Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum)

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation

TaqMan® estimates of <i>Vrn-A1</i> haploid copy number and days to ear emergence in winter wheat mapping populations.

<p>A) Copy number estimated from the <i>Vrn-A1</i>/Internal Positive Control (IPC) signal ratio. Solid circles are genotypes estimated to have one copy of <i>Vrn-A1</i>. Open circles are genotypes estimated to have two or three copies. Means and standard deviations of four measurements are shown. . T-tests showed the classes differed significantly from one another (p<0.01). B) Days to ear emergence in ‘Malacca’בHereward’ DH lines vernalized for 4 weeks (7°C; 8 h light) and grown in a heated, lit glasshouse providing 18 h light. The score for each DH line was the mean of three plants. Lines with two copies of <i>Vrn-A1</i> in white, those with three are in black. Arrows show flowering times of the parental varieties (mean of four plants). C) Days to ear emergence in ‘Claire’בHereward’ F₂ plants vernalized for 4 weeks (7°C; 8 h light) and grown as in (b). Plants homozygous C (exon 7 SNP) and with one copy of <i>Vrn-A1</i> are in grey. Plants heterozygous C/T and with intermediate <i>Vrn-A1</i> copy number are hatched (double hatched squares shows five plants with ambiguous copy number estimates between intermediate and high). Plants homozygous T and with three copies of <i>Vrn-A1</i> are in black. Arrows show flowering times of the parental varieties (mean of four plants).</p

Days to ear emergence for winter wheat plants after different vernalization times.

<p>Plants were vernalized (7°C; 8 h light) for 0 to 6 weeks (‘Claire’ – solid diamonds, dotted line; ‘Malacca’ – open circles, dashed line) or 0 to 10 weeks (‘Hereward’ – solid circles, solid line) and days to ear emergence on the main stem were recorded. Each point is the mean of five plants. Bars show standard deviations.</p

Quantitative RT-PCR analysis of <i>Vrn-A1</i> expression level in winter wheat plants vernalized for different times.

<p>Plants were vernalized (7°C; 8 h light) for 0 to 6 weeks (‘Claire’ – solid diamonds, dotted line; ‘Malacca’ – open circles, dashed line) or 0 to 10 weeks (‘Hereward’ – solid circles, solid line). A) Total <i>Vrn-A1</i> expression (C and T forms) in leaf samples at the end of the vernalization period or in unvernalized 7 day old seedlings for the 0 weeks treatment. Each time point is the mean of three biological replicates normalized against expression of an 18S rRNA control. Expression is shown as arbitrary units (AU). Bars show standard errors of means. B) Expression of the <i>Vrn-A1</i> form with the C in exon 4. C) Expression of the <i>Vrn-A1</i> form with the T in exon 4. The low level signal in ‘Claire’, which lacks the T form, was attributed to mis-priming of the assay in the absence of the correct template. At each time point letter b and c indicate significant differences (p<0.05 by ANOVA) from the ‘Hereward’ level (a).</p

Flowering time phenotypes of <i>Ppd-1a</i> introgression lines.

<p>Means and standard deviations of days to ear emergence for plants grown in short days (10 h natural light). ‘Paragon’ control (white; n = 10). The mean of two independent families (n = 20 in total) is shown for <i>Ppd-D1a</i> (hatched), <i>Ppd-A1a</i> (grey) and three <i>Ppd-B1a</i> alleles (black). Means significantly different by t-test (p<0.05) have different letters.</p

TaqMan® estimates of <i>Ppd-B1</i> haploid copy number.

<p>Solid circles are genotypes known to have a photoperiod sensitive (<i>Ppd-B1b</i>) allele and open circles are genotypes known to have a day neutral (<i>Ppd-B1a</i>) allele. References are [tp] this paper, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033234#pone.0033234-Worland2" target="_blank">[30]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033234#pone.0033234-Brner1" target="_blank">[33]</a> and others described in the text. Copy number was estimated from the <i>Ppd-B1</i>/Internal Positive Control (IPC) signal ratio. Means and standard deviations of four measurements are shown. T-tests showed the classes differed significantly from one another (p<0.001).</p

Structure of the ‘Chinese Spring’ <i>Ppd-B1a</i> allele and sequence haplotypes from other alleles.

<p>A) A 185 kb region containing ‘Chinese Spring’ <i>Ppd-B1a</i> allele. Sizes and positions of fully sequenced BACs are shown by solid lines with additional sized and end sequenced BACs as dotted lines. <i>Ppd-B1</i> copies (exons plus introns; > shows 5′ to 3′ orientation) are large dark green rectangles and the solid black line shows upstream and downstream regions with no homology to known repeated sequences. Small coloured rectangles show transposable elements annotated using the TREP database (<a href="http://wheat.pw.usda.gov/ITMI/Repeats/" target="_blank">http://wheat.pw.usda.gov/ITMI/Repeats/</a>). Vertical arrows show the junction between intact copies (black) or between the gene and transposon in the truncated copy (red). B) Haplotypes of <i>Ppd-B1</i> alleles. Intervarietal SNPs are in red. These were invariant between copies in ‘Chinese Spring’ and for clarity only the positions in copy_2 are shown. Bases distinguishing the copies in ‘Chinese Spring’ are in black (IUPAC codes). Coloured rectangles in the ‘Sonora64’/‘Timstein’ intercopy fragment are transposable elements as in (a). Vertical blue arrow shows the junction between copies. Junction sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033234#pone.0033234.s002" target="_blank">Figure S2</a>.</p