Establishment Of DNA Fingerprints For Clones Of Acer Truncatum Bunge

Acer truncatum Bunge is a deciduous species of Acer genus and native to China, which is an important colored-leaf tree for landscape use and also has high economic value in recent years. At the same time, With more attention paid, relevant scientific personnel constantly select more new clonal cultivar/lines through breeding. Therefore, it is necessary to construction of DNA fingerprints of Acer truncatum clonal cultivar /lines. In the paper, 23 clones are selected and collected, which are explored the best genomic DNA extraction way from the leaves, the optimal response of PCR amplification of SRAP, genetic diversity and genetic relationship and established of DNA fingerprints. This can give parental selection, identification and right protection of new cultivars and classification some guidance. The main results were as follows:1. Modified CTAB of DNA extraction is the most suitable genomic DNA extraction methods of Acer truncatum through comparing to the traditional CTAB method, modified CTAB method and kit method of DNA extraction and considering sample quantities and saving money.2. Orthogonal experiment design is established the reaction system for SRAP-PCR of Acer truncatum. The total 25μL reaction volume included 2.5μL of 10×PCR buffer, 1.0U of Taq enzyme, 0.3μmol/L of primer, 2.5mmol/L of Mg²⁺, 2.0 mmol/L of dNTPs, 50 ng of DNA.The influences of each factor on the PCR-reaction system is Taq enzyme, Mg²⁺, primer, dNTPs, DNA template. Amplification general program is: pre-denaturation at 94℃ for 5 min; the primer 5 reaction cycles of 94℃ for 1 min, 50℃ for 1 min, and 72℃ for 1 min; the following 35 reaction cycles of 94℃ for 1 min, 50 ℃ for 1 min, 72℃ for 1 min; then, at 72℃ for 5 min and stored at 4℃.3. 16 primers pairs were selected from 90 pairs, which are applied to analysis the genetic diversity and fingerprinting construction of 23 clones. 16 primers produced 223 bands of which 197 were polymorphic, the percentage of polymorphic bands is 88.34%, each primer produced 13.9 bands and 12.3 polymorphic bands. the Effective number of allele is 1.2653¹.5841 with an average of 1.4416; the Nei’s genetic diversity（H） is 0.2024⁰.3302 with an average of 0.3406;and the Shannon’s information index（I） is 0.3590⁰.5879 with an average of 0.2565. The results shows that materials between Acer truncatum clones has a certain degree of genetic diversity.4. By NTSYSpc2.10 and UPGMA cluster analyzed, the 23 clones can be divided into 5 groups. The first and second group are 2014-N and NO. 1 respectively. The third group is 2013-4, 5-2 and 5-3. The The fourth is 6-1, 6-2, 6-3, 5-4, The fifth group has remained 14 clones. With the increase of genetic distance, 14 clones can be divided into 4 subgroup at 0.77（L3）, The first is 2014-1; The second is 2013-3; The third is concluded nong 1, 1-1, 1-3, 1-4, 1-5, 1-6, 1-7, 1X-3, 1X-6; The fourth is jing 2, zhong 2 and tian 3, which is consistent with the classification based on the main characters and genetic relationship. The result shows that SRAP was scientific and suitable for Acer truncatum.5. Six core primer are selected to amplificated of 23 clones of Acer truncatum, which obtained rich and clear bands and fragment between 100-1500 bp. 23 clones can be identified by using the fingerprints established by primers of ME1-EM4 and ME2-EM1, which confidence probability is 99.99%.