Development Of SSR Primers And Establishment Of SCoT Molecular Marker System In Osmanthus Fragrans

Osmanthus fragrans Lour., Oleaceae, Osmanthus is one of China’s top ten traditional flowers. China is the distribution center of Osmanthus species, and has been endowed with international registration authority of Osmanthus plants and O. fragrans cultivars. Cross breeding is an effective method for creating new cultivars. O.fragrans individuals unfortunately take between 6 and 8 years to progress from gemination to blossoming, making conventional breeding a painfully slow process. Using molecular markers to construct high density genetic linkage maps can reveal markers associated with phenotype. Such markers can facilitate purposeful selection of O. fragrans individuals in the breeding process, thus substantially improving efficiency and cutting cost. The SSR marker technique is based on DNA length polymorphism. It is simple and highly specific, thus providing an extremely useful tool for analyzing population genetic structures and constructing molecular genetic maps. The ScoT technique relies on the conservatism of plant genome ATG loci to design single primers, thus generating polymorphism associated with targeted genes. This technique is very simple and highly reliable. SCoT molecular markers have been successfully applied to study rice plants, supplementing more traditional AFLP, RAPD, SRAP and ISSR markers.Seeking to support the construction of molecular genetic maps of O. fragrans, the present study used high-throughput sequenceing(Roche 454 GS FLX+) to develop Simple Sequence Repeat(SSR) primers, and constructed an amplification system of Start Codon Targeted(SCoT) polymorphism molecular markers. The results were as follows:1.This study used high-throughput sequencing to develop SSR primers for O. fragrans. We obtained 89633 reads, which were screened with MISA1.0 to search for sequences containing repeating units. SSR primers were designed using Primer5.0. Eventually 3006 SSR primers remained, 1471 of these have been submitted to the GenBank.2.We then randomly selected 100 of these primers, 20 of them were polymorphic and exhibited clear amplification bands, indicating there were numerous SSR primers that could be used to construct a saturatedmolecular genetic map of O. fragrans.3.In this study, we used a 2×Es TaqMasterMix solution optimized for O. fragrans to isolate 12 suitable ScoT primers from 36 candidates.The 12μL reaction system included 30 ng template DNA; primer concentrate on was 0.4 mol.L-1; primer annealing temperatures were 48, 49.9, 54.3, or 56 degrees Celsius.4.Using this system, we obtained 244 amplification bands from 12 O. fragrans cultivars, 238 of these bands were polymorphic. We conducted cluster analysis of the SCoT amplification bands using the UPGMA method. The results showed that flower coloration and genetic linkage were not fully correlated in O. fragrans. The SCoT marker system developed in this study could be used to construct genetic linkage maps of O. fragrans.