Ant28 gene for proanthocyanidin synthesis encodingthe R2R3 MYB domain protein (Hvmyb10) highly affectsgrain dormancy in barley

Eiko Himi • Yuko Yamashita •

Naoto Haruyama • Takashi Yanagisawa •

Masahiko Maekawa • Shin Taketa

Received: 20 July 2011 / Accepted: 7 October 2011

� Springer Science+Business Media B.V. 2011

Abstract A number of anthocyanin- and proantho-

cyanidin-free mutants (ant mutants) in barley were

induced and selected because of breeding interest to

reduce proanthocyanidins, which could cause haze

and degrade the quality of beer. Ant loci, known as

anthocyanin or proanthocyanidin synthesis genes, are

classified into Ant1 to Ant30 through allelism tests.

However, only the Ant18 gene has been molecularly

shown to encode dihydroflavonol 4-reductase (DFR),

which is involved in both anthocyanin and proanth-

ocyanidin synthesis. In this study, an R2R3 MYB gene

of barley was isolated by PCR and named Hvmyb10

due to its similarity to Tamyb10 of wheat, which is a

candidate for the R-1 gene grain color regulator. The

predicted amino acid sequences of Hvmyb10 showed

high similarity not only to Tamyb10 but also to TT2,

the proanthocyanidin regulator of Arabidopsis. Non-

synonymous nucleotide substitutions in the Hvmyb10

gene were found in all six ant28 mutants tested.

Mapping showed that a polymorphism in Hvmyb10

perfectly cosegregated with the ant 28 phenotype on

the distal region of the long arm of chromosome 3H.

These results demonstrate that ant28 encodes

Hvmyb10, the R2R3 MYB domain protein that

regulates proanthocyanidin accumulation in develop-

ing grains. The reduced grain dormancy of ant28

mutants compared with those of the respective wild

types indicates that Hvmyb10 is a key factor in grain

dormancy in barley.

Keywords Barley � Proanthocyanidin �Grain dormancy � Ant mutants

Abbreviations

CHI Chalcone isomerase

CHS Chalcone synthase

DAP Days after pollination

DFR Dihydroflavonol 4-reductase

F3H Flavanone 3-hydroxylase

GI Germination index

LAR Leucoanthocyandin reductase

Electronic supplementary material The online version ofthis article (doi:10.1007/s10681-011-0552-5) containssupplementary material, which is available to authorized users.

E. Himi (&) � Y. Yamashita � M. Maekawa � S. Taketa

Institute of Plant Science and Resources, Okayama

University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046,

Japan

e-mail: eikohimi@server.rib.okayama-u.ac.jp

N. Haruyama

Tochigi Prefectural Agricultural Experiment Station,

2920 Otsuka-cho, Tochigi 328-0007, Japan

Present Address:N. Haruyama

Tochigi Prefectural Sustainable Agriculture Extension

Center, 1032-2 Takebayashi, Utsunomiya,

Tochigi 321-0974, Japan

T. Yanagisawa

NARO Institute of Crop Science (NICS), 2-1-18

Kannondai, Ibaraki, Tsukuba 305-8518, Japan

123

Euphytica

DOI 10.1007/s10681-011-0552-5

Introduction

In wheat, barley, and rice, pre-harvest sprouting is a

very serious problem because severe degradation of

grain quality is induced. Pre-harvest sprouting is

caused by the weak dormancy of grain at the ripening

stage. In wheat, it is well known that grain dormancy is

associated with grain color; white-grained wheat

exhibits a reduced dormancy compared with red-

grained wheat (Warner et al. 2000; Flintham 2000;

Himi et al. 2002). It was also reported that the

dormancy of lighter colored seed was weaker than that

of darker colored seed in Arabidopsis (Debeaujon

et al. 2000). Although major pigments that confer the

color of the wheat grain coat (testa) and Arabidopsis

seed testa were identified as proanthocyanidins

(Matus-Cadiz et al. 2008; Lepiniec et al. 2006), the

mechanisms linking grain/seed color and grain/seed

dormancy remain unclear.

The red grain color of wheat is controlled by R-1

genes located on the distal region of the long arm of

chromosome group 3 (McIntosh et al. 1998). A

germinability test using a mutant line and near-

isogenic lines of the R-1 gene showed that the R-1

gene might enhance grain dormancy by increasing

the sensitivity of embryos to ABA (Himi et al.

2002). Recently, our group found that one of the

MYB-related genes, Tamyb10, is a strong candidate

for R-1 through chromosomal localization, sequence

analysis of different R-1 genotypes with cosegrega-

tion of the Tamyb10 gene and the R-1 gene, and

functional analysis for flavonoid biosynthesis (Himi

et al. 2011a). The Tamyb10 gene is reported to

induce the expression of flavonoid biosynthetic

genes such as chalcone synthase (CHS), chalcone

isomerase (CHI), flavanone 3-hydroxylase (F3H),

and dihydroflavonol 4-reductase (DFR). These

enzymes are also essential for the synthesis of both

anthocyanins and proanthocyanidins.

In Arabidopsis, mutants with impaired flavonoid

accumulation have been identified as transparent testa

(tt) mutants (Lepiniec et al. 2006). Identified alleles of

these mutants were classified into either structural

genes or regulatory genes for flavonoid biosynthesis.

In wheat, however, no grain color gene except the R-1

gene has been identified. Further, when more than 20

white-grained cultivars were investigated, all of them

were confirmed as recessive R-1 genotypes (Himi,

unpublished data).

In barley, a number of anthocyanin- or proantho-

cyanidin-less mutants (designated ant mutant) were

documented after intensive screening of mutagenized

populations (Jende-Strid 1993) because of breeding

interest with an aim to reduce proanthocyanidins

located in the testa layer of the grain, which can cause

haze and degrade the quality of beer (Aastrup et al.

1984; von Wettstein 2007). These mutants possessing

recessive mutations at the ‘‘Ant’’ loci were classified

into Ant1 to Ant30 (Jende-Strid 1994, 1998). In these

loci, only Ant18 was molecularly shown to encode the

DFR gene (Kristiansen and Rohde 1991); Ant17 was

considered the F3H gene (Meldgaard 1992).

The grains of the ant28 mutant are proanthocyani-

din-free, but the vegetative tissue of ant28 retained a

wild-type level of anthocyanin content (Jende-Strid

1993). Moreover, the enzymatic activities of DFR and

leucoanthocyanidin reductase (LAR), which are essen-

tial for proanthocyanidin synthesis, were reduced in the

ant28 mutant (Jende-Strid 1993). These results suggest

that Ant28 might act as a regulator of proanthocyanidin

synthesis. Garvin et al. (1998) reported the ant28 locus

on the distal region of chromosome 3HL; a similar

location on a homoeologous wheat group 3L arm was

occupied by a wheat candidate for R-1, the grain color

regulator (Himi et al. 2011a).

In this study, we attempted the molecular cloning of

barley ant28 to test its possible orthologous relationship

with wheat R-1. PCR-amplified Hvmyb10, an R2R3

MYB domain protein, conserved a domain among other

proanthocyanidin regulators. Mapping showed that

polymorphisms in the Hvmyb10 gene perfectly coseg-

regated with the ant28 phenotype in the distal region of

the chromosome 3HL arm, similar to the homoeologous

location of the R-1 gene of wheat. All of the investigated

barley ant28 mutants carried lesions in the Hvmyb10

gene. Furthermore, a germinability test indicated that

the ant28 mutants had a reduced level of grain dormancy

similar to that of white-grained wheat. These results

show that barley ant28 encodes Hvmyb10 and that it is

an ortholog of the R-1 gene of wheat.

Materials and methods

Plant materials

Wild-type barley (Hordeum vulgare L., cvs. Alexis,

Catrin, and Grit) and six proanthocyanidin-free

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mutants were used in this study: ant28-2131 and

ant28-2132 (derived from Alexis); ant28-493, ant28-

494, and ant28-495 (derived from Catrin) and ant28-

484 (derived from Grit) (Jende-Strid 1994). These

lines are two-rowed and hulled. The barley plants were

grown in a field in Kurashiki, Okayama, Japan. A total

of 187 lines derived from F2 of a cross between ‘‘Sky

Golden’’ and ‘‘ant28-494’’ (Haruyama et al. 2011)

were used for the mapping of Hvmyb10.

Spikes were tagged at anthesis, and grains were

collected from the central spikelets at 5-day intervals

after anthesis. Dry grain weight was measured after

incubation at 150�C for 3 h. Water content (%) of

grains was estimated by (fresh weight – dry weight)/

fresh weight. Germination Index (GI) of whole grains

or half grains containing embryo (embryo half grains)

was calculated according to Himi et al. (2002) with the

formula as follows:

GI = (7 9 n1 ? 6 9 n2 ? 5 9 n3 ? 4 9 n4 ? 3 9

n5 ? 2 9 n6 ? 1 9 n7 9 100)/(7 9 (total number of

grains or embryos)).

Where n1, n2 … n7 are the number of grains or

embryos that germinated on the first, second and

subsequent days until the 7th day, respectively.

All experiments were done in triplicate with 20–25

grains (from one spike) per each.

Vanillin-HCl staining

Freshly harvested immature grains (5–25 days after

pollination; DAP) of Catrin and ant28-494 were used

for Vanillin-HCl staining tests according to Aastrup

et al. (1984). Dehulled grains were stained with 1%

vanillin-6 M HCl solution for 3 h at 25�C and rinsed

with distilled water.

Genomic DNA, RNA, and cDNA preparation

DNA was isolated from 100 mg leaves according to

Murray and Thompson (1980). Total RNA was

extracted from 100 mg of grains with the RNA-suisui

S kit (Rizo). Poly (A) ? RNA was isolated with the

Oligotex -dT30 \ Super [ mRNA Purification Kit

(Takara). cDNA was obtained by reverse-transcription

reaction using SuperScript II (Invitrogen).

Isolation of the Hvmyb10 gene

Three EST clones (CK123590, CK123641, and

BU971906) of barley showed high homologies to the

wheat Tamyb10 gene. CK123590 and CK123641 were

obtained from embryosac tissue of 0-10 DAP and

BU971906 was obtained from developing caryopsis of

8–15 DAP. These EST clones were derived from

cultivar Barke. Primers for Hvmyb10 were designed

based on these EST sequences (Table 1). Hvmyb10

fragments were amplified in 20 ll reaction solution

containing 4 ng genomic DNA, 1x Ex Taq buffer,

0.2 mM dNTP, 0.5 lM of primers, 10% glycerol, and

0.25 U of Ex Taq DNA polymerase (TaKaRa). After

the DNA was denatured for 5 min at 94�C, the reaction

mixture was subjected to 35 cycles in the following

temperature profile: 94�C for 30 s, 62�C for 30 s, and

72�C for 1 min, with a final extension at 72�C for

7 min. PCR products were cloned with the pGEM-T

easy vector system (Promega). DNA sequences were

determined by the ABI 3100 genetic analyzer

(Applied Biosystems). The obtained nucleotide

sequences were analyzed using the GENETYX soft-

ware v. 10.0. The deduced amino acid sequence data

of the plant MYB proteins were aligned using

Table 1 Sequences of

primers used in this studyPrimer name Sequences

Hvmyb

Hvmyb10-LP1 CGAGAGAGAAAGGCAGAGGA

Hvmyb10-RP1 TCGGAGATGTTGCCTCTCTT

Hvmyb10-LP2 CTGAATAGGTGCGGGAAGAG

Hvmyb10-RP2 GATGGTTCCTCCTCTTGCTCAGG

Hvmyb10-LP3 GGGCGAAACAGACAATGAGAT

Hvmyb10-RP3 GCCATGCAAGTCGCAATAAT

dCAPS

dCAPS ant28 M F2 TCCAAGGAGGGCCTGAACAGAGGAGCGTT

ant28 R1 ATGTCGGGGTATGAGCAAAG

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123

ClustalW software v. 1.83 (http://clustalw.ddbj.nig.

ac.jp/top-j.html). A neighbor-joining tree was con-

structed using Kimura 2-parameter model and 1,000

bootstrap replicates with TREEVIEW (Page 1996).

Mapping of Hvmyb10 by dCAPS markers

A dCAPS primer was designed using the dCAPS

Finder ver. 2.0, provided on a website (http://helix.

wustl.edu/dcaps/dcaps.html). A single nucleotide mis-

match was introduced adjacent to the SNP position,

which created a restriction site in the amplified PCR

product of one parent but not of the other (Fig. 3a).

Reverse primers were designed approximately 160 bp

apart from the forward primers (Table 1).

PCR reactions were performed in 10 ll reaction

mixtures containing 5 ll of Quick TaqTM

HS DyeMix

(TOYOBO), 20 ng genomic DNA, and 0.2 lM of

each specific primer using the following conditions:

initial denaturation at 94�C for 2 min followed by 35

cycles of 94�C for 0.5 min, 56.3�C for 0.5 min, and

68�C for 0.5 min, with a final extension step at 68�C

for 7 min. For restriction assays, 10 ll of the PCR

products was incubated with or without 2 U of

restriction enzyme, MseI, in a final volume of 15 ll.

These fragments were separated in 3% Metaphor

agarose gels.

Other molecular markers used for mapping were

from public sources (Varshney et al. 2007; Haruyama

et al. 2011).

Results

Proanthocyanidin synthesis in developing grains

The coloring of mutant line ant28-494 and corre-

sponding wild type line Catrin were examined over a

developmental time course with vanillin staining to

compare the apparent accumulation of proanthocy-

anidins. The vanillin assay is widely employed as a

method for quantitative determination of proanthocy-

anidins in plant materials.

Developing grains of freshly harvested Catrin and

ant28-494 from 5 to 25 DAP are shown in Fig. 1a. In

both lines, grains at 5 DAP were pale green and then

became greener at 10 DAP. From 25 DPA, the green

color of the grains started to change to yellow.

After treatment with vanillin-HCl, Catrin grains

showed a dull olive color at 5 DAP, but the grain color

drastically changed to reddish brown from 10 DAP,

whereas ant28-494 showed a slight color change of

green to dark green (Fig. 1b). These results suggest

that proanthocyanidins do not accumulate in ant28-

494. In contrast, the synthesis of grain pigments in

Catrin started before 10 DAP, which coincides with

reports of the identification of a proanthocyanidin

precursor in 4 DAP grains (Kristiansen 1984; Jende-

Strid 1993).

Identification of Hvmyb10

Fragments of the Hvmyb10 gene of Alexis, Catrin, and

Grit were amplified with primers based on EST

sequences showing a high similarity to wheat Tam-

yb10 and then were sequenced. The obtained genomic

sequences of all these cultivars were exactly the same,

and the Hvmyb10 gene consisted of three exons and

two introns (Fig. 2a). Two sites of the intron–exon

boundaries were conserved among the Hvmyb10 gene

and the Tamyb10 genes (Tamyb10-A1, B1, and D1)

(Himi et al. 2011a). The Hvmyb10 gene encodes an

R2R3 MYB domain protein which showed 82, 81, and

83% identities to Tamyb10-A1, B1, and D1, respec-

tively (data not shown). The predicted amino acid

sequence of the Hvmyb10 gene shared a conserved

motif (IRTKAL/IRC) of regulatory MYB proteins of

proanthocyanidin synthesis (Himi et al. 2011a)

(Fig. 2a).

Genomic sequences of six ant28 alleles (ant28-

2131, 2132, 484, 493, 494, and 495) were examined

and compared with the wild-type sequence. In ant28-

484, 493, 494, and 495, the 51st nucleotide (guanine)

from start codon of each line changed to adenine,

which is predicted to cause a stop codon (Fig. 2a). In

ant28-2131 and 2132, the 558th nucleotide (guanine)

at the junction of the second intron was changed to

adenine, resulting in missplicing, which would prob-

ably cause a lack of five amino acid residues. This

missplicing was confirmed by cDNA sequencing

(Fig. 2a–c).

Phylogenetic analysis using the predicted amino

acid sequences of the R2R3 MYB domains of

Hvmyb10 and other plant MYB proteins showed that

Hvmyb10 was more closely related to the proantho-

cyanidin regulators (PA -clade 2) rather than to

other flavonoid regulatory MYB subgroups, such as

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123

PA-clade 1 and other anthocyanin- and phlobaphene

regulators (Fig. 2d).

Mapping of the Hvmyb10 gene

To map the Hvmyb10 gene, dCAPS primers were

designed as shown in Fig. 3a. PCR was performed,

and amplified DNA fragments were treated with/

without MseI. Since a recognition site of MseI (TTAA)

was produced by the DNA fragments derived from

ant28-484 (Fig. 3a), digested DNA fragments were

observed in an MseI-treated sample of ant28-484 and a

mixture of Grit and ant28-484 (Fig. 3b).

Mapping of the Hvmyb10 gene was carried out

along with grain color stained with vanillin-HCl

segregating among the F2 population consisting of

187 individuals derived from the ‘‘Sky Golden’’ and

‘‘ant28-494’’ cross. The ant28 grain color phenotype

and the Hvmyb10 gene cosegregated completely and

mapped to the same position (Fig. 3c). ant28 and the

Hvmyb10 gene mapped proximal to other barley

chromosome 3HL arm markers such as the SNP

marker OPAp1-1297 and the microsatellite marker

EBmag0705 (Varshney et al. 2007) (GrainGenes,

http://wheat.pw.usda.gov/GG2/index.shtml).

It was reported that the R-1 genes of wheat, which

control grain color, mapped on the distal region of the

long arms of chromosomes 3A, 3B, and 3D (Gale et al.

1995). Since the nearby 3BL and 3DL homoeoloci

from probe ABC174 were both highly linked to the

trait of grain color (Nelson et al. 1995) and BCD131

and ABC174 were identified as flanking markers for

the R-1 genes (Gale et al. 1995) (Fig. 3c), the locations

of ant28 and the R-1 gene appeared to be similar.

Effect of the Hvmyb10 gene on grain dormancy

The dry weight and water content of developing grains

of Catrin and its mutant, ant28-494, were measured

(Fig. 4a). The grain weight of the mutant was lighter

than that of the wild type through the grain develop-

mental period. Water content decreased rapidly and

a

b

5 10 15 20 25

Catrin

ant28

-494

Catrin

ant28

-494

control

Vanillin

-HCl

DAPFig. 1 Grain color of Catrin

and ant28-494. Grains were

collected at 5, 10, 15, 20,

and 25 DAP (left to right).a Freshly harvested grains of

Catrin (upper panels) and

ant28-494 (lower panels).

b Vanillin-stained grains of

Catrin (upper panels) and

ant28-494 (lower panels)

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123

reached a minimum around 15% at 40 DAP. Gener-

ally, physiological maturity is defined as when the

spikes have lost their green color and grains have

moisture content below 30%. Grains of Catrin, ant28-

494, Grit, ant28-484, Alexis, and ant28-2131 appeared

to have reached maturity at 40 DAP (Fig. 4a, Supple-

mental Fig. 1a, and e).

The germinability of the developing grain was

examined, and the Germination Index (GI) was

calculated after imbibing for 7 days (Fig. 4b). The

germinability of ant28-494 grains increased from 35

DAP. On the other hand, grains of Catrin did not

germinate during 30–40 DAP, and even at 45 and 50

DAP, the GI values were lower than those of ant28-

494. These results show that the dormant level of

Catrin was higher than that of ant28-494.

Since mechanical scarification breaks grain dor-

mancy (Baskin and Baskin 2004), the germinability of

0.5kbTGG(W) TGA (stop)ant 28-484, 493, 494, 495

AGGT AGA T (missplicing)ant28-2131, 2132

R L H R L L G N R W S L WT AGG CTC CAC AGG TTG CTA GGC AAC AG GT -(0.7kb intron)- AG G TGG TCG CTGant28-2131 AGG CTC CAC AGG TTG CTA GGC AAC AG AT -(0.7kb intron)- AG G TGG TCG CTG

WT AGG CTC CAC AGG TTG CTA GGC AAC AG G TGG TCG CTG ant28-2131 AGG CTC CAC AG G TGG TCG CTG

R L H R W S L

genomicDNA

cDNA

a

b

d

R2 R3IRTKAL/IRC

LP1 LP2 LP3RP1 RP2 RP3

0.1

882

243

Anthocyanin

Phlobaphene

PA-clade 1

PA-clade 2

Hvmyb10 LNRGAWTAMEDDILVSYINEHGEGKWGSLPKRAGLNRCGKSCRLRWLNYLRPGant28-2131 LNRGAWTAMEDDILVSYINEHGEGKWGSLPKRAGLNRCGKSCRLRWLNYLRPGTamyb10-A1 LNRGAWTAMEDDILVSYINDHGEGKWGSLPKRAGLNRCGKSCRLRWLNYLRPGTamyb10-B1 LNRGAWTAMEDEILVSYINDHGEGKWGSLPKRAGLNRCGKSCRLRWLNYLRPGTamyb10-D1 LNRGAWTAMEDEILVSYINDHGEGKWGSLPRRAGLNRCGKSCRLRWLNYLRPGOsmyb3 LNRGAWTAMEDDILVSYIAKHGEGKWGALPKRAGLKRCGKSCRLRWLNYLRPGAtTT2 LNRGAWTDHEDKILRDYITTHGEGKWSTLPNQAGLKRCGKSCRLRWKNYLRPG

Hvmyb10 IKRGNISDDEEELIVRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNTTLSKRant28-2131 IKRGNISDDEEELIVRLH-----RWSLIAGRLPGRTDNEIKNYWNTTLSKRTamyb10-A1 IKRGNISNDEEELIVRLHGLLGNRWSLIAGRLPGRTDNEIKNYWNTTLSKRTamyb10-B1 IKRGNISDDEEELIVRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNTTLSKRTamyb10-D1 IKRGNISDDEEELIVRLHGLLGNRWSLIAGRLPGRTDNEIKNYWNTTLSKROsmyb3 IKRGNISGDEEELILRLHTLLGNRWSLIAGRLPGRTDNEIKNYWNSTLSKRAtTT2 IKRGNISSDEEELIIRLHNLLGNRWSLIAGRLPGRTDNEIKNHWNSNLRKR

c

65

65

65

65

67

65

65

115

115

115

115

117

115

110

64

64

64

64

66

64

6412

12

12

12

14

12

12

Euphytica

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embryo-half grains of these lines imbibed in water or

ABA solution for 7 days was examined. As shown in

Fig. 4c, the embryo-half grains of Catrin germinated

at 30 and 35 DAP, but the germination level decreased

at 40 DAP and remained lower than that of ant28-494.

These results suggest that the immature grains around

30–35 DAP had germinability and the dormancy was

broken by scarification. However, after 40 DAP, the

grains acquired other factor-controlled dormancies,

such as embryo dormancy, which was not broken by

scarification. The germination of embryo-half grains

was repressed by ABA treatment at 30–35 DAP, but

the sensitivity level to ABA seemed to decrease

gradually during grain ripening (Fig. 4c).

Discussion

A numbers of Anthocyanin- and proanthocyanidin-

deficient mutants of barley (ant mutants) have been

selected by screening after mutagen treatment with

sodium azide (NaN3) (Jende-Strid 1978, 1993).

Selected M2 plant or M3 grains were used for diallelic

tests and classified into ant1 to ant30 (Jende-Strid

1994, 1998). We isolated Hvmyb10 gene which is a

causal gene of Ant28 and found the mutation sites in

six ant28 mutants (Fig. 2a). The mutation sites of

ant28-493, 494, and 495 were identical and these lines

were derived from Catrin. ant28-2131 and 2132 also

showed same mutation sites and these lines were

derived from Alexis. These results suggest that the

selected lines from mutagenized M3 pools were

siblings from the same origins.

Monomeric proanthocyanidins (catechin) were

found in developing grains, whereas dimeric proanth-

ocyanidins and trimeric proanthocyanidins were iden-

tified in mature barley grains (Jende-Strid 1993) also

reported that the synthesis of the monomer started

from about 4 days after pollination (DAP) of grains.

The accumulation level was highest in 18 DAP grains,

and the monomer content gradually decreased, while

the dimeric and trimeric proanthocyanidins increased.

The results of the vanillin-HCl treatment of Catrin in

the current study showed a slight color change at 5

DAP of grains and full staining at 10 DAP of grains

(Fig. 1b). Vanillin-HCl reacts with both polymeric

proanthocyanidins and monomeric proanthocyanidins

(catechin) (Price et al. 1978), which suggests that

the grains of Catrin might start the accumulation

of monomeric/polymeric proanthocyanidins from 5

DAP.

In Arabidopsis, previous studies showed that the

monomeric proanthocyanidins in seed testa (mainly

epicatechin) are transported into the vacuole by

Transparent Testa12 (TT12), a MATE family trans-

porter, and polymerized in the vacuole (Debeaujon

et al. 2001; Zhao and Dixon 2010). The polymerized

Fig. 2 Characterization of the Hvmyb10 gene. a Genomic

structure of the Hvmyb10 gene. Boxes indicate exons, and lines

indicate introns and untranslated regions. R2 and R3 repeats of

the MYB consensus region and conserved sequences (IRTKAL/

IRC) among proanthocyanidin regulators are represented in

grey boxes, black boxes, and a striped box, respectively.

Mutation sites of ant28 mutants are depicted by open arrows.

Sites and directions of primers are depicted with black arrows.

Bar shows 0.5 kb. b Partial alignments of Hvmyb10 sequences

of genomic DNA and cDNA obtained from 5 DAP grains of

wild type and ant28-2131. The deduced amino acid sequences

are shown above the DNA sequences (wild type) and below the

DNA sequences (ant28-2131). A single nucleotide substitution

(G to A) in ant28-2131 is written in bold, and spliced sequences

as the second intron are shown in italics. c Comparison of amino

acid sequences of the conserved R2 (upper) and R3 (lower)

MYB repeats of Hvmyb10 with other MYB-related proteins

from wheat, rice, and Arabidopsis. The dashed line in the ant28-

2131 sequence indicates a gap caused by missplicing. A blackarrow indicates the mutation site of ant28-484, 493, 494, and

495. White arrowheads denote the conserved W/I residues.

Essential residues of the conserved amino acid signature ([DE]-

L-x(2)-[RK]-x(3)-L-x(6)-L-x(3)-R) as the structural basis for an

interaction between MYB and bHLH proteins are shown by

black arrowheads. d Phylogenetic analysis for plant MYB

transcription factors. For construction of the tree, the R2R3

MYB domain sequences of each MYB protein were aligned

using ClustalW. A neighbor-joining tree was constructed using

Kimura 2-parameter model and 1,000 bootstrap replicates with

TREEVIEW presented with bootstrap value. The scale barrepresents 0.1 substitution per site. The GenBank accession

numbers are as follows: AtPAP1 (Arabidopsis, AAG42001),

AtPAP2 (Arabidopsis, AAG42002), AtTT2 (Arabidopsis,

AED93980), DkMYB2 (persimmon, BAI49719), DkMYB4

(persimmon, BAI49721), FcMYB251 (Japanese beech,

BAG75107), GhMYB10 (upland cotton, AAK19615),

GhMYB36 (upland cotton, AAK19617), Hvmyb10 (barley,

AB645844), LjTT2a-c (Lotus japonicus, BAG12893-

BAG12895), MdMYB9 (apple, ABB84757), MtMYB11 (apple,

AAZ20431), OsC1 (rice, BAD04030), OsMYB3 (rice,

BAA23339), PhAN2 (petunia, AAF66727), PhredMYB9 (Pha-laenopsis, ACH95795), PtMYB134 (quaking aspen,

ACR83705), SbMYB1 (sorghum, ADD18214), Tamyb10-A1,

B1, and D1 (wheat, AB599721, AB599722, AB191460),

VvMYBPA1 (grape, CAJ90831), VvMYBPA2 (grape,

ACK56131), ZmC1 (maize, P10290), and ZmP (maize,

AAC49394). Solid underlined, dotted underlined, and boxedMYB proteins are known regulators of proanthocyanidins,

anthocyanin, and phlobaphene synthesis, respectively

b

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proanthocyanidin precursors (colorless proanthocy-

anidin) are oxidized into brown by a laccase-like

oxidase, Transparent Testa10 (TT10) (Pourcel et al.

2005). However, both TT12-like transporter and TT10-

like laccase have not been found in barley. Since

proanthocyanidin deficient ant26 mutants synthesize a

wild-type amount of anthocyanin in vegetative parts

and catechin (monomer) in developing grains, Marles

et al. (2003) proposed that the Ant26 might be an

analog of TT12 of Arabidopsis.

The identified Hvmyb10 may act as a regulator of

proanthocyanidin synthesis in grains, including the R-

1 genes of wheat. R-1 genes show a similarity to TT2

of Arabidopsis, which controls proanthocyanidin

synthesis complementary to TT8 (bHLH) and TTG

(WD40) (Nesi et al. 2001). Since functional bHLH

and/or WD40 which are involved with proanthocy-

anidin synthesis have not been identified, it is still

unknown whether the synthesis of wheat and barley

requires only R-1/Hvmyb10 or R-1/Hvmyb10 with

5’-TCCAAGGAGGGCCTGAACAGAGGAGCGTt-3’Grit 22 TCCAAGGAGGGCCTGAACAGAGGAGCGTGGACGGCAAT 59ant28-484 22 TCCAAGGAGGGCCTGAACAGAGGAGCGTGAACGGCAAT 59

S K E G L N R G A W T A M /stop

Grit 130 CACACCATCGTCTCCAATCTTTGCTCATACCCCGACAT 181ant28-484 130 CACACCATCGTCTCCAATCTTTGCTCATACCCCGACAT 181

3’-GAAACGAGTATGGGGCTGTA-5’

dCAPS ant28M F2

ant28 R1

TTAA: MseIaGrit

ant28-484

Mix Gritant28-484

Mix

+MseINT

160 bp

132 bp

b

ant28Hvmyb10

OPAp1-1297

OPAp2-0505

EBmag0705OPAp2-1523

0.0

1.3

4.4

5.2

0.8

7.1

abc174

OPAp1-1297150

130

110

90

70

abc174

EBmag0705

150

130

110

90

70

Xbcd131

Xabc17422 R-1

Barley3HL

Barley3HL

Wheat3L

Barley3H

Gale et al.1995

Pilot OPA1consensus map(GrainGenes)

Varshney et al.2007

c

E31/M41

Fig. 3 Detection and mapping of the SNP between normal and

ant28-484 lines. a Partial alignments of the sequences and the

deduced amino acid sequences of Grit (wild type) and ant28-484

in exon1 of the Hvmyb10 gene. The single nucleotide-

substituted site is printed in bold. Primers for PCR are shown

in the upper (dCAPS ant28 M F2) and the lower (ant28 R1)

parts with black arrows in alignments. The dCAPS ant28 M F2

primer generates a recognition site of MseI (TTAA) with a

mismatch base printed in lower case and shown by a grey arrowin the PCR product derived from the ant28-484 allele. b PCR

products from Grit, ant28-484, and a mixture of these lines. NTrepresents non-treated DNA fragments of MseI, and ?MseI

represents MseI-treated DNA fragments. c Location of the

Hvmyb10 gene with a comparison with other genetic maps of

the long arm of barley chromosome 3H and wheat group 3

chromosomes. Left to right barley 3HL maps of the pilot OPA1

consensus map from GrainGenes (http://wheat.pw.usda.gov/

GG2/index.shtml) and the microsatellite consensus map

(Varshney et al. 2007), consensus map of wheat (Gale et al.

1995), and partial linkage map of barley chromosome 3H in the

F2 population of ‘‘Sky Golden’’ x ‘‘ant28-494.’’ The locations of

the ant28 trait and Hvmyb10 are shown in bold. The homoeol-

ogous marker loci present on two or three maps are joined by

dotted lines. Arrows indicate centromeres. Numbers on the leftside show the distance in centiMorgans, and the numbers on the

two maps on the left show the distance in centiMorgans from the

top of each chromosome

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123

other unidentified factor(s). R2R3 MYB repeats of the

deduced amino acid of Hvmyb10, on the other hand,

showed a conserved amino acid signature ([DE]-L-

x(2)-[RK]-x(3)-L-x(6)-L-x(3)-R) as the structural

basis for the interaction between the MYB and bHLH

proteins (Zimmermann et al. 2004) (Fig. 2c).

Tonooka et al. (2010) developed a proanthocyani-

din-free commercial cultivar ‘‘Shiratae Nijo’’ by the

backcross breeding method using ‘‘Nishinohoshi’’ as a

recurrent parent and ant28-494 as a donor parent.

Proanthocyanidins and catechin were hardly detected

in the grains of the cultivar. However, the cultivar with

the ant28 gene showed weaker seed dormancy than its

recurrent parent, suggesting that the gene pleiotropi-

cally impaired pre-harvest sprouting resistance in

barley (Tonooka et al. 2010).

We showed the difference in the dormancy level of

ant mutant, ant28-494, and its wild type, Catrin.

Grains of ant28-494 showed weaker dormancy during

the grain development stage (Fig. 4). Other combina-

tions of the wild type and its ant28 mutant (i.e., Grit

and ant28-484, and Alexis and ant28-2131) were used

for the germinability test. These comparisons also

show that other ant28 mutants have reduced dormancy

compared with the respective wild types (Supplemen-

tal Fig. 1).

Interestingly, embryo-half grains around 40 DAP of

the wild type showed dormancy even though grains

germinated prior to 35 DAP. In wheat, grain dormancy

was broken by scarification, resulting in the germina-

tion of embryo-half grains throughout the develop-

ment period (Himi et al. 2002). Based on these results,

barley grains appear to acquire other factor-controlled

dormancies such as embryo dormancy.

Some QTLs related to pre-harvest sprouting were

identified in wheat (Flintham et al. 2002). Two QTLs,

QPhs.ocs-3A.1 and Phs1, were shown that the QTLs

have stable and large effects (Mares et al. 2005; Mori

et al. 2005). Recently, Nakamura et al. (2011)

identified that the MOTHER OF FT AND TFL1

(MFT) gene was the causal gene of the QTL QPhs.ocs-

3A.1. Whereas TaVp1, the orthologue of the maize

viviparous gene (Vp1), and R-A1 gene are located on

the long arm of chromosome 3A, the QTL located on

the short arm of chromosome 3A. While Phs1 was

reported that the QTL was located on the chromosome

4, the DNA sequences responsible for the QTL have

not been determined.

In wheat, three possible reasons that red grains have

stronger dormancy than white grains have been

proposed: (1) R-1 genes affect unknown dormancy-

related genes directly, (2) synthesized grain color

pigments affect dormancy, and (3) an unknown

dormancy-related gene is located near the R-1 gene.

However, we previously reported that the mutant of R-

1 gene developed by EMS treatment showed lower

dormancy than the original line (Himi et al. 2002). We

also report here that the mutants of Ant28 showed the

lower dormancy than the each original line. Our

30 35 40 45 50

Ger

min

atio

n In

dex

0

10

20

30

40

50

60

Wat

er c

on

ten

t (%

)

Dry

wei

gh

t (m

g)

Ger

min

atio

n In

dex

a

b

c

50

45

40

35

30

25

20

15

10

5

080

70

60

50

40

30

20

10

080

70

60

50

40

30

20

10

0

DAP (Days after pollination)

Fig. 4 Changes in grain character during the grain develop-

ment (30–50 DAP Days after pollination) of Catrin (closedcircle) and its mutant, ant28-494 (open circle). a Dried grain

weight (solid lines) and water content (dotted lines) of Catrin

and ant28-494. b Germination index of developing grains

incubated with water at 20�C for 7 days of Catrin and ant28-

494. c Germination index of embryo-half grains incubated with

water (solid lines) or 10 lM ABA (dotted lines). Bars show

standard error

Euphytica

123

results suggested that the third possibility, an unknown

dormancy-related gene located near the R-1 gene, is

not the cause of stronger dormancy of red grains.

A direct role of R-1 in dormancy cannot be

investigated because the effects on color and dor-

mancy cannot be easily separated. We investigated

more than 20 white-grained cultivars, including arti-

ficially produced white-grained lines, but all of them

were confirmed as recessive R-1 genotypes (Himi,

unpublished data). It is possible that mutations occur

in a structural or regulatory gene other than Tamyb10

genes but that the phenotype is not changed since other

non-mutated genes may work normally. In fact, all of

the DFR and F3H genes of chromosomes A, B, and D

were expressed in grains of wheat (Himi and Noda

2004; Himi et al. 2011b). Therefore, a white-grained

line which has dominant R-1 allele(s) cannot be

obtained unless all gene copies, such as the F3H or

DFR genes, are mutated.

Despite ample data accumulated in wheat, physi-

ological mechanisms underlying grain dormancy in

relation to grain color remain largely elusive because

of triplicated nature of the wheat genome and limited

availability of grain-color mutants. To overcome such

a stagnant situation in wheat, barley could be an

excellent vehicle because it is a diploid with gene

composition similar to wheat and reserves rich

collections of mutants that impair proanthocyanidins

accumulation in grains. These advantages in barley

would also facilitate comparative studies with the

model plant Arabidopsis thaliana. In this study, we

demonstrated that the barley Ant28 gene encodes

R2R3 MYB domain protein and that ant28 is the

barley ortholog of wheat R-1 gene. Thus, the present

study employing barley has indirectly verified the

unresolved hypothesis proposed by Himi et al. (2011a)

that wheat R-1 gene encodes R2R3 MYB domain

protein. We also observed that the grains of ant28

mutant showed weaker dormancy, and Ant28, as

Hvmyb10, is also a key factor of grain dormancy in

barley. Further investigations using other barley ant

mutants will help to divulge the relation between grain

color and grain dormancy and will give a clue to

resolving the mystery of wheat dormancy.

Acknowledgments We thank Dr. Udda Lundqvist, Nordic

Genetic Resource Center, and Dr. Fredric Ottosson, Department

of Crop Science, Swedish University of Agricultural Sciences,

Alnarp, Sweden, for kindly providing the grains of the ant28

mutants. This work was supported by Ofu-Kai for the Promotion

of Education and Culture at Japan Women’s University, the

Elizabeth Arnold Fuji Foundation, and a grant from the Ministry

of Agriculture, Forestry, and Fisheries of Japan. (Development

of crop production technology for all year round multi-

utilization of paddy fields).

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