The b Gene of Pea Encodes a Defective Flavonoid 3',5'-Hydroxylase, and Confers Pink Flower Color

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Abstract
The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

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The b Gene of Pea Encodes a D efective Flavonoid
39,59-Hydroxylase, and Confers Pink Flower Color
1[W][OA]
Carol Moreau, Mike J. Ambrose, Lynda Turner, Lionel Hill, T.H. Noel Ellis, and Julie M.I. Hofer*
Department of Metabolic Biology (C.M., L.H.) and Department of Crop Genetics (M.J.A., L.T.), John Innes
Centre, Norwich NR4 7UH, United Kingdom; and Institute of Biological, Environmental, and Rural Sciences,
Aberystwyth University, Gogerddan Campus, Aberystwyth, Ceredigion SY23 3EB, United Kingdom (T.H.N.E.,
J.M.I.H.)
The inheritance of ower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes
corresponding to these classical loci remain unidentied. Anthocyanins are the main ower pigments in pea. These are
generated via the avonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher
plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 59 position of the B ring of
avonoid precursors of the anthocyanins suggested to us that the gene encoding avonoid 39,59-hydroxylase (F3959H), the
enzyme that hydroxylates the 59 position of the B ring, was a good candidate for B. In order to test this hypothesis, we
examined mutants generated by fast neutron bombardment. We found allelic pink-owered b mutant lines that carried a
variety of lesions in an F3959H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and
petunidin, the major pigments present in the progenitor purple-owered wild-type pea. These results, combined with the
nding that the F3959H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the
B gene of pea corresponds to a F3959H gene. The molecular characterization of genes involved in pigmentation in pea provides
valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that
lead to variation in pigmentation among legume species.
Flavonoids are a large class of polyphenolic second-
ary metabolites that are involved in pigmentation, de-
fense, fertility, and signaling in plants (Grotewold,
2006). Their basic skeleton consists of two six-carbon
aromatic rings, A and B, connected by ring C, a three-
carbon oxygenated heterocycle. Flavonoids are divided
into different subclasses according to the oxidation state
of the C ring, and compounds within each subclass are
characterized by modications such as hydroxylation,
methylation, glycosylation, and acylation. Anthocya-
nins, for example, the major water-soluble pigments in
owers, have a fully unsaturated C ring and are usually
glycosylated at position 3. Two important determinants
of ower colo r are the cytochrome P450 enzymes
avonoid 39-hydroxylase (F39H; EC 1.14.13.21) and a-
vonoid 39,59-hydroxylase (F3959H; EC 1.14.13.88). These
hydroxylate the B ring of the anthocyanin precursor
molecules naringenin and dihydrokaempferol, generat-
ing substrates for the production of cyanidin-3-glucoside
and delphinidin-3-glucoside, which can be seen in a
variety of pigmented owers (Grotewold, 2006).
The study of genetic loci regulating oral pigmen-
tation has a long histor y, beginning with crosses made
between white- and purple-owered varieties of gar-
den pea (Pi sum sativum; Knight, 1799; Mendel, 1866).
Later cro sses made between white-owered P. sativum
and rose-pink-owered Pisum arvense dened two
factors conferring ower color as A and B, respectively
(Tschermak, 1911). The white owers of pea anthocyanin-
inhibition (a) mutants lack anthocyanins and av ones
(Statham et al., 1972), in accordance with the role of A
as a fundamental factor for pigmentation (Tschermak,
1911; De Haan, 1930). Another locus in pea, a2, similarly
confers a white-owered phenotype lacking anthocya-
nins and other avonoid compounds (Marx et al., 1989).
It was shown that A and A2 regulate the expression of
genes encoding avonoid biosynthetic enzymes (Harker
et al., 1990; Uimari and Strommer, 1998), and recently
they were identied as a basic helix-loop-helix (bHLH)
transcriptionfactorandaWD40repeatprotein,respec-
tively (Hellens et al., 2010). They are likely to be com-
ponents of the Myb-bHLH-WD40 transc ription fact or
complex that regulates avonoid biosynthesis in all plant
species studied so far (Koes et al., 2005; Ramsay and
1
This work was supported by the European Union FP6 Grain Le-
gumes Integrated Project (grant no. FOOD CT20 04506223 to J.M.I.H.)
and b y the Department f or Environment, Food, and Rural Affairs Pulse
Crop Genetic Improvement Network (grant no. AR0711 to C.M., L.T.,
T.H.N.E., and M.J.A.).
* Corresponding author; e-mail jmh18@aber.ac.uk.
The authors responsible for distribution of materials integral to the
ndings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
Julie M. I. Hofer (jmh18@aber.ac.uk) and Mike J. Ambrose (mike.
ambrose@jic.ac.uk).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscrip-
tion.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.197517
Plant Physiology
Ò
, June 2012, Vol. 159, pp. 759768, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved. 759
Page 1
Glover, 2005). The gene encoding the Myb component
of this complex in pea, as well as genes at other loci in-
volved in pigment production, such as Clariroseus (B),
Roseus (Ce), and Fuscopurpureus (Cr; Statham et al., 1972),
remain to be identied.
The major anthocyanins found in wild-type pea
lines that contribute to their purple ower color are
delphinidin-, petunidin-, and malvidin-3-rhamnoside-
5-glucosides (Statham et al., 1972). Rose-pink b mutants
(Blixt, 1972) produce a different range of anthocyanins
(pelargonidin-, cyanidin-, and peonidin-3-rhamnoside-
5-glucosides), suggesting that the B gene controls hy-
droxylation of the anthocyanin B ring (Statham et al.,
1972) and encodes a hydroxylase. Pink-owered mutants
identied in species that are typically purple owered,
such as Petunia 3 hybrida (Snowden and Napoli, 1998;
Matsubara et al., 2005) and Gentiana scabra (Nakatsuka
et al., 2006), were found to have resulted from the in-
sertion of transposable elements into the gene encod-
ing F3959H. If anthocyanin biosynthesis in pea were to
conform to the enzymatic steps elucidated in other
plant species (Grotewold, 2006), then the activity
missing in b mutants would be predicted to corre-
spond to that of a F3959H.
In soybean (Glycine max), however, the wp locus,
which conditions a change in ower color from purple
to pink (Stephens and Nickell, 1992), was reported to
encode a avanone 3-hydroxylase (F3H; EC 1.14.11.9;
Zabala and Vodkin, 2005). Furthermore, an insertion/
deletion mutation in a gene encoding a F3959H was
associated with the white-owered phenotype of the
soybean w1 mutant (Zabala and Vodkin, 2007). These
results suggested that anthocyanin biosynthesis in le-
gumes, or at least in soybean, may differ from that in
other plant species studied, where F3959H mutations
result in pink owers (Snowden and Napoli, 1998;
Matsubara et al., 2005; Nakatsuka et al., 2006) and F3H
mutations result in white
owers (Martin et al., 1991;
Britsch et al., 1992). More recently, a Glycine soja ac-
cession carrying a w1-lp allele was described as having
pale pink ba nner petals and a ower color designated
as light purple (Takahashi et al., 2010). Our analysis
here of the b mutant of pea, which is also a legume,
addresses the complexity of these ndings in soybean.
Transposon-tagged mutations have facilitated the
isolation of genes involved in anthocyanin biosynthesis
in numerous plant species, and transposon tagging is a
useful technology for gene identication that remains
particularly re levant for species without sequenced ge-
nomes, such as pea. Endogenous retrotransposons and
DNA transposons have been identied in pea, but the
transposition rate of those studied to date has been too
low to be exploited for gene tagging (Shirsat, 1988;
Vershinin et al., 2003; Macas et al., 2007). The identi-
cation of active DNA transposons usually occurs when
sectors are found on pigmented owers or seeds. Be-
cause most cultivated pea crop varieties have white
owers, any chance identication of sectored owers
in the eld is extremely limited. A secondary purpose of
this study was to carry out a screen for sectors on
purple-owered peas with the aim of identifying an
active transposon.
We generated pink-owered fast neutr on (FN) de-
letion mutants and used these to identify the gene
corresponding to B. Among the pigmentation mutants
we obtained were several new b alleles, including
pink-sectored mutants, which we charac terized fur-
ther. Stable pink b mutants were shown to carry a
variety of lesions in an F395 9H gene, including com-
plete gene deletions. Analysis of one of these deletion
lines showed that it lacked delphinidin and petunidin,
the major anthocyanins of the progenitor wild-type
pea variety. These results, combined with the nding
that the F3959H gene cosegregates with b in a genetic
mapping population, strongly sup port our hypothesis
that the pea gene b corresponds to a F3959H.
RESULTS
Generation of New b Mutant Alleles
We used FN mutagenesis to generate pigmentation
mutants in line JI 2822, which is wild type at the ower
color loci A, A2, Albicans (Am), B, Ce, and Cr. The fully
open petals of JI 2822 owers are nonuniformly pig-
mented (Fig. 1A); the adaxial standard petal is pale
purple, the two wing petals are dark purple, and the
two fused abaxial keel petals are very lightly pig-
mented. The standard and wing petals fade to a blue
purple. The JI 2822 ower is described here as purple
to conform with previous naming conventions (De
Haan, 1930).
M2 and M3 progeny from the mutagenized po pu-
lation were screened for ower colo r variants that
differed from the wild t ype. Six FN lines were iden-
tied with pale pink standards, rose-pink wing
petals, and lightly pi gmented keel petals (Fig. 1B).
Backcrosses to JI 2822 showed that four of these lines,
FN 1076/6, FN 2160/1, FN 2255/1, and FN 2438/2,
carried stable recessive mutations that determined the
pink ower trait. These lines yielded rose-pink F1
progeny when crossed to the b mutant type line, JI
118, conrming that t hey carried allelic mutations.
Two further lines, FN 2271/3/pink and FN 3398/
2164, were stable rose-pink and allelic to b; however,
sibling individu als carried owers with pink sectors
on a purple background (Fig. 1C), suggesting they
were unstable at the b locus.
The b mutation is also known to confer paler stem
axil pigmentation than the wild type and paler pod
color in genotypes carrying the purple-podded Pur
allele (De Haan, 1930; Statham et al., 1972). All six FN b
alleles likewise differed from JI 2822 in having paler
axillary rings. No effect on pod color was observed in
the FN alleles, because JI 2822 is a green-podded
genotype (pur). The FN b mut ants are described here as
rose pink to incorporate previous conventions
(Tschermak, 1911; De Haan, 1930) yet distinguish them
from cerise-pink ce and crimson-pink cr mutants.
760 Plant Physiol. Vol. 159, 2012
Moreau et al.
Page 2
The b Mutant Lacks Delphinidin and Petunidin
Methanol-HCl extracts of anthocyanins from the wing
petals of line JI 2822 and a stable pink M3 plant, FN
2271/3/pink, were analyzed using liquid chromatogra-
phy (LC) coupled with mass spectroscopy (MS). Chro-
matograms with two major peaks showed that JI 2822
contained two major anthocyanins (Fig. 2A; 611 and 625
atomic mass units [amu]). MS data averaged across the
peaks indicated that these were anthocyanins isomeric to
delphinidin and petunidin glycosylated with deoxyhex-
ose and hexose sugars (Supplemental Fig. S1). Frag-
mentationofthesugarmoietiesasmasslossesof146and
162 amu were consistent with Rha and Glc, respectively.
Fragmentation consistent with the loss of both mono-
saccharide moieties individually was observed, which
suggested that the anthocyanidins delphinidin (303 amu)
and petunidin (317 amu) were monoglycosylated at two
different positions (Supplemental Fig. S1). These results
agree with earlier studies that identied delphinidin-3-
rhamnoside-5-glucoside and petunidin-3-rhamnoside-5-
glucoside among the anthocyanins present in wild-type
pea (Statham et al., 1972).
The peaks indicating glycosylated delphinidin and
petunidin were absent from FN 2271/3/pink samples
(Fig. 2B). A range of ions consistent with glycosylat ed
cyanidin and peonidin were present in FN 2271/3/pink
and ab sent from JI 2822 (Fig. 2, C and D). These were
isomeric to cyanidin glycosylated with deoxyhexose and
hexose sugars (595 amu), peonidin glycosylated with
deoxyhexose and hexo se sugars (609 amu), and cyani-
din glycosylated with a pento se and two hexose sugars
(743 amu; Fig. 2C). Fragmentation of the sugars attached
to cyanidin (287 amu) as mass losses of 162 , 294, and
456 amu was consistent with a pentose moiety buried
beneath a Glc moiety (Supplemental Fig. S1). No single
loss of 132 amu, expected of an exposed pentose, was
observed. These re sults conrmed earlier studies that
identied cyanidin-3-sambubioside-5-glucoside among
the anthocyanins present in b mutants (Statham et al.,
1972). Fragmentation of the sugars attached to cyanidin
andpeonidin(301amu)asmasslossesof146and162
amu was consistent with cyanidin-3-rhamnoside-5-
glucoside and peonidin-3-rhamnoside-5-glucoside, also
previously identied in b mutants (Statham et al., 1972).
The conversion of cyanidin and peonidin to del-
phinidin and petunidin requires hydroxylation at the
59 position of the B ring of the precursor avonoids.
Because the products of this conversion were not ob-
served in b mutants, it was presumed that the B gene
controls the hydroxylation of the anthocyanin B ring
(Statham et al., 1972). Our studies conrmed this
conclusion and suggested to us that the gene encoding
F3959H was a good candidate for B.
Isolation of a Pea F3959H Gene from a Purple-Flowered
Wild-Type Plant
We performed PCR on cDNA derived from JI 2822
wing petals using primers based on aligned Medicago
truncatula and soybean F3959H sequences. This yielded
a product encoding a partial open reading frame (ORF)
with extensive sequence similarity to F3959H. We used
primers based on this new pea sequence together with
primers based on the Medicago sequence for adaptor-
ligation PCR (Spertini et al., 1999), which enabled us to
isolate genomic DNA sequences and a larger cDNA
product including a TAG stop codon. Amplication
and sequencing of a single PCR product, using primers
at the 59 and 39 ends of the surmised contig, conrmed
that a 1,548-bp cDNA encoded a cytochrome P450
monooxygenase 515 amino acids long.
A BLASTP search of Medicago genome pseudomo-
lecules (version 3.5) using the chromosome visualiza-
tion tool CViT (http://www.medicagohapmap.org)
identied CU651565_9 on bacterial articial chromo-
some (B AC) CU651565, a F3959H 515 amino acids in
length, as the most similar sequence, with 89% iden-
tity. The predicted pea protein sequence is 79%, 78%,
and 75% identical to predicted full-length F3959H se-
quences from lotus (Lotus japoni cus; LjT34E09.40),
soybean (AAM51564, ABQ96218, and BAJ14024), and
buttery pea (Clitoria ternatea ; BAF49293), respectively.
The soybe an sequences are classied as CYP75A17
cytochrome P450s (Nelson, 2009). The Arabidopsis
(Arabidopsis thaliana) sequence most closely related to
the pea F3959H (48% identity) is the cytochrome P450
monooxygenase CYP75B1, encoded by TRANSPAR-
ENT TESTA7 (At5g07990; GenBank accession no.
NP196416). This 513-amino acid protein has been
demonstrated to have F39H activity (Schoenbohm
et al., 2000), and it lies within a separate clade when
compared with other plant F39 5 9H sequences (Fig. 3).
Figure 1. Pea b mutant phenotypes. A,
Purple-flowered wild-type line JI 2822.
B, Rose-pink-flowered b mutant line
FN 2271/3/pink. C, Unstable b mutant
line FN 2271/3/flecked with rose-pink
sectors on a purple background.
Plant Physiol. Vol. 159, 2012 761
The b Gene in Pea
Page 3
A 3,231-bp genomic DNA sequence was obtained
from PCR products amplied from JI 2822 DNA using
primers spanning the cDNA sequence and adaptor-
ligation PCR products corresponding to the promoter
and 3 9 untranslated region (GenBank accession no.
GU596479). The position of a single 530-bp intron, 915
bp downstream of the ATG start codon, was deter-
mined by alignment of the genomic DNA and cDNA
sequences. A single intron is predicted in Medicago
CU651565_9 at the same position, but in other legumes,
such as soybean (Zabala and Vodkin, 2007) and lotus
(LjT34E09.40), two introns are reported or annotated. In
both these species, the position of the predicted second
intron is coincident with the position of the pea intron.
The rs t introns are predicted in different positions, 331
and 348 bp downstream of their ATG, for lotus and
soybean, respectively.
Genetic Mapping of F3959H Reveals Cosegregation with b
A cleaved-amplied polymorphic sequence (CAPS)
marker for F3959H that distinguished the JI 15 and JI
73 alleles was generated by TaqI cleavage of the PCR
products ampli ed from genomic DNA. Cosegregation
of the CAPS marker with b was tested directly in a JI 15 3
JI 73 recombinant inbred population of 169 individuals,
because JI 73 carries the recessive b allele. JI 73 also carries
k, the homeotic conversion of wing petals to keel petals,
and d, the absence of pigmentation in foliage axils,
whereas JI 15 carries ce, an independent crimson-pink
ower trait. The b, ce double mutant is almost white,
sosingleanddoublemutantscanbedistinguishedeasily,
except in a k mutant background, where only the pale
standard petal gives a clue to ower color. The genotypes
Figure 2. LC-MS analysis of anthocyanins present in the wild type and
b mutant lines. A, Extracted ion chromatograms showing the summed
intensities of ions with masses corresponding to delphinidin and
petunidin, each glycosylated with Rha and Glc, present in JI 2822.
These masses are m/z = 611 (delphinin) and m/z = 625 (petunin). B,
Masses corresponding to delphinin and petunin absent from FN 2271/
3/pink. A and B are plotted to the same scale. C, Extracted ion chro-
matograms showing the summed intensities of three alternative an-
thocyanin ions, with masses based on glycosylated cyanidin (m/z =
743, m/z = 595) and peonidin (m/z = 609), present in line FN 2271/3/
pink. D, Masses corresponding to cyanin and peonin absent from JI
2822. C and D are plotted to the same scale. Chromatographic peaks
are annotated with m/z of the mass responsible for the peak.
Figure 3. Phylogenetic analysis of cytochrome P450 sequences. The
optimal neighbor-joining tree derived from the multiple sequence
alignment in Supplemental Figure S2 is drawn to scale, with the sum of
branch lengths = 4.7. The Jones-Taylor-Thornton amino acid substitution
model was used in phylogeny construction, and the scale bar indicates
the number of amino acid substitutions per site. Percentage support
for 1,000 bootstrap replicates is shown at the branch points. Labeled
lines show GenBank accession numbers as follows: LjT34E09_40,
L. japonicus; BAJ14024, soybean; BAF49293, C. ternatea;ADW66160,
P. sativum; CU651565_9, M. truncatula;ABH06585,Vitis vinifera;
BAE86871, G. scabra;P48418,Petunia 3 hybrida; CU651565_21, M.
truncatula; NP_001064333, Oryza sativa; NP196416, Arabidopsis;
ABH06586, V. vinifera; BAB83261, soybean; NP182079, Arabidopsis;
NP775426, Rattus norvegicus.
762 Plant Physiol. Vol. 159, 2012
Moreau et al.
Page 4
at b and ce are particularly difcult to distinguish in a k, d
background, where axillary pigmentation is also absent.
For these reasons, the cosegregation analysis was re-
stricted to a subset of 160 of the 169 recombinant inbred
lines. The b phenotype cosegregated exactly with the JI 73
F3959H CAPS marker lacking a TaqI restriction enzyme
site (b:B = 71:89; x
2
=2.0,notsignicant), consistent with
our hypothesis that this F3959H identi es a single gene
that corresponds to B.
Identication of Lesions in F3959H Alleles from
Pink-Flowered b Mutants
In order to provide further evidence of a corre-
spondence between the pea gene encoding F3959H and
B, we sequenced alleles from known mutants. The b
mutant type line, JI 118, carries a single nucleotide
polymorphism 332 bp downstream of the ATG. This
G/A transition would result in a single amino acid
change, G111E (Supplementa l Figs. S2 and S3). Line JI
73, the b mapping parent used above, carries a 23-bp
deletion in the ORF, 291 bp from the ATG start. This
deletion would introduce a change in the reading
frame at position 98, resulting in the inclusion of 29
residues unrelated to the wild type followed by a
premature stop codon (Supplemental Fig. S3). PCR
analysis using primers that spann ed the F3959H gene
showed that lines FN 2160/1, FN 2255/1, and FN
2438/2 as well as the stable pink line FN 2271/3/pink
all carry complete gene deletions (Supplemental Fig.
S4). FN 1076/6 contains a genomic rearrangement that
is consistent with a reciprocal break and join between
the F3959H gene and a predicted Ogre retroelement
(Neumann et al., 2003). The 59 segmentoftheOgre el-
ement lies 1,330 bp downstream of the F3959H start
codon, whereas the 39 segment lies upstream of position
1,330 at the 39 end of the F3959H gene (Supplemental
Fig. S4).
Characterization of an Unstable Pink-Sectored b Mutant
Unstable b mutants occurred in the M3 families FN
2271/3/ecked (Fig. 1C) and FN 3398/2164. It was
found that sectored pink M3 siblings gave rise to sec-
tored or stab le pink M4 progeny, whereas stable pink
M3 plants gave rise to stable pink M4 progeny only.
Wild-type purple M3 siblings gave rise to either stable
wild type, or a mix of stable wild type and stable pink,
or a mix of stable wild type, stable pink, and sectored
pink M4 progeny. Sectored pink M4 progeny gave rise
to sectored or stable pink M5 plants in the follo wing
generation. In order to study this instability further,
PCR analysis was carried out on individual owers
and progeny plants of line FN 2271/3/ecked/8.
Primers 39pinkS1 and 39pinkS2comp amplied 693
bp of genomic DNA and reporte d on exon 1 and the
intron of the F3959H gene. Primers 39pinkS2 and 39extR
amplied 683 bp of genomic DNA or cDNA and
reported on exon 2. Both pairs of primers were used in
conjunction with control primers designed to a pea
Argonaute gene, which veried that PCR amplication
had occurred, even in the absence of a F3959H PCR
product. Genomic DNA and cDNA were prepared
from the purple petals of a JI 2822 wild-type ower
and from the petals of an entirely pink ower on a FN
2271/3/ecked/8 plant that carried purple/pink-
sectored owers at other nodes. PCR using primers
39pinkS2 and 39extR showed the presence of the
F3959H gene in JI 2822 and pink ower FN 2271/3/
ecked/8 genomic DNA samples; however, cDNA
amplication occurred in line JI 2822 only, sugges ting
that the F3959H gene was present but not expressed in
the entirely pink FN 2271/3/e cked/8 ower (Fig. 4).
Stable pink-owered M4 progeny w ere grown from
seed set on that entirely pink FN 2271/3/
ecked/8
ower. When these were analyzed by PCR, exon
1andexon2ofF3959H failed to amplify from genomic
DNA, suggesting that the gene was del eted in these
progeny, as was observed previously in the stable
pink-owered line FN 2271/3/pink.
DISCUSSION
The early part of anthocyanin biosynthesis from
chalcone to anthocyanidin is well conserved in higher
plants and has been studied in detail (Grotewold,
2006). One of the key enzymes responsible for blue-
purple coloration in ower petals is F3959H, which
catalyzes hydroxylation at the 39 and 59 positions of
the B ring of naringenin and dihydrokaempferol,
yielding avanone and dihydroavonol precursors
of the chromophore delphinidin (Grotewold, 2006;
Yoshida et al., 2009). Flowers that lack this enzyme,
Figure 4. F3959H gene expression in unstable b mutant line FN 2271/
3/flecked. PCR amplification of F3959H and Argonaute (Ago) genes
from JI 2822 genomic DNA (lane 1), JI 2822 cDNA (lane 2), FN 2271/
3/flecked/8 genomic DNA (lane 3), FN 2271/3/flecked/8 cDNA (lane
4), and no-DNA control (lane 5) is shown. Lane 6 shows 100-bp
markers. The top Ago band represents PCR amplification products
spanning two introns from genomic DNA, and the bottom Ago band
represents PCR amplification products (without introns) from cDNA.
The F3959H primers do not flank an intron; therefore, F3959H PCR
products from genomic DNA and cDNA are the same size.
Plant Physiol. Vol. 159, 2012 763
The b Gene in Pea
Page 5
such as rose (Rosa hybrid) and carnation (Dianthus
caryophyllus), contain only cyanidin and/or pelargo-
nidin chromophores, so their natural coloration is re-
stricted to yellow, pink, and red but not purple or blue.
Flower color also can be affected by pH, the presence
of copigments, and whether the anthocyanidin chro-
mophores are polyacetylated or held in metal com-
plexes (Yoshida et al., 2009). For example, hydrangea
(Hydrangea macrophylla) sepals can be red, mauve,
purple, violet, or blue, yet only one anthocyanin, del-
phinidin 3-glucoside, is present. It has been proposed
that the anthocyanin and copigments in hydrangea
sepals are held in a metal complex and that color de-
pends on the concentrations of these components and
the pH conditions (Kondo et al., 2005). In wild-type
pea, the F3959H gene is intact and F3959H activity
produces delphinidin-based anthocyanidins, which
confer a purple ower color. In this paper, we have
presented genetic and biochemical evidence to show
that b mutants lack a functional F3959H gene that re-
sults in a rose-pink ower color due to the presence
of cyanidin- and peonidin-based anthocyanins. The
presence of the se latter 39-hydroxyla ted compounds in
b mutants suggests that a F39H exists in pea, contrary
to previous conclusions (Statham et al., 1972).
Lesions Present in F3959H Alleles
Plant P450 monooxygenases have not been charac-
terized structurally because they are extremely insol-
uble when puried; however, membrane-associated
mammalian P450s have been studied by homology to
the crystal structure of a soluble bacterial P450 (Ferrer
et al., 2008). P450s have only three absolutely con-
served residues: a Cys that serves as a ligand to the
heme iron, and an EXXR motif that is thought to sta-
bilize the core around the heme (Werck-Reichhart and
Feyereisen, 2000). The Cys lies within the P450 con-
sensus sequence FXXGXRXCXG in the heme-binding
loop, corresponding to FGAGRRICAG in the pea
F3959H (Supplemental Fig. S2). Another consensus se-
quence, A/GGXD/ETT/S, corresponds to a proton-
transfer groove, and this corresponds to AGTDTS in
the pea F3959H (Supplemental Fig. S2). The G111E
mutation in the b type line, JI 118, does not occur in
these conserved motifs, but the change in size and
charge at this residue presumably affects protein
function. Alignm ent of the pea F3959H sequence with
homologous plant proteins (National Ce nter for Bio-
technology Information BLASTP) shows that substi-
tutions occur at the G111 residue; however, none of the
substitutes are charged residues, supporting our pro-
posal that G111E is a detrimental change.
Line JI 73 carries a b allele with a spontaneous 26-bp
deletion that is predicted to encode a truncated version
of the F3959H protein. At the 39 end of the 26-bp de-
leted sequence, there is a 10-bp motif, ATTTCTCAAA,
that is repeated at the 59 end of the deletion break
point (Supplemental Fig. S3). This repeat pattern sug-
gests that this stable b allele may have arisen from a
spontaneous deletion event as a result of recombina-
tion and unequal crossing over. The same 26-bp dele-
tion was found in lines JI 17, JI 132, and JI 2160 in the
John Innes Pisum germplasm collection.
A genomic rearrangement consistent with a trans-
location event involving a retroelemen t was evident in
line FN 1076/6. Here, sequencing showed that a break
occurred in the F3959H gene, between nucleotides 1,329
and 1,330 downstream of the ATG, but we do not know
whether the two fragmented portions of the F3959H gene
remain on the same chromosome (Supplemental Fig. S4).
The 59 end of the geni c disjunction was 95% identical
to nucleotides 77,728 to 78,111 of a Ty3-gypsy Ogre-
like retroelement (Neumann et al., 2003) identied in
pea BAC clone JICPSV-297I9, whereas the sequence at
the 39 end of the disjunction was 95% identical to nu-
cleotides 77,213 to 77,726 of the same retroelement.
This indicates that a brea k occurred in the Ogre ele-
ment between nucleotides 77,726 and 77,728 and that
nucleotide 77,727 was missing from this copy of Ogre
or was lost during the rearrangement. The presence
of this retroelement does not necessarily implicate it in
the mechanism of translocation but more likely reects
the abundance of the Ogre retroelemen t family. Data
from 454 sequencing of cv Carerra estimated that
copies of Ogre represent up to 33% of the pea genome
(Macas and Neumann, 2007).
We gathered evidence of independent, recurring,
spontaneous deletion events derived from unstable b
alleles carried by lines FN 2271/3/ecked and FN
3398/2164. These sectored owers carried an F3959H
gene, presumably in nonepidermal tissue where it is
not expressed, but repeatedly gave rise to stable pink
deletion alleles in their progeny (Fig. 4). One possible
explanation of these unstable b alleles is that FN 2271
M1 seed carried both a deletion of the b gene and a
rearrangement of the chromosome carrying the wild-
type B allele. This rearranged chromosome would be
prone to the generation of acentric fragments that
would fail to segregate properly at mitosis, generating
sectors with a haploinsufciency for many loci, in-
cluding b. Individuals with the unstable phenotype
would give rise to pink homozygous deletion progeny
(with a wild-type karyotype). They would also gen-
erate progeny that are homozygous or heterozygous
for the unstable chromosome, but the transmission of
this unstable chromosome may be inefcient, or those
that are transmitted efciently may be selected for
stability. In this scheme, the pink-owered FN 2271
mutants derive from a simple deletion segregating in
the population and the instability is not specically
associated with the b locus.
Alternatively, the unstable alleles at the b locus in the
FN 2271 lineage may be prone to deletion, perhaps be-
cause of the action of a near by transposon activated in
the FN mutagenesis. Deletion of the b gene at one allele
would be masked by the presence of the other, wild-type
B allele, but the presence of such a deletion would reveal
subsequent deletions of the B allele, which would be
seen as pink sectors. In this scheme, deletion of b is not
764 Plant Physiol. Vol. 159, 2012
Moreau et al.
Page 6
generated directly by mutagenesis and the instab ility is
associated specically with the b gene. Pink owers of
this type could be indicators of a captured insertion el-
ement, but in no case did we nd a stable pink mutant
with the F3959H gene detectably present, even when
these derived from seed set from an entirely pink owe r
on an unstable plant where the gene, but not the tran-
script, had been detected by PCR.
F3959H Homologs in Legumes
Cytochrome P450s are one of the largest enzyme
families in plants. A search of annotated Medicago
pseudomolecules (http://www.medicagohapmap.org)
reveals 142 F3959H homologs (BLASTP, P . 1e-40), with
approximately one-third of these located on chro-
mosome 5. Gene clusters are found in man y other
organisms, and in Medicago, BACs containing ve or
more homologous ORFs occurred on chromosomes 2
(AC130800), 3 (AC145061), 5 (FP102223 and AC137079),
and 6 (AC157489), although some of t hese may be
pseudogenes. The soybean genome contains 712 cyto-
chrome P450s, of which 380 are denoted pseudogenes
(Nelson, 2009). Medicago BAC CU651565 carrying
CU651565_9, the most similar intact ORF to pea F3959H,
is unanchored in version 3.5 of the Medicago genome
pseudomolecules; therefore, we were unable to gain
any further evidence of orthology by analyzing collin-
earity with b gene-anking markers. In the previous
version of annotated Medicago pseudomolecules (ver-
sion 3.0), BAC CU651565 was located on chromosome
3, which is syntenic with pea linkage group III, where b
maps.
Another predicted Medicago F3959H gene, CU651565_21
(Fig. 3), lies only 52 kb from CU651565_9. The coding
sequence of CU651565_21 corresponds to a protein
522 amino acids in length, which is anomalous com-
pared with the lengths of related F3959Hsequences
(Supplemental Fig. S2). Multiple sequence alignment
(Supplemental Fig. S2) suggests that CU651565_21
may in fact correspond to a 506-amino acid protein
that would be 63% identical to CU651565_9 and 62%
identical to the pe a F3959H. An alternative intron-
splicingmodelderivedfromORFsannotatedin
Medicago pse udomolecu le version 3.0 is presente d
(Supplemental Fig. S5).
It is not clear whether the closest related lotus and
soybean sequences are orthologous to the pea F3959H,
because they have two introns; therefore, they are
structurally dissimilar to the pea and Medicago genes.
The Petunia 3 hybrida F3959H also has two introns,
whereas the G. scabra F3959H has one, indicating that
intron number is a variable feature of these genes.
Diversity of exon-intron structure has been noted
among genes encoding P450 enzymes, with multiple
gains and losses in their evolutionary history (Werck-
Reichhart and Feyereisen, 2000).
The amino acid sequence of CU651565_9, 89% iden-
tical to pea F3959H, is the closest match; however, the
yellow (rather than purple/blue) pigmented owers of
M. truncatula suggest that there are differences in an-
thocyanin biosynthesis between these two species. All
of the conserved P450 motifs are intact in CU651565_9,
but a comparison with homologous sequences from
other plant species shows differences that may be sig-
nicant. For example, residue Phe-350, which is Leu or
Val in aligned homologs (Supplemental Fig. S2), may
disrupt F3959HfunctioninM. truncatula.Insupportof
this possibility, overexpression of the Myb transcription
factor LAP1 in M. truncatula induced anthocyanin pig-
ments, which were identied as glycosylated cyanidins
and pelargonidins but not delphinidins (Peel et al.,
2009). The absence of glycosylated delphinidins in these
transgenic plants suggests a defect in F3959Hactivity,
especially because glycosylated delphinidins were ob-
served in white clover (Trifolium repens) overexpressing
LAP1 (Peel et al., 2009).
Three soybean sequences (AAM51564, ABQ96218,
and BAJ14024) are all 78% identical to pea F3959H;
however, they are themselves nonidentical. ABQ96218
(Zabala and Vodkin, 2007) and AAM51564 (from cv
Chin-Ren-Woo-Dou) are 99% identical and 509 and
508 amino acids long, respectively. They encode a
CYP2 subfamily cytochrome P450, also classied as
a CYP75A17 cytochrome P450 (Nelson, 2009), at lo-
cus Glyma13g04210 on linkage group F of soybean
(http://soybase.org). ABQ96218, originating from cv
Lee 68 and cloned from the Williams isoline L79-908,
carries a G305D amino acid substitution (Zab ala and
Vodkin, 2007) in the conserved P450 proton-transfer
groove motif that would likel y render this a llele
nonfunctional (Supplemental Fig. S2). BAJ14024
(Takahashi et al., 2010) is a predicted F3959Hfrom
soybean cv Clark, 509 amino acids long, with in-
variant conserved motifs and 99% identical to both
ABQ96218 and AAM51564.
Flower Pigmentation in Pea and Soybean
Soybean is believed to have been domesticated from
purple-owered G. soja (Takahashi et al., 2010). Studies
of the standard (banner) petals of purple-owered
soybean cultivars show that these have a different
sugar moiety at the 3 position of the C ring of their
anthocyanidins compared with pea: the primary an-
thocyanins detected in soybean cv Clark (W1W1 w3w3
W4W4 WmWm TT TdTd) and cv Harosoy (W1W1
w3w3 W4W4 WmWm tt TdTd) were malvidin, delphi-
nidin, and petunidin 3,5-di-O-glucoside and delphini-
din 3-O-glucoside (Iwashina et al., 2008), whereas
delphinidin and petunidin-3-rhamnoside-5-glucoside
were the major anthocyanins found in the wing petals
of pea line JI 2822 in this study, consistent with pre-
vious studies on line L 60 of pea (Statham et al., 1972).
As the intensity of coloration in pea petals indicates
(Fig. 1), the concentration of total anthocyanins in
standard petals is less than in wing petals of pea at all
stages of ower development (Statham and Crowden,
1974), whereas soybean owers often have wing petals
Plant Physiol. Vol. 159, 2012 765
The b Gene in Pea
Page 7
that are less intensely pigmented than their standard
petals.
The Wp gene of soybean lies on linkage group D1b,
corresponding to chromosome 2 (http://soybase.org).
The wp allele is reported to contain a 5,722-bp CACTA
transposable element in intron 2 of a F3H gene, F3H1,
with down-regulated expression (Zabala and Vodkin,
2005). A null mutation would result in a lack of the
substrates dihydromyricetin, dihydrokaempferol, and
dihydroquercetin required for conversion into antho-
cyanins (Grotewold, 2006; Iwashina et al., 2008);
therefore, a null mutant would be expected to have
white owers and, indeed, white-owered mutants
have been observed in other plant species (Martin
et al., 1991; Britsch et al., 1992). Analysis of a wp ge-
notype obtained by back-crossing to soybean cv Loda
showed that the wp line had a low avonoid content:
9% of the total avonol glycosides, no detectable
kaempferol 3-O-glucoside, and 28% of dihydro-
avonols compared with cv Clark (Iwashina et al.,
2008). The presence of dihydroavonols indicates that
F3H activity occurs in the wp mutant, suggesting that it
is not a null allele. Alternatively, if the CACTA ele-
ment insertion does render F3H1 null, a second F3H
gene, F3H2, may be functional (Zabala and Vodkin,
2005).
Although the presence of anthocyanins in the wp
mutant can be explained by the considerations above,
the pale pink coloration (instead of pale purple) re-
mains unexplained. Many factors such as copigments
and vacuolar pH could inuence soybean ower color,
but the presence of an additi onal defective pigmenta-
tion gene, such as the ABQ96218 allele of F3959H, for
example, would also c ause pink ower color. A com-
parison of ower color and avonoid content in
available Wp and wp near-isogenic lines (Iwashina
et al., 2008) and cosegregation analysis of F3H1 and wp
would help to conrm which structural genes were
defective.
The soybean w1 gene on chromosome 13 confers
white ower color; accord ingly, no HPLC peaks cor-
responding to anthocyanins were observed in a Clark-
w1 near-isogenic line (L63-2373, w1w1
, w3w3, W4W4,
WmWm, TT, TdTd; Iwashina et al., 2007). However, it is
not clear why a w1 encoding a defective F3959H gene
would condition white ower color in soybean, when
the pea b mutant and other F3959H mutants derived
from purple-owered wild-type plants (Snowden and
Napoli, 1998; Matsubara et al., 2005; Nakatsuka et al.,
2006) have pink owers. Genetic linkage analysis of an
F2 population segregating for w1 showed that 12
white-owered individuals out of 39 F2 progeny car-
ried an F3959H allele containing a tandem repeat in-
sertion that would result in premature termination of
the protein (Zabala and Vodkin, 2007). This linkage
evidence is consistent with w1 being less than 1.1
centimorgan (Kosambi, 1944; Allard, 1956) from the
tandem repeat-containing F3959H gene but with a high
SE: the F3959H homozygotes in the purple ower class
were not shown to be W1 homozygotes by progeny
testing, and the population size is small. Thus, it is not
clear that a mutated F3959H gene conditions white
ower colo r in soybean.
One possibility is that w1 is a separate nonfunctional
pigmentation locus, distinct from, but tightly linked to,
the F3959H gene. This w1 locus is predicted to be
functional in a G. soja line carrying the w1-lp allele ,
which has pale pink banner petals (Takahashi et al.,
2010), and nonfunctional in Clark-w1. A cross between
these two lines produced purple-owered F2 progeny
at a frequency of 0.9% (Takahashi et al., 2010), which is
consistent with recombination between a distinct w1
gene and the F3959H gene. Soybean orthologs of genes
encoding components of the Myb-bHLH-WD40 tran-
scription factor complex that regulate s anthocyanin
biosynthesis (Koes et al., 2005; Ramsay and Glover,
2005), such as a and a2 (Hellens et al., 2010), have not
yet been identied. These are good candidates for the
proposed F3959H-adjacent w1 gene.
Pigmentation loci in pea, which have been studied in
crosses for more than 100 years (Mendel, 1866;
Tschermak, 1911), represent historic anchor markers
that will aid comparative genomics between legume
species as more physical maps are generated from
sequenced genomes. Further biochemical studies,
combined with genetic and genomic analyses, will
help to elucidate the differences in anth ocyanin bio-
synthesis that lead to variation in pigmentation a mong
legume crop species such as soybean as well as im-
portant legume forage species such as alfalfa (Medicago
sativa) and clover.
MATERIALS AND METHODS
Plant Material
The garden pea (Pisum sativum) type line for b, JI 118, also known as WBH
22 (Blixt, 1972), multiple marker line JI 73 (genotype b, also known as WBH
1238), multiple marker line JI 15 (genotype B, also known as WBH 1458), F13
recombinant inbred mapping population JI 15 3 JI 73, and all FN mutant lines
were obtained from the John Innes Pisum Germplasm collection. Plants were
grown in 16-h daylength in John Innes No. 1 compost with 30% extra grit.
DNA was prepared from leaves according to Vershinin et al. (2003), and RNA
was prepared from owers according to Hofer et al. (2009).
Mutagenesis
A total of 1,400 seeds of line JI 2822 were subjected to 20 Gray FN irradiation
from a
252
Cf source at Oak Ridge National Laboratory. Irradiated M1 plants
were self fertilized, and M2 families of up to four plants were screened for
variant ower color phenot ypes. Rose-pink mutants were backcrossed to JI
2822 to generate lines FN 1076/6, FN 2160/1, FN 2255/1, FN 2438/2, FN
2271/3/pink, and FN 3398/2164. These stable pink lines segregated purple:
pink in a 3:1 ratio after backcrossing, indicating that the pigme ntation muta-
tions were recessive.
LC-MS
Purple (JI 2822) and pink (FN 2271/3/pink) wing petal tissue was harvested from
10 fully open owers, ground in liquid N
2
, and stored in methanol at 220°C. Sample
aliquots of 10 mLcontaining300mgoftissueinmethanoland0.1
M HCl were
analyzed by LC-MS using a Surveyor HPLC apparatus attached to a DecaXPplus
ion-trap mass spectrometer (Thermo Fisher). Anthocyanins were separated on a
766 Plant Physiol. Vol. 159, 2012
Moreau et al.
Page 8
100- 3 2-mm, 3-mm Luna C18(2) column (Phenomenex) using the following gra-
dient of methanol (solvent B) versus 2 m
M triuoroacetic acid in water (solvent A),
run at 230 mLmin
21
and 30°C: 0 min, 2% B; 40 min, 70% B; 41 min, 2% B; 50 min,
2% B. Anthocyanins were detected by UV A
520
and by positive electrospray ioni-
zation MS. Spray chamber conditions were 50 units of sheath gas, 5 units of aux-
iliary gas, 350°C capillary temperature, and 5.2-kV spray voltage. In order to
investigate the structure of anthocyanins, data-dependent secondary fragmentation
(MS2) spectra were collected at an isolation width of mass-to-charge ratio (m/z)=4.0
and 35% collision energy.
Isolation of Pea F3959H cDNA and Genomic DNA
Total RNA was extracted from JI 2822 wing petals using the Qiagen RNeasy
Plant Mini kit. DNA was removed from RNA samples by digestion with DNA-
free DNaseI (Ambion) in buffers according to the manufacturers protocol. Two
micrograms of RNA was reverse transcribed with SuperScript reverse tran-
scriptase (Invitrogen) from an oligo(T) primer in a 20-mL reaction. Ampli-
cation of a F3959H cDNA fragment from pea was achieved using 1 mL of 1:20
diluted rst-strand cDNA in 20-mL PCRs containing 0.25 m
M primers
mtF35HF1 and mtF35HR2 (Supplemental Table S1) for 35 cycles with an
annealing temperature of 62°C. Products were separated by electrophoresis on
a 1% agarose gel in 13 Tris-borate/EDTA buffer. A 794-bp sequence obtained
from this fragment was used to design additional primers for the amplication
of 3,231-bp genomic DNA using successive rounds of adaptor ligation PCR
(Spertini et al., 1999). The genomic DNA sequence was used to design primers
pinkmtF1 and 39extR for the amplication of a 1,595-bp cDNA clone, minus
the ATG start codon and extending 50 bp beyond the TAG stop codon. This
was cloned into a Topo4 vector (Invitrogen).
Mutation Analysis
Genomic DNA from JI 2822 and FN mutant lines was analyzed using pairs
of primers that spanned the F3959H gene sequence in order to determine the
size of deletion alleles (Supplemental Table S1). Primers PsAGO1 and
PsAGO2, anking introns 19, 20, and 21 of a pea Argonaute1 cDNA clone
(accession no. EF108450), were included in the reactions as internal controls.
For the analysis of unstable lines, wing petal cDNA and genomic DNA from JI
2822, plant FN 2271/3/ecked/8, and its progeny were analy zed. Touch-
down PCR was performed using 250 n
M primers 39pinkS2 and 39extR, 250 mM
deoxyribonucleotide triphosphates, and 1 unit of Taq polymerase in a 10-mL
volume of PCR buffer. Primers PsAGO1 and PsAGO2 were included in the
reactions as internal controls. Components were denatured at 95°C for 180 s,
before being subjected to one cycle of 94°C for 45 s, 62°C for 45 s, and 72°C for
90 s, followed by 10 further cycles with the annealing temperature 1°C lower
at each cycle. Twenty-nine further cycles of 94°C for 45 s, 50°C for 45 s, and 72°
C for 90 s were terminated at 72°C for 300 s. Reactions were held at 10°C for
300 s prior to analysis by agarose gel electrophoresis (Supplemental Fig. S4;
Supplemental Table S1).
Genetic Mapping
A CAPS marker for F3959H was generated by TaqI cleavage of the 363- and
340-bp PCR products amplied from 100 ng of genomic DNA from parental
lines JI 15 and JI 73, respectively, using primers pinkmtF1 and psf35hF2comp.
Reactions contained 250 n
M primers, 250 mM deoxyribonucleotide triphos-
phates, and 1 unit of Taq polymerase in a 20-mL volume of PCR buffer.
Components were denatured at 94°C for 120 s, cycled through 94°C for 30 s,
55°C for 60 s, and 72°C for 120 s for 35 cycles, and nally incubated at 72°C for
5 min. Cleavage products of 293 bp from line JI 15 and 340 bp from line JI 73
were separated on a 2% agarose gel. Cosegregation of b with the 340-bp
F3959H CAPS marker was tested for 160 lines out of 169 in total at the F13
generation of a recombinant inbred population derived from the cross JI 15 3
JI 73. A total of 71 lines were b/b and carried the 340-bp marker, and 89 in-
dividuals were B and carried the 293-bp marker.
Sequencing
Sequencing was performed using the BigDye Terminator version 3.1 cycle
sequencing kit (Applied Biosystems) at the John Innes Centre Genome Lab-
oratory. Genomic DNA sequence was obtained from line JI 2822 using the
primers listed in Supplemental Table S1. A 3,232-bp overlapping DNA se-
quence contig was generated using the program BioEdit (http://www.mbi o.
ncsu.edu/bioedit/bioedit.html). Overlapping DNA sequence contigs from b
mutant lines JI 118, JI 73, and FN 1076/6 and cDNA sequences from lines JI
2822, JI 118, JI 73, and FN 1076/6 were obtained in the same way.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: JI 2822 F395 9H cDNA se-
quence, GU596478; JI 2822 F3959H genomic DNA sequence, GU596479.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Ion fragmentation analysis of anthocyanins pres-
ent in the wild type and b mutant lines.
Supplemental Figure S2.F3959H sequence analysis.
Supplemental Figure S3. Sequence characterization of mutant b alleles.
Supplemental Figure S4. Characterization of mutant b alleles by PCR.
Supplemental Figure S5. Proposed splicing model for Medicago gene
CU651565_21.
Supplemental Table S1. Primers used for PCR and sequencing.
ACKNOWLEDGMENTS
We thank Andrew Davis for photography, Ruth Pothecary and Hilary Ford
for plant care, and a Nufeld scholarship student, Priyanka Tharian, for
technical assistance.
Received March 22, 2012; accepted April 3, 2012; published April 6, 2012.
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    • "Human selection for lighter seed color during crop domestication is well documented in pea (Hellens et al., 2010) and sorghum (Wu et al., 2012). Molecular genetic analyses of seed and flower color in soybean and garden pea (Hellens et al., 2010; Yang et al., 2010; Moreau et al., 2012) implicate genes in the phenylpropanoid pathway (Winkel-Shirley, 2001; Grotewold, 2006), in which a bifurcation typically leads either to the production of condensed tannins (proanthocyanidins) in seeds, giving them their dark appearance, or to the brightly colored anthocyanins characteristic of flowers. Both the biosynthetic enzymes and their transcriptional regulators have been well characterized in several plants, notably Arabidopsis, maize and Medicago, the latter of which is a close relative of chickpea (Xie et al., 2003; Baudry et al., 2004; Broun, 2005; Appelhagen et al., 2011; Dixon et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Chickpea (Cicer arietinum) is among the founder crops domesticated in the Fertile Crescent. One of two major forms of chickpea, the so-called kabuli type, has white flowers and light-colored seed coats, properties not known to exist in the wild progenitor. The origin of the kabuli form has been enigmatic. We genotyped a collection of wild and cultivated chickpea genotypes with 538 single nucleotide polymorphisms (SNPs) and examined patterns of molecular diversity relative to geographical sources and market types. In addition, we examined sequence and expression variation in candidate anthocyanin biosynthetic pathway genes. A reduction in genetic diversity and extensive genetic admixture distinguish cultivated chickpea from its wild progenitor species. Among germplasm, the kabuli form is polyphyletic. We identified a basic helix-loop-helix (bHLH) transcription factor at chickpea's B locus that conditions flower and seed colors, orthologous to Mendel's A gene of garden pea, whose loss of function is associated invariantly with the kabuli type of chickpea. From the polyphyletic distribution of the kabuli form in germplasm, an absence of nested variation within the bHLH gene and invariant association of loss of function of bHLH among the kabuli type, we conclude that the kabuli form arose multiple times during the phase of phenotypic diversification after initial domestication of cultivated chickpea.
    Full-text · Article · May 2016
    • "Several forward genetic screens of the FN population generated in JI 2822 have contributed to the identification of novel alleles for several classical genetic markers in pea, as summarised inTable 2. These studies, together with the results presented here, indicate that a range of deletion sizes exists in the population and that individual lines could carry more than one mutation. For the deletion alleles of the mutants listed inTable 2, one rearrangement (Moreau et al. 2012) that appears to result from a non-homologous exchange involving an Ogre element was found, and two were small deletions, one of 22 bp (Hellens et al. 2010) and the other of 1.4 kb (Moreau et al. 2012); the remaining 25 alleles were all larger than the gene being characterised. The number of mutations per line was estimated in an AFLP screen; for this purpose 16 FN lines were selected, eight had a clearly observable mutant phenotype and eight had no obvious mutation (Table 1). "
    [Show abstract] [Hide abstract] ABSTRACT: A fast neutron (FN)-mutagenised population was generated in Pisum sativum L. (pea) to enable the identification and isolation of genes underlying traits and processes. Studies of several phenotypic traits have clearly demonstrated the utility of the resource by associating gene deletions with phenotype followed by functional tests exploiting additional mutant sources, from both induced and natural variant germplasm. For forward genetic screens, next generation sequencing methodologies provide an opportunity for identifying genes associated with deletions rapidly and systematically. The application of rapid reverse genetic screens of the fast neutron mutant pea population supports conclusions on the frequency of deletions based on phenotype alone. These studies also suggest that large deletions affecting one or more loci can be non-deleterious to the pea genome, yielding mutants that could not be obtained by other means. Deletion mutants affecting genes associated with seed metabolism and storage are providing unique opportunities to identify the products of complex and related gene families, and to study the downstream consequences of such deletions.
    Full-text · Article · Aug 2013
    • "McGrath et al. 2011) but deleterious mutants obtained in the laboratory were excluded because they are actively maintained for research rather than from commercial use by breeders. However this later statement is not always easy to verify: for instance, it is not clear if the pea strains studied by Gregor Mendel should be considered as mutants or cultivars, but the recent mapping studies of pea color phenotypes were included here (Hellens et al. 2010; Moreau et al. 2012). Thus, the boundary between mutants and bona fide selected phenotypes can be blurry. "
    Full-text · Dataset · May 2013 · Functional Plant Biology
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