Article title: Copy Number Variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley
Journal: Plant Molecular Biology
Authors: Enrico Francia *, Caterina Morcia, Marianna Pasquariello, Valentina Mazzamurro, Justyna Anna Milc, Fulvia Rizza, Valeria Terzi, Nicola Pecchioni
*: Enrico Francia <>
Department of Life Sciences
University of Modena and Reggio Emilia
Via Amendola 2, Pad. Besta
Reggio Emilia, I 42122, Italy
Tel: +39-0522-522041
Fax: +39-0522-522027
Online Resource 1:
Supplementary Table 1 List of the 41 barley genotypes used for the study
Genotype name / Origin / Row Type(end use) a / Growth habit b / Pedigree c
Aday-2 / TUR / 6 / Winter / GK-Omega/Tarm-92
Airone / ITA / 2 (M) / Winter / Gitane//Tipper/A10
Albacete / ESP / 6 (M) / Spring / Selected from landrace in Albacete
Aldebaran / ITA / 6 (F) / Spring / Rebelle/Jaidor
Angela / FRA / 6 (F) / Spring / Borwina/Express
Arta / SYR / 2 (F) / Spring / Selected from ‘Arabi Abiad’ landrace
Avci-2002 / TUR / 6 (F) / Winter / Sci/3/Gi-72AB58,F1//WA1245141
Aydanhanim / TUR / 2 (M) / Spring / GK-Omega/Tarm92
Bazant / POL / 6 (M) / Winter / Unk
Brucker Stamm II / AUT / 6 / Facultative / Unk
Bulbul89 / TUR / 2 / Facultative / 13GTH/Line-657(selected landraces)
Bursztyn / POL / 6 / Winter / Unk
Carola / DEU / 6 / Winter / SG-402085/Franka//GW-1307
Cetin / TUR / 6 (F) / Winter / Star(Iran)/Line 4875(selected landraces)
Cometa / ITA / 2 (F) / Winter / PO202.169//Amillis/Fior 2377
Dasio / ITA / 2 (F) / Spring / Tipper/Arupos
Doria / ITA / 2 (F) / Spring / Nure/Zita//Nure/PO202.169,F8
Dura / DEU / 6 / Winter / Friedrichswerther-Berg/Ragusa//Doria
Gitane / BEL / 2 / Spring / Zéphyr//CI-1237/Cambrinus
Golden Promise / GBR / 2 (M) / Spring / Gamma ray mutant of Maythorpe
Keel / AUS / 2 (F) / Spring / CPI18197/Clipper//WI2645
Kika / ESP / 2 (F) / Spring / Kym/Albacete
Lunet / CZE / 6 / Facultative / Firlbeck-Astrid/Dea//Senta
Magda / NLD / 2 / Spring / Cebeco-6708/Apex
Mansholts-Fletumer / NLD / 6 / Winter / Selection from landrace, Netherlands
Mellori / FRA / 6 / Spring / Unk
Morex / USA / 6 (M) / Spring / Cree/Bonanza
Nure / ITA / 2 (F) / Winter / Fior-40/Alpha//Alpha///Baraka
Okal / CZE / 6 / Winter / Rubina(W-77)/HVW-860
Otis / DEU / 2 (M) / Spring / ST.08020/Europa//Atem
Pamina / DEU / 6 / Facultative / Hohenthurmer-2192-44/Mahnd.Viktoria//Dominator
Pariglia / ITA / 2 (M) / Winter / Airone/Arco
Ponente / ITA / 6 (F) / Spring / Vetulio/Arma//Express
Proctor / GBR / 2 / Spring / Kenia//Plumage-Archer
Ragusa / DEU / 6 / Spring / Selected from landrace in Dalmatia (HRV)
Steptoe / USA / 6 (F) / Spring / Washington-Selection-3564/Unitan
Tadmor / SYR / 2 (F) / Spring / Selected from ‘Arabi Aswad’ landrace
Tidone / ITA / 2 (F) / Spring / Okos/273-cat,F5//Igri
Tiffany / DEU / 2 (M) / Winter / Labea/Marinka
Tremois / FRA / 2 (M) / Spring / Dram/Aramir//Berac
U259 / HUN / 6 / Facultative / Nacktgerste/Mezohegyesi-68
a : M = malt, F = feed.
b : Genotypes were screened and classified according to von Zitzewitz et al. (2005).
c : Unk = unknown.
Supplementary Table 2 Summary of the 13 amplicons designed on target and reference genes used for RT-qPCR analysis. In silico quality control and empirical validation parameters are presented; for HvCBF2, a unique intergenic sequence tightly close at gene’s 5' end, was amplified
Amplicon a / Primer sequences / In silico QC b / Empirical validation c(5'–3') / Size / ΔG / Tm / Position in GenBank / Efficiency
(bp) / (kcal/mol) / (°C) / KF686739 (bp) / (%) / Slope / y-intercept / R2
Target genes
HvCBF9 / F-ttctacatgtcttcaggcgacc / 249 / 0.49 / 83 / 109,750–109,998 / 104.4 / -3.22 / 26.76 / 0.98
R-ctctctttcctcccaaatctgg
HvCBF4 / F-agcgccgctctgttttaca / 208 / 1.52 / 85 / 166,466–166,673 / 89.6 / -3.6 / 25.37 / 0.99
R-agcagtcgaacaaatagctcca
HvCBF2 / F-ttgtagtgcaatcattcgctcc / 101 / 0.76 / 77 / 180,778–180,878 / 94.2 / -3.47 / 27.94 / 0.98
R-cccaaggcgagatgtcttttc
HvCBF14 / F-agccgttgacgagaaggaagtc / 112 / 1.52 / 84 / 618,681–618,792 / 103.1 / -3.25 / 22.52 / 0.99
R-gtagcatgatccggcatccat
HvCBF15 / F-gacgtcgtcatccctaccttca / 107 / 1.74 / 83 / 758,676–758,735 / 87.9 / -3.65 / 28.05 / 0.99
R-atgtgtccaggtccatttccc
HvCBF12 / F-ttgtgccatgacatgttggag / 214 / 1.38 / 85 / 798,863–799,076 / 91.3 / -3.55 / 28.09 / 0.99
R-gaaataaacgaacggagcgaag
HvCBF16 / F-atgttcgagctggacttgtctg / 212 / 0.21 / 85 / 909,126–909,337 / 77.1 / -4.03 / 29.04 / 0.99
R-ttgcgacatgtgtgttgcat
HvCBF3 / F-ttgacttgttcccggaaatgg / 104 / 1.07 / 84 / 943,342–943,401 / 99.7 / -3.33 / 27.15 / 0.96
R-ttgtcttcgtacgcgtggatg
HvCBF13 / F-tgctgattcatcggagacgtc / 104 / 1.36 / 81 / Multiple hits / 163.1 / -2.38 / 29.57 / 0.99
R-ccatttccggcaaaaagtca
HvCBF10 / F-gcagaatcgtcgtctgagtctg / 102 / 1.17 / 84 / 1,022,203–1,022,304 / 111.2 / -3.08 / 26.59 / 0.96
R-catttcgatgtccattccga
HvCBF6 / F-ggaaacagagcatggcttttga / 101 / 1.52 / 79 / 1,051,128–1,051,228 / 93.4 / -3.49 / 28.78 / 0.98
R-cgcaaagattccctcgatcat / 1,088,950–1,089,050
Reference genes
HvNUD / F-gcatcctctcctgtaagcctct / 109 / 1.28 / 82 / n/a / 90.6 / -3.57 / 25.27 / 0.99
R-tctatctctcaggacacggtgg
HvSAMDC / F-tcggctacagcattgaagacg / 101 / 2.01 / 79 / n/a / 109.7 / -3.11 / 26.03 / 0.99
R-ccaaaaacgatatcaggatggc
a : Probes for HvCBFs are listed according to their physical order in KF686739 (Pasquariello et al. 2014). HvCBF6 primers target two different regions in the locus (see text for details). HvCBF13 amplicon has been excluded because of apparent lack of sequence specificity, later confirmed by abnormal primer efficiency at the empirical validation. Reference genes HvNUD and HvSAMDC correspond to GenBank accessions AP009567 and AK252992, respectively.
b : Absence of secondary structures in the region of annealing of primers has been verified using the MFOLD web server (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dnaform1.cgi).
c : Standard curves derive from ‘Nure’ genomic DNA dilution series.
Supplementary Table 3 Matrix of correlations among copy number values at ten HvCBFs
HvCBF9 / HvCBF4 / HvCBF2 / HvCBF14 / HvCBF15 / HvCBF12 / HvCBF16 / HvCBF3 / HvCBF10 / HvCBF6
HvCBF4 / 0.06
HvCBF2 / 0.15 / 0.89***
HvCBF14 / -0.09 / 0.36* / 0.43**
HvCBF15 / 0.49** / 0.18 / 0.30 / 0.38*
HvCBF12 / 0.71*** / 0.24 / 0.33* / -0.10 / 0.44**
HvCBF16 / 0.43** / 0.12 / 0.24 / 0.46** / 0.70*** / 0.38*
HvCBF3 / 0.73*** / -0.08 / 0.06 / -0.26 / 0.27 / 0.58*** / 0.19
HvCBF10 / 0.57*** / 0.14 / 0.04 / 0.08 / 0.47** / 0.45** / 0.41** / 0.59***
HvCBF6 / 0.63*** / 0.19 / 0.41** / 0.20 / 0.66*** / 0.69*** / 0.60*** / 0.41** / 0.27
*: p < 0.05, **: p < 0.01, ***: p < 0.001.
Supplementary Table 4 Frost resistance vs. copy number of HvCBF2 forms in facultative, winter and spring genotypes
HvCBF2 forms / # of individuals / CNRQ a / Fv/Fm a / Winter survival aFacultative / 5 / 9.0±2.1 a / 0.751±0.005 a / 64.99±4.61 a
2A / 5 / 9.0±2.1 / 0.751±0.005 / 64.99±4.61
2A+2B / 0 / n/a / n/a / n/a
2A/B / 0 / n/a / n/a / n/a
Winter / 14 / 4.4±0.5 b / 0.687±0.015 a / 61.56±3.51 a
2A / 2 / 7.6±0.1 / 0.741±0.004 / 72.35±0.82
2A+2B / 11 / 4.1±0.4 / 0.689±0.014 / 62.23±3.14
2A/B / 1 / 0.7 / 0.563 / 33.30
Spring / 22 / 2.6±0.4 c / 0.548±0.032 b / 40.74±7.31 b
2A / 9 / 2.5±0.3 / 0.618±0.026 / 54.24±6.26
2A+2B / 5 / 5.6±0.6 / 0.580±0.077 / 42.04±10.15
2A/B / 8 / 0.9±0.2 / 0.429±0.039 / 17.90±0.33
a : Different letters indicate least significant differences (LSD0.05) among groups mean.
Supplementary Table 5 Relationship between CNV of HvCBFs and frost resistance explored by Pearson correlation (r), simple linear regression (SLR) and simple linear regression with groups (gSLR) models. Only relevant genes or gene combinations are presented
Explanatory variable a / Corr b
( r ) / SLR c
( adj-R2 ) / gSLR d
( adj-R2 ) / Corr b
( r ) / SLR c
( adj-R2 ) / gSLR d
( adj-R2 )
HvCBF4 / 0.56*** / 0.295 / 0.395 / 0.56** / 0.290 / 0.405
HvCBF2 / 0.51*** / 0.237 / 0.400 / 0.42* / 0.145 / 0.345
HvCBF3 / -0.37* / 0.110 / 0.386 / -0.49* / 0.205 / 0.461
SUMall / 0.44 / 0.171 / 0.365 / 0.34 / n.s. / 0.301
SUMprox / 0.54*** / 0.270 / 0.400 / 0.48* / 0.199 / 0.364
SUMcent / 0.21 / n.s. / n.s. / 0.12 / n.s. / n.s.
SUMdist / -0.12 / n.s. / n.s. / -0.29 / n.s. / n.s.
SUM4+2 / 0.55*** / 0.281 / 0.403 / 0.50** / 0.222 / 0.378
SUM4-3 / 0.59*** / 0.324 / 0.411 / 0.61*** / 0.341 / 0.445
SUM2-3 / 0.53*** / 0.265 / 0.414 / 0.46* / 0.181 / 0.371
SUM4+2-3 / 0.56*** / 0.297 / 0.411 / 0.48* / 0.245 / 0.395
a : SUMall is the sum of copy numbers for all the HvCBFs at FR-H2 while SUMprox, SUMcent and SUMdist were only calculated for the HvCBFs of the proximal, central and distal clusters, respectively. SUM4+2, 4-3, 2-3, and 4+2-3 are arithmetic combinations (i.e., sum and/or difference) of the copy number for the indicated genes.
b : Pearson correlation r is followed by its two-tailed significance level; *: p < 0.05, ***: p < 0.001.
c : Linear regression adjusted R-squared is the proportion of variance accounted for by the fitted model (38 and 26 observations for Fv/Fm and winter survival, respectively).
d : The final gSLR model fits also allelic variation at VRN-H1 (‘Tremois’ susceptible allele set as reference) to produce a different constant and a different regression coefficient for the explanatory variable across winter/facultative and spring genotypes.