frizzy panicle drives supernumerary spikelets in bread ... · 11/14/2014 · 106 primary axillary...

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FRIZZY PANICLE drives supernumerary spikelets in bread wheat (T. aestivum L.) 1

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Oxana Dobrovolskaya1*, Caroline Pont2, Richard Sibout3, Petr Martinek4, Ekaterina Badaeva5, Florent 3

Murat2, Audrey Chosson2, Nobuyoshi Watanabe6, Elisa Prat7, Nadine Gautier7, Véronique Gautier2, 4

Charles Poncet2, Yuriy Orlov1,8, Alexander A. Krasnikov9, Hélène Bergès7, Elena Salina1, Lyudmila 5

Laikova1, Jerome Salse2* 6

7 1 Institute of Cytology and Genetics, SB RAS, Lavrentieva ave. 10, 630090 Novosibirsk, Russia. 8 2 INRA-UBP UMR-1095, 5 Chemin de Beaulieu, 63100 Clermont –Ferrand, Cedex 2, France. 9 3 INRA MR1318 INRA-AgroParisTech, Route de St-Cyr, 78026 Versailles, Cedex, France. 10 4 Agrotest Fyto, Ltd. Havlíčkova 2787, 767 01 Kroměříž, Czech Republic. 11 5 Vavilov Institute of General Genetics , RAS, Gubkina str., Moscow, 3119333, Moscow, Russia. 12 6 College of Agriculture, Ibaraki University, 3-21-1 Chuuo, Ami, Inashiki, Ibaraki 300-0393, Japan. 13 7 INRA, Centre National de Ressources Génomiques Végétales (CNRGV). Chemin de Borde Rouge BP 14

52627, 31326 Castanet Tolosan cedex, France. 15 8 Novosibirsk State University, Pirogova str., 2, 630090, Novosibirsk, Russia. 16 9 Central Siberian Botanical Garden SB RAS, Zolotodolinskaya str., 101, 630090 Novosibirsk, 17

Russia. 18

19 *Corresponding authors: 20

e-mail address: [email protected] 21

Tel. number +7(383)363 49 51 22

Fax number +7(383)333 12 78 23

e-mail address: [email protected] 24

Tel. number +33(0)4 73 62 43 80 25

Fax number +33(0)4 73 62 44 53 26

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Running title: FZP determines spikelet number in bread wheat. 29

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The authors responsible for distribution of materials integral to the findings presented in this article are 31

Oxana Dobrovolskaya ([email protected]) and Jerome Salse ([email protected]). 32

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ONE-SENTENCE SUMMARY 34

Wheat FRIZZY PANICLE (FZP) homoeologous genes (WFZP-A, WFZP-B, WFZP-D) located on the 35

chromosome group 2 drive the yield-related SS (supernumerary spikelets) trait. 36

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Plant Physiology Preview. Published on November 14, 2014, as DOI:10.1104/pp.114.250043

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SUMMARY 39

Bread wheat inflorescences, or spikes, are characteristically unbranched and normally bear one 40

spikelet per rachis node. Wheat mutants on which supernumerary spikelets (SS) develop are particularly 41

useful resources for work towards understanding the genetic mechanisms underlying wheat inflorescence 42

architecture and, ultimately, yield components. Here, we report the characterization of genetically 43

unrelated mutants leading to the identification of the wheat FRIZZY PANICLE gene, encoding a member 44

of the APETALA2/Ethylene Response Factor (AP2/ERF) transcription factor family, which drives the SS 45

trait in bread wheat. Structural and functional characterization of the three wheat FRIZZY PANICLE 46

homoeologous genes (WFZP) revealed that coding mutations of WFZP-D cause the SS phenotype with 47

the most severe effect when WFZP-D lesions are combined with a frameshift mutation in WFZP-A. We 48

provide WFZP-based resources that may be useful for genetic manipulations with the aim of improving 49

bread wheat yield by increasing grain number. 50

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KEY WORDS: FRIZZY PANICLE, Gene, Synteny, Grasses, Wheat, Spike, Inflorescence development, 52

Yield. 53

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INTRODUCTION 55

Bread wheat (Triticum aestivum L., BBAADD) is one of the most important food crops in the 56

world. Its yield in grain production per plants largely depends on the architecture of the inflorescences. 57

Studies on genetic determinism of inflorescence development may allow new spike architectures to be 58

designed, with the aim of enhancing grain production. The wheat inflorescence classically consists in a 59

spike with a main axis (the spike rachis) carrying lateral sessile spikelets that are directly attached to the 60

rachis, and also a terminal spikelet (Figure 1A). The spikelet constitutes the basal unit of the spike 61

inflorescence, and is a characteristic of all modern grasses, except one lineage that diverged a long time 62

ago (Malcomber et al., 2006). The number of spikelets per rachis node is a key taxonomic trait of the 63

Triticeae tribe (Muramatsu et al., 2009; Sakuma et al., 2011). 64

A wheat spike normally bears one spikelet per rachis node, which arises directly on the main 65

inflorescence axis; the formation of supernumerary spikelets (SS) is rare. The term ‘SS’ includes any 66

additional sessile spikelets developed at a rachis node, such as multirow spikes (MRS), horizontal 67

spikelets (HS), and the ramified spike (RS) or branched spike (Figure 1B, Figure S1). In wheat, the 68

development of SS is a recessive trait (Pennell and Halloran, 1983; Klindworth et al., 1990b; 69

Dobrovolskaya et al., 2009) and is modulated by a number of environmental factors (Sharman 1944; 70

Pennell and Halloran, 1983). In tetraploid wheat (BBAA), the SS trait is quantitatively inherited, 71

controlled by a single major gene acting in concert with an unknown number of minor genes (Klindworth, 72

1990b; Haque et al., 2012). It has been suggested that the SS trait in hexaploid wheat (BBAADD) is 73

determined by either two (Pennell and Halloran, 1983) or three (Koric 1980) loci. The SS trait in 74

hexaploid wheat has been reported to be a consequence of the absence of a complete chromosome 75

(Swaminathan et al., 1966). The involvement of the homoeologous chromosome group 2 in the control of 76



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SS has been suggested in both tetraploid and hexaploid wheats (Sears 1954; Klindworth et al., 1990a; 77

Peng et al., 1998; Laikova et al., 2005; Yang et al., 2005). Genetic loci linked to the SS trait have been 78

located on the chromosome 2AS in hexaploid (qTS2A-1), tetraploid (bh gene) and diploid wheats (bhm 79

gene), and on the chromosome 2D in hexaploid bread wheat (Li et al., 2011; Haque et al., 2012; Amagai 80

et al., 2014; Dobrovolskaya et al., 2009). 81

The Multirow Spike (MRS) phenotype is one of the SS traits of the bread wheat spike (Figure 1C, Figure 82

S1). The primary feature of MRS lines is that they have a large number of spikelets emerging from each 83

rachis node, in most cases in the lower third of the spike (Figure 1B, 1C). The central and upper thirds of 84

the spike generally have only three spikelets per node, similar to the spike architecture of six-row barley 85

lines (Tanno and Takeda 2004). The mrs1 gene, on chromosome 2DS, may be the major determinant of 86

the MRS trait in two genetically related bread wheat lines derived from genotypes obtained by chemical 87

mutation (Dobrovolskaya et al., 2008, 2009). This gene is also known as bh-D1 (Jia et al., 2013) as it is 88

considered to be a mutant allele of the Bh-D1 locus (http://ccg.murdoch.edu.au/cmap/ccg-live). 89

Here, we report the characterization of genetically unrelated Multirow Spike (MRS) mutants that led to 90

the identification of the wheat FRIZZY PANICLE homoeologous genes; we provide evidence that these 91

genes are responsible for SS traits in bread wheat. Structural and functional characterization of the three 92

wheat FRIZZY PANICLE homoeologous genes (WFZP) implicates WFZP as a key player in spikelet 93

development during the floret meristem transition phase. 94

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RESULTS 96

The mrs1 mutant allele affects spikelet development. 97

The 13 MRS lines used in this work all have numerous supernumerary spikelets (SS) emerging 98

from a rachis node (Figure 1B). We developed a near-isogenic line (NIL), NIL-mrs1, which carries a 17 99

cM segment that includes the mrs1 locus in the cv. Novosibirskaya 67 (N67) background (Figure S1, S2). 100

NIL-mrs1 and N67 only differ in inflorescence structure, with NIL-mrs1 showing severe SS phenotypes 101

with numerous supernumerary spikelets emerging at rachis nodes in the lower half of the spike (Figure 102

S1); the additional spikelets were not sessile as they developed at very short branches (Figure 1C). At the 103

earliest stages of spike development, NIL-mrs1 inflorescence appeared phenotypically normal (Figure 1, 104

D-E-J-M-N panels). At the spikelet differentiation stage, the inflorescence meristem (IM) produced 105

primary axillary meristems (AxM), which first initiated glume primordia (Figure 1, D panel); at the early 106

floret differentiation stage (Figure 1, E-M-N panels) secondary axillary meristems (AxM) were produced 107

that could not be distinguished from normal floret meristem (FM) in the wild-type inflorescence (Figure 108

1, L panel). In the wild type (WT), the FM first initiated lemma, and then formed the other floral organs 109

(Figures 1, O- R panels). In contrast, in the NIL-mrs1 inflorescence, basal secondary axillary meristems 110

developed as ectopic spikelet meristems and first produced spikelet organs, the glumes (Figure 1, F-P 111

panels), and then generated the other spikelet organs (Figure 1, G-Q-S panels). 112

Thus, supernumerary spikelets in NIL-mrs1 developed exactly at the location of florets of primary 113

spikelets. The development of the basal florets was replaced by ectopic spikelets in the middle part of the 114



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spike rachis, whereas the development of most or all florets of primary spikelets were replaced by ectopic 115

spikelets in the lower part of the spike rachis and, thus, branch-like structures were formed (Figure 1, H-I 116

panel). These branch-like structures were not encompassed by normal glumes, and rudimentary glumes 117

could be distinguished only at early stages of the spike development (Figure 1, P-Q panels). Most ectopic 118

spikelets developed as wild-type spikelets and produced florets. Within the ectopic spikelets, located in 119

the basal part of the branch-like structure, the development of basal florets could be replaced by 120

secondary ectopic spikelets (Figure 1C). The meristems of ectopic spikelets were formed at 90° (Figure 1, 121

P-S panels) to normal spikelets or to the previous order ectopic spikelet. In line MC1611, with a weak SS 122

phenotype, ectopic spikelets developed at the location of basal florets of the lower part of the spike 123

(Figure S3). The other genetically unrelated SS lines used in this work, Skle128, Ruc204, So164, showed 124

phenotypes similar to NIL-mrs1 and the line MC1611 (next section, Figure S1). 125

The 2DS locus is the main contributor to the SS trait in bread wheat. 126

In addition to the MRS lines (mrs1) described above, we also genetically characterized four 127

unrelated SS bread wheat lines: MC1611, Skle128, Ruc204, and So164 (Figure S1). Line MC1611 is a 128

mutant produced by NMU-mutagenesis (NMU, for NitrosoMethylUrea), line Skle128 was derived from 129

spontaneous mutation(s), and line So164 was derived from a cross with a branched tetraploid wheat (see 130

the Methods section for detailed descriptions of these lines). C-banding and SSR genotyping 131

characterization revealed structural rearrangements of chromosome 2D in three of these four lines 132

(Figures S4-S5). The C-banding analysis revealed structural rearrangements on the short arm of the 133

chromosome 2D in both Skle128 and Ruc204 (Figure S4), and that chromosome 2D was completely 134

absent from So164, making it nulli2D-tetra2A (Figure S5). The short arm of chromosome 2D in the line 135

Ruc204 was clearly short (~10% shorter than controls), associated with a diagnostic C-band at the 2DS 136

termini, proof of an interstitial deletion. The other chromosomes in Ruc204 were morphologically 137

indistinguishable from those in normal, reference lines (Figure S4). The deletion in the short arm of 138

chromosome 2D in line Skle128 was much larger than that in line Ruc204, such that the arm was half the 139

size of that in controls (Figure S4). The presence of these deletions was confirmed, and the location, of 140

deletion breakpoints were determined precisely, by SSR genotyping of the 2DS chromosomes in these 141

lines (Figure S4). 142

We performed genetic mapping with F2 populations derived from Ruc204 x Saratovskaya 29 (S29) and 143

Skle128 x S29 crosses to investigate the contributions of these deletions to the SS trait (Supplemental 144

Information, SI document). The two deletions identified were found to be related to the SS phenotype, 145

and explained ~50% of the phenotypic variation (Figure S6). The deletions observed on the chromosome 146

2D genetic map colocate with the mrs1 locus (Figure S7). In addition to the 2D QTL, the 2A QTL, which 147

explained ~ 16% of the phenotypic variation, was detected in Ruc204. The 2A QTL, also responsible for 148

the SS trait, has also been described in the TTSW line (with Skle128 originating from TTSW, Li et al., 149

2011). NIL-mrs1 was crossed to each of the two SS deletion lines to conduct genetic complementation 150

tests. All the F2s produced expressed the SS phenotype, demonstrating that the SS phenotypes of the three 151

lines share the same genetic determinant (Table S1). The segregation ratios for the F2s and BC1s crosses, 152



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as well as for the MC1611 mutant line, which was obtained in a S29 background by NMU-mutagenesis, 153

clearly indicate that the SS trait in MC1611 is due to the mutation of a single gene (Table S1). Similarly, 154

MC1611 was crossed with standard spiked cv. Skala and the observed F2 segregation was close to 155

monogenic (Table S1). Genetic mapping data indicated that the gene responsible for SS was on 2DS and 156

co-located with the mrs1 locus (Figure S7). Genetic complementation tests between MC1611 and NIL-157

mrs1 revealed that, in addition to the 2DS gene, other secondary genetic factors may also contribute to the 158

SS trait (Table S1). Nevertheless, our findings for genetically unrelated SS genetic materials (NIL, bread 159

wheat lines, bi-parental populations, and independent mutants) implicate the 2DS gene/locus as the main 160

determinant of the SS trait in bread wheat, with the driving gene (mrs1 in the current study) still to be 161

rigorously identified. 162

Dissection of the FZP locus and identification of the gene in bread wheat. 163

Accurate genetic maps that included the mrs1 gene (Dobrovolskaya et al., 2009) were used for 164

further synteny-based identification of SS candidate markers using the Conserved Orthologous Set (COS) 165

genes available in bread wheat (Quraishi et al., 2009, Pont et al., 2013). The mrs1 locus is flanked by the 166

COS1 (LOCOs07g38530-Bradi1g23970-Sb02g037070), COS2 (LOCOs07g45064) and COS3 167

(LOCOs07g48200-Bradi1g17680-Sb02g043000) markers (Figure 2A). The COS2-COS3 region shares 168

synteny with the rice chromosome 7, Brachypodium chromosome 1, and sorghum chromosome 2 regions 169

(Figure 2B). The identification of unique orthologous regions in rice, sorghum and Brachypodium 170

provided a list of candidate genes (Table S2). The rice region hosts a FRIZZY PANICLE (FZP) homolog 171

that encodes a transcription factor of the APETALA2/Ethylene Response Factor (AP2/ERF, Tanaka et al., 172

2003) family; these factors contribute to determining inflorescence development and spikelet meristem 173

identity in rice. In rice fzp mutants, the formation of florets is replaced by sequential rounds of branching 174

(Komatsu et al., 2003). The fzp mutant phenotype suggests that FZP is required to prevent the formation 175

of axillary meristems within the spikelet meristem and to allow the subsequent establishment of floral 176

meristem identity (Komatsu et al., 2003). The maize gene branched silkless1 (bd1) is an FZP ortholog, 177

and the BD1 protein is associated with a conserved function in spike development (Chuck et al., 2002; 178

Komatsu et al., 2003). Based on shared synteny between grasses at the mrs1 locus and the observed 179

similarity in mutant phenotypes between fzp/bd1 and mrs1, the bread wheat FZP gene (namely, WFZP) 180

appeared to be the strongest candidate for the MRS trait. 181

Homoeologous WFZPs were isolated from a bread wheat BAC library (T. aestivum L. cultivar ‘Chinese 182

Spring’, see Methods), using Brachypodium as a model genome for the Triticeae to develop a new set of 183

COS markers for further gene cloning and sequencing. The BLAST search did not find significant 184

orthologous wheat sequences in public databases, and therefore primers were designed from the 185

conserved regions of the orthologous FZP genes in grasses (Table S2). These primers were used to screen 186

the bread wheat BAC library from Chinese Spring genotype 187

(http://cnrgv.toulouse.inra.fr/fr/library/genomic_resource/Tae-B-Chinese%20spring). BAC clones 188

harboring WFZP genes were identified, and three carrying the WFZP homoeologs from the A, B and D 189

subgenomes were sequenced. All the WFZPs consist of single exons with an AP2/ERF domain that is 190



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conserved in grass relatives (Figure 2D, Figure S8). The WFZP-B gene carries 278-bp and 323-bp 191

transposable element (TE) insertions in the proximal promoter sequence, 114 bp and 320 bp upstream 192

from the TATA-box, respectively. The WFZPs showed organ-specific and stage-specific expression 193

patterns. WFZP transcripts were detected in young spikes, with the highest level of expression observed at 194

the early floret developmental stage (Figure 3D). Homoeologous WFZP genes were found in the 2AS, 195

2BS and 2DS chromosome bins with genome-specific primer pairs (Figure S9) and DNA from Chinese 196

Spring deletion lines. Genetic mapping indicated that WFZP-D was at the expected position on 2DS, co-197

locating with the mrs/qSS loci on all genetic maps constructed (Figures S7-9). 198

Coding and null-mutations in WFZP-D are associated with the SS phenotype. 199

We investigated the relationship between the WFZP sequences in the genetically characterized SS 200

lines and the SS trait. Analysis of the WFZP BAC clone sequences allowed us to develop a set of 201

sequence-specific primers for further re-sequencing of WFZP homoeologous genes in bread wheat 202

cultivars (Table S3, Figure S10). Direct Sanger sequencing was used to determine the sequences of the 203

PCR products of WFZP-A, WFZP-B and WFZP-D genes in eleven bread wheat lines, including seven 204

mutants (Ruc163, Ruc167, NIL-mrs1, MC1611, Ruc204, Skle128, and So164) and four normal spiked 205

lines (Renan (Rn), S29, N67, and So149). Thus, we obtained the sequences of the FZP genes in SS and 206

normal spiked parents of the populations used for mapping purposes, NIL-mrs1 and its parent (N67), the 207

NMU-induced mutant (MC1611) and the WT line (S29). 208

WFZP-D - The WFZP-D gene structure differed between mutant and normal spiked lines (Figure 3A-C). 209

The WFZP-D gene was completely deleted from three of the seven SS lines was found (Figure 3B), due 210

either to a deletion from 2DS or a substitution of the entire 2D chromosome (Figures S4-5). A mutation in 211

the GCC-box binding site of the AP2/ERF functional domain of WFZP-D was found in three genetically 212

related mutant MRS lines, including the NIL-mrs1 (Figure 3A-C, Figure S10). Genotyping, using 213

sequence-specific primers (Table S3), revealed this non-synonymous substitution in the other ten MRS 214

lines (listed in the Methods section) and in all of the MRS F2 plants resulting from Ruc167 x So149 and 215

Ruc163 x So149 crosses. This allele was named wfzp-D.1. The NMU–induced mutant, MC1611, was 216

found to carry a premature stop codon in the AP2/ERF functional domain of WFZP-D (Figure 3B-C and 217

Figure S10). The WFZP-D sequences in 96 BC1s derived from the cross between MC1611/S29//MC1611 218

were determined: all the SS plants harbor the mutant allele, now named wfzp-D.2, whereas the standard 219

spiked plants were heterozygous for the WFZP-D wfzp-D.2 allele. Thus, the mutant allele wfzp-D.2 is 220

associated with the SS phenotype in the MC1611 line (obtained by NMU-mutagenesis of S29 considered 221

here as WT). Overall, SS lines were either null-mutants for WFZP-D or harbored G-A or C-T mutations 222

in the functional AP2/ERF domain of the FZP gene (Figure 3B). 223

WFZP-B - The sequencing of WFZP-B in bread wheat lines revealed that, independent of their spike 224

morphology, all contained MITEs (miniature inverted-repeat transposable elements) inserted (114 bp and 225

320 bp upstream from the TATA-box) into the proximal promoter region of FZP; this was associated 226

with major reorganizations in the promoter structure. Expression tests revealed that the abundance of 227

WFZP-B transcripts was very low in the developing spikes of cv. Chinese Spring (as little as 5% of the 228



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amount of WFZP-D transcript; Figure 3D). Thus, in bread wheat, the WFZP-B gene is largely inactive. 229

These observations are consistent with the possibility that the insertion of the MITE into the promoter 230

region is responsible for the loss of WFZP-B homoeolog expression and even its silencing. 231

WFZP-A - In WFZP-A, there was a 14 bp-deletion 40 bp upstream from the functional AP2/ERF domain, 232

which results in a frameshift, in six of the seven SS lines (the exception was MC1611) investigated and in 233

one normal spiked line, So149 (Figure 3A-B). Primers were designed (Table S3) to test for this deletion 234

and were used to screen 48 bread wheat cultivars with standard spike morphology (listed in Table S4): 235

6.3% of these cultivars carried the deletion. Thus, the deletion is part of the natural variation in bread 236

wheat and does not by itself cause phenotypic changes; this allele was named wfzp-A.1. However, we 237

observed that the combination of wfzp-A.1 with wfzp-D.1 or with wfzp-D.null alleles resulted in a severe 238

SS phenotype in MRS lines, SS deletion lines and SS substitution lines; the combination of the wild allele 239

WFZP-A and the mutant allele wfzp-D.2 in line MC1611 led to only a weak SS phenotype (Figure 1 and 240

Figure 3A-C). The segregation patterns observed in the crosses between standard spiked lines and 241

SS/MRS lines confirmed the WFZP-A and WFZP-D allele composition of the lines (Table S1). Thus, F2s 242

derived from the crosses between the standard spiked parental line So149 (wfzp-A.1 WFZP-D) and the 243

MRS lines, Ruc163, Ruc167, (wfzp-A.1 wfzp-D.1) showed segregation of 3 (standard spike) to 1 (MRS). 244

Unlike the parent line N67 (WFZP-A WFZP-D), NIL-mrs1 harbors mutant alleles at both the wfzp-A.1 245

and wfzp-D.1 loci. The various mapping and sequencing results provide evidence that WFZP-D is 246

synonymous with MRS1, and that the wfzp-D.1 and wfzp-D.2 alleles are associated with the MRS/SS 247

phenotype in bread wheat. The WFZP-D transcript was four times more abundant than the WFZP-A/B 248

transcripts (Figure 3D), consistent with the WFZP-D homoeoallele making the largest contribution to the 249

MRS/SS trait. 250

Finally, we screened the collection of mutagenized Brachypodium lines from INRA-Versailles 251

(http://www-ijpb.versailles.inra.fr/en/plateformes/crb/index.html) for aberrant spikelets as described in 252

Dalmais et al. (2013). Two lines, Bd8202 and Bd8972 showed a supernumerary spikelets phenotype but 253

produced viable seeds (Figure 4). The full sequence of Bradi1g18580 gene was amplified by PCR from 254

the genome of Bd8202, Bd8972 and WT (Bd21-3) lines, and determined. Two transitions were detected: 255

G-232-A in Bd8972 and G-163-A in Bd8202. These mutations lead to amino acid substitutions in the 256

conserved region of the AP2/ERF domain (Figure 4). These results are consistent with the report that 257

deletion in the Bradi1g18580 promoter causes inflorescence branching (Derbyshire and Byrne 2013). Our 258

findings with the closely related Brachypodium as a validation system confirm the role of FZP and more 259

precisely AP2/ERF domain function in the determination of spike architecture in Triticeae. 260

261

DISCUSSION 262

The inflorescence of cereals includes a unique structure called the spikelet. In bread wheat, 263

spikelets are arranged as two opposite rows on the main axis, the rachis. Each spikelet is composed of 264

florets joined at the indeterminate spikelet axis (rachilla), alternately on opposite sides, and flanked by 265

two bracts, the glumes. Normally, the spikelet meristem gives rise to glume primordia and then to the 266



8

floret meristem. The floret meristem, in turn, differentiates into floret organs such as a lemma, a palea, a 267

pair of lodicules, three stamens and a pistil. Spikes in the SS mutant lines display supernumerary spikelets 268

developed on very short branches. We compared the anatomy of young spikes of the line NIL-mrs1 269

presenting a severe SS phenotype of the MRS type and its normal-spiked parent, N67: differences in their 270

morphological development were investigated by light microscopy and SEM. Ectopic spikelets developed 271

at the location of florets in NIL-mrs1, in all cases at 90° to the normal spikelets, or to ectopic spikelets of 272

the previous order, suggesting that the defect in the mrs1 mutant occurs soon after the acquisition of the 273

spikelet meristem identity. Our observations suggest that the mrs1 mutant allele affects spikelet 274

development and disturbs the establishment of the floral meristem identity. 275

In all the SS wheat lines studied in this work, the supernumerary spikelets develop in the same way, at the 276

location of florets, at 90° to normal spikelets. The number of additional spikelets and, thus, the severity of 277

the SS phenotype of these lines, depend on: (i) the number of florets that are replaced by ectopic 278

spikelets, (ii) the number of mature ectopic spikelets and (iii) the number of spikelets with hypoplasia. 279

Similar SS phenotypes have been reported in tetraploid wheats. Cofmann (1924) described spontaneous 280

mutants of the Mindum durum wheat variety characterized by the presence of one or two additional 281

spikelets at several rachis nodes in the lower part of the spike. The spike phenotype of these spontaneous 282

mutants is similar to SS in bread wheat. The Four-Rowed Spike (FRS) trait has been described in 283

Triticum turgidum L. (Zhang et al., 2013), where in FRS plants two spikelets develop at a rachis node in 284

the “tetrastichon” manner. It seems likely that the same developmental mechanisms are involved the 285

appearance of supernumerary spikelets in both hexaploid and tetraploid wheats. 286

The MRS trait has been shown to be determined by a single recessive mrs1 gene on chromosome 2DS 287

(Dobrovolskaya et al., 2009). Here, we genetically characterized four bread wheat SS lines, in addition to 288

the mrs1 mutant, that included both induced and spontaneous mutants. We show that different SS 289

phenotypes in fact reflect the same series of related traits with a common genetic determinism, and that 290

the 2DS locus is the main contributor to the SS trait in bread wheat. In 1954, Sears described the 291

“reduplication of spikelets” in hexaploid wheat plants nullisomic for chromosomes 2A or 2D (Sears 292

1954). The involvement of the homoeologous chromosome group 2 in the control of SS in both tetraploid 293

and hexaploid wheat was subsequently suggested (Klindworth et al., 1990a, 1997; Peng et al., 1998; 294

Laikova et al., 2005; Muramatsu et al., 2009). The frs1 gene for the FRS trait and the bh (branched head) 295

gene have now been mapped on chromosome 2AS of tetraploid wheats, Triticum turgidum L. (Zhang et 296

al., 2013) and Triticum durum Desf. var. ramosoobscurum Jakubz. (Haque et al., 2012). Consequently, 297

the general view is that the 2AS and 2DS homoeologous loci are responsible for the SS trait in bread 298

wheat. 299

We used a combination of molecular genetic mapping, analyses based on the conservation of synteny and 300

phenotypic characterization of independent SS mutants to study the gene underlying SS-related traits. We 301

mapped the mrs1 locus on the chromosome 2DS using COS markers and showed that the mrs1 region 302

shared synteny with the rice chromosome 7, carrying the FRIZZY PANICLE (FZP) gene. In rice fzp 303

mutants, supernumerary axillary meristems are formed in axils of bracts in young spikelet meristems, and 304



9

the formation of florets is replaced by sequential rounds of branching (Komatsu et al., 2003). Rao and co-305

authors (2008) confirmed that FZP represses axillary meristem formation. The gene branched silkless1 306

(bd1) is the maize ortholog of FZP (Chuck et al., 2002), and bd1 mutants show a similar phenotype 307

involving the initiation of extra spikelets in the tassel, and, in the ear, spikelet meristems are replaced by 308

branch meristems (Chuck et al., 2002). Derbyshire and Byrne (2013) isolated a Brachypodium mutant 309

(mos1) with similar defects in spikelet architecture; although no mutation in the coding region of FZP 310

was found in this mutant, the authors proposed that the larger number of axillary meristems is due to a 311

slightly but significantly lower expression of this AP2/ERF transcription factor than in the WT (20% less 312

transcripts in the mos1 mutant). Derbyshire and Byrne suggested that this abnormal expression may be 313

the consequence of a chromosome rearrangement affecting the promoter region of FZP. Here, we 314

reported two new mutants sharing mutations in the highly conserved AP2/ERF domain of FZP, and 315

associated with an increased number of lateral floral meristems. These findings are consistent with the 316

FZP gene acting in the same way as branched silkless1 and the FRIZZY PANICLE gene in 317

Brachypodium. The identification of two independent mutant alleles (Bd8202 and Bd8972) within the 318

AP2/ERF domain associated with a supernumerary spikelet phenotype provides clues about the role of 319

FZP and, more precisely, implicate AP2/ERF domain functionality in the maintenance of spike 320

architecture. Homologs of the BD1 gene have been isolated from sorghum BAC libraries, and from 321

Setaria italica (foxtail millet), Panicum miliaceum (common millet), Avena sativa (oats), Eleusine 322

coracana (finger millet) by PCR with degenerate primers (Chuck et al., 2002). FZP (Triticeae), bd1 323

(maize), and mos1 (Brachypodium) genes encode orthologous transcription factors of the AP2/ERF 324

family (Chuck et al., 2002; Komatsu et al., 2003) showing complete amino acid identity within the 325

AP2/ERF domain, and with up to 75% identity (Chuck et al., 2002) with Bradi1g18580, and 59% with 326

BD1 and 54% with FZP (Derbyshire and Byrne, 2013). 327

T. aestivum (AABBDD) is a natural allohexaploid species originating from three diploid ancestral 328

species: the A genome is derived from Triticum urartu, the D genome from Aegilops tauschii and the B 329

genome is from an unknown progenitor closely related to Ae. speltoides (Feldman, 2001; Feldman and 330

Levy, 2005). We isolated and sequenced WFZP-A-B-D gene copies and thereby established that they are 331

associated with a single exon structure as in rice, maize and Brachypodium. Interestingly, the loci that 332

were genetically associated with the SS trait in tetraploid and hexaploid wheats map on chromosomes 2A 333

and 2D, consistent with our finding that WFZP-B is not functional. Quantitative RT-PCR showed 334

consistently stronger expression (more abundant mRNA) of WFZP-D than WFZP-A, also in agreement 335

with the 2DS locus being the main contributor to the SS/MRS phenotypes in hexaploid wheat. Thus, our 336

analyses revealed that the three homoeologous gene copies in wheat do not all have the same roles in 337

spike morphology. In rice, the alteration of one of six conserved amino acids of the GCC box binding site 338

(fzp-2 and fzp-7 mutants) in the AP2/ERF domain leading to a premature stop codon (fzp-1 and fzp-4 339

mutants) resulted in severe fzp phenotypes (Komatsu et al., 2003). In contrast, we report that, in wheat, a 340

G-A mutation in the GCC-box binding site of the AP2/ERF functional domain (wfzp-D.1) of WFZP-D, a 341

C-T mutation in AP2/ERF (wfzp-D.2) of WFZP-D and complete deletions of the WFZP-D locus (null-342



10

mutations) are associated with SS phenotypes. In bread wheat, the wfzp-D.1 and wfzp-D-null mutations in 343

combination with a frameshift mutation in the WFZP-A gene copy resulted in severe SS phenotypes, 344

whereas the wfzp-D.2 mutation in combination with the WT WFZP-A allele resulted in only a weak SS 345

phenotype. These results agree with the 2DS locus being the main contributor to the SS phenotype. The 346

WFZP mutations described here control the development of supernumerary spikelets from ectopic 347

spikelet meristems in all the SS-related bread wheat mutants investigated in this study. However, the 348

number of supernumerary spikelets and length of branch-like structures, which distinguish the different 349

SS phenotypes (HS, RS, MRS) may be determined by other, as yet unknown, genes. Note also that the 350

function of the FZP orthologs appears to be conserved among cereal species, including bread wheat, rice, 351

maize, and Brachypodium (Figure 5). 352

Finally, we report structural and functional characterization of the WHEAT FRIZZY PANICLE gene in 353

wheat (T. aestivum L.) and demonstrate that it is one of the key regulators of inflorescence development, 354

and a determinant of supernumerary spikelet formation. Therefore, it may serve as a basis for the 355

development of providing new resources for the genetic manipulation of the grain number, one of the 356

major components of yield. Standard wheat morphotypes respond to environmental changes, such as the 357

availability of resources, using three levels of branching: tillers, spikelets in the spike, and grains in the 358

spikelet. These metameric organs allow plants to respond to environmental constraints during growth and 359

development (Acevedo et al., 2002). The hierarchical organization of plants (Nicolis et al., 1977) ensures 360

their reproduction even in unfavorable conditions where increases in apical dominance, like inter- and 361

intra-plant competition, decreases crop yield (Miralles and Slafer, 2007). The clusters of spikelets in 362

multirow spike (MRS) lines constitute another metameric structure: the increased numbers of spikelets 363

may contribute to yield stability especially when under the influence of environmental constraints. 364

Indeed, the yields of MRS lines remain more stable than those of standard varieties if the doses of 365

fertilizers and pesticides applied during wheat development are altered (Martinek and Váňová, 2012). 366

Repetitive marker assisted back-crossing of the MRS donors with selected elite varieties with standard 367

spike structure, using the WFZP markers described here, will rapidly lead to the development of new 368

breeding lines with multirow spikes, and improved genetic backgrounds and yield potential. 369

370

EXPERIMENTAL PROCEDURE 371

The experimental procedures may be found in the online version (in Supporting Information file) of 372

this article in detailing: (i) Plant materials, (ii) Light microscopy and SEM analysis, (iii) C-banding 373

procedure (iv) Primers, (v) Mapping procedure, (vi) COS-SSCP analysis, (vii) BAC clone screening, 374

sequencing and annotation (viii) Re-sequencing, (ix) RT-PCR, (x) Brachypodium mutant identification 375

and sequencing, (xi) qSS mapping and (xii) WFZP expression pattern analysis. 376

377

FUNDING AND ACKNOWLEDGEMENT 378

We thank Dr. V.M. Melnick (Altai Research Institute of Agriculture SB RAAS, Barnaul, Russia) 379

for providing the MC1611 mutant line, O.M. Popova (Institute of cytology and genetics SB RAS (IC&G 380



11

SB RAS), Novosibirsk, Russia) and P. Desray (INRA-UBP UMR-1095, Clermont-Ferrand, France) for 381

technical assistance, Dr. P. Sourdille (INRA-UBP UMR-1095, Clermont-Ferrand, France) for providing 382

DNA of F2s from the Chinese Spring x Renan cross, Dr. A. Didier (INRA-UBP UMR-1095) for 383

providing information about normal-spiked bread wheat cultivars used in our research, and Prof. T.I. 384

Aksenovich, IC&G SB RAS, Novosibirsk, Russia, for helpful suggestions on the paper. This work was 385

supported by grants from the Russian Foundation for Basic Research (Projects 10-04-01458-а and 12-04-386

00897-а), the Agence Nationale de la Recherche (Programs ANRjc-PaleoCereal, ref: ANR-09-JCJC-387

0058-01 and program ANR Blanc-PAGE, ref: ANR-2011-BSV6-00801) and the program of Presidium of 388

Russian Academy of Sciences ''Molecular and cell biology'' (project 6.14). O.D. acknowledges the French 389

Embassy in Russia, Department for Science, Technology and Space for a Mobility grant (2012). P.M. 390

thanks project RO0211 of the Ministry of Agriculture of the Czech Republic. We thank Sébastien 391

Antelme, Philippe Le Bris, and Aurélie Lemaire for taking care of plant cultures and genotyping 392

Brachypodium mutants referenced in the article. 393

394

AUTHOR CONTRIBUTIONS 395

O.D. and J.S. designed and managed the project. P.M. developed the SS lines. N.W. developed 396

the NIL-mrs1 line. R.S. carried out Brachypodium mutant identification and sequencing. O.D., L.L. and 397

Y.O. designed and performed genetic mapping experiments. E.B. performed C-banding experiments. C.P. 398

selected COS markers, O.D. and C.P. participated in the genotyping and gene cloning experiments. E.P., 399

N.G. and H.B performed the BAC clone screening and V.G. and C.P. BAC clone sequencing. F.M. and 400

A.C. performed the sequence annotation and synteny analysis. O.D. and C.P. contributed to the 401

expression analysis. A.A.K. and O.D. contributed to the SEM analysis. O.D. carried out re-sequencing. 402

E.S. contributed to the writing of the manuscript. O.D. and J.S. wrote the manuscript with input from all 403

the coauthors. 404

405

SUPPORTING INFORMATION 406

Supplemental Information. Additional and background information 407

Table S1. Segregation data for the numbers of normal-spiked (NS) and supernumerary spikelet (SS) 408

plants in the F2 and BC1 progenies. 409

Table S2. List of orthologous genes (rice, sorghum, maize, Brachypodium) at the Mrs1 locus. 410

Table S3. Primer sequences derived from FZP-A-B-D gene copies. 411

Table S4. WFZP-A alleles characterized in bread wheat cultivars. 412

Table S5. SS phenotypes and associated mutations in WFZP-D. 413

Figure S1: Spike morphology of bread wheat lines with different SS phenotyes. 414

Figure S2. Genetic mapping of the NIL-mrs1 line on the chromosome 2D. 415

Figure S3: Light microscopy image of the MC1611 inflorescence. 416

Figure S4. Deletion breakpoint mapping and ideograms for the Skle128 (a) and Ruc204 (b) lines on 417

chromosome 2D. 418



12

Figure S5. Ideogram of the So164 line. 419

Figure S6. Frequency distribution of supernumerary spikelets in Skle128 x S29 and Ruc204 x S29 F2 420

mapping populations. 421

Figure S7. mrs1 gene and QTL mapping in five F2 mapping populations. 422

Figure S8. Alignment of wheat WFZP gene copies with rice and maize orthologs. 423

Figure S9. Deletion bins and genetic positions of WFZP homoeologous genes. 424

Figure S10. Alignment of deduced WFZP-A, WFZP-B and WFZP-D amino-acid sequences. 425

426

FIGURE LEGENDS 427

Figure 1: Supernumerary spikelet (SS) phenotypes in bread wheat. (A) Schematic representations of 428

a spike (left) and of a spikelet (right) from the bread wheat cultivar Novosibirskaya 67, and of a 429

theoretical wild-type (WT) spikelet (boxed). (B) Schematic illustration of various supernumerary spikelet 430

(SS) structures: a cluster of spikelets at a rachis node referred to as a multirow spike (MRS); three 431

spikelets (triple-spikelet) and two spikelets in horizontal positions at a rachis node referred to as 432

horizontal spikelets (HS); additional sessile spikelets with a lateral branch bearing spikelets at a node 433

referred to as a ramified spike (RS); and a single spikelet at a node for the WT spike. (C) Illustration of 434

the spike structure in the near isogenic line NIL-mrs1 harboring numerous supernumerary spikelets (left); 435

a rachis node with additional spikelets (right) where supernumerary spikelets are indicated with red 436

arrows; and schematic representation of a branch-like structure (b) bearing ectopic spikelets at a rachis 437

node in the lower part of the NIL-mrs1 spike (box). (D-G) Light microscopy images of the NIL-mrs1 438

inflorescence at several developmental stages. (D) Illustration of the spikelet differentiation stage; green 439

arrows indicate glume primordia. (E-F) Early floret differentiation stage. (E) Secondary axillary 440

meristems that later produce ectopic spikelets are indicated with asterisks. (F) Spikelet meristems of 441

ectopic spikelets form glume primordia. (G) Late floret differentiation stage when all floral organs of 442

wild-type spikelets (ws) are differentiated in the upper part of the inflorescence. (H) A branch-like 443

structure dissected from a rachis node of the NIL-mrs1 inflorescence, · – ectopic spikelet (es). (I) 444

Location of ectopic spikelets at rachis nodes of the NIL-mrs1 inflorescence, Bars = 0.25 mm. (J-S) SEM 445

images of Novosibirskaya 67 (WT) and NIL-mrs1 (mrs1) inflorescences at various developmental stages. 446

(J, K) Spikelet differentiation stage in (J) WT and (K) mrs1. (L) Early floret differentiation stages when 447

the spikelet meristem produces the floret meristem in WT and (M, N) differentiation of second axillary 448

meristems (indicated by asterisks) in the mrs1 mutant; (O) Early floret differentiation stage (WT), 449

showing lemmas, and (P, Q) the development of glumes (gl* and indicated by white arrows), lemma and 450

floret meristems by second axillary meristems in the mrs1 mutant. (R) Floret differentiation stage 451

showing differentiated floral organs in a basal floret of the WT and (S) the development of ectopic 452

spikelets in mrs1, Bars =100 μm. Abbreviations: Axm – axillary meristem, b – branch-like structure, es – 453

ectopic spikelet, , es2nd – ectopic spikelet of the second order, developing at the place of a floret of the 454

first order ectopic spikelet (es), f – floret with floret organ promordia, fm – floret meristem, gl – glume, 455

im – inflorescence meristem, l – lemma, lo – lodicule, pa – palea, pi – pistil, ts – terminal spikelet, sm - 456



13

spikelet meristem, st – stamen, sm*, fm*, gl*, f*, l* – spikelet meristem, floret meristem, glume, floret of 457

an ectopic spikelet and lemma, respectively. 458

Figure 2: WFZP gene characterization in bread wheat. (A) Composite genetic map of the wheat 459

chromosome 2D, including the mrs1 locus and flanking COS markers. (B) Orthologous chromosomes in 460

rice (Chr7), sorghum (Chr2) and Brachypodium (Chr1) are shown, with conserved genes linked with 461

black lines. (C) Annotated BAC clones are illustrated with conserved genes (black boxes) linked with 462

black connecting lines; Copia (in green), Gypsy (in blue), uncharacterized (in yellow) and class II (in red) 463

transposable elements are indicated. (D) Comparison of WFZP homoeologous gene structures with red 464

boxes highlighting the conserved AP2/ERF domain. – small deletion; – small insertion; – insertion 465

of TEs; red and blue lines indicate non-synonymous and synonymous single nucleotide substitutions, 466

respectively. Red arrows indicate the positions of primers designed from the Brachypodium 467

(Bradi1g18580) gene sequence, and that were used for BAC library screening. 468

Figure 3: Structural and functional characterization of WFZPs. (A) Schematic representation of the 469

WFZP-A and WFZP-D gene structures and the various mutations. The light red box indicates the 470

AP2/ERF domain, and gray boxes indicate deletions. (B) WFZP haplotypes of SS (in red)) and standard 471

spiked (NS in green) lines. (C) Alignment of the amino acid sequences of the AP2/ERF domains of wild 472

type, WFZP-D, and wfzp-D.1, wfzp-D.2 mutants. * - amino acids that confer specificity to the GCC-box 473

binding site. (D) Relative abundance of WFZP homoeolog mRNAs as determined by quantitative RT-474

PCR with samples from spikes of cv. Chinese Spring at various developmental stages of the spike (see 475

below) and from roots and leaves. The values shown correspond to the mean values for three biological 476

and three technical replicates, normalized to the value for the Ta.304.3 gene used as a reference. Error 477

bars represent SE (standard errors) between replicates. Spike developmental stages were assigned as 478

follows: SD – spikelet differentiation stage (when spikelets are differentiated to form two opposite rows 479

at the rachis and glume primordia are initiated), EFD1 – early floret differentiation stage 1 (spikelet 480

meristems initiate floral meristems and lemma primordia are apparent); EFD2 – early floret differentiation 481

stage 2 (floral organ primordia are apparent in basal florets); LFD - late floret differentiation stage (floral 482

organs are differentiated in each floret). 483

Figure 4: Functional validation of FZP in Brachypodium. FZP mutant screening identified two 484

Brachypodium alleles with independent mutations (Bd8202 and Bd8972) located in the AP2/ERF domain 485

(A), both exhibiting supernumerary spikelets not present in the wild type (Bd 21-3) (B). Brachypodium 486

fzp mutants and wild-type plants show similar growth and architecture (C left). Spikes with different 487

penetrance of the SS phenotype can be observed in mutants (C right, stars indicate supernumerary 488

spikelets). Spikes in fzp mutants show spikelets with a single pedicel as observed in the wild type but with 489

dichotomous or trichotomous rachilla (D). 490

Figure 5: Evolutionary and functional model of FZP in grasses. FZPs are illustrated as black 491

horizontal bars showing substitutions in the AP2/ERF domain ( ), frameshift mutations ( ) and TE 492

insertions in the promoter region ( ) leading to FZP non-functionality and driving supernumerary 493

spikelets (SS) phenotypes in Brachypodium and wheat. In wild-type (WT) wheat, FZP represses the 494



14

formation of axillary meristems (AxM) from spikelet meristems (SM) and controls the floral meristem 495

(FM) identity. In contrast, in SS mutants, AxM is not suppressed and leads to ectopic spikelet meristem 496

(eSM) formation. 497

498

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Figure 1: Supernumerary spikelet (SS) phenotypes in bread wheat. (A) Schematic representations of a spike (left)

and of a spikelet (right) from the bread wheat cultivar Novosibirskaya 67, and of a theoretical wild-type (WT) spikelet

(boxed). (B) Schematic illustration of various supernumerary spikelet (SS) structures: a cluster of spikelets at a rachis

node referred to as a multirow spike (MRS); three spikelets (triple-spikelet) and two spikelets in horizontal positions

at a rachis node referred to as horizontal spikelets (HS); additional sessile spikelets with a lateral branch bearing

spikelets at a node referred to as a ramified spike (RS); and a single spikelet at a node for the WT spike. (C)

Illustration of the spike structure in the near isogenic line NIL-mrs1 harboring numerous supernumerary spikelets

(left); a rachis node with additional spikelets (right) where supernumerary spikelets are indicated with red arrows; and

schematic representation of a branch-like structure (b) bearing ectopic spikelets at a rachis node in the lower part of

the NIL-mrs1 spike (box). (D-G) Light microscopy images of the NIL-mrs1 inflorescence at several developmental

stages. (D) Illustration of the spikelet differentiation stage; green arrows indicate glume primordia. (E-F) Early floret

differentiation stage.



Figure 1 (end). (E) Secondary axillary meristems that later produce ectopic spikelets are indicated with asterisks. (F)

Spikelet meristems of ectopic spikelets form glume primordia. (G) Late floret differentiation stage when all floral

organs of wild-type spikelets (ws) are differentiated in the upper part of the inflorescence. (H) A branch-like structure

dissected from a rachis node of the NIL-mrs1 inflorescence, · – ectopic spikelet (es). (I) Location of ectopic spikelets

at rachis nodes of the NIL-mrs1 inflorescence, Bars = 0.25 mm. (J-S) SEM images of Novosibirskaya 67 (WT) and

NIL-mrs1 (mrs1) inflorescences at various developmental stages. (J, K) Spikelet differentiation stage in (J) WT and

(K) mrs1. (L) Early floret differentiation stages when the spikelet meristem produces the floret meristem in WT and

(M, N) differentiation of second axillary meristems (indicated by asterisks) in the mrs1 mutant; (O) Early floret

differentiation stage (WT), showing lemmas, and (P, Q) the development of glumes (gl* and indicated by white

arrows), lemma and floret meristems by second axillary meristems in the mrs1 mutant. (R) Floret differentiation stage

showing differentiated floral organs in a basal floret of the WT and (S) the development of ectopic spikelets in mrs1,

Bars =100 μm. Abbreviations: Axm – axillary meristem, b – branch-like structure, es – ectopic spikelet, , es2nd –

ectopic spikelet of the second order, developing at the place of a floret of the first order ectopic spikelet (es), f –

floret with floret organ promordia, fm – floret meristem, gl – glume, im – inflorescence meristem, l – lemma, lo –

lodicule, pa – palea, pi – pistil, ts – terminal spikelet, sm - spikelet meristem, st – stamen, sm*, fm*, gl*, f*, l* –

spikelet meristem, floret meristem, glume, floret of an ectopic spikelet and lemma, respectively.



Rice [chr7]

Brachy. [chr1]

Genome A

Genome B

Genome D

A

B

Wheat [Chr 2DS)

Sorghum [chr2]

C

WFZP-A

WFZP-B

WFZP-D

100 bp

3 bp 3 bp

12 bp 21 bpAP2/ERF

domain

ATG

TATA

3 bpAP2/ERF

domain ATG

TATA

-320 / -114 bp

9 bp

9 bp

15 bp

AP2/ERF

domain ATG

TATA

D

TEs

gw

m4

83

0

ba

rc1

68

CO

S3

gw

m4

84

bcd

26

2

CO

S2

gw

m5

15

C

cd

o1

37

9

gd

m1

07

gw

m8

15

wm

c4

53

fbb

27

9

gw

m1

02

gw

m9

88

gw

m4

75

6

CO

S1

gw

m4

02

9

mrs

1

WFZP-AOrtholog to

LOC_Os7g47340

Ortholog to

LOC_Os7g47350

Ortholog to

Bradi2g40380.1

Ortholog to

Bradi2g60090.1

Ortholog to

Sb07g015466

Ortholog to

Bradi4g24070.1

Ortholog to

LOC_Os7g47350

WFZP-B

WFZP-D

Amplified region

FZP

TE Class I CopiaTE Class I GypsyTE Class IITE UnknownGenes

Figure 2: WFZP gene characterization in bread wheat. (A) Composite genetic map of the wheat chromosome 2D,

including the mrs1 locus and flanking COS markers. (B) Orthologous chromosomes in rice (Chr7), sorghum (Chr2)

and Brachypodium (Chr1) are shown, with conserved genes linked with black lines. (C) Annotated BAC clones are

illustrated with conserved genes (black boxes) linked with black connecting lines; Copia (in green), Gypsy (in blue),

uncharacterized (in yellow) and class II (in red) transposable elements are indicated.https://plantphysiol.orgDownloaded on February 9, 2021. - Published by

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


Figure 2 (end): (D) Comparison of WFZP homoeologous gene structures with red boxes highlighting the conserved

AP2/ERF domain. – small deletion; – small insertion; – insertion of TEs; red and blue lines indicate non-

synonymous and synonymous single nucleotide substitutions, respectively. Red arrows indicate the positions of

primers designed from the Brachypodium (Bradi1g18580) gene sequence, and that were used for BAC library

screening.



Figure 3: Structural and functional characterization of WFZPs. (A) Schematic representation of the WFZP-A and

WFZP-D gene structures and the various mutations. The light red box indicates the AP2/ERF domain, and gray boxes

indicate deletions. (B) WFZP haplotypes of SS (in red)) and standard spiked (NS in green) lines. (C) Alignment of the

amino acid sequences of the AP2/ERF domains of wild type, WFZP-D, and wfzp-D.1, wfzp-D.2 mutants. * - amino

acids that confer specificity to the GCC-box binding site. (D) Relative abundance of WFZP homoeolog mRNAs as

determined by quantitative RT-PCR with samples from spikes of cv. Chinese Spring at various developmental stages

of the spike (see below) and from roots and leaves. The values shown correspond to the mean values for three

biological and three technical replicates, normalized to the value for the Ta.304.3 gene used as a reference. Error bars

represent SE (standard errors) between replicates. Spike developmental stages were assigned as follows: SD – spikelet

differentiation stage (when spikelets are differentiated to form two opposite rows at the rachis and glume primordia are

initiated), EFD1 – early floret differentiation stage 1 (spikelet meristems initiate floral meristems and lemma primordia

are apparent); EFD2 – early floret differentiation stage 2 (floral organ primordia are apparent in basal florets); LFD -

late floret differentiation stage (floral organs are differentiated in each floret).

WFZP-DWFZP-A

lineCs CGGCGGCGCGCGGC GAG CAG

Rn CGGCGGCGCGCGGC GAG CAG

Skle128 -------------- complete-deletion

Ruc204 -------------- complete-deletion

So164 -------------- complete-deletion

Ruc163 -------------- AAG CAG

Ruc167 -------------- AAG CAG

So149 -------------- GAG CAG

NIL-mrs1-------------- AAG CAG

N67 CGGCGGCGCGCGGC GAG CAG

MC1611 CGGCGGCGCGCGGC GAG TAG

S29 CGGCGGCGCGCGGC GAG CAG

phenotype

NS

NS

SS

SS

SS

SS (MRS)

SS (MRS)

NS

SS (MRS)

NS

SS weak

NS

wfzp-A.1wfzp-D.2

wfzp-D.1

WFZP-D RFLGVRRRPWGRYAAEIRDPTTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVY

wfzp-D.1 RFLGVRRRPWGRYAAKIRDPTTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVY

wfzp-D.2 RFLGVRRRPWGRYAAEIRDPTTKERHWLGTFDTA*EAALAYDRAALSMKGAQARTNFVY

Phenotype Lines

Frameshift mutation

G-A mutation

C-T mutation

A

C

B

x10-2

0

2

4

6

8

10

12

14

16

SD EFD1 EFD2 LFD

stage of spike development

rela

tive a

ban

den

ce o

f W

FZ

P

FZP-A FZP-B FZP-D

SD EFD1 EFD2 LFD leaf root

D

AP2

AP2/ERF AP2/ERF

0

2

4

6

8

10

12

14

16

SD EFD1 EFD2 LFD leaf root

0

2

4

6

8

10

12

14

16

WFZP

rela

tive

ab

un

da

nce

WFZP-A

WFZP-B

WFZP-D

spike



Figure 4: Functional validation of FZP in Brachypodium. FZP mutant screening identified two Brachypodium

alleles with independent mutations (Bd8202 and Bd8972) located in the AP2/ERF domain (A), both exhibiting

supernumerary spikelets not present in the wild type (Bd 21-3) (B). Brachypodium fzp mutants and wild-type plants

show similar growth and architecture (C left). Spikes with different penetrance of the SS phenotype can be observed

in mutants (C right, stars indicate supernumerary spikelets). Spikes in fzp mutants show spikelets with a single pedicel

as observed in the wild type but with dichotomous or trichotomous rachilla (D).

Bd8972Bd8202

A

B

WT-Bd21-3

AP2/ERF domainN- -COOH

Bd21-3: RRRTQEPG RFLGVRRRPWGRYAAEIRDP TTKERHWLGTFDTA QEAALAYDRAALSMKGAQARTNFVYAAPASCBd8202 : RRRTQEPG RFLGVRRRPWSRYAAEIRDP TTKERHWLGTFDTA QEAALAYDRAALSMKGAQARTNF VYAAPASC

Bd8972 : RRRTQEPG RFLGVRRRPWGRYAAEIRDP TTKERHWLGTFDT TQEAALAYDRAALSMKGAQARTNFVYAAPASC

CWTMutants MutantsWT

pedicel

glume

lemma

palea

mutantD

*

*

*

*

*

*

**

WT



Figure 5: Evolutionary and functional model of FZP in grasses. FZPs are illustrated as black horizontal bars

showing substitutions in the AP2/ERF domain ( ), frameshift mutations ( ) and TE insertions in the promoter region

( ) leading to FZP non-functionality and driving supernumerary spikelets (SS) phenotypes in Brachypodium and

wheat. In wild-type (WT) wheat, FZP represses the formation of axillary meristems (AxM) from spikelet meristems

(SM) and controls the floral meristem (FM) identity. In contrast, in SS mutants, AxM is not suppressed and leads to

ectopic spikelet meristem (eSM) formation.

FZP ancestor

Brachypodium Bread wheat

A

B

D

AP2/ERF

SM

FM

AxM (eSM)

FZP

SS SS WTWT

Wheat spikeBrachypodium spike

EVOLUTION

NETWORK

PHENOTYPE



frizzy panicle drives supernumerary spikelets in bread ... · 11/14/2014 · 106 primary axillary...

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