Cicer.

A cladistic analysis of tribes Cicereae, Trifolieae, and Vicieae was carried out using 33 morphological, anatomical, karyological, and chemotaxonomical characteristics. Transformation polarities of the character states were developed by comparison with the character states of the genus Galega, the sister group of the tribes. Cicereae and Vicieae were found to be a monophyletic group, and Trifolieae was its sister group. The seven synapomorphic characters of Cicereae and Vicieae were presumed to be: germination hypogeal vs. epigeal; first leaf in seedlings scarious vs. laminous; leaves paripinnately and tendrilous vs. imparipinnately and etendrilous; postchalazal vascular bundle present in seed coats vs. absent; a cotyledon flap present at antihilar side vs. absent; radicles short in seeds vs. long; and plumules long in seeds vs. short.

Changes in α- and β-amylase activities in the cotyledons of germinating clover were examined. The activity of α-amylase decreased during germination and growth of seedling, while β-amylase activity increased. These changes are different from those of germinating seeds of many other plants, but similar to the germinating seeds of alfalfa, which belongs to the same tribe (Trifolieae) as clover.

Delimitation of Medicago from its close relatives in tribe Trifolieae subtribe Trigonellinae remains controversial. Scanning electron microscope studies of the seed surface of Medicago, Trigonella, and Melilotus showed consistent differences between Medicago and the other two genera. Smooth and rough seeds occur in all three genera, the rough seeds proving taxonomically useful. The roughness of Medicago seeds is due to rugose surface bumps (usually wrinkled, always with rounded sides), except in the monotypic section Orbiculares. The roughness of seeds of Trigonella, Melilotus, and Medicago section Orbiculares is due to tuberculate surface bumps. The structure of the Malpighian layer shows completely parallel taxonomic differences. The Malpighian cells are traversed by the light line, and the length of the portion of the cell above the light line (a) and that below the light line (b) distinguishes smooth, rugose, and tuberculate seeds. In smooth seeds (of Medicago, Melilotus, and Trigonella), a and b are constant. In rugose seeds (in Medicago only), a is constant while b is greater under the testa bumps, varying with the height of the portion of the bump occupied. In tuberculate seeds (in Medicago section Orbiculares, and in Trigonella and Melilotus), b is constant while a is greater under the testa bumps. These studies confirm the appropriateness of circumscribing Medicago in a wide sense, except for the inclusion of Medicago section Orbiculares.

International audience; Pea albumin 1b (PA1b) is a small sulphur-rich peptide from pea seeds, also named leginsulin because of the binding characteristics of its soybean orthologue. Its insecticidal properties were discovered more recently. By using a combination of molecular, biochemical and specific insect bioassays on seed extracts, we characterised genes from numerous Papilionoideae, but not from Caesalpinioideae or Mimosoideae, although the last group harboured species with partially positive cues (homologous biological activities). The A1b defence peptide family, therefore, appears to have evolved relatively late in the legume lineage, maybe from the sophoroid group (e.g. Styphnolobium japonicum). However, unambiguous sequence information is restricted to a group of tribes within the subfamily Papilionoideae (Psoraleae, Millettieae, Desmodieae, Hedysareae, Phaseoleae, Vicieae, and the now clearly polyphyletic "Trifolieae" and "Galegeae"). Recent diversification by gene duplications has occurred in many species, or longer ago in some lineages (Medicago truncatula), as well as probable gene or expression losses at different taxonomic levels (Loteae, Vigna subterranea).

A new species, Trigonella bakhtiarica, from the Iranian province Chahar Mahal Va Bakhtiari is described, illustrated and compared to its most closely related species, T. aphanoneura. Trigonella bakhtiarica has a longer corolla and differs in the shape, surface and size of its pods, which are taxonomically informative characters in Trigonella sect. Ellipticae. Chromosome counts and meiosis assays show that both species are diploid, and that their euploid plants possess a somatic chromosome number of 2n = 2x = 16, which is consistent with the predicted base number of x = 8.

ferent tribes or subtribes: Candolle (1825) to the Phaseoleae DC., based on fruit characters; Wight & Arnott (1834) to the Phaseolinae (DC.) Wight & Arn.; Bentham & Hooker (1865) to the Trifolieae Bercht. & J. Presl, based on habit; Schulz (1901) to the Trifoliinae DC., along with Trifolium L. and Ononis L., based on ovule morphology and seed position within the pods. Small (1987, 1989) and Small & Jomphe (1989) pointed out the anomalous position of Parochetus and Ononis in the Trifolieae and their not being closely related to the other genera of that tribe. Further, studies were therefore essential to elucidate the definite affinity of Parochetus with other papilionoid genera. While revising the Trifolieae (excluding Ononis) of India we also reached the conclusion that Parochetus differs considerably from the other genera of the tribe in many morphological characters of the stipules, leaves, bracts and flowers. As the genus does not fit within either of the described subtribes of the Trifolieae, Trifolii

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.