| Genus Lolium includes about nine species, all of them are diploid species (2n=2x=14) except some modern cultivars in the L. perenne and L. multiflorum, the most common used species in the Lolium. L. perenne and L. multiflorum belong to out-breeding species, it easily cross each other naturally, and also can be cross with some Festuca species and product fertility hybrids. Because of the population heterogeneity of the out-breeding species, the genotype of each seed in the same cultivar may be different. This out breeding habit ensures a high degree of genetic variation in natural populations and cultivars, it provide primely genetic resource for breeding. Study of the differentiation of the Lolium well increase breeding efficiency by using the genetic resources of this species. On the other hand, L. temulentum (Darnel ryegrass) is most important one in the five inbred species of genus Lolium, it was considered as a model grass species for genetic analysis and genomic studies, because it is a self-fertility, and has a short life cycle (2-3months), also they have no vernalization requirements for flowering, In addition, non-shattering habit of L. temulentum can be improve the easy-shattering nature of L. multiflorum and increase its seed yield, moreover, the self-compatibility in L. temulentum also can be introduce into L. perenne and L. multiflorum to produce inbred lines for heteros is breeding in these two important species. Therefore, construction of linkage map and introgression lines of L. temulentum will be useful for QTL analysis and gene cloning of important agronomic traits and genes. This objectives of this study were:1) phylogenetic analysis of the Lolium genus;2) Linkage map construction using a L. multiflorum×L. temulentum BC1F1population; and3) Construction of introgression line (ILs) of L. multiflorum on L. temulentum background. The main results of this study are as followed:(1) To assess genetic diversity and differentiation among Lolium species, two kinds of markers including32nuclear SSR and7cytoplasm gene markers were used to analyze357individuals from160accessions. It revealed a high level of polymorphism at the DNA level, the average number of alleles per nuclear SSR locus was23.6, the average PIC value was0.83; the average number of alleles per cytoplasm gene markers was5.3, the average PIC value was0.54. Using STURCTURE and PowerMarker software, we conducted clustering analysis based on32nuclear SSR markers data using357individuals, and7cytoplasm gene markers using160individuals, the results showed that there are very clear population structure, especially when using nuclear SSR markers, the three inbreeding species can clustered into one group and other out-breeding species can also clearly divided. The combination of two kinds of markers could more accurately reflect the genetic relationship among materials used, and this will facilitate the use of the Lolium germplasm for improvement and to increase the effectiveness of Lolium breeding.(2) To construct a linkage map for ryegrass using a L. multiflorum x L. temulentum BC1F1population consisting of88individuals, SSR marker and CISP marker which were developed based on EST sequence of cereal genome database were successfully employed. The genetic linkage map covered488.8cM in seven linkage groups including108SSRs and84CISPs, ranging from42.2cM to98.9cM for individual linkage groups. The average density of markers was2.5cM. Syntenic relationships between ryegrass and rice were also studied based on the position of CISP markers, the results almost agreed with previously reported but also with some different. The rice grain size gene GS5homologous in L. temulentum was sequenced and was mapped on ryegrass LG1. In addition, we also found three QTLs for seed number on LG4and6. This information found in present study will be useful in linkage map construction, QTL analysis for important agronomic traits for IRG and related species such as perennial ryegrass.(3) To construct introgression line (ILs) of L. multiflorum on L. temulentum background, a total of125BC3F1individuals carrying L. multiflorum segments in L. temulentum background were created using L. multiflorum as a donor and L. temulentumas recurrent parent. The mean number of donor segments per individual was4.5in the BC3F1population when using84polymorphic SSR markers. We selected60individuals from125initial ingression lines for final ILs, and the60ILs represented43.75%of the genome of L. multiflorum. Using the final60ILs, we detected two QTLs for plant height and tiller number on LG6and LG2, respectively, and the two QTLs showed reduction effect on L. multiflorum parent. This lay a foundation for subsequent construction of introgression line, and provide material basis for fine mapping and cloning of genes, such as the grain weight, the seed shattering, vernalization requirements habits and selfing incompatibility. |
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