Objective

2

To conduct phylogenetic analysis of internal transcribed spacer 2 （ITS2） and chloroplast gene segments including

psbA-trnH

，

rbcL

， and

matK

of

Sophora japonica

cv.

jinhuai

resource samples from different geographical sources， and to explore the genetic diversity of

S. japonica

cv.

jinhuai

.

Method

2

Polymerase chain reaction （PCR） method was used to amplify the nucleic acid sequences of ITS2，

psbA-trnH

，

rbcL

， and

matK

of

S. japonica

cv.

jinhuai

. Neighbor joining （NJ） method was used to construct phylogenetic trees， and Kimura 2-Parameter （K2P） model was used to calculate the genetic distance of different samples. MEGA and BIOEDIT softwares were applied for mutiple alignment and analysis of ITS2，

psbA-trnH， rbcL

， and

matK

sequences of

S. japonica

cv.

jinhuai.

Result

2

The lengths of ITS2 sequence were 278-279 bp. The lengths of

psbA-trnH

were 289 bp. The lengths of

rbcL

sequence were 673 bp. The lengths of

matK

sequences were 786-792 bp. There were 3 mutation points in ITS2 and

psbA-trnH

， no mutation point in

rbcL

， and 13 mutation points in

matK

. The samples of

S. japonica

cv.

jinhuai

were clustered into two groups based on the phylogenetic tree constructed by ITS2 sequences. The sample of seedling tree in Baibao was clustered into one group， while the other 25 samples were clustered into another group. For the

psbA-trnH

sequence， the success rate of PCR amplification of 28 samples of

S. japonica

cv.

jinhuai

was 100%. The 28 samples of

S. japonica

cv.

jinhuai

were clustered into three groups based on the clustering results of

psbA-trnH

sequence. The sample of seedling tree in Shaoshui was clustered into one group. The five samples of grafting tree and seedling tree in Miaotou， grafting trees in Jiantang， Wenqiao， and Daxu， and seeding tree in Xianshui were clustered into one group. The other 21 samples were clustered into another group. The 26 samples of

S. japonica

cv.

jinhuai

were clustered into two groups based on the phylogenetic tree constructed by

matK

sequences. The sample of seedling tree in Xianshui was clustered into one group， while the other 25 samples were clustered into another group. The clustering results of the

rbcL

sequence of

S. japonica

cv.

jinhuai

could not distinguish 28 resource samples. The phylogenetic tree constructed by the combined sequence of ITS2+

psbA-trnH

+

rbcL

+

matK

divided

S. japonica

cv.

jinhuai

resource samples into 4 groups. The 13 samples of seedling trees in Qiyang， Daoxian， Miaotou， Shaoshui， Shitang， Xianshui， Jiantang， and Xiangli， and grafting trees in Qiyang， Miaotou， Yongsui， Wenqiao， and Yangtang were clustered into one group. The sample of seedling tree in Wenqiao was clustered into one group. The sample of seedling tree in Daxu was clustered into one group. The remaining samples were clustered into another group.

Conclusion

2

Phylogenetic and mutation analysis provide the theoretic foundation to investigate the evolution of the resources of

S. japonica

cv.

jinhuai

， and evaluate their genuineness. The results of mutation points can be used to identify the related

S. japonica

cv.

jinhuai

resources. The findings of this study show that the combination of different gene sequences has an optimal effect on plant identification.