Genetic Linkage Map And Flowering Time Gene Mapping In Orchardgrass (Dactylis Glomerata L.)

Orchardgrass (Dactylis glomerata L.) is indigenous to Eurasia and northern Africa, has been naturalized on nearly every continent, and is one of the top perennial forage grasses grown worldwide, owing to its good nutrition, high yield and good adaptability. Orchargrass gene resources are diverse and rich and have a great potential for breeding use. But few tools and information are available for molecular breeding, gene mapping and genetic improvement of the species. Accordingly, this research aimed to offer an important information and basis for further molecular assisted selection (MAS) breeding, improve economically important traits, enhance breeding efficiency and shorten breeding process of the speices.In this study, SSR molecular markers were used to detect the genetic diversity and variation between and within diploid and tetraploid orchardgrass accessions collected in Asian. A combination of simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular markers was used to construct the first diploid orchardgrass map. Orchardgrass EST-SSR markers and AFLP markers were employed to construct a high-density tetraploid orchardgrass maps. QTLs associated with heading date in this species were reported first. Based on454high-throughput sequencing, we obtained amounts of sequences of flowering candidate genes in orchardgrass. Finally, Vrn3gene was successfully mapped on previous maps. These results were as follows:1. Diversity comparison and phylogenetic relationships of diploid and tetraploid orchardgrass(Dactylis glomerata L.) germplasm as revealed by SSR markersPhylogenetic relationships among or within16accessions collected from Asia were investigated using SSR markers. The21SSR primer pairs generated a total of143polymorphic alleles, with an average allele per locus of6.8. The average polymorphic rate (P) for this species was90.7%. The polymorphic information content (PIC) ranged from0.17(A01F24) to0.45(A01E02) with an average of0.33. And the Shannon’s information index of diversity (Ⅰ) ranged from0.0463to0.3547, suggesting a high degree of genetic diversity. Analysis of molecular variation (AMOVA) revealed larger genetic variation within accessions (65.75%) and geographical regions (75.58%) rather than between them, while the ploidy level variance among the accessions contributed only2.62%. The16accessions were grouped into two major clusters (GI=0.876). Specifically, all tetraploid accessions originating from different regions were grouped into the same cluster whereas the diploid accessions were grouped into the two major clusters. The dendrogram was concordant with the morphological variability, agronomic traits and karyotype.2. Genetic linkage maps of diploid orchardgrass (Dactylis glomerata L.)The objective of this study was to construct a diploid (2n=2x=14) orchardgrass genetic linkage map useful as a framework for basic genetic studies and plant breeding. A combination of simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular markers were used for map construction. The linkage relationships among164simple sequence repeats (SSR) and108sequence-related amplified polymorphism (SRAP), assayed in a pseudo-testcross F1segregating population generated from a cross between two diploid parents were used to construct male (01996) and female (YA02-103) parental genetic maps. The paternal genetic map contained90markers (57SSR markers and33SRAP) over9linkage groups and the maternal genetic map was comprised of87markers (54SSR and33SRAP) assembled over10linkage groups. The total map distance of the male map was866.7cM representing81%genome coverage, whereas the female map spaned772.0cM, representing75%coverage. The average map distance between markers was9.6cM in the male map and8.9cM in the female map. The level of segregation distortion observed in this cross was15%. Homology between the two maps was established between five linkage groups of the male maps and five of the female map using10bridging markers. The information presented in this study establishes a foundation for extending genetic mapping in this species and will serve as a framework for mapping QTLs and provide basic information for further molecular breeding studies.3. A genetic linkage map of tetraploid orchardgrass (Dactylis glomerata L.) and QTL analysis for heading dateA combination of EST-based SSR markers and AFLP markers were used to genotype an F1population of284individuals derived from a very late heading D. glomerata ssp. himalayensis parent (him271) and an early to mid-heading D. glomerata ssp. aschersoniana parent (asch621). Two parental maps were constructed with28cosegregation groups and seven consensus linkage groups each, and homologous linkage groups were tied together by38bridging markers. The him271map consisted of183markers with a total length of974cM, the mean density between markers was5.2cM. The asch621map consisted of193markers and spanned a total length of1038cM with mean density between markers of5.5cM. Parental linkage group lengths varied from98to187cM. All but two mapped SSR markers had homologies to physically mapped rice (Oryza sativa L.) genes, and seven orchardgrass linkage groups were assigned based on this putative synteny with rice.7QTLs were detected for heading date on linkage groups2,5, and6in both parental maps, explaining between7.85%and24.19%of the penotypic variation. These results laid a theoretical foundation for further molecular assisted selection (MAS) breeding, improvement of important agronomic traits, later-mature cultivar release for mixture pasture and functional and comparative genetic analysis.4. Flowering gene identification and mapping in orchardgrassThe conserved sequences of flowering candidate genes (VRN, Hd, FT) in rice, wheat, ryegrass and tall fescue were used to design28baits. Solution capture method was employed to capture flowering genes in orchardgrass. Approximately21,000reads were generated from454sequencing, which were assembled into2,094contigs varying in size from105bp to1975bp. After BLASTn searches, contig404, contig441and contig558had hits to these bait sequences, including the Vrn, Hd, and FT candidate genes.20pairs of candidate gene primers were designed and amplified in F1mapping population to test segregation ratio. Finally, Vrn3with1:1segregation ratio was successfully mapped on LG3of the him271parental map.