Supplemental data

Table S.1. Primer sequences of RFLP probes, STS markers, and gene-based markers derived from GrainGenes. Bin localization of markers (in brackets) according to

Marker / Primer forward (5’3’) / Primer reverse (5’3’) / Localization
Aba2 / ctggggttggttctgg / ggcagcaacatggcattc / 1HL, 5AL
Abc254 / ctcaccaccgaggccaagaa / aagaaagctgaaggcacatt / 5H(11), 7H(7)
Abg702b / tcatgtacgtcggccatctc / ccgagaaaacatcaaacctg / 1H(13), 5H(11)
Abg712 / aaatatggttggtcaaagtt / gcttctggtctgtgttcttg / 5H(11)
Mwg2230 / aatgatgttgctttcctgtttgctc / acagatgatgatggcgtgcagcttt / 5H(10)
Mwg514 / gtgcattaatacagtgccca / cacaaacgttacgagtacag / 5H(11), 6H(14)
Mwg549b1 / cttaacttttgaagggcacg / gcaggtgtgtcatcagtgta / 3H(15), 5H(10), 6H(9)
Mwg549b2 / tacgtgttggctaagctaagttcag / ataggtggagaacagaagggagatg / 3H(15), 5H(10), 6H(9)
Mwg550 / ctgacaatgtcgcctgtatgtgta / gacgcatttgttcatacaacctga / 5H(10)
Mwg553a / tacaagaactcgagaaagatgcca / ctgaatttgtgtggtgtcttgtgt / 2H(5),5H(10)
Mwg63b / caacagcagcacaaggtcgctgcct / ggtggaagccaagtctgacatcgtc / 5H, 3H, 1H, 7H, 4H, 2H
Mwg858.1 / cgcctcgtctgtcatactca / aaagaggatggcagccgt / 2H(4)
Mwg858.2 / tctgtcatactcaaagccgaatgcc / catacgcaatgctgctgctcccctc / 2H(4)
Mwg862 / aatacttacgcacgagagcc / gctatgcacaaacgattgg / 5H(11)
Mwg865 / gccctgttgctgtactaaaa / ctaacgaactgtgcatgatt / 2H(10)
Mwg877 / ccgtgatacacctggtgatatatg / cacaagaaagcagctcctccatt / 5H(12)
Mwg900 / cgtgtaactttactgtgcaaatctg / ccaaggagaagatccacacaatatg / 5H(11)
Mwg914 / gccatgaccgtcttccc / ccccataacattcgggtatg / 5H(10)
Mwg933 / aaaggagtctgggtggaaagc / atggattgcctggaaactgc / 5H(11)
Psr115 / gttttcccagtcacgac / caggaaacagctatgac / 5H(14)
Psr128 / tgccatgtttagaataacacgc / agaacaaggagaaggagctcg / 5H(8)
Wg583 / tgtacgcagggaaacaggtcgttc / gtccaagcagagagagagattccg / 5H
Mwg2062.1 / tctcgctggtattcagggtcc / aaacgatagcaagaggaaccg / 7H(12)
Mwg2062.2 / tactatctcgctggtattcagggtc / cttggttgttttgtttccttctttc / 7H(12)

Table S.2. Primer sequences of RFLP probes, STS markers, and gene-based markers designed for respective sequences. Bin localization of markers (in brackets)according to

Marker / Primer forward (5’3’) / Primer reverse (5’3’) / Localization / Sequence No
Abg3 / agtacctgatgaggcaggag / agatcatctggacagcatca / L43972
Af236869 / ccccagaaccctagatacaa / cgtcattccatcattcagaa / 5H(11) / AF236870.1(CDO504)
Aj440540 / agaagctgtgggtggggc / taggcaactacactttgcca / AJ440540.1(PSR120)
Bcd265 / aaaacaaacaccacaaagca / ggtatcaactaccagccacc / 1H(12), 4H(5), 5H(11) / BE438696, BE438788
Bcd266 / taataacaatggcagctcca / atggcgatagctatgctctc / 2H(13) / BE438697, BE438789
Bcd351 / ggcgtttgatgaagattctt / caggaatcaaccttgtcaca / BE439015.1, BE438985.1
Cdo771 / atgatggatcctagcagctc / atggcaaggaacactctttc / 7H(6), 1H(10), 5H(9) / BE439173, BE439243
Mwg894 / gttctgccttctctggtacc / caggttcgattacatgcttg / 5H(10) / AJ234629, AJ234628
Mwg922 / ttttgatttgattgggaggt / ctgagtccgagtggtctctt / 5H(12) / AJ234649, AJ234648
Mwg989 / gacgaaactgaagaagggaa / cgaggaaggtgttgaaatct / 2H(15) / AJ234703
Psb140 / agtgactgattcttggcatg / ctgaggatcttcatggtggt / AZ048464.1
Psb85.1 / ctgaaaaggaacgacttcgc / gcagaaaattgcggaaacat / AZ048452.1
Psb85.2 / acttcgcttggagcaaatag / agctctaacatggaaggcag / AZ048452.1
Psr150 / caacatggagaggatcatga / caccaggtccttgacagact / 7H(7), 2H(3) / AJ440577.1, AJ440576.1
Psr360.1 / aattgacatggctgatgatg / aactatcagcaggaccaagc / AJ440688.1
Psr360.2 / gagcttcactaccattggct / gaaaaccagtgggtataccg / AJ440688.1
Psr637 / ccccaaaactgtagttttcc / gcccgaccttgaattaaata / AJ440636.1, AJ440637.1
Psr911 / tagatcggtgcactttctca / ccccaatttgttgatcatta / AJ440655.1, AJ440654.1
Rz395 / cagagctggcatctacaaga / gttcaacggctaaagcaact / AA231885.1, AA231767.1
Wac4a / gaatattttccggtaagcga / cacctgcaatgaaccaataa / M58753.1
Wac4b / cgcttcggtttaagatttgt / atgccctaggtgttgctaac / M58753.1
Wg1026 / caagctgcataatcagcagt / ggtcggagaagaaagcataa / 4H(5,6), 5H(9,10) / L44007.1
Wg364 / atttatcataccactatatcgtaaa / gcgttgttttagttaccgct / 5H(9) / BH854373.1
HvCBF4-Mo / tgcctcaacttcgccgactc / atcccctgcgccaagctc / 5H(9) / AF298230.1, AF418204.1, AF376136.1
TaVrn1-pr / tcctaggactggcgagtatc / cagagtttttcctttggcat / 5H(11) / AY616454.1
TaVrn1-i1a / cttgccggctttattttctt / tcgatcaatgctgattttgt / 5H(11) / AY747606.1, AY747604.1, AY747601.1
TaVrn1-i1b / ggaggctttacaaagaatga / gttcgacaagcttcccttca / 5H(11) / AY747604.1

Table S3. Components of PCR reactions used for generation of different markers from Table1.

PCR / Volume (L) / Spermidine / Taq (U) / DNA quantity (ng)
SSR_1 / 15 / 0.4 mM / 0.5 / 35
SSR_2 / 15 / 0.4 mM / 1.0 / 35
STS_1 / 25 / 0.6 mM / 0.7 / 60
STS_2 / 20 / 0.4 mM / 0.7 / 65
STS_3 / 20 / 0.4 mM / 0.9 / 65
STS_4 / 25 / 0.6 mM / 0.5 / 35

Primers design and BLAST homology of target sequences

Forward primer of marker HvCBF4-Mo primer was designed for consensus coding sequence of putative CRT/DRE-binding factor of barley mRNA cv. Morex (Choi et al. 2002), cv. Halycon (Xue 2002) and wheat cv. Winoka (Jaglo et al. 2001). BLAST search with respective fragment of AF298230.1 sequence showed that primer HvCBF4-Mo1 with conserved motive 5’-GCCGAC-3’ targeted loci of barley (HvCBF4A, HvCBF4B, HvCBF4D, HvCBF14, HvCBF5, and HvCFB3),Triticum monococcum(FR-Am2, and CBF14), Triticum aestivum (TaCBF4a, TaCBF4b, TaCBF4c, TaCBF2), and Sorghum bicolor(SbCBF6). Similarly, reverse primer of marker HvCBF4-Mo2 derived from ‘Morex’ (Choi et al. 2002) annealed to barley and wheat genes CBF4A, CBF4D, and CBF9.

Primers for the first intron of TaVrn1-i1a were designed on the base of sequences AY747606, AY747604, AY747601 of TaVrn-D1, TaVrn-B1, and TaVrn-A1 genes, respectively. Marker TaVrn-i1b was derived fromsequence AY747604 (Fu et al. 2005). BLAST search revealed that primers of markerTaVrn1-i1a matched homologous barley sequences while no barley sequence were found in silico for TaVrn1-i1b marker.TaVrn1-pr marker was designed to sequence AY616454 from promoter region of Aegilops tauschii VRN-D1 gene (Yan et al. 2004). Blast search with the target sequence revealed that primers were conserved across Triticum aestivum, T.monococcum and Hordeum vulgare BM5A gene, and 5 size differences (from 379 – 340 bp) of fragment flanked by primers in barley could be predicted for sequences reported by von Zitzewitz et al. (2005).

Supplemental references

Choi DW, Rodriguez EM, Close TJ (2002) Barley Cbf3 gene identification, expression pattern, and map location. Plant Physiol 129:1781–1787

Fu D, Szucs P, Yan L, Helguera M, Skinner JS, von Zitzewitz J, Hayes PM, Dubcovsky J (2005) Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Mol Genet Genomics 273:54-65

Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, Thomashow MF (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol 127: 910-917.

Xue GP (2002) An AP2 domain transcription factor HvCBF1 activates expression of cold-responsive genes in barley through interaction with a (G/a)(C/t)CGAC motif. Biochim Biophys Acta 1577:63-72.

Yan L, Helguera M, Kato K, Fukuyama S, Sherman J, Dubcovsky J (2004) Allelic variation at the VRN-1 promoter region in polyploid wheat. Theor Appl Genet 109:1677–1686.

von Zitzewitz J, Szucs P, Dubcovsky J, Yan L, Francia E, Pecchioni N, Casas A, Chen TH, Hayes PM, Skinner JS (2005) Molecular and structural characterization of barley vernalization genes. Plant Mol Biol 59:449-467.

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