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LÜ, R-H, XU, Y-H, Boyd, R., Zhang, X-Q, Broughton, S., Jones, M., LI, C-D and Chen, Y-F (2013) Barley and wheat share the same gene controlling the short

basic vegetative period. Journal of Integrative Agriculture, 12 (10). pp. 1703-1711.

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Copyright: © 2013 CAAS It is posted here for your personal use. No further distribution is permitted.

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Journal of Integrative Agriculture Advanced Online Publication: 2013 Doi: 10.1016/S2095-3119(13)60351-2

Barley and Wheat Share the Same Gene Controlling the Short Basic

Vegetative Period

LV Rui-hua1, 3, 4*, XU Yan-hao2, 3, 4*, Rodger Boyd5, ZHANG Xiao-qi4, Sue Broughton3, Michael Jones4, LI

Cheng-dao3, 4, 5 and CHEN Yao-feng1

1 College of Agronomy, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, P.R.China

2 College of Agriculture, Yangtze University, Jingzhou, Hubei 434000, China

3 Department of Agriculture and Food, Government of Western Australia, 3 Baron-Hay Court, South Perth WA

6151, Australia

4 The State Agricultural Biotechnology Centre, Murdoch University, Murdoch WA 6132, Australia

5 Natural and Agricultural Sciences, The University of Western Australia, Crawley WA 6009, Australia

Abstract

Basic vegetative period (BVP) is an important trait for determining flowering time and adaptation to variable

environments. A short BVP barley mutant is about 30 days shorter than its wild type. Genetic analysis using 557

F2 individuals revealed that the short BVP is governed by a single recessive gene (BVP-1) and was further

validated in 2090 F3 individuals. The BVP-1 gene was first mapped to barley chromosome 1H using SSR markers.

Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice

chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with

candidate genes in rice and Brachypodium; seven new gene-specific markers were developed and mapped in the

mapping populations. The BVP-1 gene co-segregated with the Mot1 and Ftsh4 genes and was flanked by the

gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM). Further analysis demonstrated that barley

and wheat share the same short BVP gene controlling early flowering.

Key words: Basic vegetative period, Candidate genes, Hordeum, Triticum1

INTRODUCTION

The switch from vegetative to reproductive growth is a critical developmental transition in the life of a plant

(Bäurle and Dean 2006). Discovering the control of time to flowering is a common goal of plant breeders to

produce novel varieties that are better adapted to local environments and changing climatic conditions (Boyd et al.

2003; Jung and Müller 2009). Studies using model plants such as Arabidopsis (Yant et al. 2009) and important

cereal crops such as rice (Tsuji et al. 2011) and wheat (Distelfeld et al. 2009) have provided insight into the

molecular network control of flowering and indicated that the mechanism for control of flowering might be

LV Rui-hua, E-mail: vvlv07@gmail.com; XU Yan-hao, E-mail: xyh09@yangtzeu.edu.cn; Correspondence LI

Cheng-dao, Tel: +61-8-93683843, E-mail: chengdao.li@agric.wa.gov.au; CHEN Yao-feng, Mobile: 13060391299,

E-mail: chenyf3828@126.com

* These authors contributed equally to this study.

mailto:vvlv07@gmail.commailto:xyh09@yangtzeu.edu.cnmailto:chengdao.li@agric.wa.gov.aumailto:chenyf3828@126.com

2

partially conserved among higher plants ( Colasanti and Coneva 2009; Greenup et al. 2009; Brkljacic et al. 2011).

Barley is an important crop and genetic model of the Triticeae tribe within the Poaceae. Photoperiod,

vernalization and ‘earliness per se’-or the duration of the basic vegetative period (BVP)-are three major genetic

factors determining flowering time in barley (Boyd et al. 2003; Hay and Ellis 1998). Vernalization (Vrn) genes

determine the need for a period of cold temperature to induce flowering. Photoperiod (Ppd) genes determine the

need for long days for flowering. Additional loci that account for variation in heading time independent of

photoperiod and vernalization are termed ‘earliness per se’ (Eps). In barley, Eps is also defined as genes

controlling the developmental phase from sowing until the plant is responsive to photoperiod, or the duration of

the pre-inductive period, or the duration of BVP (Boyd et al. 2003; Hay and Ellis 1998).

The vernalization genes (Vrn1, Vrn2, Vrn3) (Yan et al. 2003, 2004, 2006) and the photoperiod gene (Ppd-H1)

(Turner et al. 2005) in barley have been cloned; their physiological and biochemical functions, along with their

roles in genetic pathways leading to heading, are well documented. Previous conventional genetic studies

identified some possible Eps loci including eam7 (syn ea7), eam8 (eak), eam9 (eac), and eam10 (easp), located on

chromosomes 6HS, 1HL, 4HL, and 3HL, respectively (Gallagher et al. 1991). In addition, Laurie et al. (1995)

identified eight QTLs, located on 2H, 3H, 4H, 5H, 6H (×2), and 7H (×2), that contribute to variation in flowering

time for reasons other than photoperiod or vernalization. In contrast to the vernalization and photoperiod response

genes, the precise genetic map locations and physiological effects of Eps or BVP genes in barley are poorly

understood.

Although significant progress has been made to map important genes in barley, isolating the genes using a

position cloning strategy has been limited, as the barley genome is replete with repetitive sequences (Appels et al.

2003). Fortunately, there is good colinearity among species in the grass family (Devos 2010; Mayer et al. 2011).

The available rice (Yu et al. 2002) and Brachypodium distachyon (Vogel et al. 2010) genome sequences provide a

pivotal system to generate markers in targeted regions of the barley genome. This study conducted genetic

analysis of the short BVP gene to develop new molecular markers using a comparative genomic approach in order

to precisely map the gene and understand its relationship between wheat and barley.

RESULTS

Genetic analysis of the BVP-1 gene

Heading occurs at about 90 days in Mona and at about 154 days in Turk. The heading date in the F2 population is

clearly divided into early and later groups with dates similar to the parents (Fig. 1). The ratio of early type (147

plants): late type (410 plants) fit with the 1:3 ratio expected for one-locus segregation (χ2=0.23, P=0.63). This

result confirms that early heading date is governed by a single recessive mutant gene.

The phenotype of each F2 individual was further confirmed by the progeny test using F3 lines. In the F3 generation,

heading occurs at about 70 d in Mona and at about 116 d in Turk. A similar distribution of heading dates was

observed in the F3 population (Fig. 2).

Mapping the BVP-1 gene

3

The short BVP gene was initially mapped to bin 14 of chromosome 1H (Lundqvist et al. 1997). Barley DNA

markers were mined from the integrated genetic map (Aghnoum et al. 2010) and DNA sequences were retrieved

from the GrainGenes database (http://wheat.pw.usda.gov). Sixty-four available markers were identified at the

corresponding region (Table 1). PCR primers were designed based on known DNA sequences. Among these

molecular markers, polymorphisms were identified between the two parents in seven microsatellite markers

GMS149, WMC1E8, Bmag0579, GBM1434 and GBM1461, scssr02748 and scssr04163, and one

RFLP-converted PCR marker ABG373. The short BVP gene was mapped to 2.8 cM towards the telomere from

marker ABG373 on chromosome 1H (Fig. 5-C).

Comparative analysis of barley BVP-1 gene region in rice and Brachypodium genomes

Molecular markers in bin 14 of barley chromosome 1H were compiled with known DNA sequences via the

GrainGenes database (http://wheat.pw.usda.gov). A BLASTN search using sequences of the BVP gene region

identified a syntenic region of 443 kb on rice chromosome 5 (Os05) from gene Os05g50980 to gene Os05g51754

and a syntenic region of 216 kb on Brachypodium chromosome 2 (Bd2) from gene Bradi2g14190 to

Bradi2g14450. Twenty-five barley EST sequences were identified by using all the rice and Brachypodium putative

genes in the syntenic region (Fig. 3).

Fine-mapping the BVP-1 gene using gene-specific molecular markers

The identified barley EST sequences (including the 3_UTR, 5_UTR and introns) were used as a template for the

design gene-specific markers for further mapping of the BVP-1 gene. In all, seven new markers were developed

from the six genes listed in Table 2. Examples of gene-specific markers Mot1-M9 (Fig. 4-A) and Ftsh4-F1 (Fig.

4-B) in Mona, Turk and some of the F2 individuals are shown in Figure 4. A linkage analysis was conducted to

combine known molecular markers GMS149, WMC1E8, Bmag0579, GBM1434, GBM1461, ABG373 and

scssr02748 from the above preliminary mapping and seven newly-developed gene-specific markers (Table 2). The

linkage analysis results indicate that the barley BVP-1 gene co-segregated with Mot1 and Ftsh4 genes, flanked by

the gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM) in the distal region of the long arm of

chromosome 1H (Fig. 5-C).

Furthermore, the barley BVP-1 gene genetic map was compared to the physical map of the collinear regions

of rice (Fig. 5-A), Brachypodium (Fig. 5-B), Aegilops tauchii (Fig. 5-D) and Triticum monococcum (Fig. 5e). It is

interesting to note that some of the identified barley ESTs displayed strong similarities to genes in the Eps-1

region from diploid wheat Triticum monococcum (AK251606 similar to Adk gene, AK354960 similar to Ftsh4

gene, Barley1_13126 similar to Mot1 gene, BY855287 similar to wg241 gene, Barley1_14512 similar to CA60858,

AK249839 similar to Vaptc gene, AK357891 similar to Unp30 gene, AK251854 similar to Pp2c gene, and

Ak252402 similar to cdo393 gene) (Fig. 3). Thus, the short BVP gene may be shared among Hordeum, Triticum

and Aegilops.

Further analyses of the predicted Mot1 protein across the barley, wheat, rice and Brachypodium revealed higher

amino acid identity between barley and wheat (96% identity) than between barley and rice (84% identity) or

barley and Brachypodium (89% identity) (Fig. 6-A). The combined exon regions for this gene were more alike

http://wheat.pw.usda.gov/http://wheat.pw.usda.gov/

4

between barley and wheat (96% identity) than between barley and rice (85% identity) or barley and

Brachypodium (90% identity) along the 4.9-6.2 kb high-scoring segment pairs (HSP) aligned by BLASTN (Fig.

6-B).

DISCUSSION

Comparative genomic analysis is a powerful tool to make positional cloning feasible in large genome crops such

as barley and wheat. In this study, seven gene-specific markers were developed from six barley EST sequences

using a comparative strategy, which significantly narrowed the region of the BVP-1 gene. In previous research, a

comparative genomic strategy was used to identify and clone target genes in barley ( Li et al. 2004; Jia et al. 2009;

Liu et al. 2011; Ye et al. 2011; Silvar et al. 2012) and in wheat (Valárik et al. 2006; Kuraparthy et al. 2008;

Faricelli et al. 2010).

Gene order is well-conserved between wheat and barley. Flowering time and vernalization response are

shared with wheat and the causal genes are located at conserved genomic regions ( Fu et al. 2005; Turner et al.

2005; Yan et al. 2006; Beales et al. 2007). Some disease resistance also shares conserved genetic elements in

barley and wheat (Jordan et al. 2010; Liu et al. 2011; Silvar et al. 2012). Previous research confirmed earliness

per se loci in collinear regions on wheat and barley homologous groups 2 and 5 (Snape et al. 1996; Worland 1996;

Kato et al. 1999, 2002). An earliness per se gene, Eps-1, has been identified and mapped to chromosome 1Am L

in einkorn wheat T. monococcum (Bullrich et al. 2002). Recently, the Eps-1 region was compared in diploid wheat,

rice and Brachpodium: Mot1 and FtsH4 genes were completely linked to the earliness per se phenotype in T.

monococcum and candidates for the Eps-1 gene (Faricelli et al. 2010). In this study, we demonstrated that genes in

both T. monococcum and Brachpodium Eps-1 regions were collinear to the barley genes in the short BVP-1 region

by comparative genomic analysis. Some of these genes (CA60858 and wg241) were mapped to the barley BVP-1

gene region. Furthermore, the Ftsh4 and Mot1 genes co-segregated with the short BVP phenotype in barley. This

indicates that the barley BVP-1 gene might be orthologous to the diploid wheat Eps-1 gene.

In this study, the Mot1-M4 polymorphic marker is 2816 bp downstream from the Mot1 gene coding region and the

Mot1-M9 polymorphic marker is 794 bp downstream from the Mot1 gene coding region. These two polymorphic

markers were selected from 42 pairs of primers which cover the full length of the barley Mot1 gene (12812 bp)

and 2816 bp downstream sequence (data not shown). The Ftsh4-F1 polymorphic marker is 155 bp downstream

from Ftsh4 gene coding region, which was selected from 12 pairs of primers covering four exons (exon 3, 4 , 5,

and 6) and the 263 bp downstream sequence (total 2722 bp) (data not shown). We could not detect any difference

in the coding regions of Mot1 and Ftsh4 genes in this study. It is not clear if these polymorphisms are responsible

for the different phenotypes. Further research is needed to understand if there is any difference in the upstream

regions of Mot1 and Ftsh4 genes among varieties with different BVPs.

Although a high level of collinearity between barley and Brachpodium was detected in the syntenic BVP-1

gene region, gene inversions and deletions (Ftsh4 and Mot1) were detected between barley and rice in the syntenic

BVP-1 gene region (Fig. 3). The comparative genomic study also provided additional evidence to support the

notion of treating Triticeae as a single genetic system (Mayer et al. 2011).

Previous researchers have identified numerous Eps gene loci and QTLs. The relationship between these two

nomenclatures is still unclear. Franckowiak and Konishi (2002) identified the Eam6 locus on chromosome 2HS,

5

which might be synonymous with the eps2 locus identified by Laurie et al. (1995). Recently, a QTL on

chromosome 1H was located at the interval between ABC261 and ABG055 markers controlling heading time

from the Azumamugi (JP17209) Kanto RIL population, which is supposedly closely linked or allelic to eam8

(Sameri and Komatsuda 2004; Sameri et al. 2006). However, comparing our mapping results with the barley

consensus map, ABC261 and ABG055 markers were not entirely consistent with the BVP-1 gene closely-linked

markers in this study. Further work is needed to clarify this relationship.

Heading time is a major factor determining adaptation of barley genotypes to diverse growing season

conditions and their associated cultural practices. Given this, it is no surprise that the precise regulation of heading

timing is a major goal in barley breeding programs. The BVP-1 gene is impendent of environmental clues, which

provides a target for precise regulation of heading time. A better understanding of the BVP gene could help in the

development of gene-specific molecular markers for marker-assisted selection and improve the manipulation of

heading time during breeding.

CONCLUSION

We developed 557 F2 and 2090 F3 populations between Mona × Turk to reveal that the short BVP is governed by

a single recessive gene (BVP-1); this gene was first mapped to barley chromosome 1H using SSR markers.

Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice

chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with

candidate genes in rice and Brachypodium and seven new gene-specific markers were developed from six barley

EST sequences-this significantly narrowed the region of the BVP-1 gene-which were mapped in the mapping

populations.

Materials and methods

Plant materials and DNA extraction

Mona, which exhibits an extremely short basic vegetative period, independent of photoperiod and vernalization,

was derived from the barley cultivar Bonus by x-ray radiation. The phenotypic difference between early-flowering

Mona and late-flowering genotypes Bonus and Turk was large enough to map the mutant gene as a single

Mendelian locus (Boyd et al. 2003). Turk is a long basic vegetative genotype barley variety which does not

respond to vernalization but responds to extended photoperiod (Boyd et al. 2003). An F2 population from the cross

between Mona and Turk, comprising 557 individual plants, was subjected to genetic analysis for heading time

grown under natural photoperiod conditions at The University of Western Australia field station. The phenotype of

each F2 individual was further validated using the F2:3 families with 2090 individual plants. Phenotype

identification and sowing date were performed as described by Boyd et al. (2003) to minimize the photoperiod

effect.

Molecular marker data mining and comparative genomic analysis

The short BVP gene in Mona has been previously mapped to bin 14 on chromosome 1H (Boyd et al. 2003;

6

Lundqvist et al. 1997). Barley DNA markers were mined from the integrated barley genetic map (Aghnoum et al.

2010). Marker information was obtained from the GrainGenes database (http://wheat.pw.usda.gov); 64 available

markers were identified at the corresponding region (Table 1).

Bioinformatics and molecular genetic approaches were used to identify the putative syntenic region in rice

and Brachypodium genomes with three major steps: (1) molecular markers in barley BVP-1 gene region were

matched to corresponding DNA sequences retrieved from public databases; (2) corresponding sequences were

aligned to rice and Brachypodium genomes by BLASTn similarity searches using the Phytozome v7.0 database

(http://www.phytozome.net/) to identify potential syntenic regions; and (3) rice (http://rice.plantbiology.msu.edu/)

and Brachypodium (http://www.brachypodium.org/) annotated genes in the corresponding syntenic regions were

used to search for barley EST sequences via the NCBI database (http://www.ncbi.nlm.nih.gov) and released barley

genome sequences (http://webblast.ipk-gatersleben.de/barley/viroblast.php). The resulting barley EST and genome

sequences were used to develop new gene-specific molecular markers.

Molecular marker analysis

Genomic DNA was extracted from young leaves using the standard CTAB protocol. DNA samples were

quantified using the Nanodrop equipment and adjusted to a final concentration of 50 ng μL–1 for PCR.

All primers used in this study were synthesized by Gene Works Pty. Ltd. (Australia). PCR reactions consisted of

50 ng genomic DNA as template, 0.1 µmol L-1 of each primer, in a final volume of 10 µL containing 1 PCR

buffer, 1.5 mmol L-1 MgCl2, 0.2 mmol L-1 dNTP and 0.5 U Taq polymerase (Bioline, Australia). PCR reactions

were performed with the following program: denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30

s, annealing for 45 s, 72°C for 40 s, and a final extension at 72°C for 5 min. The optimal annealing temperature of

each primer pair combination was determined by gradient-PCR.

Molecular markers with size differences between Mona and Turk were run on 6-8% PAGE gels. The single

nucleotide polymorphic (SNP) markers were detected on SSCP gels using a previously described procedure (Yang

et al. 2009) and was optimized with a 12% polyacrylamide gel (acrylamide/bisacrylamide ratio of 37.5:1) in

0.5×Tris-borate-ethylenediaminetetraacetic acid and run at room temperature for 22-32 h.

Data analysis

Deviations of observed phenotype data from theoretically-expected segregation ratios were analyzed by a

chi-square (χ2) test using the SPSS 13.0 for Windows package. The linkage map was constructed using JoinMap

4.0.

Acknowledgements

This project was supported by the Grain Research and Development Cooperation. RL was sponsored by the

Chinese Scholarship Council.

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rice genome (Oryza sativa L. ssp. indica). Science, 296, 79-92.

Fig. 1 Frequency distribution of days to heading in the F2 population from the cross Mona Turk. Arrow

indicates the point which divides the population into early and later heading groups.

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Fig. 2 Frequency distribution of days to heading in the F2-derived F3 generation from the cross Mona Turk.

Arrow indicates the point which divides individuals into early and later heading groups.

Fig. 3 Comparative map of syntenic regions of barley BVP gene on chromosome 1H in relation to rice

chromosome 5 (Os05) and Brachypodium chromosome 2 (Bd2).

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Fig. 4 Polymorphism of six new genome-specific markers segregating in Mona × Turk F2 population. A,

Mot1-M9 polymorphism marker. B, Ftsh4-F1 polymorphism marker, C. CA608558-C1 polymorphism marker. D,

AK250075-H1 polymorphism marker. E, AK252360-H4 polymorphism marker. F, WG241-W2 polymorphism

marker.

Fig. 5 A, rice physical map of barley BVP1 gene collinear region; B, Brachypodium physical map of barley

BVP1 gene collinear region; C, linkage map of BVP1 gene on barley chromosome 1H (linkage analysis performed

on Mona × Turk F2 population. Distances (in cM) between loci listed on left side of diagram); D, Aegilops tauchii

physical map of barley BVP1 gene collinear region; E, Triticum monococcum physical map of barley BVP1 gene

collinear region.

Fig. 6 Comparisons of the Mot1 protein identity (%, A) and exons identity (%, B) among barley, wheat, rice and

Brachypodium.

Table 1 Summary of available barley markers mapped in 1HL. Bold-typed markers were mapped in the current

population

Marker type Marker name

AFLP E39M61-346, E39M61-332, P15M52-592, P16M51-390, E42M51-389,

E42M32-202

DArT bPt-0618, bPt-1221, bPt-7696, bPb-1487, bPb-3756, bPt-8107,

bPt-7975, bPt-0677, bPt-6792, bPt-7356, bPb-2260, bPt-1815,

bPt-5090, bPb-5550, bPt-4627, bPt-4925, bPt-3986, bPt-2481,

bPb-8307, bPb-3201

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Microsatellite GMS149, WMC1E8, BMAG0579, GBM140, GBMS184, GBMS143,

GBM1434, GBM1461, GBM1314, GBM1204, scssr02748,

scssr08238, scssr04163

RFLP Aga7, GBR1390, BCD340A, GBR1441a, ABG373, GBR1146,

ABG387a

RFLP STS ABG055, MWG912

SNP GBS0383, GBS0554, GBS0469, GBS0450

TDM HVSMEl0003B06r2_at, Contig5061_at, Contig14590_at,

Contig7056_at, HS03C23r_at, Contig25736_at, Contig18440_at,

Contig17647_at, Contig2541_at, Contig7727_at, Contig17218_at

HvHDAC2_1

Table 2 Summary of new gene-specific molecular markers used for mapping BVP gene on chromosome 1H

Marker

name

Derivation Primers sequences (5' to 3') Marker

type

Tm

(°C)

Amplicon

(bp) Forward Reverse

H1 AK250075 TTACAAACGAAC

ACGGAAAA

GAACATGCACG

CATCTGG

SSCP 55 434

H4 AK252360 GAAGGAAGAAA

CAACCCAACT

GGAATTCTAGG

AGGTTGGTGT

SSCP 55 323

M4 Mot1 CGATGGTGAACT

TTTATCTCA

CGATGGTGAAC

TTTTATCTCA

SSCP 55 423

M9 Mot1 CATTTCTGTCCA

CTCCTTAG

TTACCTTGATG

TTATGCTTAG

SSCP 55 404

F1 Ftsh4 GTGTTCCTGGCT

GTTGGT

TCTAATTGATG

CGGAGATTAC

In/Del 55 107

C1 CA608558 GAAGAGGCTGTC

CAACTAT

TTGACCTCTTC

CCTTTTAT

SSR 55 253

W2 wg241 AAAGCCATTGTC

TTAGCAGAG

GGACTCCCCAC

CCAAAAC

In/Del 55 346