2012 Vol.14 No.1 * ** ** 550002 / 100193 PCR 6 matk psbk-psbi ITS ITS2 DNA 11 20 matk psbk-psbi matk 85% matk+psbk-psbi 100% matk+psbk-psbi DNA matk+psbk-psbi doi: 10.3969/j.issn.1674-3849.2012.01.005 Aspidistra Lili- [7~10] aceae [1] 93 67 44 18 [2~6] 20 80 [11] 2012-01-13 * ** 2012-02-13 D08080203640901 E-mail hesz8899@126.com Tel 010-57833194 E-mail scauyaoh@sina.com E-mail slchen@implad.ac.cn 1166
20 1 A. longiloba [12] [5] A.Pingtangensis 2. 1 DNA PCR 2001 Hebert [13] 11 13320 COI Retsch MM 400,Germany 2 min 30 r s -1 COI DNA Tiangen Biotech Co. COI China DNA PCR 25 μl PCR buffer 2.5 μl 10 MgCl 2 2.0 μl 25 mmol L -1 dntp 2.0 μl 2.5 mmol L -1 1.0 μl 2.5 2009 μmol L -1 Taq 1.0 U DNA1.0 μl 30 ng Consortium for the Barcode of Life, CBOL 25 μl +matk 2 ABI3730XL [14] 11 DNA Applied Biosystems Co.,USA ITS 2 Kress [15] Fazekas [16] CodonCode Aligner V 2.06 CodonCode Co. USA ClustalX V 2.0 Higgins 4800 6600 D.G. PCR ITS2 1 90% ITS2 [17~19] 1 A. pingtangensis 071002 COI 2 A. chishuiensis 0307045 3 A. fungilliformis 100920 rpoc1 matk ps- 4 A. guizhouensis 110602 ba-trnh psba -trnh matk atpf - 20 mg DNA 5 A. liboensis 090307 atph matk atpf-atph 6 A. sichuanensis psbk-psbi [15 7 100523 8 11 9 100520 20 6 DNA 10 matk psba -trnh psbk - 11 psbi ITS ITS2 12 DNA 13 14 15 A. caespitosa 16 17 A. fasciaria 18 A. retusa 19 A. linearifolia 1. 20 A. cerina 11 1167
2012 Vol.14 No.1 psbk-psbi matk Song [22] MEGA 4.0 K2P psbk-psbi matk Ross [23] BLAST1 3. Nearest distance 5 matk 85% psbk-psbi 1. PCR 65% 50% PCR DNA matk+psbkpsbi 100% PCR ITS ITS2 0 matk+ +matk + 95% matk psba -trnh psbk -psbi 100% matk psba -trnh psbk - fwd PA psbi rev TH 100% 2. matk KIM_3F KIM_1R 1f psbk -psbi 394 ~ 724r 398 bp matk 849~855 bp psbk-psbi 3 4 ITS psbk -psbi matk psba -trnh ITS2F ATGCGATACTTGGTGTGAAT ITS2 ITS3R GACGCTTCTCCAGAC TACAAT 2 PCR 5'-3' 3 PK PI ITS5F ITS4RITS5 af ITS4R GTTATGCATGAACGTAATGCTC CGCGCATGGTGGATTCACAATCC CGTACAGTACTTTTGTGTTTACGAG ACCCAGTCCATCTGGAAATCTTGGTTC GTTTCTGTTTGTGGTGACAT GCTACTGCAGGTACATGCGA CTTGCCAAACAAAGGCTAA ATGCTTACTCTCAAACTCT GGAAGTAAAAGTCGTAACAAGG TCCTCCGCTTATTGATATGCCCTTAT- CATTTAGAGGAAGGAG TCCTCCGCTTATT GATATGC 4 94 5 min 94 1 min 55 1 min 72 1.5 min 30 72 7 min 94 1 min 94 30 s 52 20 s 72 50 s 35 72 5 min 95 2 min 94 1 min 55 30 s 72 1 min 36 72 7 min 94 5 min 94 1 min 55 1 min 72 1.5 min 30 72 7 min 94 5 min 94 1 min 50 1 min 72 1.5 min + 3sec/ 30 72 7 min 94 5 min 94 30 s 56 30 s 72 45 s 40 72 10 min matk psbk-psbi / % / % / % / % 11/0.2 0.2 0.7 0.4 0.2 0.5 11/0.3 0.0 0.0 0.0 0.0 0.0 10/0.0 0.2 0.0 0.0 0.0 0.2 11/1.6 1.6 1.3 1.3 1.9 1.1 0.1 0.1 0.1 0.5 0.2 0.7 0.4 0.2 0.5 0.1 0.1 0.1 0.0 0.3 0.0 0.5 0.3 0.3 0.3 0.3 0.3 0.3 0.7 0.3 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.2 1.6 1.3 1.6 1.3 1.1 0.8 0.8 0.3 1.1 1.1 0.8 0.0 0.5 0.7 0.4 0.2 0.5 0.1 0.9 0.0 0.0 0.0 0.0 0.0 0.2 0.2 0.3 0.2 0.2 0.2 0.8 0.8 0.8 1.3 1.1 0.5 Aspidistra 0.1 0.1 0.5 0.4 0.7 0.2 0.3 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.8 1.1 0.8 0.0 1.1 0.3 11 spp. 0.6 0.6 0.6 0.2 0.4 0.1 0.0 0.3 0.0 0.5 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.2 0.8 0.0 0.8 0.0 1.1 0.3 0.2 0.2 0.2 0.1 0.5 0.1 0.1 0.1 0.5 0.4 0.4 0.4 0.0 0.0 0.0 0.4 0.0 0.4 0.0 0.3 0.0 0.5 0.0 0.0 0.3 0.0 0.5 0.0 0.3 0.0 0.5 0.0 0.3 0.5 0.3 0.3 0.5 0.0 0.0 0.0 0.2 0.2 0.2 0.0 0.0 0.8 0.0 0.8 0.0 1.3 1.3 1.1 0.3 1.1 1.1 0.3 1.1 0.3 0.8 1.1 0.8 0.8 0.0 0.8 % 0.3 0.2 0.1 0.8 bp 849~855 587 663 394~398 1168
4 4 matk psbk-psbi % % % % A. sichuanensis (9) A. caespitosa (2) 0.7,0.8,0.9,0.5,0.8,0.9,1.2,0.7, 0.1,0.2,0.5,0.1,0.2,0.5,0.2,0.1, 0.6,0.0,0.1,0.4,0.1,0.7,0.1,0.0, 0.5,0.2,0.6,0.7,0.9,0.5,0.1,0.4, 0.1,0.5,0.2,0.5 (0.4) 0.3,0.0,0.0,0.2,0.2,0.2,0.2,0.5, 0.3,0.3,0.2,0.5,0.5,0.5,0.9,0.0, 0.2,0.2,0.2,0.2,0.5,0.2,0.2,0.2, 0.2,0.5,0.3,0.3,0.3,0.7,0.0,0.3, 0.7,0.3,0.7,0.7 (0.3) 0.2,0.2,0.2,0.2,0.2,0.2,0.2,0.2, 0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0, 0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0, 0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0, 0.0,0.0,0.0,0.0 (0.0) 0.8,0.8,0.5,0.5,0.5,0.5,0.8,0.8, 1.0,0.3,0.3,0.8,0.3,0.5,1.0,0.8, 0.8,0.3,0.8,0.5,0.5,0.0,0.5,0.0, 0.3,0.8,0.5,0.0,0.3,0.8,0.5,0.8, 0.3,0.3,0.8,1.0 (0.5) 0.0 (0.0) 0.5 (0.5) 0.0 (0.0) 0.0 (0.0) 5 matk % 20 85.0 [17 20] matk CBOL 20 55.0 KIM_3F/KIM_1R matk psbk-psbi +matk matk+ matk+psbk-psbi + 20 45.0 20 45.0 19 21.1 19 15.8 20 65.0 20 45.0 19 84.2 19 63.2 20 90.0 20 80.0 20 100.0 20 85.0 19 52.6 19 57.9 100% psbk-psbi 6 matk psba - 100% 85% [26] 100% 45% 21.1% psbk-psbi Ki-Joong Kim [21] Lahaye [27] 18 31 101 psbk -psbi 98% matk+psbk-psbi+atpf-atph 100% 65% DNA [28] trnh psbk-psbi ITS ITS2 ITS +psba -trnh matk +psba -trnh ITS1 ITS2 5.8S 3 + matk matk+psbk-psbi 5.8S ITS1 matk+psbk-psbi ITS2 [24] ITS1 DNA 100% + 52.6% ITS ITS2 DNA DNA barcoding [17 PCR 25] DNA [29] ITS ITS2 GenBank ITS ITS ITS2 matk psbk-psbi 1169
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Abstract: To test the DNA molecular markers (, matk,, psbk-psbi, nr ITS and ITS2) to identify medicinal plants in Aspidistra,the efficiency of PCR amplification and sequencing, intraspecific variation and interspecific divergence and identification efficiency were used to evaluate these loci. The results showed that through analysis of 20 samples of 11 species of Aspidistra, matk and psbk -psbi had high efficiency of PCR amplification and sequencing and the success identification rate of matk was the highest (85%) in single sequence. The discrimination ability of different sequences was shown higher using the sequence combinations, in which the success identification rate of matk+psbk-psbi was 100%. Therefore, matk+psbk-psbi could be used to identify the medicinal plants in Aspidistra. Key words: DNA molecular markers; Aspidistra; identification; matk+psbk-psbi 瑀 1171