Evaluation Of Application Of ITS Sequences On Analysis Genetic Diversity In Three Cherryspecies

In this paper,95 materials, from Chinese cherry (Cerasus pseudocerasus Lindl.), sweet cherry(Cerasm avium Linn.)and Nanking Cherry (Cerasus tomentosa Thunb.), were used to study the rDNA internal transcribed spacer sequence, analyze their polymorphic sites, and study on the interspecific and intraspecific relationships. The main results are as follows:(1) Sequence characteristics. ITS sequence amplification lengths of Chinese cherry, sweet cherry and Nanking Cherry were 702-703bp,704-705bp and 709bp, with small length variation. After aligning, all amplified sequences length is 712bp, and the fragment of CG nucleotideaccounts for 58.1% of total nucleotide. The 712bp ITS sequence detected 71 mutation sites in the 95 materials, of which 54 genera of parsimony informative sites. There are 40 mutations in the ITS1 (37-270bp) region,3 in 5.8S (271bp-373bp) region,28 in ITS2 (374bp-651bp) region. The 71 mutation sites in the 95 individuals defined 37 haplotypes. Processing these 37 haplotype sequences obtained data matrix and calculate the haplotype diversity (Hd=0.911), nucleotide diversity (π=0.01444), separation of polymorphism average (θw=0.01954).(2) Calculation of the 37 ITS haplotypes obtained sequences evolutionary distance by genetic distance model with Kimura-2. It is 0.011-0.027 between Chinese cherry and sweet cherry,0.047-0.051 between Chinese cherry and Nanking cherry, and 0.044-0.057 between sweet cherry and Nanking cherry. Though the ITS sequences are high homology in Chinese cherry and sweet cherry, they are all low homology with the ITS sequence of Nanking cherry.(3) 37 haplotypes divided into 3 branches with the NJ phylogenetic analysis. The Clade I includes 21 Chinese cherry haplotypes with 93% bootstrap support rate, Clade II includes 11 haplotypes of sweet cherry with 96% bootstrap support rate, andthe Clade III have 5 haplotypes of Nanking cherry with 100% bootstrap support rate.(4) In the haplotype network map, topological structure of the 37 haplotypes showed three arms spiral structure distribution. The 5 haplotypes at one end of the long arm is Nanking cherry. There are 11 conflicts in this network map, located in 79,88,100,130, 135,136,153,185,538,641. In Chinese cherry branches, H2 is located in the center position of most groups and is the ancient and core haplotype. Other haplotypes are scattered in the branches periphery, as an external node in network map.(5) Predict and analysis secondary structures. In the observation of molecular phylogenetic tree, according to the number of haplotypes of represent samples, we selected HI, H2, H22, H37 four haplotypes to take secondary structure prediction analysis. Secondary structure of the 5.8S region isalmost same, however, the two stages structure differences between ITS1 region and ITS2 region are large. The results showed the minimum free energy of Nanking cherry haplotype H37 is-256.5 kcal/mol, followed by sweet cherry haplotype H22 (-251.60 kcal/mol) and 2 Chinese Cherry haplotypes H2(-248.50 kcal/mol), H1 (-248.00 kcal/mol). The data of minimum free energy obtained by T-detection, the three species haplotype differences were significant (P<0.05). In the secondary structure map, the half of the stem loop structure is similar and the rest is great differences. Combining the secondary structure map, the minimum free energy and structure of map, the phylogenetic tree based on ITS sequences was supported and the minimum free energy difference is not significant while P<0.01, indicating that ITS secondary structure in the taxa of varieties credibility is not high.