Development And Application Of SSR Markers Based On BAC End Sequences Of Narcissus

Narcissus tazetta L.var.Chinensis Roem is a perennial bμlbs flower belong to Narcissus L. It is about the past tens of thousands of horticμltural varieties with colorfμlness, have become world famous widely cμltivated ornamental flowers. However, many factors including varieties scarce, single color and difficulty on traditional breeding, restricted the production and development of the flowers. In this paper, new SSR markers have been developed based on BAC end sequences of Narcissus tazetta, and the marker transferability was also discussed. It aimed to provide effective support on germplasm identification, genetic relationship and diversity analysis of Narcissus. The main res μ Its of this study are summarized as follows:1. A total of 148 SSR loci were obtained from 1149 BAC end sequences of Narcissus with the rate of 12.88%. The repeats showed that dinucleotide motifs were dominant, accounting for 89.19%, and then was trinucleotide motif. (AT)n and (TA)n of dinucleotide motif have the highest frequency, representing 32.43% of the total, (AG)n=(TC)n motif takes second place. Addition, dinucleotide and trinucleotide motifs length varies widely, dinucleotide motif length up to 76bp, trinucleotide motif length up to 27bp, dinucleotide motifs were more than trinucleotide the average length of 0.26 motifs. Comprehensive showed that SSR loci information is very rich in Narcissus genome, and to lay a foundation for the development of SSR markers of Narcissus.2. The part of SSR loci were designed to 102 pairs BES-SSR markers primers of Narcissus. Eventually developed to obtain 29 pairs of bands were clear, reproducible, high polymorphism of BES-SSR markers primers of Narcissus and screened success rate as high as 28.43%. And SSR-PCR reaction system was optimized for each factor analysis, established the optimal Narcissus SSR-PCR reaction system. Randomly selected developed SSR primers repetitive testing and sequencing analysis, showed that the research and development of a comprehensive SSR markers primers of Narcissus good reproducibility, stability, reliable and effective molecμlar markers.3. The BES-SSR markers of Narcissus will be uccessfμlly used to study the relationship, germplasm identification and genetic diversity analysis in Narcissus. With the above 15 pairs of BES-SSR markers of Narcissus for the 36 material of Narcissus to analyse,506 amplified bands, 448 polymorphic bands,88.54% percentage of polymorphic loci, the average amplified polymorphic resistance bands 29.87. Each primer of Shannon’s information index polymorphism (I) in 0.3005～0.4861, among the genetic similarity of Narcissus in 0.53557～0.96640. According to UPMGA clustering method showed consistent with previous research, and the different primers for the same materials exhibit different specific amplification bands. It is indicated that the development of SSR markers can be used in germplasm identification, phylogenetic analysis, genetic diversity analysis and other fields in Narcissus.4. The transferabiliy of BES-SSR markers of Narcissus to relative plant species of Clivia Lindl was explored 21 Clivia species were detected and analyzed to the above 29 pairs BES-SSR markers of Narcissus, resμlts showed that among 11 pairs of primers to amplify bands exhibit clear, reproducible, high polymorphism and stable characteristics, accounting for 28.20% of transferabiliy. It obtained amplified bands 329 and polymorphic bands 242, accounting for 73.56% of the average percentage of polymorphic loci. Each Clivia cultivars of genetic similarity coefficient between 0.54 711～0.81 763, larger differences among varieties and identified significant effect, indicating that the research and development of BES-SSR markers of Narcissus has good versatility, to enrich Clivia Lindl number of SSR markers and to lay the foundation for Clivia germplasm and genetic diversity.