Studies On Genetic Relationship Based On Sequencing NrDNA ITS Of Some Plants In Genus Buxus

Most plants of genus Buxus are used as ornamental plants. There are more than seventy species in genus Buxus and its taxonomy is confusion. In this study, the sequences of rDNA ITS (including 5.8S r DNA) region of five species in genus Buxus,and five plant populations of Buxus sinica var. parvifolia which was a rare endangered and endemic tree species in China, were sequenced based on optimizing ITS-PCR reaction system, comparing the method of cloning and sequencing and the direct sequencing method. The ITS sequences of the other nine species of genus Buxus, Sarcococca ruscifolia and Pachysandra terminalis(used as outgroup) were originated from Genbank. The phylogenetic tree of genus Buxus was constructed, by which to analyse the genetic relationship among the plants of genus Buxus. The results were as follows:①In 50μL reaction volume, the best one was as followed: 2.0mmol/L of Mg2+, 0.3μmol/L of primer, 0.375mmol/L of dNTP, 240ng/50μL of DNA template, 1.75U of qualified Taq DNA polymerase. The optimal anneal temperature in this reaction was 56℃. It could assure the purity and quality of ITS-PCR product using this optimal volume. The length of the ITS fragment of Buxus sinica var. parvifolia was 642bp by PCR product cloning and sequencing.②The method of cloning and sequencing successfully sequenced both the ITS sequences of Buxus henryi and Buxus sinica var. parvifolia, while the direct sequencing could only successfully sequenced ITS1 region sequence of Buxus henryi and the ITS1 and ITS2 region sequences of Buxus sinica var. parvifolia. Compared with direct sequencing, the approach of cloning and sequencing was more convenient and easier, and it could obtain the sequencing results quickly and accurately. In short, sequencing of cloning PCR products was more suitable than direct sequencing of PCR products for sequencing ITS sequences of nuclear rDNA from genus Buxaceae .③Compared with B. sempervirens, the genetic relationship between B. sinica var. parvifolia and B. sinica was more similar. But Buxus sinica var. parvifolia was not the variety of Buxus sinica. Buxus microphylla var. japonica and B. riparia might be the same specie. B.balearica which was assempled from Genbank and B.bodinieri might be the the same specie as well.