Bernadette Julier

Construction of two genetic linkage maps in cultivated tetraploid alfalfa (Medicago sativa) using microsatellite and AFLP markers

BackgroundAlfalfa (Medicago sativa) is a major forage crop. The genetic progress is slow in this legume species because of its autotetraploidy and allogamy. The genetic structure of this species makes the construction of genetic maps difficult. To reach this objective, and to be able to detect QTLs in segregating populations, we used the available codominant microsatellite markers (SSRs), most of them identified in the model legume Medicago truncatula from EST database. A genetic map was constructed with AFLP and SSR markers using specific mapping procedures for autotetraploids. The tetrasomic inheritance was analysed in an alfalfa mapping population.ResultsWe have demonstrated that 80% of primer pairs defined on each side of SSR motifs in M. truncatula EST database amplify with the alfalfa DNA. Using a F1 mapping population of 168 individuals produced from the cross of 2 heterozygous parental plants from Magali and Mercedes cultivars, we obtained 599 AFLP markers and 107 SSR loci. All but 3 SSR loci showed a clear tetrasomic inheritance. For most of the SSR loci, the double-reduction was not significant. For the other loci no specific genotypes were produced, so the significant double-reduction could arise from segregation distortion. For each parent, the genetic map contained 8 groups of four homologous chromosomes. The lengths of the maps were 2649 and 3045 cM, with an average distance of 7.6 and 9.0 cM between markers, for Magali and Mercedes parents, respectively. Using only the SSR markers, we built a composite map covering 709 cM.ConclusionsCompared to diploid alfalfa genetic maps, our maps cover about 88–100% of the genome and are close to saturation. The inheritance of the codominant markers (SSR) and the pattern of linkage repulsions between markers within each homology group are consistent with the hypothesis of a tetrasomic meiosis in alfalfa. Except for 2 out of 107 SSR markers, we found a similar order of markers on the chromosomes between the tetraploid alfalfa and M. truncatula genomes indicating a high level of colinearity between these two species. These maps will be a valuable tool for alfalfa breeding and are being used to locate QTLs.

Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a breeding program, using SSR markers.

Alfalfa (Medicago sativa) is an autotetraploid, allogamous and heterozygous species whose cultivars are synthetic populations. The breeders apply selection pressure for some agronomic traits within a breeding pool to increase the frequency of favorable individuals. The objective of this study was to investigate the differentiation level among seven cultivars originating from one breeding program, and between these cultivars and the breeding pool, with eight SSR markers. These highly polymorphic and codominant markers, together with recent population genetic statistics extended to autotetraploids, offer tools to analyse genetic diversity in alfalfa. The number of alleles per locus varied between 3 and 24. All loci were at a panmictic equilibrium in the cultivars, except one, probably because of null alleles. With seven SSR loci, each cultivar was at panmictic equilibrium. The mean gene diversity was high, ranging from 0.665 to 0.717 in the cultivars. The parameter FST indicated a low but significant diversity among cultivars. Among 21 pairs of cultivars, 15 were significantly different. The breeding pool also had a high diversity, and was significantly different from each cultivar except the most recent one. Considering the characteristics of the breeding program and the mode of cultivar elaboration, we found that they were unable to generate a large variety differentiation. Estimation of population genetics parameters at SSR loci can be applied for assessing the differences between cultivars or populations, either for variety distinction or the management of genetic resources.

Identification of quantitative trait loci influencing aerial morphogenesis in the model legume Medicago truncatula

In many legume crops, especially in forage legumes, aerial morphogenesis defined as growth and development of plant organs, is an essential trait as it determines plant and seed biomass as well as forage quality (protein concentration, dry matter digestibility). Medicago truncatula is a model species for legume crops. A set of 29 accessions of M. truncatula was evaluated for aerial morphogenetic traits. A recombinant inbred lines (RILs) mapping population was used for analysing quantitative variation in aerial morphogenetic traits and QTL detection. Genes described to be involved in aerial morphogenetic traits in other species were mapped to analyse co-location between QTLs and genes. A large variation was found for flowering date, morphology and dynamics of branch elongation among the 29 accessions and within the RILs population. Flowering date was negatively correlated to main stem and branch length. QTLs were detected for all traits, and each QTL explained from 5.2 to 59.2% of the phenotypic variation. A QTL explaining a large part of genetic variation for flowering date and branch growth was found on chromosome 7. The other chromosomes were also involved in the variation detected in several traits. Mapping of candidate genes indicates a co-location between a homologue of Constans gene or a flowering locus T (FT) gene and the QTL of flowering date on chromosome 7. Other candidate genes for several QTLs are described.

Achievements and Challenges in Improving Temperate Perennial Forage Legumes

The expected move towards more sustainable crop-livestock systems implies wider cultivation of perennial forage legumes. Alfalfa (Medicago sativa subsp. sativa) is the main perennial legume in most temperate regions, especially where farm systems rely largely on forage conservation. White clover (Trifolium repens) and red clover (Trifolium pratense) are dominant in specific regions and farm systems. Although breeding progress for disease and insect resistance has been achieved, these crops have shown lower rates of genetic gain for yield than major grain crops, owing to lower breeding investment, longer selection cycles, impossibility to capitalize on harvest index, outbreeding mating systems associated with severe inbreeding depression, and high interaction of genotypes with cropping conditions and crop utilizations. Increasing yield, persistence, adaptation to stressful conditions (drought; salinity; grazing) and compatibility with companion grasses are major breeding targets. We expect genetic gain for yield and other complex traits to accelerate due to progress in genetic resource utilization, genomics resource development, integration of marker-assisted selection with breeding strategies, and trait engineering. The richness in adaptive genes of landraces and natural populations can be fully exploited through an ecological understanding of plant adaptive responses and improved breeding strategies. Useful genetic variation from secondary and tertiary gene pools of Medicago and Trifolium is being increasingly accessed. Genome sequencing projects in alfalfa and white clover will enrich physical, linkage and trait maps. Genome sequences will underpin fine mapping of useful loci and subsequent allele mining, leveraging the synteny of these crops with M. truncatula. Low-cost genome-wide markers generated through genotyping-by-sequencing will make genomic selection for adaptation and forage yield possible for these crops. Genetic markers will also be used for dissecting quantitative traits and developing toolboxes of functional markers for stress tolerance and other traits. Under current regulatory policies, transgenic approaches are likely to be limited to a few breakthrough traits. The key challenge for future applications of genomics technologies is their seamless integration with breeding system logistics and breeding schemes.

Genetic diversity in red clover (Trifolium pratense L.) revealed by morphological and microsatellite (SSR) markers

The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is little described. The goal of the present study was to evaluate the diversity of a set of accessions from the core collection at the morphological and molecular level in order to extract some valuable accessions for Brazilian red clover breeding programs. Twenty-one morphological traits, collected in field and greenhouse in South Brazil, and seven SSR markers were used to describe 57 accessions from the U.S. core collection and one population cultivated in Southern Brazil. Variation between accessions was large for most of the 21 morphological traits. A cluster analysis based on the morphological traits revealed five distinct clusters that separated the populations according to flowering earliness, as already described, but also according to persistency, growth habit and dry matter productivity. Over seven SSR loci, the number of alleles averaged 11.1 alleles per locus. Genetic diversity measured with SSR markers was high, with a mean expected heterozygosity of 0.86. An analysis of molecular variance revealed that the largest proportion of variation (83.6%) resided at the within population level. Although the molecular markers also separated accessions into five clusters, there was no coincidence between the composition of groups found with morphological and molecular data. Use of genetic diversity in breeding programs requires to use the most promising populations, to combine positive traits such as persistency and forage yield, and probably to use within population variation to detect valuable genotypes that could be used as parents of synthetic varieties.

Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt

Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

A CONSTANS-like gene candidate that could explain most of the genetic variation for flowering date in Medicago truncatula

Flowering is a critical period during a plant’s life cycle, and thus the identification and characterization of genes involved in flowering date control are of great importance in agronomy, breeding, population genetics and ecology. The model species Medicago truncatula can be used to detect genes explaining the variation for flowering date, which could also explain this variation in legume crops. The objective of this study was to identify the most promising candidate gene explaining a major quantitative trait locus (QTL) for flowering date previously found in three M. truncatula mapping populations. Fine mapping and bioinformatic analysis of bacterial artificial chromosomes (BACs) in the confidence interval of the QTL showed six genes potentially involved in the control of flowering date. Two of these genes, similar to CONSTANS and FT of Arabidopsis thaliana respectively, had genomic mutations when compared to the parents. The transcriptomic study of these genes by semi-quantitative RT-PCR in leaves and flowers sampled at two developmental stages showed that the CONSTANS-like gene was differentially expressed in the two parental lines. A gene belonging to the CONSTANS-like family could explain the major QTL for flowering date segregating in M. truncatula progenies.

Detection of favorable alleles for plant height and crown rust tolerance in three connected populations of perennial ryegrass (Lolium perenne L.)

Plant height, which is an estimator of vegetative yield, and crown rust tolerance are major criteria for perennial ryegrass breeding. Genetic improvement has been achieved through phenotypic selection but it should be speeded up using marker-assisted selection, especially in this heterozygous species suffering from inbreeding depression. Using connected multiparental populations should increase the diversity studied and could substantially increase the power of quantitative trait loci (QTL) detection. The objective of this study was to detect the best alleles for plant height and rust tolerance among three connected populations derived from elite material by comparing an analysis per parent and a multipopulation connected analysis. For the studied traits, 17 QTL were detected with the analysis per parent while the additive and dominance models of the multipopulation connected analysis made it possible to detect 33 and 21 QTL, respectively. Favorable alleles have been detected in all parents. Only a few dominance effects were detected and they generally had lower values than the additive effects. The additive model of the multipopulation connected analysis was the most powerful as it made it possible to detect most of the QTL identified in the other analyses and 11 additional QTL. Using this model, plant growth QTL and rust tolerance QTL explained up to 19 and 38.6% of phenotypic variance, respectively. This example involving three connected populations is promising for an application on polycross progenies, traditionally used in breeding programs. Indeed, polycross progenies actually are a set of several connected populations.

Seed yield and yield stability of determinate and indeterminate autumn-sown white lupins ( Lupinus albus ) grown at different locations in France and the UK

The seed yields and maturity dates of an indeterminate cultivar (Lunoble) and a determinate line (CH304/70) of Lupinus albus L. were measured at three locations in France (Lusignan, Dijon and Gotheron) and at Rothamsted, UK, in 1989/90 and 1990/91. Different combinations of sowing dates, plant densities and irrigation treatments were tested at some sites. Averaged over all sites, CH304/70 yielded more than Lunoble (3.26 v. 2.98 t/ha) but there were significant genotype x location interactions for yield and date of maturity. Both genotypes gave similar yields at the three locations in France (3.13 and 3.06 t/ha, respectively) (.)

Among and within-cultivar variability for histological traits of lucerne (Medicago sativa L.) stem

The histological structure of lucerne stems is a limiting factor for digestibility of the forage. Two experiments were carried out to compare cultivars and genotypes within cultivars for histological traits of the stems. In the first one, the among-cultivar genetic differences were assessed on 6 cultivars differing indigestibility. In the second one, the within-cultivar differences were evaluated on twenty genotypes belonging to two cultivars. Two sets of quantitative histological traits were measured using image analysis on the basal portion of the stems corresponding to the mature zone: the proportions of the different tissues of the lucerne stems (cortex, xylem, pithparenchyma), and the traits describing the xylem in vascular bundles (xylem cell wall thickness, xylem cell wall surface density, mean area of xylem vessel and mean area of fibre lumen). Few significant differences were observed among-cultivars, where as numerous within-cultivar differences were observed for histological traits. When the stem length was introduced as covariate, the differences among- and within-cultivars remained significant. Xylem proportion was negatively correlated to pith parenchyma proportion only. Xylem cell wall surface density and thickness were correlated to the cross-section radius but not to xylem proportion. As digestibility of lucerne is limited by histological structures, the large within-cultivar differences for histological traits of lucerne stems could be used in a breeding program for improved digestibility.