Studies On RAPD And Chloroplast Genomic DNA For Germplasm Resources Of Genus Mulberry, Mori L.

The purpose of this paper was to clarify phylogenetic relationships in the genus mulberry, Mrous L. based on the analysis of RAPD. Meanwhile the extraction , clone and trnL-trnF intergenic spacer sequences of cpDNA of mulberry were studied. In addition, a method for extracting mulberry mitochondrial DNA was described. 1. DNA extracting method from vegetative tissue of mulberry The quality of the isolated DNA is one of the most important factors that influence the result of DNA analysis. Here we describe two simple DNA isolation protocols. Highly purified DNA from different tissue can be obtained following these procedures. 2. RAPD Analysis for the Germplasm Resources of Genus Mulberry, Morus L. 44 mulberry accessions,which belong to 12 species and 3 varieties, and 1 B. papyrifera were examined for RAPD analysis. Using 24 lObp random primers selected from 182, total 157 bands, which sizes are approximately between lOO-3500bp, were produced from 44 mulberry accessions. Of them, 113 showed polymorphism, with the ratio 72.0%. Based on RAPD data, Nei抯 genetic similarity coefficients and genetic distance were analysied and reconstructed the UPOMA dendogram . Additionally phylo genetic relationship among 45materials was determined. The results from cluster analysis indicated they are basically consistent with the morphological classification. 3. A Rapid cpDNA Isolation of Mulberry ,Moris L. and Analysis of Part Sequence According to modified Milligan method, the chloroplast (.Ienomic DNA of mulberry was prepared. The result of amplification with special primer of trnL- trnF and random primer indicated the prepared DNA extracted was chloroplast Genomic DNA of mulberry In addition, the cpDNA of mulberry was digested by XbaI and HindIII restriction endonucleases and 5 restriction fragment were cloned, of which the 700bp fragment was sequenced. The analysis of sequence showed that cloned 700bp fragment was probably the intron of trnK gene of mulberry chloroplast. 4. Studied on the cpDNA trnL-trnF mtergenic spacer sequence m the genus mulberry The cpDNA trnL-trnF intergenic spacer sequence in the genus mulberry, Yu71-1 and Sanglian, were amplified and sequenced successfully, the size of which are about 414 and 417 base pairs, respectively. The spacer region are characterized by a AT-rich insertion and the mean nucleotide compositions are 0.3 168(A)0.3345(T)0. 1673(G)0. 1914(C). There were 17 mutations because of mainly many indels between the trnL-trniF intergenic spacer of Yu71-1 and Sanglian and the sequence identity was 84.5%. By evolutionary relationships between these 2 and other 19 materials based on the trnL-tmF intergenic spacer in Genbank by cluster method, we found that genus mulberry shared high homology with family Moraceae ,followed by family Rosebush. and family Composite. These relationships are consistent with previous morphological groupings. 5. A Simple and Fast Method for the Mitochondrial DNA from mulberry, Morus L. According to isolation method of mfDNA from plant , the mtDNA of mulberry was isolated and purified0 While this method proved is a fast and inexpensive one, the extracted mtDNA of mulberry stands up to the needs of restriction endonucleases and RAPD analysis0...