Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

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1 PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 ) DNA DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus Fisch. Bge 1 1 DNA 2-3 DNA 9 ITS 10 Hedysarum polybotrys Hand. -Mazz. PCR PCR PCR BA128B X1002 * Tel li _ yahoo. cn * Tel zhoulishe@ sohu. com CTAB 1 TAE Promega Fluka DNA Taq Takara bp DNA Markar Takara PCR Eppendorf 5332 DYY-12 Eppendorf 5810R 2581

2 Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astragalus membranaceus var. mongholicus 2 2 AMM2011_NMGGY A. membranaceus var. mongholicus 2 3 AMM2011_NMGCF A. membranaceus var. mongholicus 2 4 AMM2011_NMG A. membranaceus var. mongholicus 10 5 AM2011_SXHY A. membranaceus 5 6 AM2011_GSLX A. membranaceus 5 7 AM2011_NMGCF A. membranaceus 2 8 AM2012_AH A. membranaceus 2 9 HP2011_GSWD Hedysarum polybotrys 2 10 HP2011_GSTC H. polybotrys HP2011_NMGTY H. polybotrys trnl-trnf 14 BioEdit Primer Premier PCR 2 Fig. 1 The specific primer design schematic diagram of Astragali Radix and Hedysari bp Radix 2 Table 2 PCR Primer and PCR reaction conditions 5'-3' PCR trnl GGAAATCGGTAGACGCTACG 94 5 min s 56 30s 72 1 min 30 s 72 7 min trnf ATTTGAACTGGTGACACGAG AM-F GTTCAAATCAGTCACTCCGA AM-R CGACGGACTTTCCTCTTAAT 95 5 min s s 72 1 min 40 s 72 7 min HP-F AATCAGTCACTCCACCCT HP-R TAAGTAAGTTTCAGTAGTAGTAATG 2. 2 DNA PCR DNA PCR 100 mg CTAB PCR PCR DNA DNA 10 mg 95 5 min s L - 1 trnl trnf PCR s 72 1 min 40 s DNA 20 μl min 3 μl 10 buffer 2 μl dntp1. 6 μl 1% 100 ~ 150 V μl 5 pmol L - 1 EXTaq 0. 2 μl 5 U 15 min μl - 1 DNA 0. 5 μl μl10 ng PCR 2582

3 PCR dntp PCR 3. 1 trnl PCR trnf PCR PCR Clastul W 2 PCR 20 μl PCR 10 buffer 2 μl dntp1. 6 μl PCR μl 5 pmol L - 1 EXTaq 0. 2 μl 5 U μl - 1 DNA 0. 5 μl μl min 95 PCR 1 30 s s 72 1 min 40 s 72 7 min PCR 2. 4 PCR 52 DNA PCR PCR 10 buffer 2 μl dntp1. 6 μl AM-F AM-R 0. 3 μl 5 pmol L - 1 HP-F HP- R 0. 2 μl EXTaq 0. 2 μl 5 U μl - 1 DNA 0. 5 μl μl PCR dntp 0. 8 μl 2. PCR 5 μl 6 loading AM-F /AM-R 1. 2 buffer EB 1. 5% 0. 3 μl HP-F /HP-R SYNGENE μl PCR 1% 5% 10% 20% 40% dntp PCR 60% 80% DNA PCR dntp 3 PCR PCR dntp dntp 1 /2 3 3 Table 3 PCR Optimization of PCR amplification AM1 AM2 AM3 AM4 AM5 AM6 HP1 HP2 HP T m / / pmol 0. 2 / AM-F /R /HP-F /R / / dntp / nmol AM1. AM2. AM3. AM4. AM5. AM6. HP1. HP2. HP

3. 3 PCR trnl-trnf 1 000 bp PCR 136 bp PCR 323 bp 2 PCR 1% 5% 250 100 bp 1 ~ 7. 1% 10% 8. M. DNA 2 000 1 000 750 500 5% 10% 20% 40% 60% 80% 20% 40% 60% 80% Fig.

C. M. DNA 4 PCR PCR PCR 10% PCR 1. S. 2010 283. 2. J. 2012 37 21 3203. 3. J. 2 000 1 000 750 500 250 100 bp 1. 2. 3. 4. 5. 2010 29 844. 4. J. 2002 12 6 26. 6. 7. 8. 5. J. 2007 25 9.

4 3. 3 PCR trnl-trnf bp PCR 136 bp PCR 323 bp 2 PCR 1% 5% bp 1 ~ 7. 1% 10% 8. M. DNA % 10% 20% 40% 60% 80% 20% 40% 60% 80% Fig. 3 Agarose gel electrophoresis of specific PCR amplification 3 products amplified from different percentage mixture of Astragali 10% Radix and Hedysari Radix 3 - PCR A. trnl-trnf B. C. M. DNA 4 PCR PCR PCR 10% PCR 1. S J J bp J J PCR J Fig. 2 Agarose gel electrophoresis of specific PCR amplification 7. products amplified from Astragali Radix and Hedysari Radix J

5 8. J DNA 13 Wang H Kim M K Kim Y J et al. Molecular authentication of J ITS J PCR J Wang H Kim M K Kwon W S et al. Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region J. J Pharm Biomed Anal the Oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers J. Gene Kress W J Wurdack K J Zimmer E A et al. Use of DNA barcodes to identify flowering plants J. Proc Natl Acad Sci USA Study on identification of Astragali Radix and Hedysari Radix by PCR amplification of specific alleles LONG Ping CUI Zhan-hu LI Qian-quan Xu Jian-ping ZHANG Chun-hong ZHOU Li-she * LI Min-hui * Baotou Medical College Inner Mongolia Baotou China Abstract To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted trnl-trnf sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature dntp etc were optimized. All samples were amplified by PCR with specific primer DNA from Astragali Radix would be amplified 136 bp whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix. Key words Astragali Radix PCR amplification of specific alleles molecular identification doi /cjcmm

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis