Dissecting Earliness per se and Photoperiod heading date effects in bread wheat (Triticum aestivum)

Vernalization, photoperiod and the poorly defined earliness per se (Eps) genes regulate flowering in plants. This thesis used elite hexaploid wheat varieties and focused on an Eps QTL on 1DL, and photoperiod QTLs on 1BL and 5AL short day QTL using doubled haploid (DH) populations. Four independent backcross two F5 (BC2F5) populations with 70 lines each of a cross between Spark and Rialto derived from BC2F2 by single seed decent (SSD), and four independent pairs of near isogenic lines (NILs) in the background Rialto were used for 1DL. Genomics and bioinformatics approach was used to design genome specific primers for genes on 1DL, 1BL and 5AL QTL regions using the Brachypodium distachyon syntenic genes. Resequencing genes on 1DL revealed that Spark and Cadenza have a chromosomal deletion including several genes. The equivalent region in Rialto and Avalon is intact. The DH, SSD and NILs all showed that the 1DL deletion was associated with early flowering. Recombinants from the BC2F5 populations indicate that the 1DL deletion likely contains the candidate gene(s). The 1DL QTL was defined as a discrete Mendelian factor. The 1BL QTL was short day specific for Spark X Rialto and Avalon X Cadenza but the QTL was observed under both short and long day conditions for Charger X Badger. The gene TaFT3 a homologue of the HvFT3/Ppd-H2 is suggested as the candidate for the 1BL QTL because two mutant alleles, a deletion and a conserved amino acid change seem to account for most of the variation under short days. Twenty-five varieties from Sweden segregate for the FT3-1B and loss of function mutation results in delayed flowering under short days. This thesis describes evidence that has allowed prioritization of candidate genes for the QTLs. The data from this thesis is useful for understanding wheat adaptation and marker assisted selection

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

Quantitative RT-PCR analysis of <i>Ppd-B1</i> expression in short days.

<p>A) Gene expression in short days (light period 0–9 h; white bar) sampled at 3 h intervals over a 24 h time course comparing ‘Mercia’ (<i>Ppd-B1b</i> allele; dotted line, open circles) with a ‘Mercia(Chinese Spring 2B)’ single chromosome substitution line (<i>Ppd-B1a</i>; solid line, closed squares). B) Gene expression in short days (light period 0–9 h; white bar) sampled at 3 h intervals from 0 to 9 h comparing ‘Paragon’ (<i>Ppd-B1b</i>; dotted line) with BC₄F₃ introgression lines homozygous for <i>Ppd-B1a</i> alleles from ‘Chinese Spring’ or ‘Sonora64’ or BC₃F₃ introgression lines homozygous for <i>Ppd-B1a</i> from ‘Récital’ (solid lines). 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). Standard errors of means are indicated by error bars and asterisks show probabilities from one-way ANOVAs of expression levels comparing the respective <i>Ppd-B1a</i> alleles to the ‘Paragon’ <i>Ppd-B1b</i> allele (* p<0.5; ** p<0.01; *** p<0.001).</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