Conceived and designed the experiments: RPH CM JMIH THNE. Performed the experiments: CM KLW KES SJT MWEJF TJF SRM JMEJ KMD ACA AB GMTV. Analyzed the data: RPH CM KLW MWEJF TJF SRM JMIH JMEJ KMD GMTV THNE. Contributed reagents/materials/analysis tools: AB CJC. Wrote the paper: RPH CM JMIH THNE.
The authors have declared that no competing interests exist.
The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea.
We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the
We have identified the pea genes
The segregation of flower color in the progeny of pea crosses is well known in genetics because of Mendel's experiments
White flowered cultivated forms of pea are common and were available to Knight and Mendel, but wild peas have purple flowers, presumably as did the earliest cultivated forms. This raises the question of when white flowered types arose. An early description of ‘white peas’ in agriculture appears in Ruralia Commoda, written at the end of the 13th century or beginning of the 14th century by Pietro de Crescenzi in which he describes when to sow ‘large white’ peas
The purple color that accumulates in wild type pea flowers is due to anthocyanin pigments; compounds derived from phenylalanine. Mutations in either structural or regulatory genes have been shown to lead to a loss of pigmentation in several plant species
Two cDNA-RFLP markers (CD72 and PEAPCF1) are closely linked to the
(
Degenerate primers designed to the
Primers specific to the pea
The equivalent
(
In plants, the GT splice donor site is present in almost all introns
The presence of a premature stop codon may reduce mRNA accumulation through nonsense-mediated decay. Accordingly, we studied transcript accumulation from the
Intron six has ∼5770 bases with ∼190 single nucleotide changes. The synonymous mutation rate for pea has been estimated as 7±2.6×10−9 nucleotides/yr
We were interested in whether we could find any examples of
A systematic search of exotic
We analyzed the sequence of the
Synteny analysis was used to identify gene candidates for
The nearby BAC clone MTH2-28N4 (CR940305), contains sequence that encodes a WD40-like protein. The corresponding pea sequence was obtained from JI 2822 (
We characterized the
In order to test the hypothesis that the
Particle bombardment of petals of Greenfeast (PI 250447 that carries the G to A splice donor mutation) with [left to right] the BAC containing the pea
We have established that a gene encoding a bHLH transcription factor is present at the
The cultivar Greenfeast carries the
There are many sequence variants of dominant
Seven sequences represent the JI 1987 indel variant (
Both Mendel
The following JIC lines were used: colored flower lines were, JI 15, JI 281, JI 813 and JI 2822, white flowered lines were JI 4, JI 399, JI 1194, JI 1201, Torsdag (JI 992) the Greenfeast accessions: JI 504, JI 1189, JI 1229 and JI 2737 and Knight's lines: JI 2462 and JI 2479. Exotic white flowered lines were; JI 232, JI 616, JI 817, JI 1497, JI 1512, JI 1782, JI 1987, JI 2647 and JI 3003; these correspond to white flowered
A selection of PI lines from USDA ARS (see USDA ARS Germplasm Resources Information Network at:
The RIL mapping population derived from the JI 281×JI 399 cross corresponds to the accessions numbered JI 2868 to JI 2939.
In addition, cultivar Caméor (white) and line PI 269818 (colored)
A TBLASTN
Full length
Primers RPH-445 – RPH-454 (
BAC clone 452H2 was identified using primers PsbHLHintF and PsbHLHintR. BAC clone 112D23 was identified from BAC pools
Sanger sequencing of pea BAC clones 112D23 and 452H2 was performed by GATC (Germany) and sequences were assembled using both ContigExpress and the Staden Pregap package as previously described
The sequence GU132941 corresponds to the BAC clone 112D23 derived from PI 269818 (
PCR products containing the junction of exon 6 and intron 6 of 129 PI lines (
Total RNA was isolated from flower tissue of both colored and white flowered pea varieties (Plant RNA Reagent, Invitrogen). All RNA samples were treated with DNase (DNA-free Kit, Ambion) to remove contaminating DNA. First strand cDNA synthesis was carried out using SuperScript III and Oligo dT according to manufacturer's instructions (Invitrogen).
Quantitative real-time PCR (qPCR) amplification of cDNA and analysis was carried out using the LightCycler 480 System (Roche), with LightCycler software version 1.5. Primers KL244F and KL246R (
PCR analysis of lines examined for mRNA accumulation was carried out using Platinum Taq (Invitrogen). Primers KL255F and KL256R were designed to amplify a cDNA region from 40 bp upstream to 39 bp downstream of the intron 6 splice donor site. Reaction conditions were 95°C, 5 min followed by 35 cycles of 30 s at 95°C, 30 s at 57°C and 20 s at 72°C. PCR products were separated on 3% agarose gels along with 1 kb ladder (Invitrogen) and stained with ethidium bromide.
DNA was bombarded into the abaxial surface of wing petals from white flowered Greenfeast grown in the glasshouse. DNA (5 µg with 2 µg of internal control GFP construct) was precipitated onto 5 mg of gold particles (1.0 µm diameter, in 50 µl sterile water), and tissue was surface sterilized prior to bombardment
Synteny between the pea genetic map and
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Sequence alignment of the A gene. (A) Alignment of genomic DNA of pea bHLH genes from colored flowered A line PI 269818 BAC 112D23 [GU132941] and white flowered a cultivar Caméor BAC 452H2 [GU132942]. The sequence shown for the exons is that of PI 269818 as this is known to be a functional allele. Exons 1–7 are marked and nucleotide differences between PI 269818 (A) and Caméor (a) alleles are highlighted. The G to A mutation in a at base 5005 that leads to the mis-splicing of intron 6 is marked with an asterisk (*). (B) Alignment of predicted mRNA from PI 269818 (A) and Caméor (a) genotypes. ATG start codons and TAG stop codons are marked and in bold. Nucleotide differences are highlighted. The first 8 bases of intron 6 that are included in mRNA of the Caméor (a) genotype are marked with asterisks (********). (C) Alignment of the predicted bHLH coding sequence from purple flowered A (PI 269818 and JI 2822) and white flowered a genotypes (Caméor and JI 1987). Amino acid sequence differences are highlighted and stop codons are marked with asterisks (*).
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Sequence divergence of the bHLH gene in germplasm and exotic pea lines. (A) Sequence of exotic pea lines. For JI 4 and 9 exotic white flowered pea lines; JI 232, JI 616, JI 817, JI 1497, JI 1512, JI 1782, JI 1987, JI 2647 and JI 3003 the G to A mutation is highlighted and marked with *. Three independent individuals of the white flowered JI 1987 (n1, n2 and n3) do not have the G to A transition, they do, however, have a number of SNPs that are highlighted in light blue and an additional A residue in exon 6, identified by the # and highlighted in red. (B) Association of pea flower color with the G to A mutation at base 5005 (highlighted) in 148 lines (including 129 single plant representatives from the refined core of PI lines see USDA ARS Germplasm Resources Information Network at:
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Annotated sequence of the A2 gene from JI2822 and showing differences in various lines. The sequence from nt 160 to nt 217 (bold italic) is a direct duplication, only one copy of which is found in the PI 198074 allele. From nucleotide 530 to 644 (bold italic) is also deleted in the PI 198074 allele. Note the GCACA sequence is repeated at either end of this deleted segment. The G to A SNP at nucleotide 1207 in JI2673 that changes TGG (W) to TGA (stop) is indicated in bold, cv Melrose in which this mutation arose has the sequence TGG as expected. The deletions from nucleotide 1360 to 1381 in FN3171 and from nucleotide 1510 to 1514 in JI 3062 are highlighted in bold italics. The line JI 3061, which carries the progenitor allele of this mutation, has the same sequence as JI 2822 at that point. The encoded amino acid sequence and other features of note are indicated. Four SNPs identified in the lines PI 193578 PI 198074 and PI 266070 are indicated in bold and ‘in PIs’ (all carry the same SNP with respect to JI2822).
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Complementation of white pea petals by particle bombardment. Particle bombardment of petals of Greenfeast (PI 250447 that carries the G to A splice donor mutation) with [left to right], over-expression cassettes for PhAN1 (AN1), reproduced from
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Alignment of the predicted amino acid sequence of the N-terminal region of bHLH proteins.
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A) Primers used in the identification and characterization of the bHLH gene from pea (Ps) [GU132941] and
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We thank Ian Ferguson, Jo Putterill and William Laing for critical discussions and reading of the manuscript, Javier Paz-Ares for suggesting BAC complementation experiments and K. West, M. Zuccoli and R. Tuberosa for their assistance in finding early references.