High genetic differentiation among wild populations of alien Medicago sativa in Lithuania

Alfalfa (Medicago sativa; =M. sativa ssp. sativa) in Lithuania is sown as albuminous forage for cattle due to favourable climatic condition. Over many generations, alfalfa plants have escaped from cultivation fields into natural ecosystems and established wild populations. We collected and analyzed individuals from seventeen wild populations of M. sativa. Using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses, 117 RAPD and 64 ISSR reproducible and highly polymorphic (90.8% for RAPD and 86.3% for ISSR) loci were established. AMOVA showed a high genetic differentiation of M. sativa populations for both types of DNA markers utilized. According to RAPD markers, the genetic variability among populations was 63.1% and 57.0% when ISSR markers were used. Taken together, these results demonstrate that wild populations of M. sativa possess a high potential of genetic variability, that could potentially result in colonization of natural ecosystems. The UPGMA cluster analysis also showed that the DNA markers discovered in this study can distinguish between M. sativa and M. falcata (=M. sativa ssp. falcata) populations and therefore may be used to study the genetic impact of M. sativa on the native populations of M. falcata.     

The evolution of hemolytic saponin content in wild and cultivated Alfalfa (Medicago sativa,Fabaceae)

Hemolytic saponin content was determined of the leaves of 1213 plants of different variants ofMedicago sativa s.l. (including wild and cultivated alfalfa), and a close ally,M. papillosa. The latter species had a much higher content than any of the groups ofM. sativa. Medicago sativa ssp. caerulea, the most important ancestor of alfalfa, had a very low content of hemolytic saponins. The most primitive forms of cultivated alfalfa examined, from Turkey, and wildM. sativa ssp. sativa of Turkey, also both had very low contents of hemolytic saponins. This is consistent with, and likely explained by, a direct origin of the two Turkish groups from sympatricM. sativa ssp.caerulea. The second most important ancestor of alfalfa,M. sativa ssp.falcata, had the highest content of any of the examined groups ofM. sativa. Modern “Western” (European, NorthAmerican) cultivars and Western ruderal populations had intermediate levels of hemolytic saponins. This is consistent with, and likely explained by, their origin by hybridization and introgression between the low saponin groups noted above andM. sativa ssp.falcata.     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Chromosomal and molecular rearrangements in somatic hybrids between tetraploid Medicago sativa and diploid Medicago falcata

The aim of this study was to produce somatic hybrids between tetraploid (2n=4x=32) M. sativa and diploid (2n=2x=16) M. ?falcata and analyse their genomic structure. Protoplasts from genotypes selected for regeneration ability from the cultivar Rangelander of M. sativa and Wisfal-1 of M. falcata were electrofused. Seven somatic hybrid calli were produced and one of them regenerated plants. The hybrid nature of these plants and their genetic composition were assessed with morphological, cytological, and molecular analyses. The resulting plants were hyper-aneuploid (2n=33) and contained one extra long chromosome, indicating that a translocation had taken place. The presence of both types of parental sequences in the RAPDs analysis confirmed the true hybrid nature of the plants. Rearrangements within the parental genomes and the presence of somaclonal variation among hybrid plants were observed through an RFLP analysis of the nucleolar organizing region (NOR). The possible causes for the gross genomic alterations, and the suitability of this method for transferring useful agronomic traits from wild species to cultivated alfalfa, are discussed.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

Simulating the impact of genetic diversity of Medicago truncatula on germination and emergence using a crop emergence model for ideotype breeding

Background and Aims

Germination and heterotrophic growth are crucial steps for stand establishment. Numerical experiments based on the modelling of these early stages in relation to major environmental factors at sowing were used as a powerful tool to browse the effects of the genetic diversity of Medicago truncatula, one of the model legume species, under a range of agronomic scenarios, and to highlight the most important plant parameters for emergence. To this end, the emergence of several genotypes of M. truncatula was simulated under a range of sowing conditions with a germination and emergence simulation model.

Methods

After testing the predictive quality of the model by comparing simulations to field observations of several genotypes of M. truncatula, numerical experiments were performed under a wide range of environmental conditions (sowing dates × years × seedbed structure). Germination and emergence was simulated for a set of five genotypes previously parameterized and for two virtual genotypes engineered to maximize the potential effects of genetic diversity.

Key Results

The simulation results gave an average value of 5–10 % difference in final emergence between genotypes, which was low, but the analysis underlined considerable inter-annual variation. The effects of parameters describing germination and emergence processes were quantified and ranked according to their contribution to the variation in emergence. Seedling non-emergence was mainly related to mechanical obstacles (40–50 %). More generally, plant parameters that accelerated the emergence time course significantly contributed to limiting the risk of soil surface crusting occurring before seedling emergence.

Conclusions

The model-assisted analysis of the effects of genetic diversity demonstrated its usefulness in helping to identify the parameters which have most influence that could be improved by breeding programmes. These results should also enable a deeper analysis of the genetic determinism of the main plant parameters influencing emergence, using the genomic tools available for this model plant.     

Inferring population structure and genetic diversity of broad range of wild diploid alfalfa (Medicago sativa L.) accessions using SSR markers

Diversity analyses in alfalfa have mainly evaluated genetic relationships of cultivated germplasm, with little known about variation in diploid germplasm in the M. sativa–falcata complex. A collection of 374 individual genotypes derived from 120 unimproved diploid accessions from the National Plant Germplasm System, including M. sativa subsp. caerulea, falcata, and hemicycla, were evaluated with 89 polymorphic SSR loci in order to estimate genetic diversity, infer the genetic bases of current morphology-based taxonomy, and determine population structure. Diploid alfalfa is highly variable. A model-based clustering analysis of the genomic data identified two clearly discrete subpopulations, corresponding to the morphologically defined subspecies falcata and caerulea, with evidence of the hybrid nature of the subspecies hemicycla based on genome composition. Two distinct subpopulations exist within each subsp. caerulea and subsp. falcata. The distinction of caerulea was based on geographical distribution. The two falcata groups were separated based on ecogeography. The results show that taxonomic relationships based on morphology are reflected in the genetic marker data with some exceptions, and that clear distinctions among subspecies are evident at the diploid level. This research provides a baseline from which to systematically evaluate variability in tetraploid alfalfa and serves as a starting point for exploring diploid alfalfa for genetic and breeding experiments.