The Development Of Two Molecular Markers In Wintersweet（Chimonanthuspraecox） And The Evaluation Of Segregation Patterns In An F₁Hybird Population

Wintersweet (Chimonanthus praecox) belongs to the Calycanthaceae family, it is a fragrant flower shrub originated in China, and it is have a long cultivation history, it has always been loved by the people. Wintersweet will be flowering in the end of the winter and the beginning of the spring, it is a cross-pollination and diploid plants, it have a major reproduction way is seed breeding, mostly native to the southeastern China, distributed less elsewhere. Throughout the researches of wintersweet, mainly focus on propagation, species classification, germplasm resources and landscape applications, and so on. But in recent years, with the development of molecular biology, more and more researches have appearing, such as cloning and molecular markers, which provides a great development space for wintersweet.In the study, SSR markers and SNP markers were developed by use the information of wintersweet transcriptome sequencing library, and we also build a hybrid F1group by H29and H64as parents, it come from Huazhong Agricultural University campus. The separation mode were studied by SSR markers, which use the "two-way pseudo-testcross" mapping strategy, and did a preliminary study for genetic map of wintersweet. And we also analyzed the twelve wintersweet materials by SNP markers.The main results were as followes:1. Development of SSR markers and evaluate the separation mode of the hybrid F1groupAid the information of Wintersweet transcriptome sequencing library on SSR markers, which randomly selected160pairs of primers, and amplificated by H29and H64genomic DNA, at last, six pairs of primer are useful.We optimize the SSR-PCR amplification system, and we also analyzed the different systems, the annealing temperature, the different grinding merhod and sample volume. The study has found that: the optimizing SSR-PCR system: primers (10μmol/L)1μ L; DNA (100ng/mol)1μ L;2×PCR Mix10μ L(3mM MgCl2;0.4mMdNTP;0.1U Taq), adding ddH2O to20μ L; screened the optimal annealing; the quality of DNA has little effect to amplification; the sample volume in2～4μ L/pore is best.Six SSR markers were used to research the separation mode of the Fi hybrid group. The results showed that: SSR marker have35, which has27polymorphic markers, and has8markers with a1:1segregation ratio in the α=0.05, the percentage is22.9%, the no segregation and abnormal segregation both are12, the percentage is343%, the distorted segregation has3, the percentage is8.6%.2Development of SNP markerAfter contrast more than1000SNP information sites, and designed CAPS primers to ampligication twelve wintersweet materials to screen the SNP loci which can be converted into CAPS marker. The results showed that:forty-two primers were designed, at last, only thirteen SNP loci successfully converted into CAPS marker.Use those thirteen CAPS primers to amplify and cleave twelve wintersweet materials, the results showed that: the average heterozygosity (Ave Het) between0.3000-0.5000, it has a higher heterozygosity; most of Shannon’s information index (1) have reached0.6000; polymorphism information content (PIC) ranged between0.25and0.5, showing the mid-polymorphism.Clustering analysis for eleven Wintersweet materials, they were divided into two groups, H29, M, Z23, Chimonanthus salicifolius, Chimonanthus nitens were clustered into one group, H64, L1, L2, L3, L4and J1were clustered in another group.