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/ SCAR Sequence characterized amplified region DNA DNA 34 ( GSW100 SP2-002 SR2-015 SR3-004 11-22-1 11-22-2 11-24-3 11-24-4 11-191-1 12-217-1 12-249-1 14-218-21 04/05-29 04/05-66 04/05-73 12-202-2 (LP) 12-205-1 12-218-17 12-267-4 12-327-1 12-342-1 14-203-1 14-203-3 14-210-2 14-218-8 14-218-10 14-222-5 14-248-7 15-93-1) 3 3 DNA DNA SCAR GSW100 SP2-002 SR2-015 SR3-004 11-22-1 11-22-2 11-24-3 11-24-4 11-191-1 12-217-1 12-249-1 14-218-21 04/05-29 04/05-66 04/05-73 12-202-2 (LP) 12-205-1 12-218-17 12-267-4 12-327-1 12-342-1 14-203-1 14-203-3 14-210-2 14-218-8 14-218-10 14-222-5 14-248-7 15-93-1 3

SCAR PCR DNA SCAR DNA PCR SCAR ( 2008; Shimada et al. 2009) DNA SCAR SCAR (Nishihara et al. 2008) 20 PCR SCAR DNA 9 DNA -80 SCAR 4

DNA U (upper) L (Lower) ( ) ANS Primer Length Sequence ANSintU683 24 mer 5'-ATG TCA ACT TTT TAT TGG TCC TAA-3' ANSintL1142 24 mer 5'-GAG CAC AGC AAG AAC TTT GGT AGC-3' CHI 3 Primer Length Sequence CHI3rdU173 24 mer 5'-GAG TGG TTA AGC AGA TGA CAC GAC-3' CHI3rdL763 24 mer 5'-AAG AAA ATT GAC AAC ATG CAG AAG-3' FHT 1 (U24 & L356) Primer Length Sequence FHTintU24 24 mer 5'-GAT TCA TTG TTT CCA GCC ATT TGC-3' FHTintL356 24 mer 5'-GTC ATG GAT CAC AAA ACA AGA TCA-3' FHT 1 (U906 & L1896) Primer Length Sequence FHTintU906 24 mer 5'-TTA CAC AAA AAT AGG GTC AGT TCC-3' FHTintL1898 24 mer 5'-TCG TTA TAA ATA GAT GTG GTC CTC-3' FHT 1 (U1253 & L1631) Primer Length Sequence FHTintU1253 24 mer 5'-TTG CAC CTG AAG TAG AAT TTT ACA-3' FHTintL1631 24 mer 5'-TTC TGA CAG AAC TTC AAG CAA TTT-3' F3'5'H Primer Length Sequence F3'5'HintU1363 24 mer 5'-TCC ATT GAT TAA AAT GAG GGA CCA-3' F3'5'HintL1974 24 mer 5'-TAT GGT AAG TTG GGG ATG TCT GAT-3' DFR Primer Length Sequence DFRintU853 24 mer 5'-GAG CAC TTG CAT TAT TGT TCA GAA-3' DFRintL1267 24 mer 5'-GTA GTG TGC TTC ATT CCC TAT GGA-3' FLS Primer Length Sequence Alta_gFLS_U2724 24 mer 5'- TAG TAT TTG ATT TGA TGA AGA CAG-3' 5

Alta_gFLS_L3287 24 mer 5'- CAT GGC TAC CTA CTA CCT ATG AGT-3' bhlh1 Primer Length Sequence bhlh_u336 24 mer 5'- AAG GTG ATC GTT GTG AAA ATG TCT-3' bhlh_l629 24 mer 5'- GGC CGT CTA GTT TGG TGG TTG GTT-3' GenElute Plant Genome DNA Miniprep Kit (SIGMA) 1 10 (code: G2N10-1KT) 70 (code: G2N70-1KT) 350 (code: G2N350-1KT) 99.5% 500 ml (Wako, code:057-00456) 2.0 ml ( code: 2330-00) 2 x 500 2.0 ml ( code: 2001-00) 2 x 500 ( 2 mm) ( code: 5-4043-01) 1 kg ExTaq DNA polymerase (TaKaRa Bio) 250U (code: RR001A) 1000U (250U x 4) (code: RR001B) 3000U (250U x 12) (code: RR001C) PCR 0.2 ml 8 (greiner bio-one) 125 (code: 301401) SeaKem GTG Agarose ( : CMR. TaKaRa Bio) 25 g (TaKaRa code: F0071) 125 g (TaKaRa code: F0070) 500 g (TaKaRa code: F0074) 100 Base-Pair Ladder (GE Healthcare) 100 µg (code: 27-4007-01) * 1 µg/µl TE buffer, 6 x ( 10 x) (XC, BPB ) 20 ( 0.05 µg/µl) 50 x TAE () 500 ml (code: 313-90035) 6

(and/or ) TE (ph 8.0) () 500 ml (code: 316-90025) 1.5 ml (TreffLab, TOHO code: 30050) 1,000 (RAININ) GPS GPS-1000S ( ) 960 GPS-250S ( ) 960 GPS-10GS ( ) 960 121 C20 DNase/RNase 10mg/mL () 10mL (Code315-90051) GenElute TM Plant Genome DNA Miniprep (SIGMA) Wash Solution Micro Smash MS-100 (TOMY SEIKO) ABI Mupid-2plus (ADVANCE, code: M-2P) (65 C ) (TOMY MX105 ) GenElute TM Plant Genome DNA Miniprep (SIGMA) 7

GenElute TM Plant Genome DNA Miniprep (SIGMA) 65 C Elution buffer ( + 1 ) 65 C 1 GenElute Filtration Column 1 GenElute Nucleic Acid Binding Column 1 collection tube 3 1) (70-80 mg) 2 ml [ ( 2 mm) 1/3 ] DNA 80 C 80 C 2) Micro Smash TM MS-100 (TOMY) 5,500 rpm 20 [ (4-5 )] 3) 350 µl Buffer part A 50 µl Buffer part B (SDS SDS ) 4) 65 C 10 ( Buffer part A Buffer part B 5) precipitation solution 1.5 ) 5) Precipitation solution 130 µl 5 6) 5) GenElute Nucleic acid Binding Column 500 µl column preparation solution 15,000 rpm 1 9) 7) 15,000 rpm 5 GenElute Filtration Column 8) 15,000 rpm 1 700 µl Binding Solution 9) 700 µl 6) GenElute Nucleic acid Binding Column 15,000 rpm 1 ( 8

) 10) collection tube ( ) 500 µl wash solution 15,000 rpm 1 collection tube 500 µl wash solution 15,000 rpm 3 11) collection tube 65 C elution buffer 100 µl 15,000 rpm 1 15,000 rpm 1 12) DNA 20 C µl 6 x loading dye 1 µl 4 µl µl 10 ng lambda phage DNA TAE 0.7% CMR Seakem GTG Agarose (Takara Bio) µl 100 V 30 (1 g/ml ) 30 Lambda phage DNA 10-100 ng/µl 7) 20 C DNA 9

lambda phage DNA 15 lambda phage DNA DNA 10

1) 100 pmol/µl PCR 20 pmol/µl 2) PCR (1 ) 14.8 µl 10 x ExTaq reaction buffer (ExTaq ) 2.0 µl 2.5 mm dntp mix (ExTaq ) 1.6 µl Upper primer (20 pmol/µl) 0.5 µl Lower primer (20 pmol/µl) 0.5 µl ExTaq DNA polymerase (Takara Bio) 5U/µl 0.1 µl DNA (10-100 ng/µl ) 0.5 µl 20.0 µl SCAR PCR ( DNA ) PCR + 1 DNA 1 µl 13.8 µl [10 PCR (10 + 1 )] 162.8 µl 10 x ExTaq reaction buffer (ExTaq ) 22.0 µl 2.5 mm dntp mix (ExTaq ) 17.6 µl Upper primer (20 pmol/µl) 5.5 µl Lower primer (20 pmol/µl) 5.5 µl ExTaq DNA polymerase (Takara Bio) 5U/µl 1.1 µl 214.5 µl PCR 19.5 µl PCR DNA (50-100 ng/µl ) 0.5 µl 11

3) PCR 94 C 1 30 94 C 20 60 C 40 35 72 C 1 72 C 5 10 C PCR 5 µl 6 x loading dye 1 µl TAE 1.6% DNA 100 bp (GE Healthcare ) DNA 12

PCR DNA 1) ANS (ANSintU683&ANSintL1142) A: 300 bp 500 bp B: 500 bp C: 300 bp 350 bp D: 300 bp 2 M M M ANS (ANSintU683&ANSintL1142) M 100 bp 13

2) CHI 3 (CHI3rdU173&CHI3rdL763) A: 500 bp 600 bp B: 600 bp 900 bp C: 600 bp D: 900 bp E: 700 bp F: 600 bp, 700 bp 3 500 bp 600 bp 700 bp 900 bp M M M 3 CHI (CHI3rdU173&CHI3rdL763) M 100 bp 14

3) FHT 1 (FHTintU24 & FTHintL356) A: 400 bp 2 B: 400 bp 1 900 bp C: 400 bp M 1 D: 400 bp 2 900 bp 4 3 B 4 FHT 1 (FHTintU24 & FTHintL356) M 100 bp 15

4) FHT 1 (FTHintU906 & FTHintL1898) A: 330 bp B: 330 bp 750 bp C: 750 bp 5 3 B M M 5 FHT 1 (FTHintU906 & FTHintL1898) M 100 bp 16

5) FHT 1 (FHTintU1253 & FHTintL1631) A: 400 bp 600 bp B: 600 bp C: 400 bp 6 3 A 6 FHT 1 (FHTintU1253 & FHTintL1631) M 100 bp 17

6) F3'5'H (F3'5'HintU1363& F3'5'HintL1974) A: 300 bp B: 650 bp C: 300 bp 650 bp 7 C A 7 F3'5'H (F3'5'HintU1363& F3'5'HintL1974) M 100 bp 18

7) DFR (DFRintU853& DFRintL1267) A: 420 bp B: 350 bp 420 bp 500 bp D: 350 bp 420 bp 500 bp 8 A B 350 bp 420 bp 500 bp 12-267-4 350 bp 420 bp 500 bp 8 DFR (DFRintU853& DFRintL1267) M 100 bp 19

8) FLS (Alta_gFLS_U2724& Alta_gFLS_L3422) A: 320 bp B: 370 bp C: 420 bp D: 320 bp 370 bp E: 370 bp 420 bp F: 600 bp G: 320 bp 420 bp 9 C G 9 FLS (Alta_gFLS_U2724& Alta_gFLS_L3422) M 100 bp 20

9) bhlh1 (bhlh_u336&bhlh_l629) A: 850 bp 1.3 kb B: 1.3 kb C: 850 bp 10 3 A 850bp 1.3kb 10 bhlh1 (bhlh_u336&bhlh_l629) M 100 bp 21

SCAR ANS CHI 3 FHT (U24 & L356) FHT (U906 & L1898) FHT (U1253 & L1631) F3'5'H DFR FLS bhlh 1 A A B B A C A C A 2 C F B B A C B G A 3 A C B B A A A C A PCR () 9 37 3 3 3 1 3 ANS CHI 3 FHT (U24 & L356) FHT (U906 & L1898) FHT (U1253 & L1631) F3'5'H DFR FLS bhlh B B A B B A A F C A C A A A A A B B A A B A A F A B A A B A C A B A B A A A A B B C B C A A A C B A A B B A A A A A SCAR 22

GeneCleanII kit( ) DNA pcr4topo(invitrogen ) pgem-teasy(promega ) llustra TempliPhi 100 Amplification Kit (GE Healthcare ) DNA PCR PCR M13RV M13M4 BigDye(R) Terminators version 1.1 Cycle Sequencing Kit ABI 3130xl ABI 10 4 Genetyx-Mac ver. 14 23

GenElute TM Plant Genome DNA Miniprep (SIGMA) DNA 10-100ng/ l 94 C 1 30 94 C 20 60 C 40 35 72 C 1 72 C 5 10 C 1.6% 30

DNA DNA DNA 2 Nishihara, M., Nakatsuka, T., Mizutani-Fukuchi, M., Tanaka, Y., Yamamura, S. (2008) Gentians: From gene cloning to molecular breeding. Floricultural and Ornamental Biotechnology, published by Global Science Books, UK. pp 57-67. Shimada, N., Nakatsuka, T., Nakano, Y., Kakizaki, Y., Abe, Y., Hikage, T. and Nishihara M. (2009) Identification of gentian cultivars using SCAR markers based on intron-length polymorphisms of flavonoid biosynthetic genes. Scientia Horticulturae 119: 292-296 2008 31