Study of the transferability of microsatellite markers derived from bread wheat (T. aestivum) or rice ( O. sativa) ESTs (EST-SSRs) to their close and wild relative and evaluation of their potential for the organisation of genetic resources in the grass family
Etude de la portabilité de marqueurs microsatellites issus d'EST de blé tendre (T. aestivum) ou de riz (O. sativa) vers des espèces apparentées et évaluation de leur intérêt pour la structuration des ressources génétiques chez les graminées
Résumé
Despite recent progress in wheat (T. aestivum L.) genomics, only few efforts have been made in the genomics of wild and closely related wheat species. The first aim of this PhD was to develop new molecular markers useful on wheat and transferable to other cultivated as well as wild grass species. We focused our work on microsatellites (SSRs) located in expressed gene sequences (ESTs) to exploit the large number of wheat ESTs present in the databases and also because the genes are the best conserved regions of the genome between the different species. From about 1,000 EST contigs bearing a microsatellite, we observed that the trinucleotide motifs were the most frequent (~80%). We developed 301 primer pairs (EST-SSRs) that were evaluated on a set of eight wheat cultivars and eight related species. About 80% of the primer pairs gave an amplification product on wheat. Most of the time, we had high quality patterns. The level of polymorphism was estimated to be 25.4% on wheat (mean of 3.1 allele/locus and PIC value of 0.40). Among the 240 EST-SSRs that gave an amplification product on wheat, 177 were assigned to wheat chromosomes using aneuploid lines and 81 new loci were added and integrated to the reference wheat genetic maps. Transferability of wheat EST-SSRs to related species was very good for wheat subspecies (100% for T. aestivum ssp compactum) and decreased while the phylogenetic distance increased. However, transferability reached 28% with rice. The alignment of the selected wheat EST-SSRs on the rice genome sequence showed a preferential localisation in the telomeric regions and a better similarity of wheat ESTs on rice chromosomes 2, 3 and 5. Moreover, a thorough study of the EST-SSRs giving an amplification product in wheat, rice and barley indicates that the motif is present and is frequently similar in the three species suggesting a common ancestral origin. The allelic diversity of the EST-SSRs among the wheat related species was higher in allogamous species such as Ae. speltoides, Lolium perenne and maize (respectively 0.423, 0.388 and 0.352) while autogamous species such as bread and durum wheats showed lower values (respectively 0.108 et 0.093). Reciprocal development of 106 rice EST-SSRs originating from genes of the rice chromosome 1 gave similar results concerning the transferability to wheat (28.5%) as those observed for wheat EST-SSRs and confirmed the known syntenic relationships between rice chromosome 1 and wheat homoeologous group 3. The second aim of the PhD was the exploitation of the EST-SSRs showing a good transferability to validate their capacities for phylogenetic analyses among the Triticeae and the grasses. The results indicate that these markers are able to properly classify the lines and the species according to the known data about their phylogeny and their pedigrees. The results also confirmed that T. monococcum ssp urartu, Ae. speltoides and Ae. tauschii species are closely related to the ancestral donors of the A, B and D genomes of bread wheat respectively. Thus, we can conclude that wheat EST-SSRs are interesting and powerful markers to study wheat related orphan species and to make phylogenetic and comparative genomics analyses among the Triticeae tribe and more generally among the grass family.
Le premier objetif de cette thèse a été de développer de nouveaux marqueurs moléculaires utilisables sur le blé et transférables vers un nombre important d'espèces cultivées ou sauvages de graminées. Nous avons observé une portabilité des EST-SSR excellente pour les sous-espèces de blé qui diminue avec l'éloignement phylogénétique pour atteindre encore près de 30% avec le riz. Le deuxième objectif de cette thèse était d'exploiter les EST-SSR montrant une bonne portabilité pour valider leur capacité dans le cadre d'analyses phylogénétiques chez les Triticés et les graminées. Les résultats confirment que les espèces T. monococcum ssp urartu, Ae. speltoides et Ae tauschii sont respectivement apparentées aux donneurs des génomes A, B et D du blé tendre. Nous pouvons donc conclure que les EST-SSR sont des marqueurs intéressants et puissants pour étudier les espèces sauvages orphelines et pour faire des analyses phylogénétiques chez les graminées
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