Development And Application Of Microsatellite Markers In Betula Luminifera

Betula luminifera H. Winkler of the genera Betuleae of the family Betulaceae is an endemic deciduous monoecious tree in China with great timber value. The wood which is hard, close grained, and fine textured, is used for construction and making agricultural tools and furniture. B. luminifera plays a very important role in development of mountainous areas, adjusting the stand structure and improving the ecological environment. In the present study, microsatellites were identified from EST data source of B. luminifera. Moreover, these non-abundant SSR resources were used to design informative SSR primers and were applied in studies of genetic diversity and linkage mapping.In total,3692bi-to hexa-type SSR-containing sequences and4419SSRs were identified from37662Unigene ESTs with total length about1687.3kb, resulted from the pretreatment and clusting of53799ESTs of B. luminifera. On an average, one SSR was found per3.8kb of EST sequence, with the tri-nucleotide motifs as the main type, accounting for50.03%in all SSRs. Compared with CTT/GAA (5.45%in all SSRs), the dominant repeat type in tri-nucleotide motifs, the frequency of AG/TC, the most abundant SSR, was11.00%. Furthermore, primer sequences flanking SSR motifs were successfully identified from2609B. luminifera ESTs.3085primer pairs were picked.Orthogonal design was used to optimize EST-SSR PCR amplification system for B. luminifera in terms of five factors containing Taq DNA polymerase, Mg2+, DNA template, dNTPs and primers in five levels. An optimal EST-SSR PCR system for B. luminifera was obtained as4ng·μL-1DNA template,0.500μmol·L-1primers,1.25mmol·L-1Mg2+,0.250mmol·L-1dNTPs,0.0725U·μL-1Taq DNA polymerase. Under the optimal reaction system, we can acquire stable and clear polymorphic bands. And the optimal annealing temperature for SSR PCR reaction system is determined by gradient PCR.Primers were synthesized and tested for308of these loci with repeat lengths of≥16bp. Of these,260markers were validated for consistent amplification in seven diverse B. luminifera clones;131were found to be polymorphic on non-denaturation polyacrylamide gel electrophoresis. Genetic diversity analysis was done on77B. luminifera clones using60highly polymorphic EST-SSR markers. UPGMA dendrogram showed distinct separation of the different groups of B. luminifera.A genetic map for B. luminifera is presented here. It is based on a F1progeny of78individuals allowing the construction of a female and a male map according to the pseudo-testcross strategy. Two separate female and male maps were constructed using77and107EST-SSR markers, respectively, containing14female linkage groups and17male linkage groups. The female map covered a941.65cM distance, with an average distance of12.23cM and maximum map distance of99.7cM between two loci; the male map covered a1282.4cM distance, with an average distance of11.99cM and a maximum map distance between two loci is of72.3cM.