Sandrine Flajoulot

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.

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.

Sample size for diversity studies in tetraploid alfalfa (Medicago sativa) based on codominantly coded SSR markers

The number of genotypes investigated per population is important for the reliability of diversity studies. The objective of this study was to determine the sample size for the identification of differences among populations of an outcrossing autotetraploid species, alfalfa (Medicago sativa), using codominantly coded SSR markers. One hundred and twenty genotypes from each of two closely related populations were analysed with two markers. Twenty random subsamples for each of three sample sizes (10, 20 and 40 genotypes) were built. Compared to the populations with 120 genotypes, alleles that were no longer present in subsamples with 40 genotypes were mainly rare, whereas abundant alleles were also excluded in subsamples with 10 genotypes. FST values for pairs of subsamples between the two populations were always significantly different based on 40 genotypes, whereas for 10 genotypes more than half of the pairs were not significantly different. We concluded that 40 genotypes are a reasonable sample size for diversity studies with closely related populations of tetraploid alfalfa investigated with SSR markers. Twenty genotypes may be an economical alternative for large scale studies, but 10 genotypes were a too low number for reliable results.

Genetic Diversity Assessment of Two Medicago sativa Genes: CAD and WXP1

Alfalfa (Medicago sativa L.) is a major perennial forage legume crop with numerous nutritional and environmental benefits. This allogamous and autotetraploid species whose varieties are synthetic populations, has a highly polymorphic genome and a short linkage disequilibrium. Allele mining strategy on targeted candidate genes is an option to select valuable parents for breeding. In this paper, we assessed allelic diversity of CAD and WXP1 genes, involved in lignin biosynthesis and drought tolerance respectively, in a set of 384 individuals. For CAD which had two splice forms, 30 and 31 variants (out of 1077 and 906 base pairs of the coding sequences, respectively) have been observed including 37 and 52% of non-synonymous mutations. More variants were observed in WXP1 sequence, with 157 observed in WXP1 coding sequence (coding sequence of 1116 base pairs), including 60% non-synonymous mutations. Among the non-synonymous mutations, some probably affected protein function. Both genes were under purifying selection, especially CAD with dN/dS rate of 0.05 and 0.12 for both splice forms, against a rate of 0.26 for WXP1. Difference in variant proportion is probably explained by differential selective pressure that may be induced by contrasted expression levels. Indeed, CAD is highly and continually expressed whereas WXP1 is induced in some specific conditions. Further studies assessing the impact of variants on phenotype will help to conclude on the allele mining strategy in alfalfa breeding.

Vegetative Plant Height QTLs in Elite Perennial Ryegrass Material

Leaf length in perennial ryegrass meadows is a limiting factor for feeding grazing cows. In spite of the phenotypic selection efficiency to improve this trait, synthetic varieties hamper fixing favourable alleles. Our aim was to detect QTLs of vegetative plant height which is highly correlated to leaf length in order to start Molecular Assisted Selection (MAS). Two hundred plants from a cross between two elite plants were used to build a genetic map for each parent with 39 SSR and 47 AFLP markers. The maps consisted of seven linkage groups for both parents, with a length of 408 cM and 548 cM. Plant height was measured in a nursery on spaced plants during spring 2008. Using the Composite Interval Mapping method, we detected two QTLs of plant height in the parent RA958 B on linkage group 2 and 5. The one on LG2 co-localized with a QTL of earliness in vegetative growth after winter explaining 21% of variance. Moreover, two QTLs of plant growth rate were found on linkage groups 4 (RA958 F) and 7 (RA958 B), each of them explaining 11% of variance.

Impact of Four Decades of Breeding on Molecular Differentiation Between Forage and Turf Cultivars of Lolium Perenne

How much differentiated are forage and turf type cultivars within L. perenne? To estimate this, we used 10 SSR/STS markers for genotyping a collection of 7 natural populations, 50 forage and turf cultivars and 4 old cultivars of dual usage registered since 1965–2004. We showed that differentiation between usage types has steadily increased since the opening of a turf national list in France and that it has mostly involved 3 markers, among which 2 were mapped onto linkage group 1 in L. perenne. Relative to natural populations, assumed to sample genetic diversity in perennial ryegrass when breeding started, turf cultivars were found to be more distantly related than forage cultivars, especially those which were recently registered. However, genetic differentiation remained primarily between cultivars whatsoever type they were. Differentiation between cultivars has increased to be about twice higher on average than between natural populations, even of quite distant geographical origin. Loss of genetic variability after 40 years of breeding was found to be very low. All alleles present in natural populations were sampled again in the collection of cultivars we investigated. The results are briefly discussed in conclusion as respect to phenotypic differentiation and efficiency of breeding methods in the grasses.

Genetic Diversity in Tall Fescue Using AFLP Markers

Despite some data on tall fescue diversity using molecular markers, mainly from United States accessions, there is a lack of knowledge on the global diversity all over the world for this species. The objectives of this study were to evaluate the sub-structure of tall fescue world wide variability and to place French cultivars into this sub-structure using AFLP markers. One plant per accession from 32 ecotypes and 40 plants from six French cultivars were used. A total of 116 polymorphic fragments were scored from two primers combinations. Clustering analysis on 32 ecotypes of tall fescue from all over the world showed two distinct clusters: European and Mediterranean types. In the Mediterranean type two clusters could be distinguished probably illustrating different colonization origins. The analysis of six French cultivars representing the French seed market showed six genetically distinct subsets which were included in the European type.