Authors: Naoko Kitamoto1, Susumu Yui2, Kazuhiro Nishikawa3, Yoshihito Takahata1, Shuji Yokoi1
Authors: Naoko Kitamoto1, Susumu Yui2, Kazuhiro Nishikawa3, Yoshihito Takahata1, Shuji Yokoi1
Title: A naturally occurring long insertion in the first intron in the Brassica rapaFLC2 gene causes delayed bolting.
Addresses: 1Laboratory of Plant Breeding, Faculty of Agriculture, University of Iwate, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan, 2National Agricultural Research Center for Tohoku Region, 4 Akahira, Shimo-kuriyagawa, Morioka, Iwate 020-0123, Japan, 3Kimitsu Research Station, Sakata Seed Corporation, 358 Uchikoshi, Sodegaura, Chiba 299-0217, Japan.
Corresponding author: Shuji Yokoi
Telephone number: +81-19-621-6152
Fax number: +81-19-621-6177
E-mail:
Supplemental Table 1. Sequences of primers used in expression analyses and sequencing analyses of BrFLCs.
Supplemental Table 2.Mean ± SD of number of days to bolting under long day after vernalizaion in the expression analysis. An extremely late-bolting breeding line “Tsukena No. 2”, a mid-bolting commercial F1 variety “Muso” and an early-bolting parent of commercial F1 varieties, “Early” (Sakata Co.) were grown with daily cycles of 16 h of light at 22°C and 8 h of dark at 18°C. Fourteen days after sowing, plants with two or three true leaves were transferred to 4°C under a 24-h photoperiod for 40 days. After this cold treatment (vernalization), the plants were transferred to an original condition. In each genotype, eight plants were grown and leaf samples for the expression analyses were collected from the all plants individually.
Genotype / Mean / SDMuso / 7.0 / 1.9
Early / 8.0 / 0.5
Tsukena No. 2 / 252.0 / 15.4
Supplemental Figure 1. Locations of primers used in this study. Black boxes and lines represent exons and introns, respectively. The inverted triangles in the BrFLC2, BrFLC3 and BrFLC3’ denote inserted sequences in Tsukena No. 2. Numbers in the 
figure represents the corresponding ID in the Supplemental Table 1.
Supplemental Figure 2.Comparison of genomic sequences between Arabidopsis FLC (AtFLC; AT5G10140) and BrFLCs of Tsukena No. 2. Although the large insertions in the first intron of Tsukena No. 2 were deleted from the sequence for comparison, the location of the insertion were indicated by arrows. Dot with a gray shadow representssame sequence with AtFLC, and "–" indicates gap. Boxes represent exon 1, 2 and VRE (the vernalization response element) in the AtFLC, and sequences with a gray shadow in the AtFLC indicate common sequences between AtFLC and BrFLCs of Tsukena No. 2. Sequence alignment were conducted using CodonCode Aligner ver.3.7.1 (CodonCode Corporation, and this figure was drawn using GeneStudio Professional ver.2.2.0.0. (GeneStudio com,
10 20 30 40 50 60
AtFLC ATGGGAAGAAAAAAACTAGA AATCAAGCGAATTGAGAACAAAAGTAGCCGACAAGTCACC
BrFLC2 ...... G...... G......
BrFLC3 ......
BrFLC3’......
70 80 90 100 110 120
AtFLC TTCTCCAAACGTCGCAACGGTCTCATCGAGAAAGCTCGTCAGCTTTCTGTTCTCTGTGAC
BrFLC2 ...... A.....T...... A...... C..T
BrFLC3 ...... A....G...... T...... C..T
BrFLC3’...... A....G...... T...... C..T
130 140 150 160 170 180
AtFLC GCATCCGTCGCTCTTCTCGT CGTCTCCGCCTCCGGCAAGCTCTACAGCTTCTCCTCCGGC
BrFLC2 ...... T.....A...... T....A...... G.....
BrFLC3 ...... G...... T...... T...... G....T
BrFLC3’...... G...... T...... T...... G....T
190 200 210 220 230 240
AtFLC GATAAGTACG CCTTTTCCTT ACCTGGGTTTTCATTTGTTC CCCCTTTTAT CTTCTGTTTT
BrFLC2 ...... T. A...C....C TT...... C...C...A..T G...... CG ....C...CC
BrFLC3 ...... TA A...C.T..C TT...T.CC...C...A..A G..T...A.G ...... A
BrFLC3’...... AA G...C....C TT...T.CC...C...A..A G..T...A.G ...... A
250 260 270 280 290 300
AtFLC GTGCTCTTTT ACTTTTCTGA GAAAATA------AAAATAA AAAAACAA-T
BrFLC2 TCTTCTC...... A..TAT..G...ATAAA ATAAAGTACA AAT...... T..A..A.
BrFLC3 -----..... -..GA.AAA. T..T-...... T..T....
BrFLC3’-----..... -..GA.AAA. ------...... --- ..T..T....
310 320 330 340 350 360
AtFLC TAATATACCG TTTGGTTTTT TTCCGGCGGATCTCTTGTTG -TTTCTCGGTTCTGTGTTTG
BrFLC2 ...A.G..AC ....A..-.. --.....A...... A...G. A...... T......
BrFLC3 ...A.A..AC ....A..C.. --...A.AA...... A..GA A.....T....T...... T
BrFLC3’...A.A..AC ....A..C.. --...A.AA...... A..GA A.....T....T...... T
370 380 390 400 410 420
AtFLC TTTGTGTTTT ------TT TCTGCGA--- CCATGATAGA TACATGAGAT AACCAAATTT
BrFLC2 ..AT.....C TTCTATGAAC ATGAGAGATA .ATGAGGTA. CCAGATTTGA G.AAG..CAA
BrFLC3 ...T.C.... CAAAATTG.....T.T.TGG ...... G...... C.
BrFLC3’...T-C.... CAAAATTG.....T.T.TGG ...... G...... C.
430 440 450 460 470 480
AtFLC AAGGAAGAAC AATGTCGTGA AGAAGCTTTT TAGCTTCTTA CTTTTGTTCA TTTCTCTCTC
BrFLC2 TGTCCT..TG .GCTAAAGCT TCTTA....G .TCA..TC.C TC.C.CAATC ------
BrFLC3 G...... C.. T..-...AA- -.C...... C-......
BrFLC3’ G...... C.. T..-...AA- -.C...... C-......
490 500 510 520 530 540
AtFLC TATTTCT--- --TAAAAAAA AAAATTCTGCATGGATTTCA TTATTTCCTT GGAAAAAAA-
BrFLC2 ------.....------TTT...... TT ..T...--.. T.T...... G
BrFLC3 .C...T.CGT TC.TCT.TTG .TTT...... T...T.A.--.. T.T.....T.
BrFLC3’.C...T.CGT TC.TCT.TTG .TTT...... T...T.A.--.. T.T.....T.
550 560 570 580 590 600
AtFLC -TTGCATGTC ATTCACGATT TGTTTGATAC GATCTGATGC GTGCTCGATGTTGTTGAGTG
BrFLC2 A...... T .....A.G.C .T.-.....A .C.G...... A.A..T...... GT....
BrFLC3 ...... CG...A...C .T.C...A...T.G...... A.A..T...... T....
BrFLC3’...... CG...A...C .T.C...A...T.G...... A.A..T...... T....
610 620 630 640 650 660
AtFLC AAGTTTCAAG CCATCTTTGATTGTTTCTTA CCTTTAGAGA TTCCTTAAGT TTTTGAAGAG
BrFLC2 ...... T A...... AA.T GGA..TAG.. C....A.TTA AG.ATCT..A
BrFLC3 ...... TG...... AG..C ATCAGTTGTT AGTTAGCTT. AGGG.TTTTC
BrFLC3’...... TG...... AG..C ATCAGTT.TT AGTTAGCTT. AGGG.TTTTC
670 680 690 700 710 720
AtFLC TTAATTATAT ATCACAAGAC TAATGATTAA TGTCTCTTTT CAGAGTGATT AAAATTCATT
BrFLC2 GAGT.CC.TA TCAGA.T..G ..T.AG.GGT .T...T...A G..TAATT.. GGTCGATGGA
BrFLC3 ..TTGAGA...ATTG.TT...G.ATT..TT .T.TCAG.GG TTACTGAT.A .T.C...TG.
BrFLC3’..TTGAGA...ATTG.TT...G.ATT..TT .T.TCAG.GG TTACTGAT.A .T.C...TG.
730 740 750 760 770 780
AtFLC GGATCTCTCG GATTTGTATG CAATGCACTT ACGGGAGATC TATAGAGTTG CTATGGGGTT
BrFLC2 TCTCT.GATT TG.------AA TGCAAT.CAC
BrFLC3 TTT.AGAGT..T.AAAATCA TTGGAGCT...G.ATTTT.A .G..AT.CAA TGCACT.CAC
BrFLC3’ TTT.AGAGT..T.AAAATCA TTGGAGCT...G.ATTTT.A .G..AT.CAA TGCACT.CAC
790 800 810 820 830 840
AtFLC AATGCTGAAC AATGTATATA TACCACATTG TGCAGCTATT GACTATATAA GACTATTGTT
BrFLC2 TTC.GG..GA TC.A..A.A. ATGTGGGA.T AATGCT..A. A.TAGAT.....G.T..AA.
BrFLC3 TCC.G.T.GA TC.A..A.AT .G.TGA..A..AG.T.A.GA ATT.TG.CCT ..A.G.AACG
BrFLC3’ TCC.GAT.GA TC.A..A.AT .G.TGA..A..AG.T.A.GA ATT.TG.CCT ..A.G.AACG
850 860 870 880 890 900
AtFLC AATCTTCTAT GAATTCCTAT CTTTGCTGTG GACCTATTAC TTGGTGATTA TCCAAATTAG
BrFLC2 TCGAA.G....CCACAT.T. GCAGCAAA.. TGAACTCCGT AGAACTCC.T AAGT.TC.GA
BrFLC3 .CAT.GTGCA .CTA.TGACA AAAA.A.ACT .TAGATA.T. AAATAA..A..AT.TT.ACC
BrFLC3’.CAT.GTGCA .CTA.TGACA AAAA.A.ACT .TAGATA.T. AAATAA..A..AT.TT.ACC
910 920 930 940 950 960
AtFLC TGTTTTTAAT TGATTCATAT TTTTCATACA CAGTAGTT------
BrFLC2 A.AG..C.T. AT.AGA...A A.AATGATT. AT..TTC.AT TTTTAGAGTA ATTCAAGTCA
BrFLC3 ATGGA..T...ACA.ATCTA GA..ATA.GT TTTCTAA.TG GTTCATAATT TTTACATACA
BrFLC3’ ATGGA..T...ACA.ATCTA GA..ATA.GT TTTCTAA.TG GTTCATAATT TTTACATACA
970 980 990 1000 1010 1020
AtFLC ------
BrFLC2 ATGAATCTCT TGGAGTTCGA TGCAATGCAC TTCGGGGAGA TCTATAAAAT TGATGTAGGA
BrFLC3 TATATGTATA TATAATAGAA CG......
BrFLC3’ TATATGTATA TATAATAGAA CG......
1030 1040 1050 1060 1070 1080
AtFLC ------
BrFLC2 TTAATGCAGA ATAATAGATC AAGAGTTTTA ATATGAATGC ATATATGCCA CATTGTGCAG
BrFLC3 ......
BrFLC3’......
1090 1100 1110 1120 1130 1140
AtFLC ------
BrFLC2 CCATTAACTT AAAAAGATAA TTGTAGATCT TCAAAAAAAA ATTACCTCTT TGCCATGGAT
BrFLC3 ......
BrFLC3’......
1150 1160 1170 1180 1190 1200
AtFLC ------
BrFLC2 TTATTGCTTG AAGATTATCC AAATTATAAG GCTACTAGTT GGTTCTTAGT ATCCACATAT
BrFLC3 ......
BrFLC3’......
1210 1220 1230 1240 1250 1260
AtFLC ------TTGAA TTTTGGTAGC TTCAAAAA-ACTCAGCCTCACAATTAGTAC
BrFLC2 ATGTGTAAAG TAATT..... A...... A ..T..G..A......
BrFLC3 ...... C.A ...T.G..C....G...... T......
BrFLC3’...... C.A ...T.G..C....G...... T......
1270 1280 1290 1300 1310 1320
AtFLC TTACCGCACA TATGCTACTT CAGTAACATA ATCTGGTTAT CGATTGCGAT TCTTTGAATC
BrFLC2 ...A---C.. C.CATATGC. ACACT..T.. TGT.CTCCGC GATCGC.AT. A.AA..GTGT
BrFLC3 ...ATTAC.G C.CATATGC. ACACTTA..T ..G..A.GTG GT..CTATT. ATA..CTT.A
BrFLC3’...ATTAC.G C.CATACGC. ACACTTA..T ..G..A.GTG GT..CTATT. ATA..CTT.A
1330 1340 1350 1360 1370 1380
AtFLC ACAATCGTCG TGTGCTATAT ATATAACACC TTTTGCTGTA CATAAACTGG TCTAATTTTA
BrFLC2 TG..CAAGGA .TA....GT. G.T.TGAT.T .AACTTAT.T TG.TTTG.CT C.------
BrFLC3 .TC.CAA....TGTG.GA...AT.G.TTGT CA.GTAAA.. TTCCTTAGAA AGC.T.AGAG
BrFLC3’.TC.CAA....TGTG.GA...AT.G.TTGT CA.GTAAA.. TTCCTTAGAA AGC.T.AGAG
1390 1400 1410 1420 1430 1440
AtFLC GACTAATTAA ATTTCATTGT TCTCTTGGAT TTGTATATGC ACGTCCGGGA GATTTATAAA
BrFLC2 ------
BrFLC3 CGTGCTAA.. G.AAG.AGTG GAAA.CCA.A .GTGCGGCTG CTTGAACATG AT.ACTAC..
BrFLC3’ CGTGCTAA.T G.AAG.AGTG GAAA.CCA.A .GTGCGGCTG CTTGAACATG AT.ACTAC..
1450 1460 1470 1480 1490 1500
AtFLC TAAAATTAGT ATGAGGTTAA TGGTAAAAAG GATCAAGAAG TTTGGTTTTA AATGTAAGCC
BrFLC2 ------
BrFLC3 .TTTT.CCTG .AATTT.AGC AAA.CTT.GT .G..CG...A ..AACA.C.T GG.T...AAT
BrFLC3’.TTTT.CCTG .AATTT.AGC AAA.CTT.GT .G..CG...A ..AACA.C.T GG.T...AAT
1510 1520 1530 1540 1550 1560
AtFLC ACATTAATTG GGAAACTATG ACTAAAAGAT GAATTGGTAA TATATACATA ATATTTTTAA
BrFLC2 ------
BrFLC3 GT--CTTC.. ------
BrFLC3’ GT--CTTC.. ------
1570 1580 1590 1600 1610 1620
AtFLC CGAATTTCTC TCCTTTTTAT GGGATATGCT ATTTAAAGAT TGTCCAAAGG TTTATAGTTT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1630 1640 1650 1660 1670 1680
AtFLC CCCACTCTTG CAGTTACACA CATAGATTTG CCTCATATTT ATGTGATTGT ATATCAATTA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1690 1700 1710 1720 1730 1740
AtFLC TCGCCCTTAA TCTTATCATC GTTGTGTTCA TTTATGACTT TGTTCCTATT CGTTAAAATT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1750 1760 1770 1780 1790 1800
AtFLC GACAATCCAC AACCTCAATC TTTTGTTGTG AAAATCGACA ATCACACAAC CTTTGTATCT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1810 1820 1830 1840 1850 1860
AtFLC TGTGTCTTTT GTCACACAAC CTTTGTATCT TGTGTCTTTT GTCATGGAAA TTGTCATTCA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1870 1880 1890 1900 1910 1920
AtFLC CACAGCCTTG TTTCTTTGGT GCCTCTAGGA AATTGAAAAT CCCACAACAC TTGTCTTCAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1930 1940 1950 1960 1970 1980
AtFLC GTAAGAAATA CCAACCTCTT TGGTACGGAT CTATAATGAA TCAATATAAT CCTATATATA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
1990 2000 2010 2020 2030 2040
AtFLC AGTTGTCAAA ATTGAATCTG GTGTAGTGTC TACTACAACC CTCCAATATA ATAACCAAAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2050 2060 2070 2080 2090 2100
AtFLC GGTTGTAGTA GTTTGGCCAT GTTGGTCAAG ATCGCTGGCC GATTCTCACT TGATGCATAC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2110 2120 2130 2140 2150 2160
AtFLC TTTGTTAGGA TTTGTTCACC CCTAGTTAGG TCCAGCCTTG GAATTGTCGA GACACCTGAC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2170 2180 2190 2200 2210 2220
AtFLC TAGAACTCCT GGTCTTAATT ATGATTTAAT AAAGAAGAAG CCTTTTAGAA CGTGGAACCC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2230 2240 2250 2260 2270 2280
AtFLC TTAGTTACTC AGTTACTCTT TTTGCATACT TAGGTTGATG CAAAGAGCTT AACTTCACAA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2290 2300 2310 2320 2330 2340
AtFLC TAGGACTGAT ATCTATTAAC AAAACAAATT AAGTGAAGTT TTGTCAAAAT TGTTGGATCT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2350 2360 2370 2380 2390 2400
AtFLC TCTAGGTCAA TATGTAGTTT AGTTTTTATC TGTCTTAGTC GCTTCCTTCT ATGGAAGATA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2410 2420 2430 2440 2450 2460
AtFLC TATTTATAGA TATGTGTGAT AAGTTTCCTA CTAATATTAG TTAGTGTAAC TTCAAGGGCA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2470 2480 2490 2500 2510 2520
AtFLC GAAAACTCTT TACTTTTATT GCTTGATTAA TTTGGGGTTT AAATATAGGA AATTGGAACC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2530 2540 2550 2560 2570 2580
AtFLC TCACAGTTTC TATAAACGAG TAAGTAATTG AATGTGAAAT AACAAAATGG AAGACCGGCT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2590 2600 2610 2620 2630 2640
AtFLC TCCTATTCTT AGGAGTCTTT TGATATTTGC AAAAAAAACA TATACAATAG AAATATGAGT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2650 2660 2670 2680 2690 2700
AtFLC TTTGTCTAAG ACTCGGTCCA TGTATTTGGA GTTTGGCTTC CTCATACTTA TGGTTATCTG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2710 2720 2730 2740 2750 2760
AtFLC GTTACCGCCA CATCATCATT ATCATCTTAT GGGTCATCAA TACTAGCTCT ATCGCTGGAA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2770 2780 2790 2800 2810 2820
AtFLC AAAACCTTGT CCTCAAGGTT CATTGAAAAA TCCGAAAAGT TTTCTCGTAT ATGTTGATAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2830 2840 2850 2860 2870 2880
AtFLC GGTATTACTT ACAAACAAAG AGCTGATGTT ACCAATTTTG ACACGAGATT ACTAATGAAC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2890 2900 2910 2920 2930 2940
AtFLC TCATGAAAGA GGCGTTTTTA AAAAATTCTT TTTAAAACTG GGATACAAAA AGAAAAGAGG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
2950 2960 2970 2980 2990 3000
AtFLC TAACTAATAA TTTGATACCA TTGTTCGTAG TCCTGATCAA ATGTTATAAG GGTAAACATG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3010 3020 3030 3040 3050 3060
AtFLC ATAGAAAATA GAGGGTAAAT AGGTTTTGTT CTTATAATGG TTTTGATAAC ACGCTTTGTA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3070 3080 3090 3100 3110 3120
AtFLC AAGGATATAG GTGTTTTTTG ATGCTAAAAG TTGTGGTATG GATCAAAACC AAAATGGAAG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3130 3140 3150 3160 3170 3180
AtFLC CTCTGAATCT CTGATAGAGG TTGCAATTAG AATTATATAA GTTAATTTGC AAATGAATTG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3190 3200 3210 3220 3230 3240
AtFLC GAAGCAGTCT TCCACTATTT GCTATTGTTA GGGAAGTCTT TCAGTTAATT TCAGAAAATT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3250 3260 3270 3280 3290 3300
AtFLC AAGAGAAATA TGACTTTCTA GACTCAGTCT GTGTACTTGG AATTTTACTT CGGTTTACTT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3310 3320 3330 3340 3350 3360
AtFLC CCATGTCATC ACATTGTGGC TCATCAATAT ATGTGTGTAT ATACATTCAT GAGTATATAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3370 3380 3390 3400 3410 3420
AtFLC GATTTCTGGA AAAATAAAAA TTGCTTGTTT GCATTTAAGA TTGGGGCTGC GTTTACATTT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3430 3440 3450 3460 3470 3480
AtFLC TATATTGCAT CAATTATTTC AACATAGATT CACAAACATA AATGCATAGA AACAATCTGG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3490 3500 3510 3520 3530 3540
AtFLC ACAGTAGAGG CTTATGTTTA GGGTTCTTAT GTACCTTAAC TAGTTTGACT TTAAGTTAAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3550 3560 3570 3580 3590 3600
AtFLC CAAAGCCAGC GCTATCACTA AACTTTATCT GTATGCCTTT GTATGACTTT TCTTTGAGGG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3610 3620 3630 3640 3650 3660
AtFLC AAAATGTCAT TTTCAATCTG CCGAAATATA TAATAAATAC ATGTTAGCCC ACATAATTCA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3670 3680 3690 3700 3710 3720
AtFLC TTGGATAACT AATCTTTGAG CAATTTTTGG TAAATGTTTT GGTTCTTTTC TTTTCTTGAG
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3730 3740 3750 3760 3770 3780
AtFLC AGAGAAAAAA AATATCAGAT ATTATTAAAT ATTGCTTACA AAGCTAAGAA CAAGTTAAAA
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3790 3800 3810 3820 3830 3840
AtFLC CTTTTTTGAA AAAGTGGAAA TTCAGATGTG CTACTGCTTA AACATGAATA TTAAGATTAT
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3850 3860 3870 3880 3890 3900
AtFLC TGTTTTTCTG AAATGTTACG AATACTAGCG TGTTATATAT ATGTAAAAGG TAAGGTGTTC
BrFLC2 ------
BrFLC3 ------
BrFLC3’ ------
3910 3920 3930 3940 3950 3960
AtFLC TCTCAATGTT TCATAGTTTC CAGTGGCCTT TTCAAGGGTT AGCTAGTAGT TTTGATCCTA
BrFLC2 ------
BrFLC3 -----...... A...... TG...... -...... A...... T..G..
BrFLC3’ -----...... A...... TG...... -...... A...... T..G..
3970 3980 3990 4000 4010 4020
AtFLC ACATATTTTT ATTTTTTTTG TCATCTCTCCAGCCTGGTCA AGATCCTTGATCGATATGGG
BrFLC2 ------...T...... A
BrFLC3 ....----.A ...G.....C .--.-...... G...... A
BrFLC3’....----.A ...G.....- .--.-...... G...... A
4030 4040 4050
AtFLC AAACAGCATGCTGATGATCTTAAAGCCTTG 3736
BrFLC2 .....A...... TC.. 1362
BrFLC3 ...... G...... C.. 1351
BrFLC3’........ G...... C.. 1345
Supplemental Figure 3. Comparison between the present linkage map and the previously published linkage maps; AGF2 (Suwabe et al., 2006) and KHF2 (Hatakeyama et al., 2010). The information about the previously published linkage maps was downloaded from VegMarks ( The Result of the QTL analysis was shown. V+; in the vernalization condition, V-; in the non-vernalized condition. DB; the number of days to bolting, DF; the number of days to flowering, NL; the number of leaves to flowering.
Supplemental Figure4. Involvement of the BrFLC2 polymorphism in flowering-time variation in the field condition. a) Frequency distribution of bolting date in a F2 population derived from a cross between a late-flowering commercial variety “Haruwarai” and an extremely late-flowering selected line derived from Tsukena No. 2. In total, 200 seeds were sown in pots in an unheated green house on 9 September 2009 and transplanted to an open field on 5 October. The bolting date of each plant was evaluated after a long, severe winter in northern Japan. b) Agarose gel electrophoresis of the genotypes of the 10 earliest or 10 latest F2 plants in BrFLC2, BrFLC3 and BrFLC3′ 1st intron. Lane 1 (left) is a 1 Kb DNA Ladder. H:Haruwarai, T:Tsukena No. 2.