Supplementary Tables
Supplementary Table S1. Effect of DDM1-knockdown and TSA-treatment on recombination around centromeres.
Chr. / left marker (cM) / right marker (cM) / line/treatment / recombinant / non-recombinant / observed cM / P value*3 / RM1940 (86.0)1 / C60465 (87.1)2 / wild type / 11 / 345 / 3.09
TSA / 10 / 342 / 2.84 / 0.845
asDDM1 / 26 / 340 / 7.15 / 0.014
11 / RM7463 (54.8)1 / RM3428 (56.2)1 / wild type / 5 / 375 / 1.31
TSA / 7 / 373 / 1.84 / 0.561
asDDM1 / 7 / 373 / 1.84 / 0.561
*Significance of difference between the wild type and the asDDM1 or TSA-treated F2 progenies by chi-square test.
1 McCouch SR et al (2002) Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.) (supplement) DNA Res 9:259-279
2 http://rgp.dna.affrc.go.jp/E/publicdata/caps/index.html
Supplementary Table S2. Segregation of the parental alleles in the centromeric region of chromosome 2 and 11.
marker / RM5210 / RM3443 / RM7463 / RM3428
F2 (NB x KS) / N/N / 26.0** / 23.8 / 21.0 / 21.7
K/K / 26.6 / 25.9 / 27.6 / 28.3
N/K / 47.5 / 50.3 / 51.5 / 50.0
F2 (asDDM1 x KS) / N/N / 24.8 / 25.1 / 20.9 / 21.0
K/K / 24.8 / 27.4 / 30.3 / 30.6
N/K / 50.3 / 47.5 / 48.7 / 48.4
* N/N, homozygous for Nipponbare; K/K, homozygous for Kasalath; N/K, heterozygous.
** % frequency of indicated genotype.
Supplementary Table S3. Primers used for mapping of recombination sites.
RM5210 / 2 / 54.6 / SSR / 155 / CTAAATGACAAAAGATAGCA / AAATTCTGACTTGTATGACA / 1
RM3443 / 2 / 54.6 / SSR / 118 / AGGCCGCACATGATAGTACC / TACACGCCTGTAGCTCGTTG / 1
RM4108 / 3 / 1.1 / SSR / 142 / GTCCCTCGCTTTATATCTAG / CAACTCTGCTAAACGAATTA / 1
RM4683 / 3 / 14.8 / SSR / 155 / TACAGCAACAATCTTTAACC / TAGAGGGAGTATTGTGCTAG / 1
RM1338 / 3 / 40.3 / SSR / 143 / AGAGGGAATTAGATTGGATT / GGTCCACTTCTTCCTTCTAT / 1
RM6676 / 3 / 66.2 / SSR / 145 / GTTCACGGTCCAATAAGAAT / CTTTCAAGCTTACGAAAACA / 1
C53358 / 3 / 69.7 / STS / 268 / GGAGGAATGAATGAATGAAG / GCAGTATTCATACATAGGAG / 2
RM2346 / 3 / 85.2 / SSR / 168 / TTCAGGGATGTGAAATATAC / ATCGTGCTTTTATTGAAATA / 1
RM1940 / 3 / 86.0 / SSR / 172 / ACTATCGATCAAAATGCTAG / AAACGAATGGTTAAATGTTA / 1
I860 / 3 / 86.0 / STS / 233 / GACACTCTTGTAGCTAGGTCAAACTA / CATTGTCACGGTGACGGCATTGTCA / 3 / +27 in NB
D3806 / 3 / 86.0 / STS / 199 / CATTGGCATCGAGGATCTCTAGCAT / GAACCAGCTTCATGTTCCACAA / 3 / -24 in NB
YH0302 / 3 / 86.0 / STS / 182 / CTGAGAGTACGCCCCTCCATATATA / CCTTGCAAGTTGTGGTACCTACAAC / 3 / -39 in NB
YH0309 / 3 / 86.0 / STS / 156 / CAACAATACAGTTGATACCGCCTAAC / TACTAAGATAATTTCCTCCACGTGGG / 3 / +18 in NB
YH0320 / 3 / 86.0 / CAPS / 134 / GCGTGCCAACATGTACAATCCGTTGT / TCTTCATCAATCAGCCCCAGTTGTTC / 3 / HinfI in NB
YH031821Bg / 3 / 86.0 / CAPS / 17,313 / ACCATAAACCCTCACCTCTACCGCT / TTAGCAACCAAGTCGCCTTAGGGAT / 3 / BglII in NB
YH0312 / 3 / 86.0 / CAPS / 270 / CCAAGCCTGAAAACAGCAGCGAATT / CGACCTGAACCTCCTTAGATATGTA / 3 / TaqI in KS
YH0317 / 3 / 86.0 / CAPS / 265 / GGGGATTGCAATTTCCCAATTGTGC / TAGGCTTGGAACAGCTAAGAGGAAG / 3 / TaqI in NB
D5711 / 3 / 86.0 / STS / 255 / CGGGCAAAAGGAAAGATGATGGAGT / GAATGTGGGCAATGCTAGAAAGTCC / 3 / +54 in NB
D7722 / 3 / 86.0 / STS / 286 / GCATCGTCGTGTCATGTTTGTTGGT / TAGGTATTGGCGGCATAGAATTGGC / 3 / +67 in NB
D3104 / 3 / 86.0 / STS / 231 / CTGACTTGTCTTCAGATGGATGACC / GGATGCGTTTTCACTTGAGTCCTTC / 3 / -25 in NB
D1521 / 3 / 86.0 / STS / 207 / GGCTGACATATGTATGGCACCATGT / AACACGGCCTAGAGTTCCATGTTAG / 3 / -23 in NB
C60465 / 3 / 87.1 / STS / 214 / TTAACCGTGATAGCCCGTGC / CTTGACTGGCCCTGTTCTGG / 2
RM5626 / 3 / 99.0 / SSR / 96 / ATCAGTCGGTCATAAACGCC / ACCTTCCTCTTCTGCTGCTG / 1
RM2334 / 3 / 115.6 / SSR / 155 / CATGCATCTGATCTGATTAT / TGTGAAGAGTACAAGTAGGG / 1
RM3856 / 3 / 127.7 / SSR / 140 / TTGCATAACAAGATGAACAA / TTAATTGCGGATTTTTTATT / 1
RM7076 / 3 / 151.1 / SSR / 198 / CTCCACCAACAACTCGTATC / AAGCTATTCACAAGCAGCTC / 1
spl-1AB / 3 / 158.2 / CAPS / 320 / TGCATTGACCCTAATGGTGAGGACAT / TCAGGTATAAGTCACTGCCATCCAC / 3 / HhaI
CK1354AC / 3 / 158.2 / CAPS / 392 / CCGGTTGATATTCAATCCACCCATC / TAAACAACTACGTGACGATACCGCG / 3 / HhaI
RM7463 / 11 / 54.8 / SSR / 187 / CGAGGATCACACCAGTTTTG / TGCAGAGCCAAGAAAGGAAG / 1
YH1106 / 11 / 55.1 / STS / 209 / GGCAATGCAAATCGTATTTGCGATAG / TGTCTCTTCTGGCCTGGACCATTTA / 3 / +40 in NB
YH1105 / 11 / 55.1 / STS / 129 / CAAGTTGGTGGGCTAGCCGATCTA / CAATCCAGGGATTCAAATGCTAGGC / 3 / -23 in NB
YH1102 / 11 / 55.9 / STS / 181 / CGGACATTGATGACGAGCACTCATT / CCCTAAACGTATGCCTGTCACCAAT / 3 / +40 in NB
RM5857 / 11 / 55.9 / SSR / 155 / ACAGCTTGTCTTTATTTCCTG / GATGGTAATCCAGGTTGTTG / 1
YH1101 / 11 / 55.9 / STS / 182 / AGCCTCATTGATTTGCCAGGAATCG / TTGCAGATCATCTTGCAGTGGTTACA / 3 / +37 in NB
RM3428 / 11 / 56.2 / SSR / 172 / ACTATCGATCAAAATGCTAG / AAACGAATGGTTAAATGTTA / 1
1 bp in Nipponbare; 2 NB, Nipponbare
References
1. McCouch SR et al (2002) Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.) (supplement) DNA Res 9:259-279
2. http://rgp.dna.affrc.go.jp/E/publicdata/caps/index.html.
3. this work
Supplementary Table S4. Primers used for chromatin immunoprecipitation.
MgT1 / 3 / 28,927,966 - 28,928,109 / OSJNBb0024B16 / 144 / MgTF / CCTAGATCAGGGGTGTCTCC
MgTR2 / TCACATGCCTGAAAAATGCT
actin2 / 3 / 29,886,559 - 29,886,706 / OSJNBa0078A17 / 148 / OsAcF / GAGCTGTTATCGCCGTCCT
OsAcR / CAAGTGAGAACCACAGGTAGCA
rRNA / 9 / not identified / 109 / OsrRNA spcF2 / GGGAGAGAACGAGAAAGCTG
OsrRNA spcR2 / GCTAGTGCCGTGAGTCGTTT
Cen3-653 / 3 / 20,526,953 - 20,527,077 / OSJNBa0053G10 / 125 / Cen3-65F / CCACAACGCATTTTATTTTGA
Cen3-65R / TCAGATTCTCCTGGAACTGCT
Cen3-663 / 3 / 20,538235 - 20,538,375 / OSJNBa0053G10 / 141 / Cen3-66F / AAGTTGCCGTTGTCAAGGAG
Cen3-66R2 / GCCCACGCTCAAGATCAA
Cen3-673 / 3 / 20,548,025 - 20,548,157 / OSJNBa0053G10 / 133 / Cen3-67F2 / AGGGCTTTGGCAAGCTATC
Cen3-67R / CCATATGCATCAGCTTGCTC
Cen3-683 / 3 / 20,560,709 - 20,560,812 / OSJNBa0057E11 / 104 / Cen3-68F / CCGGTCTGCTTTAGGAAACTC
Cen3-68R / GGAGAGAGCAAGGAAGACGA
Cen3-693 / 3 / 20,576,206 - 20,576,316 / OSJNBa0057E11 / 111 / Cen3-69F2 / GGAGAAACGACCTCACAAGG
Cen3-69R / TTCTTTTTCTTTAAAACGCGAAT
Cen3-733 / 3 / 20,669,680 - 20,669,803 / OSJNBa0057E11 / 124 / Cen3-73F / AGGATGGGGGTCTTTCAAGT
Cen3-73R / AACAAGAAATTGCATGGTTGC
3C003 / 3 / 20,036,339 - 20,036,462 / OSJNBb0047D08 / 124 / 3C003F / GGAACAAGCCATTTGGAGAA
3C003R / GGGCTAGGCCTTTTAAGGAG
3C004 / 3 / 20,036,556 - 20,036,688 / OSJNBb0047D08 / 133 / 3C004F / TCCTCCGTTTAGCGTCATTT
3C004R / CGAAGGGTTCAGGGAAAGAT
3C007 / 3 / 20,157,339 - 20,157,463 / OSJNBb0047D08 / 125 / 3C007F / CGACGCTGGTAACAAGAACA
3C007R / AATTCTCGGGTGCTTCACAG
3C008 / 3 / 20,094,905 - 20,095,040 / OSJNBb0047D08 / 136 / 3C008F / GTGGCAATTGCAAAAGGAGT
3C008R / TAGCAACTGGCTTGGTTCCT
3C015 / 3 / 20,392,622 - 20,392,771 / OSJNBb0038E06 / 150 / 3C015F / TTTCCAAGTTTCCATCTAGCA
3C015R / AATTGTGAATATGAGGCTGGA
3C016 / 3 / 20,394,190 - 20,394,343 / OSJNBb0038E06 / 154 / 3C016F / CCCTTCCAGCACTTATTTTCA
3C016R / TGTTAGTGTGGCAGCGTTGT
8C0184 / 8 / 13,545,756 - 13,545,964 / OJ1120_B07 / 209 / chr8cen18U / CTTGAGTCTCGTCCTGCTTTGC
chr8cen18L / TGCTGTTCTTCCTTTCGTTTGA
1 A reference region for ChIP encoding a manganese transport protein (Yan et al 2006)).
2 A reference region for ChIP encoding actin.
3 Centromeric regions on chromosome 3 (Yan et al 2006). Primers were designed to amplify single regions in the genome and to amplify DNA fragments smaller than 150 bp.
4 A CENH3-positive region on chromosome 8 (Nagaki et al 2004).
References
1. Yan H et al (2006) Transcription and histone modifications in the recombination-free region spanning a rice centromere. Plant Cell 17:3227-3238
2. Nagaki K et al (2004) Sequencing of a rice centromere uncovers active genes. Nat Gene. 36:138-145
Supplementary Table S5. Primers used for RT-PCR.
RCS2 / RCS2F1 / CGCGAATCACGAAGTGAGTTTTTGC / RT and PCR
RCS2R1 / CGGCGAACTTTTGTCAATTAATGCC / RT and PCR
ACTIN / OsAc2 / GTACCCGCATCAGGCATCTGATTAA / RT and PCR
OsAc1 / TTCTTCGGACCCAAGAATGCTAAGC / PCR
Supplementary Table S6. Genotypes of markers around the recombination hotspot.
RM1940 / C60465 / F1 (NB x KS) (179)3 / TSA-treaed F1
(NB x KS) (176) / F1 (asDDM1 x KS) (179)
NB / hetero / 2 / 3 / 2
KS / hetero / 6 / 1 / 21
hetero / NB / 3 / 5 / 2
hetero / KS / 1 / 1 / 1
NB / KS / 0 / 0 / 0
KS / NB / 0 / 0 / 0
1 Genotypes at RM1940 (left side of the hotspot) and C60465 (right side of the hotspot) are shown. NB, homozygous for Nipponbare; KS, homozygous for Kasalath; hetero, heterozygous.
2 Number of F2 plants carrying recombination between RM1940 and C60465 are shown.
3 Total numbers of F2 plants are shown in parentheses.
6