Six-rowed spike4 (Vrs4) controls spikelet determinacyand row-type in barleyRavi Koppolua,1, Nadia Anwarb,1, Shun Sakumab, Akemi Tagirib, Udda Lundqvistc, Mohammad Pourkheirandishb,Twan Ruttend, Christiane Seilere, Axel Himmelbacha, Ruvini Ariyadasaa, Helmy Mohamad Youssefa,f, Nils Steina,Nese Sreenivasulue,g,h, Takao Komatsudab, and Thorsten Schnurbuscha,2

aDepartment of Genebank, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben D06466, Germany; bPlant Genome Research Unit,National Institute of Agrobiological Sciences (NIAS), Tsukuba 3058602, Japan; cNordic Genetic Resource Center, Alnarp SE-23053, Sweden; dDepartment ofPhysiology and Cell Biology, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben D06466, Germany; eDepartment of MolecularGenetics, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben D06466, Germany; fDepartment of Plant Physiology, Faculty ofAgriculture, Cairo University, Giza 12613, Egypt; gResearch Group Abiotic Stress Genomics, Interdisciplinary Center for Crop Plant Research (IZN), Halle (Saale)D06120, Germany; and hGrain Quality and Nutrition Center, International Rice Research Institute (IRRI), Metro Manila 1301, Philippines

Edited by George Coupland, Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved June 26, 2013 (received for reviewDecember 16, 2012)

Inflorescence architecture of barley (Hordeum vulgare L.) is com-mon among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meri-stem is one of the unique features of barley spikes, in which threespikelets (one central and two lateral spikelets) are produced ateach rachis internode. Fertility of the lateral spikelets at triplespikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increasedgrain yield per spike, compared with two-rowed spikes with sterilelateral spikelets. Thus, far, two loci governing the row-type phe-notype were isolated in barley that include Six-rowed spike1(Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.)inflorescence architecture gene RAMOSA2 (RA2). Eighteen codingmutations in barley RA2 (HvRA2) were specifically associated withlateral spikelet fertility and loss of spikelet determinacy. Expres-sion analyses through mRNA in situ hybridization and microarrayshowed that Vrs4 (HvRA2) controls the row-type pathway throughVrs1 (HvHox1), a negative regulator of lateral spikelet fertility inbarley. Moreover, Vrs4 may also regulate transcripts of barley SISTEROF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phospha-tase involved in trehalose-6-phosphate homeostasis implicated tocontrol spikelet determinacy. Our expression data illustrated that,although RA2 is conserved among different grass species, itsdown-stream target genes appear to be modified in barley andpossibly other species of tribe Triticeae.

cytokinin | EGG APPARATUS1 | grain number | yield potential

Inflorescence architecture is highly diverse among members ofthe grass family (Poaceae), in which spikelets represent the

fundamental building blocks, comprising one or more floretsenclosed by two glumes. Economically important grass species ofthe tribe Triticeae, such as wheat (Triticum spp.), barley, triticale(×Triticosecale Wittm. ex A. Camus), and rye (Secale cereale L.),typically possess a branchless spike-shaped inflorescence, whereasinflorescences of tropical species, e.g., maize, Sorghum spp., andrice (Oryza sativa L.), have highly branched tassels or panicles.Inflorescence branching in maize appears to be largely regulatedthrough the RAMOSA gene network, which involves the RAMOSA1(RA1), RA2, RA3, and RAMOSA1 ENHANCER LOCUS2 (REL2)genes (1). In maize, determinacy of the spikelet pair meristems(SPMs) is mediated through RA2, which encodes a lateral organboundaries (LOB) domain-containing transcriptional regulatorthat functions upstream of the RA1 and REL2 determinacy-providing complex (2–4). Expression of RA1 is dependent uponRA3, which encodes a phosphatase involved in trehalose metab-olism (5). Orthologs of RA1 and RA3 are unique to the grass tribeAndropogoneae (paired spikelets arise from SPMs), but closeparalogs of RA3, with an as of yet unknown function, exist in barley(HvSRA) and other grasses (5).

The barley inflorescence is an indeterminate spike that pro-duces three single-flowered spikelets in a distichous manner ateach rachis internode that develop into one central and twolateral spikelets (Fig. 1A) (6, 7). Based upon lateral spikelet/floret size and fertility barley is classified into two different row-types; i.e., two-rowed and six-rowed barley (8). In two-rowedbarley, the central spikelet is fertile and produces grain, and thetwo lateral spikelets remain sterile (Fig. 1G). In six-rowed barley,all three spikelets are fertile and develop into grains (Fig. 1B).The six-rowed phenotype is controlled by at least five indepen-dent loci that include Six-rowed spike1 (vrs1), vrs2, vrs3, vrs4, andIntermedium-C (Int-c). Vrs1 encodes a homeodomain-leucinezipper class I transcription factor that is a negative regulator oflateral spikelet fertility (9). Mutant vrs1.a promotes lateralspikelet fertility resulting in a complete six-rowed spike (Fig. 1B).Alleles at the locus int-c, which is an ortholog of the maize do-mestication gene TEOSINTE BRANCHED1 (HvTB1) (10), modifylateral spikelet development with respect to allelic constitutionat vrs1 (Fig. 1F). Loss-of-function vrs1.a is generally accompa-nied by Int-c.a in six-rowed barley and the functional Vrs1.b byint-c.b in two-rowed barley. The remaining vrs loci vrs2, vrs3, andvrs4 show varying levels of lateral spikelet fertility with completelateral spikelet fertility observed in vrs4 mutants (Fig. 1 C–E).Apart from lateral spikelet fertility, vrs4 mutants show inde-terminate triple spikelet meristems (TSMs), thereby producingadditional spikelets/florets. The apparent indeterminacy of theTSM in vrs4 mutants suggests that vrs4 is involved in a geneticpathway that regulates the highly conserved determinate natureof the TSM inherent to Hordeum species.In the present study, we isolated the vrs4 locus through genetic

mapping and extensive mutant analysis. Our findings showedthat Vrs4 underlies HvRA2, an ortholog of maize transcriptionfactor RAMOSA2, which is important for inflorescence de-velopment in grasses. Tissue localization through mRNA in situhybridization in immature spikes showed that HvRA2 is ex-pressed very early during inflorescence development and mostprobably confines barley’s TSM meristem to three spikelets. Geneexpression analysis in combination with microarray experiments

Author contributions: N. Sreenivasulu, T.K., and T.S. designed research; R.K., N.A., S.S.,A.T., U.L., M.P., T.R., C.S., A.H., R.A., and H.M.Y. performed research; N. Stein contributednew reagents/analytic tools; R.K., N.A., S.S., A.T., U.L., M.P., N. Sreenivasulu, T.K., and T.S.analyzed data; and R.K., N.A., S.S., N. Sreenivasulu, T.K., and T.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos.: KC854546–KC854554).1R.K. and N.A. contributed equally to this work.2To whom correspondence should be addressed. E-mail: thor@ipk-gatersleben.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221950110/-/DCSupplemental.

13198–13203 | PNAS | August 6, 2013 | vol. 110 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1221950110

demonstrated that HvRA2 may likely function in two distinctpathways, thereby establishing spike architecture through regu-lation of TSM determinacy and controlling the Hordeum-specificrow-type pathway.

Resultsvrs4 Mutants Display Indeterminate TSM and Six-Rowed Phenotype.Unlike maize or sorghum, in which spikelets develop from SPMs,immature barley spikes develop a TSM that arises from the firstaxillary meristem right after double ridge stage (first reproduc-tive stage). TSMs are determinate and form three distinct moundsproducing three secondary axillary meristems (AMs), one centralspikelet meristem (CSM) and two lateral spikelet meristems(LSMs) (Fig. 2A and SI Appendix, Fig. S1A). Each spikelet meri-stem initially differentiates into a pair of glume primordia (Fig. 2A),and then into one floral meristem (FM). Scanning electronmicroscopy (SEM) analysis of wild type and vrs4 mutants at

double ridge stage revealed no morphological differences, sug-gesting that there is no change in determinacy of AM that givesrise to triple spikelet primordia (Fig. 2 A and B). The appearanceof two additional mounds on either side of the LSMs was the firstvisible deviation observed in vrs4 mutant inflorescences (Fig.2B). The additional spikelets/florets emerging at rachis intern-odes (SI Appendix, Fig. S1 B and C) were frequently fertile anddeveloped into grains (Fig. 1 L and N). Additional spikelets hadthe same orientation (lemma primordia on abaxial side) asnormal spikelets (Fig. 1M and SI Appendix, Fig. S1 A, B, D, andE). However, additional florets were oriented opposite to thenormal spikelet (lemma primordia on the adaxial side showingthe alternate orientation of floret formation on the rachilla) andlacked glumes (Fig. 1M). Apart from the additional spikelets/florets(Fig. 1O and SI Appendix, Fig. S1G) we rarely found additionalpistils (SI Appendix, Fig. S1I), suggesting that FM determinacywas also affected in vrs4 mutants. Loss of determinacy became

Fig. 1. Spike morphology of different row-type loci andvrs4 phenotype. (A) Two-rowed spike (Vrs1): fertile centralspikelets (CS) and sterile lateral spikelets (LS). (B) Six-rowedspike1 (vrs1): completely fertile CS and LS. (C) Six-rowedspike2 (vrs2): LS fertility observed at the spike base withoccasional additional spikelets [additional spikelet (AS)fertile or sterile, AS in light green color]; along the spike, LSare occasionally enlarged and set seed, (D) Six-rowed spike3(vrs3): spike base appears two-rowed, and the remainingportion appears six-rowed. (E) Six-rowed spike4 (vrs4): spikesimilar to vrs1, with frequent awn bearing AS (light greencolored). (F) Intermedium-C (int-c): LS are enlarged, setseed, which are usually smaller than in vrs1, LS withoutawns. (G) Two-rowed wild-type spike (Piroline). vrs4mutantspikes: (H) vrs4.l, (I) BW-NIL(mul1.a). (J) BW-NIL(vrs4.k): six-rowed-like appearance and formation of AS and additionalflorets (AF). (K) Spikelet triplet at one rachis internode.(L and M) BW-NIL(vrs4.k) (L) and vrs4.l (M) showing AS andAF; initial triplet: CS, LS with lemma (L) and palea (P). AFdeveloped on the rachilla of LS. (N) Seed formation in-volving AS and AF. (O) Classes of axillary structures pro-duced by vrs4 mutant vrs4.l and wild-type Piroline spikes.Total number of fertile or sterile axillary structures fromautumn grown plants is shown. Data were recorded on fiveplants with on average two spikes per plant. (see SI DatasetS1A for SE and significance of t test).

Fig. 2. Scanning electron microscopy and transcript localiza-tion of Vrs4 mRNA in immature barley spikes. (A) Wild-typeinflorescence at lemma primordium (LP) stage showing in-florescence meristem (IM) forming double ridge (DR), upperridge containing triple spikelet meristem (TSM), and lower leafridge (LR). TSM forms a triple mound (TM), which transitionsinto one central spikelet meristem (CSM) and two lateralspikelet meristems (LSMs). A pair of glume primordia (GP) anda single floral meristem (FM) are produced by each SM. (B) vrs4at LP initiating additional spikelet meristems (ASMs) (red arrows).CSMs develop into branch-like inflorescence meristem (BIM).Occasionally, CSMs initiate additional florets on rachilla (as-terisk). (C) vrs4 at stamen primordium (SP) stage showing BIMat DR and a developing additional floret meristem (AFM). (D)vrs4 at awn primordium (AP) stage showing BIM at TM. (E–H) Insitu RNA hybridization of HvRA2 in two-rowed barley cv. Bo-nus. Transverse sections at DR (E), TM (F), GP (G), and SP (H). cs,central spikelet; ls, lateral spikelet; gl, glume. (I) Transcriptlevels of HvRA2 determined by quantitative RT-PCR in cv.Bonus. Constitutively expressed HvActin was used for nor-malization. X-axis represents spike developmental stages.Mean ± SE of three biological replicates. WA, white anther; GA,green anther. (J) Vrs1 expression in BW-NIL(vrs4.k) and wild-type MFB104. Mean ± SE of three biological replicates. Expression values are given at the bottom of the graph. (K) Vrs1 (yellow) and Vrs4 (green) areexpressed in overlapping domains of lateral spikelets. (L) Spikes of cv. Montcalm carrying vrs1.a1 allele and vrs4 mutant allele mul1.a. Additional spikelets inmul1.a are indicated by red triangles. (Scale bars: 200 μm in A and B; 333 μm in C and D; and 100 μm in E–H.)

Koppolu et al. PNAS | August 6, 2013 | vol. 110 | no. 32 | 13199

PLANTBIOLO

GY

even more apparent when CSMs frequently produced branch-like inflorescence meristems (BIM) with ridges (resemblingpredouble ridge stage of primary inflorescence) (Fig. 2C andSI Appendix, Fig. S1C), which later differentiated into triple mounds(Fig. 2D) and eventually produced a vrs4-like inflorescence meri-stem (SI Appendix, Fig. S1J). The majority of BIMs aborted asgrowth continued (SI Appendix, Fig. S1 B and C), and only fewmeristems at the base developed into additional spikelets. Sim-ilar developmental abnormalities were observed in two other vrs4Bowman near isogenic alleles BW-NIL(vrs4.k) and BW-NIL(mul1.a) (SI Appendix, Fig. S2 A–E). SEM analysis revealed thatvrs4 mutants lost determinacy of the TSMs, and subsequentlySMs, thus producing supernumerary spikelets and florets. Thespring-grown mutant allele vrs4.l showed an enhanced vrs4 phe-notype with a significantly higher number of spikelets and branch-like structures than autumn grown plants (SI Appendix, Fig. S1 Band C and SI Dataset S1A).Apart from the indeterminate nature of TSMs, vrs4 mutants

displayed another important feature of complete fertility anddevelopment of lateral spikelets resulting in a six-rowed pheno-type. The six-rowed phenotype observed in vrs4 mutants wasanalogous to that of vrs1 mutants. In the present study, all vrs4mutant alleles, except int-e.4, int-e.20, and int-e.72, showedcomplete or partial six-rowed phenotype (Fig. 1 H–J and SIAppendix, Fig. S3).

Genetic Mapping and Mutant Analysis Reveals That Vrs4 Underlies theBarley Ortholog of Maize RAMOSA2. We initially mapped vrs4 tothe short arm of chromosome 3H using five F2 mapping pop-ulations comprised of 188–214 gametes (SI Appendix, Table S1).Mapped markers were derived from syntenic Brachypodium(chromosome 2) and rice (chromosome 1) gene sequences basedon the virtual gene order reported in the barley genome zipper(11) (SI Dataset S1B). In all five mapping populations tested, thevrs4 phenotype cosegregated with a cluster of markers (barleyorthologs of Brachypodium/rice genes) derived from the shortarm of 3H (37.17–41.68 cM in genome zipper) (SI Appendix, Fig.S4). Flanking markers of the corresponding marker clustercontained 68 predicted Brachypodium genes (Bradi2g03717 toBradi2g04380) (SI Appendix, Table S2) within the interval. Usingrecombinant screens in 2,172 gametes (vrs4.k × Golden Promisepopulation), we further refined the interval and mapped vrs4 toa single BAC contig (1,073 kb) containing at least six predictedgenes, including barley RAMOSA2 (HvRA2), RESURRECTION1(RST1),TRYPTOPHANAMINOTRANSFERASE (TPA),EMBRYOSAC/BASAL ENDOSPERM TRANSFER LAYER/EMBRYOSURROUNDING REGION (EBE), STACHYOSE SYNTHASE,and RECEPTOR LIKE PROTEIN KINASE (INRPK1) (Fig. 3D).Of the six predicted genes, we consideredHvRA2 as an interestingcandidate gene for theVrs4 locus, because it was identified as beingessential for imposing determinacy on SPM identity in maize (3).Because vrs4 mutants in general showed loss of determinacy of

TSMs, we sequenced HvRA2 in 20 vrs4 mutants, and found that18 showed molecular lesions within the HvRA2 ORF (Fig. 3Eand SI Appendix, Fig. S5). It had been proposed that the numberof meristem primordia giving rise to spikelets is partially un-restricted in vrs4 mutants resulting in formation of additionalspikelets/florets (7). From the mutant analysis, we found thatirrespective of the type of lesion in various mutant alleles of vrs4,all showed indeterminacy of the TSM to a certain degree (Fig.1 H–J and SI Appendix, Fig. S3). The molecular nature of thelesions in mutants clearly correlated with the severity of vrs4phenotype. Five mutants with the strongest spikelet phenotypehad either a premature stop codon [BW-NIL(vrs4.k), BW-NIL(mul1.a), vrs4.l (syn. Xc 41.5), int-e.26 and int-e.128 (syn. hex-v.48)] or a putative gene deletion (MHOR 318 and MHOR 345;Figs. 1 H–J and 3E and SI Appendix, Fig. S3). Weaker allelespossessed amino acid substitutions either within the LOB do-main [int-e.23, BW-NIL(int-e.58), int-e.72] or in other conservedregions (see below) of the protein (int-e.65, int-e.66, int-e.87, int-e.89, int-e.90, int-e.91, int-e.92, int-e.101) (Fig. 3E and SI Ap-pendix, Fig. S3). The vrs4 mutants int-e.4 and int-e.20 did not

show any lesions, suggesting possible transcriptional or post-transcriptional regulation in these alleles (allelism data for int-e.4and int-e.20 with vrs4 are in SI Appendix). The collection of 18ORF mutants strongly indicated that HvRA2 underlies theVrs4 locus.

HvRAMOSA2 Encodes a LOB Domain Transcription Factor with Grass-Specific Domains. HvRA2 is a class I LOB domain protein con-sisting of a cysteine-rich repeat (CX2CX6CX3C), a highly con-served GAS block, and a Leucine-zipper coiled-coil motif(LX6LX3LX6L) (SI Appendix, Fig. S6), signature features con-served in all class I LOB domain proteins (12). Apart from thecanonical LOB domain, grasses (monocots) posses a uniqueC-terminal putative activation domain (44 aa) that is not presentin Arabidopsis and other eudicots (3) (SI Appendix, Fig. S6). Basedon sequence alignments generated from orthologous/homolo-gous RA2 proteins from a range of monocot and eudicot species,we identified another conserved grass specific N-terminal region(16 aa) preceding the LOB domain (Fig. 3E and SI Appendix,Fig. S6). The last four amino acids of the N-terminal domain(PGAG) are strictly conserved across different grass species.Interestingly, seven of the vrs4 mutants possessed amino acidsubstitutions in the last four amino acids of the conserved N-terminal region, signifying its putative functional role in grasses.Phylogenetic analysis of RA2 homologs/orthologs in eudicotsand monocots grouped them into monocot- and eudicot-specificclades (SI Appendix, Fig. S7). Among 23 LOB domain-encodinggenes (LBD; 19 class I and four class II) from barley (SI Appendix,Table S3), HvRA2 was unique with conserved N- and C-terminaldomains and clearly separated from other barley and eudicot LBDfamily members (SI Appendix, Fig. S7 and Table S3).

Resequencing of HvRA2 in diverse Barley Genotypes Shows ReducedNucleotide Diversity. In an attempt to identify row-type-specificalleles at Vrs4 in natural variation, we conducted sequenceanalysis of the HvRA2 ORF and parts of the 5′ and 3′ regulatoryregions (promoter, UTRs) in a set of 77 diverse two-rowed andsix-rowed barley genotypes (SI Dataset S1C). Unlike Vrs1 andInt-c, which showed row-type-specific alleles in natural variation(9, 10) Vrs4 appeared to be conserved across diverse two-rowedand six-rowed germplasm, revealing very low natural nucleotidevariation in the coding region (SI Dataset S1C). Haplotypeanalysis using these sequence data resulted in one major andseven minor nucleotide haplotypes (GenBank accession nos.KC854546 to KC854553) without specificity toward a particulargeographic region or row-type (SI Appendix, Fig. S8 and SIDataset S1C). Among the eight nucleotide haplotypes identified,only one haplotype comprising of the two-rowed genotype, Pal-mella Blue, coded for an amino acid substitution in an uncon-served region of the protein without showing any effect on wild-type phenotype (see SI Dataset S1C for protein sequencealignment of nucleotide haplotypes). We found complete con-servation in the 3′UTR, suggesting that it may be a target site forcis-regulatory elements or involved in mRNA metabolism.

HvRA2 Is Expressed Early in Spike Development and Functions Upstreamof Vrs1. In situ hybridization analyses revealed that the first ex-pression of HvRA2 was detected during the double ridge stage,when spikelet primordia begin to differentiate (Fig. 2E), makingHvRA2 one of the earliest expressed genes during AM differ-entiation. At triple-mound stage, when spikelet primordia dif-ferentiate into three distinct spikelet meristems, HvRA2 mRNAsignals were abundant all over the lateral spikelet primordia withweaker expression in the central spikelet primordia (Fig. 2F),indicating a role in providing determinacy and confinement tothe TSM. At glume primordium stage, during which floret pri-mordia initiate, HvRA2 mRNAs were detected in both centraland lateral spikelets (Fig. 2G). Transcript levels of HvRA2 indeveloping spikes at triple mound and glume primordium stageswere relatively higher than during later stages (Fig. 2I).Analyzing different six-rowed mutants, Sakuma et al. noticed

that Vrs1 transcripts were particularly down-regulated in the vrs4

13200 | www.pnas.org/cgi/doi/10.1073/pnas.1221950110 Koppolu et al.

mutant background (13). We also found that Vrs1 transcripts inBW-NIL(vrs4.k) were significantly lowered at different spikedevelopmental stages (Fig. 2J). Unlike HvRA2 (Fig. 2E), Vrs1expression in wild-type plants starts at triple-mound stage withno detectable expression during double ridge stage (9). Thus,HvRA2 expression is temporally ahead of Vrs1. Furthermore,HvRA2 transcripts at triple-mound stage were mainly localized tolateral spikelets (Fig. 2F), suggesting an expression domain overlapbetween VRS1 and HvRA2 proteins (Fig. 2K). Consistent with this,most of the vrs4 mutants showed a complete six-rowed phenotypesimilar to vrs1 mutants (Fig. 1 H–J and SI Appendix, Fig. S3), in-dicating that Vrs1 transcripts require HvRA2 action. Doublemutant analysis supported this view, because vrs4 allele BW-NIL(mul1.a), isolated in a six-rowed background (progenitor: Mont-calm, vrs1.a1), displayed the six-rowed spike with complete lateralspikelet fertility, as observed in Montcalm, and also indeterminacyof the TSM, diagnostic of vrs4 mutants (Fig. 2L).

Microarray Analysis of vrs4 Reveals HvRA2 as an Important Regulatorof Inflorescence Development. Because Vrs4 encodes a LOB do-main transcription factor HvRA2, we performed microarrayanalysis in two vrs4 deletion mutants, MHOR 318 and MHOR345, and their respective wild types, Ackermann’s Donaria andHeine’s Haisa, to identify its potential downstream target genesin barley. From the microarray data, we found compelling evi-dence for Vrs4-mediated regulation of Vrs1 in both mutantsanalyzed, with highly significant down-regulation of Vrs1 in vrs4mutants (SI Appendix, Fig. S9A). Importantly, among othergenes significantly down-regulated in both mutants, we identifiedtrehalose-6-phosphate (T6P) synthase and HvSRA (a putativeT6P phosphatase) (SI Appendix, Fig. S9A), both involved intrehalose biosynthesis, implying possible regulation of T6P ho-meostasis by Vrs4 during inflorescence development and growth(14, 15). From the microarray expression data, we identifiedother significant differentially regulated genes in both vrs4mutants that might likely function in establishing row-type andspikelet determinacy in barley.The other important differentially regulated gene in both vrs4

mutants was EGG APPARATUS1-LIKE (EA1-LIKE). EA1 en-codes a secretary protein that attracts pollen tube growth towardegg apparatus (16). The significant down-regulation of the EA1-LIKE gene in wild-type two-rowed inflorescence (SI Appendix, Fig.S9B) suggests that both Vrs1 and EA1-LIKE may function in asimilar pathway. Consistent with this phenomenon, Vrs1 transcriptsshow localization to pistils of lateral spikelets in barley (13).

Alternatively, elevated expression of EA1-LIKE could be dueto the proliferating additional spikelets/florets in vrs4 mutants.The differentially regulated genes likely involved in enhanced

meristematic activity of vrs4 mutants include LONELYGUY-LIKE (LOG-LIKE), which encodes cytokinin nucleotide phos-phoribohydrolase, a cytokinin biosynthesis enzyme, required tomaintain meristem activity (17). Up-regulation of LOG-LIKEgenes in vrs4 mutants indicated that higher amounts of LOG-LIKE transcripts promoted meristematic activity in vrs4 mutants(SI Appendix, Fig. S9B). LOG1 is required for the accumulationof KNOTTED1-type homeobox (KNOX) transcripts in rice (17).Thus, overproduction of cytokinin results in increased steadystate levels of KNOX gene transcripts (18). Conversely, KNOXproteins were shown to activate cytokinin biosynthesis in theshoot apical meristem of Arabidopsis and rice (19, 20). Thus,KNOX and cytokinin may act in a positive feedback loop, withKNOX genes promoting cytokinin biosynthesis and cytokininsup-regulating KNOX biosynthesis. Our data showed up-regula-tion of KNAT3 (KNOX) in vrs4 mutants (SI Appendix, Fig. S9B),suggesting a possible coregulation of LOG-LIKE and KNOXproteins in vrs4 mutants. The microarray analysis for these dif-ferentially regulated genes is supported by quantitative RT-PCRdata (SI Appendix, Fig. S10).Independent quantitative RT-PCR analyses of a few impor-

tant genes (not present on the microarray and identified basedon extrapolation of vrs4 mutant phenotype with known mutantinflorescence architecture genes characterized in rice or maize)revealed differential regulation for cytokinin oxidase/dehydrogenase(CKX2), which is involved in cytokinin degradation and meristemsize (21). HvCKX2 transcript levels in BW-NIL(vrs4.k) and MHOR318 were lowered (SI Appendix, Fig. S11), consistent with highermeristematic activity in the mutant inflorescence. Transcripts ofanother gene, PINFORMED1-LIKE (PIN1-LIKE) (22), were alsoup-regulated in the vrs4 mutants, suggesting that altered auxintransport affected lateral organ development in vrs4 mutants (SIAppendix, Fig. S11).

DiscussionThe regulation of inflorescence development has been broadlyelucidated in plant species, such as Arabidopsis and maize (23),with the help of a wealth of mutants that display abnormalinflorescence development. However, in barley, functional knowl-edge of genes and genetic mechanisms involved in spike develop-ment has only started to emerge relatively recently, with the cloningof genes such as Vrs1 (9) and Int-c (10). Mutants of vrs4, like otherbarley row-type mutants, produce a six-rowed phenotype, but the

Fig. 3. High-resolution linkage and physical map of vrs4and analysis of vrs4 mutants. (A–C) Rice (A) and Brachy-podium (B) chromosomal regions syntenic with barleychromosome 3H (C) in the vrs4 region. Predicted genes inrice, Brachypodium and barley chromosomes are indicatedby ovals (Gene names start with Os01g (rice) or Bradi2g(Brachypodium) followed by five digit number). Solid blackovals mark the genes from which markers were derived formapping in the vrs4 region. Numbers of recombinants areindicated between mapped markers on the barley chro-mosome. (D) Single BAC contig sequenced in the vrs4 re-gion (22 overlapping BAC clones covering 1,073 kb) (see SIAppendix, Table S5 for corresponding BAC names). Pre-dicted genes identified from the BAC sequences are in-dicated as circles. (E) HvRA2 gene structure showinga distinct grass-specific domain (red box), and RA2 as wellas the LOB domains. Lesions in 18 ORF-mutant alleles areindicated below the gene structure (see also SI Appendix,Fig. S5). :, Nucleotide substitution leading to prematurestop codon; *, INDELS leading to frame shift; >, non-synonymous SNP in the coding region.

Koppolu et al. PNAS | August 6, 2013 | vol. 110 | no. 32 | 13201

PLANTBIOLO

GY

most interesting phenotype is the indeterminate TSM, resulting inthe formation of additional spikelets/florets that frequently de-velop into grains. Thus, actions of vrs4, possibly along with other asof yet unknown genes, may help elucidating how to increase yieldpotential in barley and other Triticeae species.Our resequencing data showed very limited natural variation in

HvRA2 across diverse barley germplasm with complete conserva-tion in the 3′UTR. A low level of polymorphisms in the 3′ regula-tory region was also observed in maize domestication loci RA1 (24)and GRASSY TILLERS1 (25). We hypothesize that naturally oc-curred variations at this locus might have undergone purifying se-lection in two- and six-rowed barleys, as severe mutations woulddisrupt determinacy of the TSM, leading to disordered spike for-mation. However, weaker natural mutant alleles of vrs4 might stillbe hidden among intermedium barleys, which represent a large anddiverse phenotypic row-type class within cultivated barley.Another important finding from the present study is the

transcriptional regulation of Vrs1 by HvRA2. Our microarrayanalysis confirmed that Vrs1 is significantly down-regulated invrs4 mutants. The HvRA2 mRNA in situ hybridization resultsshowed that HvRA2 and Vrs1 are expressed in highly overlappingdomains of lateral spikelets, suggesting a putative interaction.Circumstantial evidence supporting this view came from theLOB domain consensus DNA recognition motif 5′-CCGGCG-3′(26) present in the Vrs1 promoter region close to the transcrip-tional start site (−60 to −65 bp) and also in the 5′UTR. Previousin vitro protein binding assays showed strong binding affinity ofLOB domain proteins to this consensus motif (26). However, thephysical interaction between HvRA2 and Vrs1 cis elements needsto be established. Clearly, reduction of Vrs1 transcripts in vrs4mutants indicated that HvRA2 directly or indirectly controlsVrs1 transcripts, and thus the row-type pathway (Fig. 4 A and B).In maize, inflorescence morphology is largely governed by the

RAMOSA pathway under the coordinated regulation of RA1,RA2, and RA3. Maize RA3 shows a highly localized expressionpattern at the base of inflorescence branches and encodes afunctional T6P phosphatase, suggesting a role for trehalose sig-naling in meristem determinacy (5). A clear ortholog of RA3 isnot present in barley. However, a homolog of RA3, termed SISTEROF RAMOSA3 (SRA), has been identified in barley (HvSRA) andrelated grasses (5). The rice homolog of RA3 (OsSRA) also showedhighly localized expression pattern at the base of inflorescencebranches (5), suggesting a role in inflorescence patterning. Inmaize, RA1 transcripts are under the control of RA2 and RA3,whereby RA2 controls the fate of SPMs through the RA1–REL2determinacy complex (4). The SPM is specific to the tribe Andro-pogoneae, as is RA1 (1, 2). Thus, HvRA2 seemed to have main-tained a conserved function (control over meristem determinacy),but diversified the downstream targets for executing this function

in barley (as the ortholog/homolog of RA1 is missing in barley).In the large barley mutant collection, several spike mutants forthe multifloret or branched spike, such as extra floret (flo)-a, flo-b,flo-c, compositum1 (com1), com2, and multiflorus2 (mul2), werereported, but the underlying genes for these mutants have stillnot been identified. These loci could be potential candidatetargets of Vrs4 for conferring spikelet determinacy. AlthoughRA2 and RA3 act independently in maize, we found thatHvRA2 may regulate transcripts of HvSRA and T6P synthase,which in turn may regulate specific growth responses throughsynthesis of T6P (Fig. 4C) (27). Moreover, HvRA2 coopted agenus-specific function for the row-type pathway through regu-lation of Vrs1 (control over lateral spikelet fertility), therebyclearly indicating different gene sets and networks for inflo-rescence development compared with maize (Fig. 4C). In addi-tion, we showed transcript differences for HvCKX2 and auxintransporters in vrs4 mutants, further suggesting a role in inflo-rescence growth and development (Fig. 4C).Taken together, our results suggest that Vrs4 is a central player

in establishing inflorescence architecture of barley spikes, as wellas in determining yield potential and grain number. Thus,a better understanding of the underlying gene regulatory net-works during spike formation may help to improve future grainyields of small-grain cereals.

Materials and MethodsPlant Material. Allelic vrs4 mutants (SI Appendix, Table S4) were obtainedfrom the Nordic Genetic Resource Center, the National Small Grains Collec-tion (US Department of Agriculture), and the IPK gene bank. Seventy-sevendiverse barley accessions were obtained from IPK gene bank for haplotypeanalysis. The mutant alleles vrs4.l (syn. Xc 41.5), its two-rowed progenitorPiroline, and Bowman isolines BW-NIL(vrs4.k) and BW-NIL(mul.1a) were usedfor phenotypic descriptions and SEM analysis. Plant material used for gen-erating the mapping populations is discussed in SI Appendix.

Vrs4 Phenotype Characterization. vrs4.l was selected as a reference for com-paring inflorescence development with the wild-type Piroline. Piroline andvrs4.l were grown in fields in Tsukuba, Japan, during the autumn (October2011 to June 2012) and spring (March 2012 to June 2012) seasons. For theanalysis shown in Fig. 1O and SI Dataset S1A, data were recorded in fiveplants (two spikes per plant) from each of the genotypes. Probability valueswere determined using t test. The number of rachis internodes and numberof seeds produced by mutant and wild type were counted. A spikelet tripletconsisting of three spikelets was considered a basic set of axillary structuresand the remaining axillary structures were considered additional axillarystructures. Each triplet or multispikelet was divided into one central and twolateral units, and the number of additional spikelets/florets was counted ineach unit. An additional axillary structure was counted as a supernumeraryfloret when the rachilla of the first axillary structure was not visible. Thetotal number of branch-like structures and additional lemma-like structures

Fig. 4. Models of Vrs4 interactions showing HvRA2 putativetargets. (A) Functional Vrs4 suppresses additional spikelet for-mation and activates Vrs1 transcription. (B) Mutant vrs4 cannotcontrol determinacy of the TSM; thus, additional spikelets areformed. Transcriptional activation of Vrs1 expression is omitted(red cross) resulting in lateral spikelet fertility. (C) HvRA2 mayregulate transcripts of T6P synthase (T6PS) and HvSRA, a puta-tive T6P phosphatase, thereby maintaining T6P homeostasisand spikelet determinacy. In Vrs4, transcript levels of PIN1-LIKEare maintained at constant level but are up-regulated in vrs4.In wild-type plants, HvRA2 functions upstream of HvHox1,which in turn may down-regulate transcripts of EA1-LIKEresulting in abortion of lateral spikelets, possibly producinga two-rowed spike (hypothetical). However, up-regulation ofEA1-LIKE in vrs4 mutants may likely be due to enhanced mer-istematic activity related to the six-rowed spike phenotype andadditional spikelets/florets. Transcript levels of genes involvedin meristematic activity [e.g., cytokinin oxidase (CKX2), LONELYGUY-LIKE, and KNOX genes] are also regulated by HvRA2.

13202 | www.pnas.org/cgi/doi/10.1073/pnas.1221950110 Koppolu et al.

produced by a spike were counted. Fertility of axillary structure was scoredby counting the number of seeds produced by each type of axillary structure.

Marker Development. For initial mapping, BOPA SNP markers (28) or SSR andEST based markers (SI Dataset S1B) were selected from the high-densitytranscript map of barley chromosome 3H (29). For further marker de-velopment in the defined vrs4 interval, we relied on the barley genomezipper (11). Gene sequences from the syntenic interval in rice or Brachypo-dium were extracted from respective genome browser servers (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/ and http://jbrowse.brachypodium.org/JBrowse.html). The obtained gene sequences were BLASTed against the IPKBarley BLAST server (http://webblast.ipk-gatersleben.de/barley/viroblast.php)to obtain barley sequences. SNP polymorphisms identified from the primersdesigned were converted to restriction enzyme based CAPS (30) (http://tools.neb.com/NEBcutter2/) or dCAPS (31) markers (http://helix.wustl.edu/dcaps/).

Quantitative RT-PCR. Total RNA was extracted from immature spike tissues(double ridge, triple mound, glume primordium, lemma primordium, stamenprimordium, and awn primordium stages) using the PureLink RNA Mini kit(Invitrogen). RNase-free DNase (Invitrogen) was used to remove genomicDNA contamination. The RNA integrity and quantities were measured usingthe Agilent Bioanalyzer and NanoDrop (Peq Lab), respectively. Reversetranscription and cDNA synthesis were carried out with 1 μg of RNA using theQuantiTect Reverse Transcription kit (Qiagen). Real-time PCR was performedusing the QuantiTect SYBR Green PCR kit (Qiagen) and the ABI Prism 7900HTsequence detection system (Applied Biosystems). RT-PCR results were ana-lyzed using SDS2.2 software (Applied Biosystems). Quantitative RT-PCRprimer sequences are listed in SI Dataset S1B.

mRNA in Situ Hybridization. The Vrs4 gene segment comprising parts of thefirst and second exons (395 bp) was amplified from cDNA isolated from cv.Bonus immature spikes using specific primers (SI Dataset S1B). The PCRproduct was cloned into the pBluescript II KS (+) vector (Stratagene). Cloneslinearized by EcoRI or NotI were used as templates to generate antisense(EcoRI) and sense (NotI) probes using T3 or T7 RNA polymerase. In situ hy-bridization was conducted as described (9).

BAC Sequencing, Assembly, and Annotation. A single BAC contig spanning1,073 kb of the minimum tiling path of barley chromosome 3H was shotgunsequenced using 454 technology (SI Appendix, Table S5) (32) and assembled asdescribed (33). ORFs from the BAC sequences were predicted using the Gen-escan web server (34). The predicted genes were annotated using the BLASTXfunction of the National Center for Biotechnology Information (NCBI).

Phylogenetic Analysis. Barley LBD genes were extracted from the IPK barleyBLAST server using HvRA2 as a query sequence. HvRA2 protein was used asNCBI BLASTp query to retrieve RA2 and RA2-LIKE proteins (E-value cutoff 10-30)from monocots and eudicots, respectively (SI Appendix, Table S6). For phyloge-netic analysis, the protein sequences were initially aligned using the MUSCLEalgorithm implemented in MEGA 5.1 (35). A maximum likelihood (ML)phylogenetic tree was constructed using the ML heuristic method NearestNeighbor Interchange (NNI) implemented in MEGA 5.1. The bootstrap con-sensus tree inferred from 1,000 replicates is taken to represent the evolu-tionary history of the sequences analyzed. Branches corresponding to partitionsreproduced in less than 50% bootstrap replicates are collapsed.

Scanning Electron Microscopy. Immature spike tissues at five stages (triplemound, glume, lemma, stamen, and awn primordium) from field-grown aswell as greenhouse-grown plants were used for scanning electronmicroscopy(SEM). SEM was conducted as described (36).

ACKNOWLEDGMENTS. We thank Dr. S. R. Palakolanu (International CropsResearch Institute for the Semi-Arid Tropics) for help with phylogenetic anal-ysis; Dr. B. Kilian (IPK Gatersleben) for providing germplasm for haplotypeanalysis; H. Bockelman (US Department of Agriculture–Agricultural ResearchService) and IPK gene bank for providing initial mutant germplasm; I. Waldefor helpwith theVeraCode experiment; P. Gawro�nski for helpful discussions; andH. Ernst, H. Koyama, S. König, K. Lipfert, M. Püffeld, M. Pürschel, C. Trautewig,and C. Weißleder for excellent technical support. This work was supported bygrants from the Ministry of Education (IZN), Saxony-Anhalt (to N. Sreenivasulu),the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics forAgricultural Innovation Grant TRG1004; to T.K.), the Deutsche Forschungsge-meinschaft (DFG Grant SCHN 768/2-1; to T.S.), and the BMBF (German FederalMinistry of Education and Research, GABI-FUTURE Start_Young Investiga-tor Program Grant 0315071; to T.S.).

1. Kellogg EA (2007) Floral displays: Genetic control of grass inflorescences. Curr OpinPlant Biol 10(1):26–31.

2. Vollbrecht E, Springer PS, Goh L, Buckler ES, Martienssen R (2005) Architecture offloral branch systems in maize and related grasses. Nature 436(7054):1119–1126.

3. Bortiri E, et al. (2006) ramosa2encodes a LATERALORGANBOUNDARYdomainprotein thatdetermines the fate of stem cells in branch meristems of maize. Plant Cell 18(3):574–585.

4. Gallavotti A, et al. (2010) The control of axillary meristem fate in the maize ramosapathway. Development 137(17):2849–2856.

5. Satoh-Nagasawa N, Nagasawa N, Malcomber S, Sakai H, Jackson D (2006) A trehalosemetabolic enzyme controls inflorescence architecture in maize. Nature 441(7090):227–230.

6. Sreenivasulu N, Schnurbusch T (2012) A genetic playground for enhancing grainnumber in cereals. Trends Plant Sci 17(2):91–101.

7. Forster BP, et al. (2007) The barley phytomer. Ann Bot (Lond) 100(4):725–733.8. Mansfeld R (1950) Das morphologische System der Saatgerste, Hordeum vulgare

L.s.l. Der Züchter 20:8–24.9. Komatsuda T, et al. (2007) Six-rowed barley originated from a mutation in a homeo-

domain-leucine zipper I-class homeobox gene. Proc Natl Acad Sci USA 104(4):1424–1429.10. Ramsay L, et al. (2011) INTERMEDIUM-C, a modifier of lateral spikelet fertility in

barley, is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1. NatGenet 43(2):169–172.

11. Mayer KF, et al. (2011) Unlocking the barley genome by chromosomal and compar-ative genomics. Plant Cell 23(4):1249–1263.

12. Shuai B, Reynaga-Peña CG, Springer PS (2002) The lateral organ boundaries genedefines a novel, plant-specific gene family. Plant Physiol 129(2):747–761.

13. Sakuma S, et al. (2013) Divergence of expression pattern contributed to neofunctional-ization of duplicated HD-Zip I transcription factor in barley. New Phytol 197(3):939–948.

14. Schluepmann H, Berke L, Sanchez-Perez GF (2012) Metabolism control over growth: Acase for trehalose-6-phosphate in plants. J Exp Bot 63(9):3379–3390.

15. Eveland AL, Jackson DP (2012) Sugars, signalling, and plant development. J Exp Bot63(9):3367–3377.

16. Márton ML, Cordts S, Broadhvest J, Dresselhaus T (2005) Micropylar pollen tubeguidance by egg apparatus 1 of maize. Science 307(5709):573–576.

17. Kurakawa T, et al. (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445(7128):652–655.

18. Rupp H-M, Frank M, Werner T, Strnad M, Schmülling T (1999) Increased steady statemRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducingArabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem. PlantJ 18(5):557–563.

19. Yanai O, et al. (2005) Arabidopsis KNOXI proteins activate cytokinin biosynthesis. CurrBiol 15(17):1566–1571.

20. Sakamoto T, et al. (2006) Ectopic expression of KNOTTED1-like homeobox proteininduces expression of cytokinin biosynthesis genes in rice. Plant Physiol 142(1):54–62.

21. Ashikari M, et al. (2005) Cytokinin oxidase regulates rice grain production. Science309(5735):741–745.

22. Gälweiler L, et al. (1998) Regulation of polar auxin transport by AtPIN1 in Arabidopsisvascular tissue. Science 282(5397):2226–2230.

23. Malcomber ST, Preston JC, Reinheimer R, Kossuth J, Kellogg EA (2006) Developmentalgene evolution and the origin of grass inflorescence diversity. Adv Bot Res 44:425–481.

24. Sigmon B, Vollbrecht E (2010) Evidence of selection at the ramosa1 locus during maizedomestication. Mol Ecol 19(7):1296–1311.

25. Whipple CJ, et al. (2011) grassy tillers1 promotes apical dominance in maize and re-sponds to shade signals in the grasses. Proc Natl Acad Sci USA 108(33):E506–E512.

26. Husbands A, Bell EM, Shuai B, Smith HM, Springer PS (2007) LATERAL ORGANBOUNDARIES defines a new family of DNA-binding transcription factors and can in-teract with specific bHLH proteins. Nucleic Acids Res 35(19):6663–6671.

27. Eveland AL, et al. (2010) Digital gene expression signatures for maize development.Plant Physiol 154(3):1024–1039.

28. Close TJ, et al. (2009) Development and implementation of high-throughput SNPgenotyping in barley. BMC Genomics 10:582.

29. Sato K, Nankaku N, Takeda K (2009) A high-density transcript linkage map of barleyderived from a single population. Heredity (Edinb) 103(2):110–117.

30. Vincze T, Posfai J, Roberts RJ (2003) NEBcutter: A program to cleave DNA with re-striction enzymes. Nucleic Acids Res 31(13):3688–3691.

31. Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single nucleotidepolymorphism analysis. Trends Genet 18(12):613–615.

32. Meyer M, Stenzel U, Hofreiter M (2008) Parallel tagged sequencing on the 454platform. Nat Protoc 3(2):267–278.

33. Steuernagel B, et al. (2009) De novo 454 sequencing of barcoded BAC pools forcomprehensive gene survey and genome analysis in the complex genome of barley.BMC Genomics 10:547.

34. Burge C, Karlin S (1997) Prediction of complete gene structures in human genomicDNA. J Mol Biol 268(1):78–94.

35. Tamura K, et al. (2011) MEGA5: Molecular evolutionary genetics analysis usingmaximum likelihood, evolutionary distance, and maximum parsimony methods. MolBiol Evol 28(10):2731–2739.

36. Lolas IB, et al. (2010) The transcript elongation factor FACT affects Arabidopsis veg-etative and reproductive development and genetically interacts with HUB1/2. Plant J61(4):686–697.

Koppolu et al. PNAS | August 6, 2013 | vol. 110 | no. 32 | 13203

PLANTBIOLO

GY

Supporting Information (SI) Appendix for

Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley Ravi Koppolu

a,1, Nadia Anwar

b,1, Shun Sakuma

b, Akemi Tagiri

b, Udda Lundqvist

c, Mohammad

Pourkheirandishb, Twan Rutten

d, Christiane Seiler

e, Axel Himmelbach

a, Ruvini Ariyadasa

a, Helmy

Mohamad Youssefa,f

, Nils Steina, Nese Sreenivasulu

e,g,h, Takao Komatsuda

b and Thorsten

Schnurbuscha,2

aDepartment of Genebank, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK),

Gatersleben, D06466, bPlant Genome

Research Unit, National Institute of Agrobiological Sciences

(NIAS), Tsukuba, Japan, 3058602, cNordic Genetic Resource Center, Alnarp, Sweden, SE-23053,

dDepartment of Physiology and Cell Biology, Leibniz-Institute of Plant Genetics and Crop Plant

Research (IPK), Gatersleben, D06466, eDepartment of Molecular Genetics, Leibniz-Institute of Plant

Genetics and Crop Plant Research (IPK), Gatersleben, D06466, fDepartment of Plant Physiology,

Faculty of Agriculture, Cairo University, Giza, Egypt, 12613, gResearch Group Abiotic Stress

Genomics, Interdisciplinary Center for Crop Plant Research (IZN), Halle (Saale) D06120, Germany; hGrain Quality and Nutrition Center, International Rice Research Institute (IRRI), Metro Manila

1301, Philippines; 1These authors contributed equally to this work.

Corresponding author: thor@ipk-gatersleben.de

Tel: +49 (0)39482 5-341

Fax: +49 (0)39482 5-595

SI Text

Characterization of deletion in MHOR 318 and MHOR 345.

Because the MHOR 318 and MHOR 345 mutants are X-ray induced and are not back-crossed to an

isogenic background we characterized the extent of their deletions in the Vrs4 region using primers

derived from the syntenic gene sequences from Brachypodium. The synteny in the Vrs4 region

appears to be highly conserved between barley, Brachypodium and rice (main text, Fig. 3). The table

below shows the list of Brachypodium orthologs of barley genes in the Vrs4 region and their presence

or absence in MHOR mutants. Brachy gene Annotation MHOR 318 MHOR 345

Bradi2g04110.1 proactivator polypeptide-like 1-like Intact Intact

Bradi2g04117.1 mini-chromosome maintenance complex-binding protein-like Intact Intact

Bradi2g04130.1 uncharacterized membrane protein At1g06890-like Intact Intact

Bradi2g04140.1 hypothetical protein Intact Intact

Bradi2g04150.1 Putative expressed protein Intact Intact

Bradi2g04160.1 pentatricopeptide repeat-containing protein At5g66631-like Intact Intact

Bradi2g04170.1 Putative expressed protein Intact Intact

Bradi2g04180.3 Putative expressed protein Intact Intact

Bradi2g04190.1 Putative expressed protein Intact Intact

Bradi2g04197.1 zinc finger, C3HC4 type family protein Deleted Intact

Bradi2g04210.1 autophagy 18D-like protein Deleted Intact

Bradi2g04220.1 DUF246 domain-containing protein At1g04910-like Deleted Intact

Bradi2g04230.1 catalytic/ hydrolase Deleted Intact

Bradi2g04240.1 flavin-containing monooxygenase YUCCA10-like * *

Bradi2g04247.1 flavin-containing monooxygenase YUCCA10-like * *

Bradi2g04257.1 flavin-containing monooxygenase YUCCA10-like * *

Bradi2g04270.1 ramosa2 Deleted Deleted

Bradi2g04280.1 RST1 Deleted Deleted

Bradi2g04290.1 tryptophan aminotransferase-related protein 2-like Intact Intact

Bradi2g04297.1 Putative expressed protein Intact Intact

Bradi2g04310.1 galactinol--sucrose galactosyltransferase-like Intact Intact

Bradi2g04317.1 D111/G-patch domain-containing protein Intact Intact

Bradi2g04330.1 probable LRR receptor-like serine/threonine-protein kinase

At4g26540-like

Intact Intact

*Indicates non-syntenic genes in barley. A nucleotide BLAST search against gene rich survey

sequences from Morex, Barke and Bowman deposited in IPK barley BLAST server indicated that the

orthologous barley genes of Brachypodium YUCCA10-like are located somewhere else in the barley

genome (Bradi2g04240.1 & Bradi2g04257.1 on chromosomes 2HL and Bradi2g04247.1 on 1H).

Grey highlighted genes are present in the sequenced BAC contig (main text, Fig. 3)

In MHOR 318 the deletion is comparatively larger than in MHOR 345. In both MHOR mutants

HvRA2 and RESURRECTION1 (RST1) were commonly deleted. RST1 has a putative function in lipid

synthesis and embryo development (1).

qRT PCR in vrs4 mutants BW-NIL(vrs4.k), MHOR 318 and MHOR 345.

Expression analysis of HvRA2 showed lower transcript abundance in BW-NIL(vrs4.k) (frame shift

mutation) compared to its progenitor MFB104, whereas MHOR 318 and MHOR 345 (deletion

mutants) showed complete absence of HvRA2 transcripts compared to their progenitors Ackermann’s

Donaria and Heine’s Haisa, respectively (Figure below).

Relative expression of HvRA2 in BW-NIL(vrs4.k), MHOR 318 and MHOR 345 compared to their

progenitors. Values in the table below the graph indicate expression of HvRA2 in wild-types and

respective vrs4 mutants. Error bars indicate mean±S.E. of three replicates.

Allelism test for int-e.4 and int-e.20.

Because the int-e.4 and int-e.20 mutant alleles of vrs4 did not show any molecular lesion within the

HvRA2 ORF the allelism test data is presented below which confirms that these two mutants are in

fact allelic to vrs4 locus. The int-e.4 and int-e.20 were crossed to vrs4.m and the phenotypes were

recorded for F1 and F2 stages.

Crossing analyses were done at Svalöv 1989 – 1990.

F1 : vrs4.m × int-e.4 Intermedium types

F2: vrs4.m × int-e.4 22 vrs4.m: 26 int-e.4: 61 Intermedium types stronger than int-e.4 but not so

pronounced than vrs4.m

F1 : vrs4.m × int-e.20 Intermedium types

F2: vrs4.m × int-e.20 48 vrs4.m: 34 int-e.20: 68 Intermedium types stronger than int-e.20 but not so

pronounced than vrs4.m

Triple Mound Glume Primordium Lemma Primordium Stamen Primordium Awn Primordium

MFB104 1.78 1.55 1.19 0.94 1.00

vrs4.k 1.16 1.01 0.48 0.33 0.33

AD 2.30 2.14 1.55 1.17 1.00

MHOR318 0.00 0.00 0.00 0.00 0.00

HH 2.01 1.66 1.31 1.45 1.00

MHOR345 0.00 0.00 0.00 0.00 0.00

0.00

0.50

1.00

1.50

2.00

2.50

3.00

HvR

A2/H

vA

cti

n

Additional phenotypic descriptions for vrs4 mutants.

In wild-type Vrs4 inflorescence, each spikelet meristem (SM) produces a pair of glumes (inner and

outer glumes) (Fig. S1A,D,F). The vrs4 mutants showed defects in glume development (Fig. S1C,G)

(SI Dataset S1A), including the occasional absence or rudimentary formation of one or both the

glumes and failure in separation of inner and outer glumes (Fig. S2G). In some cases the glumes

became indeterminate with more than two per spikelet (Fig. S2F) and also the glumes were modified

to produce additional spikelets/florets (Fig. S2G). Additional lemma-like structures with partially or

completely developed awns were also observed in both the central (Figure below (B), Fig. S1H) and

lateral spikelets. In case of BW-NIL(vrs4.k) and BW-NIL(mul1.a) spikelets were pedicelled

compared to sessile spikelets of their wild-types (Figure below (A)) (2). Another notable phenotype

observed in BW-NIL(vrs4.k) and BW-NIL(mul1.a) was the prostrate growth of tillers compared to

erect growth in wild-type Bowman (Figure below (C)).

(A) (B)

(C)

Differentially regulated genes in microarray analysis. Based on the 60K microarray analysis in two vrs4 deletion mutants MHOR 345 (wild-type-Heine’s

Haisa) and MHOR 318 (wild-type-Ackermann’s Donaria) at glume, stamen and awn primordium

stages we identified 752 genes that showed robust expression changes with a fold change of >2 at an

adjusted P-value of <0.05. Among them, 337 genes were down-regulated and 415 genes were up-

regulated in the mutants at two or more developmental stages. For the hierarchical clustering analysis

shown in SI Dataset S1D, all 36 arrays were pre-processed and quantile normalized, the co-

expression relationships were calculated using Spearman correlation test and complete linkage

algorithm implemented in Genespring 12.0. Interestingly, more than 70% of the differentially

regulated gene sets are reproduced in the two deletion mutants (SI Dataset S1D and S1F) and the

remaining 30% of the changes seen mainly in MHOR 318 could be related to higher extent of

deletion beyond Vrs4. The genes encoding C3HC4 zinc finger, AUTOPHAGY18D-LIKE in MHOR

318 and RST1 in MHOR 318 and MHOR 345 were highly down-regulated compared to their wild-

type counterparts. The down-regulation of these genes is most likely due to the deletion of these

genes in the respective MHOR mutants.

SI Materials and Methods

Genetic mapping. Two F2 mapping populations were generated by crossing mutant parent vrs4.k with two-rowed

parents Golden Promise (vrs4.k × Golden Promise) and Barke (Barke × vrs4.k) at IPK, Germany and

another three F2 mapping populations were generated by crossing two-rowed parent OUH602

(Hordeum vulgare ssp. spontaneum) with mutant parents Xc 41.5 (syn. vrs4.l) (Xc 41.5 × OUH602),

int-e.23 (int-e.23 × OUH602) and int-e.128 (int-e.128 × OUH602) at NIAS, Japan. F2 genotypes of

Xc 41.5 × OUH602 were tested by F3 analysis (SI Dataset S1H). For further high resolution mapping,

vrs4.k × Golden Promise population was utilized. For initial mapping of vrs4, 94 to 107 F2

individuals (Table S1) were analyzed. Segregation between mutant and Wt F2 plants fitted well with a

3:1 ratio typical for a monogenic recessive trait (Table S1).

The vrs4 locus was initially reported on chromosome 3H long arm 27.5 cM distal to uzu locus (3).

The recent genotyping of vrs4 Bowman introgression lines with BOPA markers showed SNP

polymorphisms spanning from 3H short arm until extreme long arm (4). BW-NIL(int-e.58) is

associated with SNP markers 1_0672 to 2_1083 (positions 38.56 to 156.06 cM), BW-NIL(mul1.a)

with 1_0762 and 2_0115 (positions 38.56 and 126.83 cM), BW-NIL(vrs4.k) with 1_0863 to 1_0926

(positions 64.85 to 85.26 cM) and with 2_1493 to 1_1330 (positions 161.43 to 178.12 cM). In an

effort to map vrs4 to a particular chromosomal region on 3H we developed a VeraCode SNP

genotyping assay with 113 BOPA SNPs mapping at regular intervals on 3H. Mapping of 36 and 26

polymorphic markers in vrs4.k × Golden Promise and Barke × vrs4.k populations localized vrs4

between markers 3_0571 (32.83cM) and 2_0276 (58.64cM) in Barke × vrs4.k and between 3_0953

(40.0cM), 1_1401 (58.01cM) in vrs4.k × Golden Promise populations, indicating its presence either

on the short arm or close to the centromere. Further marker development in this interval showed strict

co-segregation of vrs4 phenotype with the marker DQ327702 (41.68 cM on the short arm) in all five

mapping populations tested (Fig. S4). Linkage analysis of segregation data was carried out using

maximum likelihood algorithm of either joinmap 3.0 or MAPMAKER 3.0. Kosambi mapping

function was used to convert recombination fractions into map distances. Subsequently 1,086 F2

individuals were screened to identify recombinant individuals between flanking markers Hv03717

and Hv45740 (SI Dataset S1B). The 140 recombinant individuals identified were used for further fine

mapping of the vrs4 interval.

DNA preparation. For DNA preparation, leaf samples at the three leaf stage were collected from all the genotypes (F2

plants of five mapping populations, fine mapping population from vrs4.k × Golden Promise, 20 vrs4

mutants, respective Wts and 77 diverse barley genotypes used for haplotype analysis). DNA was

prepared using either Doyle and Doyle’s protocol (5) or the protocol described in Komatsuda et al.

1998 (6).

Haplotype analysis. Three primer pairs with overlapping fragments (SI Dataset S1C) covering 2046 bp (987 bp of 5’

region, 774 bp ORF and 285 bp 3’UTR) were amplified for direct PCR sequencing using the Sanger

method (BigDye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems). Fluorescently

terminated extension products were separated using a capillary-based ABI3730xl sequencing system

(Applied Biosystems). Multiple sequence alignments were performed using the ClustalW method (7).

Haplotype analysis was conducted using the median joining algorithm implemented in NETWORK

4.6.1.0. A default weight of 10 was applied for all substitutions and indel polymorphisms.

Microarray hybridization and data analysis.

By utilizing 50,000 consensus EST assembly (HarvEST) we synthesized multiple oligos for

individual sequences, generated a 244K microarray (Agilent Technologies). A pilot study was

conducted to identify and omit oligos with potential for cross hybridization across gene family

members from spike meristem tissues. Upon selecting oligos with best Agilent base composition

score, a custom eArray was designed to generate 56K Agilent barley microarray (60K × 8 plex

format). All unigene sets have been annotated and functionally classified into MapMan functional

categories.

Total RNA was isolated from spike meristems collected at glume, stamen and awn primordium stages

from two deletion mutants (MHOR 345 and MHOR 318) and respective wild-types (Heine’s Haisa

and Ackermann’s Donaria) using the RNA-queous MircroKit (Invitrogen). RNA concentrations were

measured using NanoDrop UV-VIS spectrophotometer (Peqlab) and quality of RNA samples was

measured using RNA Nano 6000 kit (Agilent) and Bioanalyzer (Agilent). The Low Input Quick Amp

Labeling kit for One-color Microarray-Based Gene Expression Analysis was used for labeling of

RNA samples (50ng) using Cyanine3 (Cy3) fluorescent dye following manufacturer's instructions

(Agilent). Labeled cRNA samples were subsequently purified using RNeasy mini spin columns

(Qiagen) according to the manufacturer’s protocol. Quantity of cRNA was measured using

NanoDrop, with which specific activity and yield of cRNA were calculated. 600ng of Cy3-labeled,

amplified cRNA with a specific activity of above six was used for subsequent hybridization following

the steps of the one-color microarray-based gene expression analysis protocol (Agilent).

Hybridizations were carried out at 65°C for 17h. Slides were washed and scanned at a high resolution

of 2 microns using Agilent DNA Microarray Scanner G2565CA (Agilent). The resultant 36

microarray TIF images were processed to run batch extractions by choosing appropriate grid using

Agilent’s Feature Extraction Software version 11.0. The quantified feature text file is first analysed

for quality checks using Agilent QC chart tool. The qualified experiments were further processed, at

first raw data (gene expression) was quantile normalized and fold changes were calculated between

mutant and wild-type samples for all developmental stages. The differentially regulated gene sets

(>2.0 folds), with multiple correction using Bonferroni method (P<0.05) were identified. Top

regulated genes were subjected to the co-expression analysis using spearman correlation test and

complete linkage algorithm implemented in Genespring 12.0. Only differentially regulated genes in

common between both mutants (MHOR 318 and MHOR 345) were considered for further analysis

and interpretation.

SI Figures

Fig. S1. SEM analysis of wild-type (Piroline) and vrs4 mutant (vrs4.l) spikes.

(A-C) Lateral view of inflorescences at awn primordium stage (A) Wt inflorescence shows triple

spikelet meristem (TSM) producing one central spikelet meristem (CSM) and two lateral spikelet

meristems (LSMs), leading to central spikelet (CS) and lateral spikelets (LSs). Gp, glume

primordium of LS (B) vrs4 inflorescence (Autumn-sown) showing LSs at an advanced stage of

development, and additional spikelet meristems (ASMs) which either remain rudimentary as aborted

additional spikelets (AASs) or form additional spikelets (ASs). SASM, Secondary additional spikelet

meristem. (C) vrs4 inflorescence (Spring-sown) showing branch-like inflorescence meristem (BIM),

ASs, and glume defects (upper half). (D) Wt inflorescence showing two LSs and a CS having two

glumes (G), and one floret containing one lemma (abaxial), three stamens (S) and one carpel. (E) vrs4

inflorescence showing advance staged LSs, and ASs with LSs and CSs. ASs show glumes, lemma

(abaxial) and a floral meristem (FM) (bottom), or a floret (top) having an additional floret (AF) on its

rachilla. Asterisk indicates further development on rachilla of FM or AF. (F) Wt inflorescence

showing LSs containing a single FM or floret with lemma (abaxial) and a pair of glumes. (G) vrs4

inflorescence showing AF on rachilla of floret in opposite orientation. LSs having no glumes

(asterisk) or a single glume (right-headed arrows) are visible. (H) vrs4 inflorescence showing an

additional lemma-like structure (arrows) with the lemma in CSs. (I) vrs4 inflorescence showing an

additional carpel (arrow) in opposite orientation to carpel in CS. (J) vrs4 inflorescence showing two

AAS

ASM

B

SASM

AS

BIM

C

AS

C

I

CS

L

H

CS LSLS

J

ASM

AFM

AFM

GD

GD

BIM

BIM

CS

CSD

S SSC

LCS LSLS

GG

GAF

F

G

*

F

CS

AS

AF

E

G GL

FM

LS

AS AS

AS GG

F

*

LS

*

L

CSLS

GFM

F

FL

A IM

L

LSM

CS

TSM

FMCSM

TMDR

A

Gp

LS

BIMs having ASMs, AFMs and glume defects (GD). Scale bars: 500µm in a-c; 100µm in d-e, h;

142µm in f-g, i; 200µm in j.

Fig. S2. SEM analysis of BW-NIL(mul1.a) and BW-NIL(vrs4.k).

(A) BW-NIL(mul1.a) mutant inflorescence at lemma primordium stage, showing initiation of

additional spikelet meristem (ASMs) indicated by red triangles, on each side of the triple mound. (B)

BW-NIL(vrs4.k) mutant inflorescence showing high proliferation of branch-like inflorescence

meristems (BIMs) indicated by red triangles at stamen primordium stage. (C-D) BW-NIL(vrs4.k) (C)

and BW-NIL(mul1.a) (D) mutant inflorescences showing ASMs, additional spikelets (ASs), aborted

additional spikelets (AASs), secondary additional spikelet meristems (SASM), and branch-like

inflorescence meristems (BIM) at awn primordium stage. (E,F) Pictures show loss of determinacy in

glume development resulting in more than two glumes per spikelet. Three glumes per spikelet are

seen at awn primordium stage (E) and at maturity (F). (G) Spikelet showing partially differentiated

inner and outer glumes at maturity. Scale bars: 400µm in A and 700µm in B-E.

ASM

AAS

ASM

SASM

AS

BIM BIM

SASM

ASM

AS

BIM

A B C D

E

F G

Fig. S3. Different vrs4 mutant alleles showing loss of spikelet determinacy (Red triangles mark the

presence of additional spikelets due to the indeterminate nature of the TSM). The MHOR mutants

MHOR 318 and MHOR 345 show a high degree of indeterminacy of the TSM probably due to the

complete deletion of HvRA2.

int-e.4 int-e.20 int-e.23 int-e.26 int-e.58 int-e.65 int-e.66 int-e.72 int-e.89 int-e.90

int-e.91 int-e.92 int-e.101 int-e.128 MHOR 318 MHOR345

Fig. S4. Linkage maps for the vrs4 locus on barley chromosome 3H.

(A-E) Linkage maps were developed based on five F2 mapping populations. The scale of the maps

has been reduced in the lower parts for C,D,E. The number of gametes analyzed in the upper parts

(above Bmag225) are 214 (C), 210 (D) and 214 (E). 58 gametes were analyzed in the lower parts

(below Bmag225) for C,D,E. Map distances are shown in centiMorgans. Arrow shows the location of

centromere. Xc 41.5 syn. vrs4.l.

2_0976 0.0 2_0595 2.8

Hv 04140 21.0 Hv 4130 Hv 0167800 22.0 Hv 04180 2_1145 22.1 3_0953 23.1 Hv 03717 Hv 04360 Hv 04380 DQ 327702 vrs4.k Hv 04317

23.2

1_1401 Hv 45400 1_0225

25.1 Hv 45740 25.2 1_0728 27.4 1_0281 28.3 103_104 28.6 2_1502 30.2 1_1391 1_0335 33.1 3_1153 2_1305 36.7 3_0616 3_0788 1_1394 2_0931

37.9

3_1242 Hv 49520 Hv 49560

41.5 12_31529 46.3 149_150 47.6 3_0170 51.5 3_0278 57.0 2_0063 59.1 3_0250 67.0 2_0999 139_140 69.9 53_54 71.1 2_1381 73.2 1_0662 74.8

3_0927 85.2 Barke × vrs 4 . k

2_0252 0.0 2_0976 3.2 2_0595 7.6 2_0742 15.9 3_0571 40.7 Hv 03717 45.1 Hv 04360 Hv 04430 DQ 327702 vrs4.k Hv 04380

47.9

Hv 45740 52.6 2_0276 Hv 45400 3_1011 1_1401 1_0225 3_1393

52.7

1_0653 1_1016 53.8 1_0728 1_0281 103_104

56.1

3_1153 1_1391 3_0788 2_1502 2_0931 3_0616 1_0335

57.2

1_1314 2_0017 3_1242 12_31529 Hv 49520

58.3

Hv 49870 58.4 1_0609 59.4 149_150 101_102 3_0170

60.5

3_0278 2_0063 65.0 3_0250 72.2 2_0999 76.0 53_54 76.1 2_1381 78.3 1_0662 83.1 2_1161 90.2 1_0312 92.6 3_0927 94.7 3_0953 99.3

vrs 4 . k × Golden Promise

3HS

3HL

3HS

3HL

int - e.23 × OUH602 int - e.128 × OUH602 Xc 41.5 × OUH602

2g 03617 Hv04317 Hv04360 DQ327702 int - e.128 Hv04180 2g 05710 Bmac209 2g10140

Bmag225

7.2 1.5 2.0 5.1

5.1

13.8

BJ457769 BJ475501 BJ466373 AV936694 BJ477206 AV921509 BJ460273

6.0 17.8 6.0

10.2 16.0 16.2 10.8

AV837005

HvLTPPB

Hv04317 Hv 04360 DQ327702 Xc.41.5 Hv 04180 2g 05710

Bmag225

21.3

18.4

0.5 1.4 6.5

29.8

BJ460273 AV921509

BJ457769

BJ475501 BJ466373 AV936694 BJ477206

16.2

31.4

9.5 7.5 7.5

13.7 7.7

HvLTPPB

2g03617 Hv04317 Hv04360 DQ327702 int - e.23 Hv04180 2g 04130 2g 05710 Bmac209 2g10140

Bmag225

14.0

4.6 1.0 2.4 7.0

5.5

2.0

15.5

BJ457769

BJ475501 BJ466373 AV936694 BJ477206 AV921509 BJ460273

5.7

28.7 8.3

12.9 7.5

11.7 5.6

A B

C

D

E

Fig. S5. HvRA2 (Vrs4) nucleotide and protein sequences showing mutated positions and effects for

sixteen Vrs4 mutant alleles. Small deletions and SNPs in the vrs4 mutant alleles leading to frame

shifts, non-synonymous changes and premature stop codons have been indicated in respective colors

with that of mutant alleles. The sequence in the boxed area represents the LOB domain. The blue

highlighted sequence at the 3’ end marks the 3’-UTR, the underlined region within the 3’-UTR

indicates the intron.

G -- --- (int-e.128-SNP-Nonsense mutation)

1 ATG GCA TCC CCG TCG AGC ACC GGC AAC TCC ATC GTC TCC GTG GTG GTT 48

1 M A S P S S T G N S I V S V V V 16

(mul1.a-11bp deletion-Nonsense mutation)

(int-e.65, .89, .90, .91, .92- Missense)

(vrs4.k-1bp deletion-Nonsense mutation)

--- --- A - A (int-e.87/int-e.101-Missense mutation)

49 GCA GCG GCC ACG ACA CCG GGG GCC GGG GCG CCG TGC GCT GCG TGC AAG 96

17 A A A T T P G A G A P C A A C K 32

E E

97 TTC CTG CGG CGC AAG TGC CTC CCC GGC TGC GTG TTC GCG CCC TAC TTC 144

33 F L R R K C L P G C V F A P Y F 48

145 CCG CCG GAG GAG CCG CAG AAG TTC GCC AAC GTG CAC AAG GTG TTC GGC 192

49 P P E E P Q K F A N V H K V F G 64

T (int-e.23-SNP-Missense mutation)

193 GCC AGC AAC GTG ACC AAG CTG CTC AAC GAG CTG CCG CCG CAC CAG CGG 240

65 A S N V T K L L N E L P P H Q R 80

V

T (int-e.58/int.e-72-SNP-Missense mutation)

241 GAA GAC GCC GTG AGC TCG CTG GCC TAC GAG GCG GAG GCG CGG GTC AAG 288

81 E D A V S S L A Y E A E A R V K 96

V

289 GAC CCC GTC TAC GGC TGC GTC GGC GCC ATC TCC GTG CTC CAG CGC CAG 336

97 D P V Y G C V G A I S V L Q R Q 112

337 GTC CAC CGC CTC CAG AAG GAG CTC GAC GCC GCG CAC ACC GAG CTC CTC 384

V H R L Q K E L D A A H T E L L 128

T (int-e.66-SNP-Missense mutation)

385 CGG TAC GCC TGC GGC GAG CTC GGC AGC ATC CCC ACC GCG CTC CCC GTT 432

129 R Y A C G E L G S I P T A L P V 144

L

433 GTC ACG GCC GGC GTC CCC AGC GGC AGG CTC TCA TCC GCC GTA ATG CCC 480

145 V T A G V P S G R L S S A V M P 160

T - (int-e.26-SNP-Nonsense mutation)

481 TGC CCC GGC CAG CTC GCC GGC GGC ATG TAC AGC GGT GGC GGT GGC GGT 528

161 C P G Q L A G G M Y S G G G G G 176

--- --- --- --- --- --- (vrs4.l-19bp deletion-Nonsence mutation)

529 GGC TTC CGG AGG CTC GGG CTT GTG GAC GCG ATA GTA CCA CAG CCC CCT 576

177 G F R R L G L V D A I V P Q P P 192

577 CTT TCC GCC GGC TGC TAC TAC AAT ATG CGG AGC AAC AAC AAC GCT GGA 624

193 L S A G C Y Y N M R S N N N A G 208

625 GGC AGC GTC GCT GCC GAC GTG GCG CCC GTT CAG ATC CCC TAC GCC TCC 672

209 G S V A A D V A P V Q I P Y A S 224

673 ATG GCG AAT TGG GCC GTG AAC GCC ATT AGC ACC ATT ACC ACC ACC TCA 720

225 M A N W A V N A I S T I T T T S 240

721 GGA TCA GAG AGC ATT GGG ATG GAT CAC AAG GAA GGA GGC GAC AGC AGC 768

241 G S E S I G M D H K E G G D S S 256

769 ATG TGA ACTGACGCGCCCAACCAACAgtgagtggcccgtttgagtaaatcgttgcactgta 829

257 M *

830 atcatgcgtgtatatatgctagcttcatgagttatctaatgctgcattcttgccttgtgcag 891

892 TGAATGTGGATTACCTCTCCTACTCTCAAGATTTATGGATGTGGAGAATGATATGCGCTTTGG 954

955 CCGGCCTTGACGCTCAAGGAAGGAAGCTTCGGGGAGACTGATGGGATCGAGCTAGCTCATTCC 1017

1018 CAGGATAATTAAGCTAAGAAGCAATCTATCTATGAATCTATCTATATATAGCCAACATGAATT 1080

1081 ATATATGTCAGTGTCTCCCTTTTTGCATGTTGTGCAAGCCATACTTTGTGTCGACTTTGGAGT 1143

1144 TCTATATGTAATCGAATCGAGATCTAGCTTGATCTCTCTTCTTT 1187

Fig. S6. RA2 and RA2-like proteins in grasses and eudicots.

Multiple sequence alignment of RA2 proteins in monocotyledonous and dicotyledonous species. The

unique grass specific domains at N-terminal and C-terminal are indicated with a combination of

dashed and dotted lines; LOB domain is marked with a triple line (The components of LOB domain

such as Cysteine rich region, GAS block and Leucine Zipper coiled coil motif are marked with

double, dotted and dashed lines respectively. GenBank accession numbers for RA2 & RA2-like

proteins are listed in Table S6.

Fig. S7. Phylogenetic analysis of RAMOSA2 orthologs/homologs and other LBD proteins.

A maximum likelihood phylogenetic tree (condensed tree) of RAMOSA2 orthologs (RAMOSA2

proteins from grasses) and homologs (RA2-LIKE proteins from eudicot species) together with other

LBD proteins from barley and eudicots. From the phylogenetic tree two separate clades are evident;

one clade carrying the LBD proteins other than RAMOSA2 orthologs/homologs and the other with

RAMOSA2 orthologs and homologs. The clade carrying RAMOSA2 orthologs/homologs has two

sub clades, one with the RAMOSA2 orthologs from grasses and the other with RAMOSA2

homologs. Eudicot homologs of RAMOSA2 are indicated by black triangles, orthologs from

monocots are indicated by green circles, class I barley and Eudicot LBD proteins other than

RAMOSA2 orthologs/homologs are marked by red squares. Class II barley LBD proteins indicated

by blue squares are clearly separated from all the remaining class I LOB domain proteins. For

descriptions of class I and class II LOB domain proteins refer to Shuai et al. 2002 (8). Td: Triticum

durum; Ae.ta: Aegilops tauschi; Ta: Triticum aestivum; Hv: Hordeum vulgare; Bd: Brachypodium

distachyon; Bs: Brachypodium sylvaticum; Os: Oryza sativa; Ls: Loudetia spp.; Pd: Phacelurus

digitatus; Ah: Andropogon hallii; Zm: Zea mays; Cg: Chrysopogon gryllus; Cf: Cymbopogon

flexosus; Sb: Sorghum bicolor; As: Andropterum stolzii; Ss: Schizachyrium sanguineum. (Please refer

to Table S6 for the details of protein accessions used in phylogenetic analysis).

Fig. S8. HvRA2 nucleotide-haplotype analysis.

Circles denote the relative number of samples represented in each Vrs4 haplotype. Number of

accessions within a particular haplotype is indicated in brackets. Pie slices within the circles indicate

geographic distribution of haplotypes. Lines connecting haplotypes denote number of mutations

(SNPs or deletions) that distinguish different haplotypes. (See SI Dataset S1C for haplotype structure

of the eight haplotypes identified).

Haplotype1 (51)

Haplotype2 (1)

Haplotype3 (1)

Haplotype4 (1)

Haplotype5 (1)

Haplotype6 (3)

Haplotype7 (8)

Haplotype8 (11)

EuropeAsiaAustraliaSouth AmericaNorth AmericaAfrica

1 SNP/INDEL

Fig. S9. Transcriptome analysis of vrs4 using microarray.

(A-B) Microarray heat maps show genes conjointly down-regulated (A), up-regulated (B) in two vrs4

deletion mutants (MHOR 318 and MHOR 345) and their respective wild-types (Ackermann’s

Donaria and Heine’s Haisa). GP: glume primordium, SP: stamen primordium, AP: awn primordium.

The scale at the bottom of the heat maps indicates the level of differential regulation observed for

different genes between Wt and mutant (green color indicates down-regulation and red color indicates

up-regulation).

HarvEST ID MAPMAN DESCRIPTION FUNCAT35_5446 Expansin-B11-like Cell wall.modification

35_18300 Mannose-6-phosphate isomerase-like Cell wall precursor synthesis

35_16305 Probable aldehyde oxidase2 Hormone metabolism

HvAAO4 Probable aldehyde oxidase2 Hormone metabolism

35_23690 GA 20 oxidase 3-like Hormone metabolism

35_20192 Glutathione S-transferase GSTF1-like Glutathione S-transferases

35_10423 LOG-like (LONELY GUY) Hormone biosynthesis

35_15636 EGG APPARATUS1-LIKE Protein secretion

35_20517 Predicted protein No ontology

35_21218 Predicted protein No ontology

35_23482 Predicted protein No ontology

35_26367 NAC transcription factor No ontology

35_27102 Predicted protein No ontology

35_30795 LOGL8-like Hormone biosynthesis35_36927 Predicted protein No ontology

35_38706 Predicted protein No ontology

35_49749 Cys-rich receptor-like kinase25 No ontology

35_7553 Predicted protein No ontology

35_9790 LOGL8-like Hormone biosynthesis35_9810 LOGL8-like Hormone biosynthesis35_49886 Ser-type peptidase/ trypsin Protein degradation

35_12664 Protein phosphatase2C 21-like Postranslational modification

35_23158 Vacuolar-sorting receptor 7-like Protein secretion

35_18932 ERF110-like Transcription regulation

HB26N23r_at AP2 transcription factor Transcription regulation

35_7835 bHLH128-like Transcription regulation

35_10104 Knotted-1-like 3 (KNAT3) Transcription regulation

Contig25238_at Knotted-1-like 3 (KNAT3) Transcription regulation

35_21780 GTP binding protein Rab1A Signalling.G-proteins

Contig15661_at GTP-binding protein RAB1C Signalling.G-proteins

35_3221 Random slug protein 5-like Transport misc

35_27283 Sugar transport protein 5-like Transporter sugars

Contig24094_at Sugar transport protein Transporter sugars

HH

-GP

1

HH

-GP

2

HH

-GP

3

HH

-SP

1

HH

-SP

2

HH

-SP

3

HH

-AP

1

HH

-AP

2

HH

-AP

3

MH

OR

345-G

P1

MH

OR

345-G

P2

MH

OR

345-G

P3

MH

OR

345-S

P1

MH

OR

345-S

P2

MH

OR

345-S

P3

MH

OR

345-A

P1

MH

OR

345-A

P2

MH

OR

345-A

P3

AD

-GP

1

AD

-GP

2

AD

-GP

3

AD

-SP

1

AD

-SP

2

AD

-SP

3

AD

-AP

1

AD

-AP

2

AD

-AP

3

MH

OR

318-G

P1

MH

OR

318-G

P2

MH

OR

318-G

P3

MH

OR

318-S

P1

MH

OR

318-S

P2

MH

OR

318-S

P3

MH

OR

318-A

P1

MH

OR

318-A

P2

MH

OR

318-A

P3

HarvEST ID MAPMAN DESCRIPTION FUNCAT35_37618 FRA1 (FRAGILE FIBER 1) Cell organisation

35_3905 Stem-specific protein TSJT1-like Hormone metabolism

Contig1679_s_at Jasmonate-induced protein1 Hormone metabolism

35_26361 Acyl desaturase4 Lipid metabolism

35_7759 Sister of ramosa3 Minor CHO metabolism

Contig21324_at Trehalose-6-phosphate phosphatase Minor CHO metabolism

Contig12019_at Trehalose-6-phosphate synthase Minor CHO metabolism

35_9180 Cytochrome P450 78A3-like Misc.cytochrome P450

35_9423 Cytochrome P450 CYP78A52 Misc.cytochrome P450

Contig1648_at Glutamine synthetase N-metabolism

35_22300 Methyltransferase-like protein 7A No ontology

35_20005 Predicted protein No ontology

35_21469 Predicted protein No ontology

35_21588 Anthocyanin 5-acyltransferase No ontology

35_37998 Predicted protein No ontology

35_7815 Predicted protein No ontology

35_9809 No description No ontology

35_34639 Vrs1 Homeobox transcription factor Transcription Regulation

AB259783 Vrs1 Homeobox transcription factor Transcription Regulation

35_8337 Weakly similar to WRKY28, partial Transcription Regulation

35_49439 LRR receptor-like ser/thr-kinase Transcription Regulation

HH

-GP

1

HH

-GP

2

HH

-GP

3

HH

-SP

1

HH

-SP

2

HH

-SP

3

HH

-AP

1

HH

-AP

2

HH

-AP

3

MH

OR

345

-GP

1

MH

OR

345

-GP

2

MH

OR

345

-GP

3

MH

OR

345

-SP

1

MH

OR

345

-SP

2

MH

OR

345

-SP

3

MH

OR

345

-AP

1

MH

OR

345-A

P2

MH

OR

345

-AP

3

AD

-GP

1

AD

-GP

2

AD

-GP

3

AD

-SP

1

AD

-SP

2

AD

-SP

3

AD

-AP

1

AD

-AP

2

AD

-AP

3

MH

OR

318

-GP

1

MH

OR

318

-GP

2

MH

OR

318

-GP

3

MH

OR

318

-SP

1

MH

OR

318-S

P2

MH

OR

318-S

P3

MH

OR

318

-AP

1

MH

OR

318-A

P2

MH

OR

318

-AP

3

A

B

A

C D

B

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Glume Primordium Stamen Primordium Awn Primordium

LO

NE

LY

GU

Y L

IKE

/H

vA

CT

IN

LONELY GUY-LIKE (35_9790)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Glume Primordium Stamen Primordium Awn Primordium

LO

NE

LY

GU

Y L

IKE

/H

vA

CT

IN

LONELY GUY-LIKE (35_10423)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Glume Primordium Stamen Primordium Awn Primordium

KN

OX

/HvA

CT

IN

KNOX (35_10104)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Glume Primordium Stamen Primordium Awn Primordium

EG

G A

PP

AR

AT

US

1-L

IKE

/Hv

AC

TIN

EGG APPARATUS1-LIKE

Legend

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Glume Primordia Stamen Primordia Awn Primordia

HvH

OX

1/H

vA

cti

n

Vrs1/HvHOX1 (35_34639)

AD

MHOR318

HH

MHOR345

Fig. S10. qRT-PCR validation of differentially regulated genes identified in the vrs4 microarray experiment. (A-D) qRT-PCR validation of

genes up-regulated in MHOR 318, MHOR 345 in comparison to their respective wild-types Ackermann’s Donaria (AD) and Heine’s Haisa

(HH). The validated genes include EGG APPARTUS1 (A), LONELY GUY-LIKE (B-C) and KNOX (D). (E-G) qRT-PCR validation of genes

down-regulated in mutants in comparison to their respective wild-types, include Vrs1 (E) TREHALOSE PHOSPHATE SYNTHASE (F),

SISTER OF RAMOSA3 (G). RNA samples from three spike developmental stages (glume primordium, stamen primordium and awn

primordium) were analyzed. Mean ± S.E of three biological replicates is shown.

E

G

F

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Glume Primordium Stamen Primordium Awn Primordium

HvH

OX

1/H

vA

cti

n

Vrs1/HvHOX1 (35_34639)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Glume Primordium Stamen Primordium Awn Primordium

TR

EH

AL

OS

EP

HO

SP

HA

TE

SY

NT

HA

SE

/HvA

CT

IN

TREHALOSE PHOSPHATE SYNTHASE(Contig12019_at)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Glume Primordium Stamen Primordium Awn Primordium

SIS

TE

R O

F R

AM

OS

A3 /

HvA

CT

IN

SISTER OF RAMOSA3 (35_7759)

Legend

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Glume Primordia Stamen Primordia Awn Primordia

HvH

OX

1/H

vA

cti

n

Vrs1/HvHOX1 (35_34639)

AD

MHOR318

HH

MHOR345

Fig. S11. Relative expression of HvCKX2, HvSRA and HvPIN1-LIKE in BW-NIL(vrs4.k), MHOR

318 and their respective wild-types Bowman and Ackermann’s Donaria. RNAs sampled at awn

primordium were used to quantify the expression. Constitutively expressed HvActin gene was used to

normalize expression of target genes. Error bars indicate mean±S.E. of three replicates.

0.00

0.50

1.00

1.50

2.00

2.50

HvCKX2 HvSRA PIN1-LIKE

Ex

pre

ss

ion

re

lati

ve

to

Hv

Ac

tin

Bowman

vrs4.k

Ackermann's Donaria

MHOR 318

SI Tables Table S1. Segregation analysis of vrs4 mutants in five F2 mapping populations.

Crosses # of F2 individuals

(gametes)

Wild-type Mutant χ2/P-value

for 3:1

Xc 41.5 × OUH602 107 (214) 84 23 0.701/0.402

int-e.23 × OUH602 105 (210) 84 21 1.400/0.236

int-e.128 × OUH602 107 (214) 88 19 2.994/0.083

vrs4.k × Golden Promise 94 (188) 71 23 0.014/0.905

Barke × vrs4.k 94 (188) 71 23 0.014/0.905

Table S2. Annotations of putative genes present in the vrs4 syntenic interval of Brachypodium based

on initial mapping.

Brachypodium gene Annotation e-value

Bradi2g03717.1 peptidase M48 like 0.0

Bradi2g03730.1 pentatricopeptide repeat-containing protein 0.0

Bradi2g03740.1 sphingosine-1-phosphate lyase-like 0.0

Bradi2g03750.1 Putative expressed protein 0.0

Bradi2g03760.1 40S ribosomal protein S5-2-like 7E-149

Bradi2g03770.1 Putative expressed protein 0.0

Bradi2g03777.1 monocopper oxidase-like protein SKU5-like isoform 1 0.0

Bradi2g03790.1 BFR2-like 0.0

Bradi2g03800.1 cysteine synthase, chloroplastic/chromoplastic-like isoform 1 0.0

Bradi2g03807.1 GDSL esterase/lipase 0.0

Bradi2g03820.1 FKBP12-interacting protein of 37 kDa-like 0.0

Bradi2g03825.1 embryonic abundant protein 1-like 4E-46

Bradi2g03830.1 transcription factor bHLH51-like 2E-154

Bradi2g03840.1 pyruvate decarboxylase isozyme 2-like 0.0

Bradi2g03850.1 wall-associated receptor kinase 2-like 0.0

Bradi2g03860.1 E3 ubiquitin-protein ligase SIAH1-like 4E-116

Bradi2g03870.1 E3 ubiquitin-protein ligase SIAH1-like 7E-77

Bradi2g03880.1 receptor-like protein 12-like 2E-170

Bradi2g03890.1 receptor-like protein 12-like 0.0

Bradi2g03900.1 receptor-like protein 12-like 2E-165

Bradi2g03910.1 receptor-like protein 12-like 0.0

Bradi2g03920.1 LRR receptor-like serine/threonine-protein kinase GSO1-like 0.0

Bradi2g03930.1 Putative expressed protein 2E-52

Bradi2g03940.5 Putative expressed protein 0.0

Bradi2g03947.1 DEAD-box ATP-dependent RNA helicase 0.0

Bradi2g03960.1 DEAD-box ATP-dependent RNA helicase 18-like 0.0

Bradi2g03970.1 probable protein phosphatase 2C 1-like 7E-177

Bradi2g03980.1 Golgi to ER traffic protein 4 homolog 0.0

Bradi2g03990.1 breast cancer 2 -like 0.0

Bradi2g04000.1 dehydration-responsive element-binding protein 2A-like 0.0

Bradi2g04010.1 E3 ubiquitin-protein ligase RING1-like 0.0

Bradi2g04020.1 zinc transporter 1-like 6E-113

Bradi2g04030.1 uncharacterized protein 0.0

Bradi2g04040.3 KEAP1 2E-163

Bradi2g04050.1 Putative expressed protein 0.0

Bradi2g04060.1 Putative expressed protein 0.0

Bradi2g04070.1 Putative expressed protein 0.0

Bradi2g04080.1 Putative expressed protein 0.0

Bradi2g04090.1 Putative expressed protein 0.0

Bradi2g04100.1 probable ubiquitin-conjugating enzyme E2 25-like 1E-154

Bradi2g04110.1 proactivator polypeptide-like 1-like 1E-163

Bradi2g04117.1 mini-chromosome maintenance complex-binding protein-

like

0.0

Bradi2g04130.1 uncharacterized membrane protein At1g06890-like 0.0

Bradi2g04140.1 hypothetical protein 0.0

Bradi2g04150.1 Putative expressed protein 0.0

Bradi2g04160.1 pentatricopeptide repeat-containing protein At5g66631-like 0.0

Bradi2g04170.1 Putative expressed protein 0.0

Bradi2g04180.3 Putative expressed protein 0.0

Bradi2g04190.1 Putative expressed protein 0.0

Bradi2g04197.1 zinc finger, C3HC4 type family protein 0.0

Bradi2g04210.1 autophagy 18D-like protein 0.0

Bradi2g04220.1 DUF246 domain-containing protein At1g04910-like 0.0

Bradi2g04230.1 catalytic/ hydrolase 6E-125

Bradi2g04240.1 flavin-containing monooxygenase YUCCA10-like 3E-128

Bradi2g04247.1 flavin-containing monooxygenase YUCCA10-like 0.0

Bradi2g04257.1 flavin-containing monooxygenase YUCCA10-like 0.0

Bradi2g04270.1 ramosa2 3E-91

Bradi2g04280.1 RST1 0.0

Bradi2g04290.1 tryptophan aminotransferase-related protein 2-like 0.0

Bradi2g04297.1 Putative expressed protein 0.0

Bradi2g04310.1 galactinol--sucrose galactosyltransferase-like 0.0

Bradi2g04317.1 D111/G-patch domain-containing protein 1E-88

Bradi2g04330.1 probable LRR receptor-like serine/threonine-protein kinase

At4g26540-like

0.0

Bradi2g04340.1 Putative expressed protein 0.0

Bradi2g04350.1 F-box domain containing protein 8E-98

Bradi2g04360.1 putative ZmEBE-1 protein 0.0

Bradi2g04370.1 Putative expressed protein 0.0

Bradi2g04380.1 universal stress protein A-like protein-like 1E-117

Table S3. List of barley LOB domain (LBD) containing genes used for phylogenetic analysis.

LBD gene contig LBD Class Partial/Complete

Ctg2547112_3HS Class I Complete

Ctg45166_1H Class I Complete

Ctg51474_4HS Class I Complete

Ctg135924_5HL Class I Complete

Ctg54974_1H Class I Complete

Ctg46032_6HL Class I Complete

Ctg1572307_3HL Class I Complete

Ctg62778_4HL Class I Complete

Ctg47723_4HL Class I Complete

Ctg1591863_7HS Class I Complete

Ctg38897_5HL Class I Complete

Ctg61827_3HL Class I Complete

Ctg98873_3HL Class I Complete

Ctg42541_2HL Class II Complete

Ctg50476_4HL Class II Complete

Ctg1561909 Class II Complete

Ctg36895 Class II Complete

Ctg39781_4HL Class I Partial

Ctg63283_3HL Class I Partial

Ctg1559770_4HL Class I Partial

Ctg39524_1H Class I Partial

Ctg41408_4HS Class I Partial

Ctg40527_1H Class I Partial

Contig IDs represent LBD gene containing Morex contigs available from IPK Barley BLAST server

Table S4. vrs4 mutant descriptions.

# Mutant allele Progenitor Background/

corresponding Vrs1

allele

Mutagen Mapping study Type of

mutation

Polymorphism/Position*

1 int-e.4 Bonus Bonus/Vrs1.b3 (9)** Neutrons Lundqvist 1991(10) No mutation —

2 int-e.20 Foma Foma/Vrs1.b3 (9) Neutrons Lundqvist 1991(10) No mutation —

3 int-e.23 Foma Foma/Vrs1.b3 (9) Propyl Methane

Sulfonate

Lundqvist 1991(10) Mis-sense Transition (C to T) 194bp

4 int-e.26 Foma Foma/Vrs1.b3 (9) Neutrons + Ethyl

Methane Sulfonate

Lundqvist 1991(10) Non-sense Transition (C to T)/490bp

5 BW-NIL(int-e.58) Kristina Bowman/Vrs1.b3 (11) Ethyl Methane Sulfonate Lundqvist 1991(10)

Druka et al 2011(4)

Mis-sense Transition (C to T) 248bp

6 int-e.65 Bonus Bonus/Vrs1.b3 (9) Sodium Azide Lundqvist 1991(10) Mis-sense Transition (G to A)/68bp

7 int-e.66 Kristina Kristina/Vrs1.b3 (9) Ethyl Methane Sulfonate Lundqvist 1991(10) Mis-sense Transition (C to T)/428bp

8 int-e.72 Bonus Bonus/Vrs1.b3 (9) X-rays Unpublished Mis-sense Transition (C to T) 248bp

9 int-e.87 Bonus Bonus/Vrs1.b3 (9) Sodium Azide Unpublished Mis-sense Transition (G to A)/74bp

10 int-e.89 Hege Hege/Vrs1.b Sodium Azide Unpublished Mis-sense Transition (G to A)/68bp

11 int-e.90 Hege Hege/Vrs1.b Sodium Azide Unpublished Mis-sense Transition (G to A)/68bp

12 int-e.91 Hege Hege/Vrs1.b Sodium Azide Unpublished Mis-sense Transition (G to A)/68bp

13 int-e.92 Hege Hege/Vrs1.b Sodium Azide Unpublished Mis-sense Transition (G to A)/68bp

14 int-e.101 Hege Hege/Vrs1.b Sodium Azide Unpublished Mis-sense Transition (G to A)/74bp

15 int-e.128 Foma Foma/Vrs1.b3 (9) X-rays Unpublished Non-sense Transition (A to G)/1bp

16 BW-NIL(vrs4.k) MFB104 Bowman/Vrs1.b3 (11) Gamma rays Fukuyama et

al.(12); Druka et

al.2011(4)

Non-sense Deletion (1bp)/72-72bp

17 BW-NIL (mul1.a) Montcalm Bowman/Vrs1.b3 (11) Gamma rays Kasha & Walker

(13); Druka et

al.2011(4)

Non-sense Deletion (11bp)/44-54bp

18 vrs4.l (syn. Xc 41.5) Piroline Piroline/Vrs1.b X-rays Fukuyama et al.

(12)

Non-sense Deletion (14bp)/528-

546bp

19 MHOR318 Ackermann’s

Donaria

Ackermann’s

Donaria/Vrs1.b

X-rays Unpublished Gene

deletion

—

20 MHOR345 Heine’s

Haisa

Heine’s Haisa

/Vrs1.b3 (14)

X-rays Unpublished Gene

deletion

—

*Nucleotide positions of lesions in different vrs4 mutant alleles with respect to reference sequence of HvRA2. See Fig. S5 for HvRA2

reference sequence (GenBank accession KC854554) and mutations in different vrs4 alleles mapped to the ORF.

**Reference stating the allelic status at Vrs1 locus.

Table S5. BAC clones sequenced in Vrs4 gene region on 3HS

BAC Clone # BAC Clone name BAC length (Kb)

1 HVVMRXALLeA0166J14 81

2 HVVMRXALLmA0525L04 155

3 HVVMRXALLrA0402J12 157

4 HVVMRXALLmA0524F02 116

5 HVVMRX83khA0179C14 94

6 HVVMRXALLhC0084K10 69

7 HVVMRXALLrA0075M21 49

8 HVVMRXALhLB0083N17 83

9 HVVMRX83KhA0016A06 69

10 HVVMRXALhLB0066O05 100

11 HVVMRXALLrA0390E03 120

12 HVVMRXALLeA0100K09 145

13 HVVMRXALLeA0170A17 98

14 HVVMRXALLmA0292B07 138

15 HVVMRXALLeA0349L07 111

16 HVVMRXALLeA0332L11 95

17 HVVMRXALLeA0217N06 138

18 HVVMRXALLmA0279J11 122

19 HVVMRXALLmA0260B08 105

20 HVVMRXALLmA0383N08 95

21 HVVMRXALLeA0271J13 111

22 HVVMRXALLeA0253H10 88

Table S6. Monocot RAMOSA2 orthologs, RAMOSA2-LIKE sequences from eudicots and LBD proteins used for phylogenetic analysis. # Annotation Query

cover (%)

E-value Max

identity (%)

Accession/ Morex

contig

Phylogenetic clade

1 RAMOSA2 [Hordeum vulgare] 100 0.0 100 ABC54561.1 RAMOSA2 ortholog/homolog

2 RAMOSA2 [Triticum durum] 100 5E-165 93 ADJ67794.1 RAMOSA2 ortholog/homolog

3 RAMOSA2 [Aegilops tauschii] 100 2E-169 95 EMT25999.1 RAMOSA2 ortholog/homolog

4 RAMOSA2 [Oryza sativa Japonica Group] 93 2E-125 79 ABU44495.1 RAMOSA2 ortholog/homolog

5 RAMOSA2 [Loudetia sp. MCE-2012] 94 2E-122 79 AFJ42326.1 RAMOSA2 ortholog/homolog

6 RAMOSA2 [Chrysopogon gryllus] 93 6E-112 72 AFJ42327.1 RAMOSA2 ortholog/homolog

7 RAMOSA2 [Triticum aestivum] 100 3E-158 94 ABK79907.1 RAMOSA2 ortholog/homolog

8 RAMOSA2 [Schizachyrium sanguineum var. hirtiflorum] 93 2E-101 69 AFJ42332.1 RAMOSA2 ortholog/homolog

9 RAMOSA2 [Sorghum bicolor] 94 3E-104 69 AFJ42334.1 RAMOSA2 ortholog/homolog

10 RAMOSA2 [Andropogon hallii] 94 1E-99 69 AFJ42331.1 RAMOSA2 ortholog/homolog

11 RAMOSA2 [Zea mays] 100 2E-112 73 NP_001131918.1 RAMOSA2 ortholog/homolog

12 RAMOSA2 [Phacelurus digitatus] 100 1E-110 73 AFJ42333.1 RAMOSA2 ortholog/homolog

13 RAMOSA2 [Andropterum stolzii] 94 8E-103 70 AFJ42330.1 RAMOSA2 ortholog/homolog

14 RAMOSA2 [Brachypodium distachyon] 100 5E-117 77 XP_003569245.1 RAMOSA2 ortholog/homolog

15 RAMOSA2 [Brachypodium sylvaticum] 100 1E-108 75 CCF55439.1 RAMOSA2 ortholog/homolog

16 RAMOSA2 [Cymbopogon flexuosus] 100 2E-101 67 AFJ42328.1 RAMOSA2 ortholog/homolog

17 ELONGATED PETIOULE1 (ELP1) - [Glycine max] 48 2E-67 77 AFK81066.1 RAMOSA2 ortholog/homolog

18 LBD PROTEIN-LIKE [Glycine max] 48 3E-67 77 XP_003527004.1 RAMOSA2 ortholog/homolog

19 ELONGATED PETIOULE2 - [Glycine max] 48 7E-67 75 XP_003523096.1 RAMOSA2 ortholog/homolog

20 LBD PROTEIN-LIKE [Cicer arietinum] 45 5E-67 81 XP_004515877.1 RAMOSA2 ortholog/homolog

21 LBD PROTEIN-LIKE [Glycine max] 49 5E-66 77 ACU23406.1 RAMOSA2 ortholog/homolog

22 SLEEPLESS (SLP) - ortholog of ELP1 - [Lotus japonicus] 63 5E-68 63 AFK81064.1 RAMOSA2 ortholog/homolog

23 ELONGATED PETIOULE1 (ELP1) - [Medicago truncatula] 45 4E-66 79 AFK81063.1 RAMOSA2 ortholog/homolog

24 LBD PROTEIN-LIKE [Cicer arietinum] 45 4E-64 78 XP_004501886.1 RAMOSA2 ortholog/homolog

25 APULVINIC (APU) - ortholog of ELP1 - [Pisum sativum] 45 4E-65 78 AFK81065.1 RAMOSA2 ortholog/homolog

26 LBD PROTEIN-LIKE [Cucumis sativus] 45 2E-67 81 XP_004173060.1 RAMOSA2 ortholog/homolog

27 LBD PROTEIN-LIKE [Ricinus communis] 51 9E-69 78 XP_002511341.1 RAMOSA2 ortholog/homolog

28 LBD PROTEIN-LIKE [Populus trichocarpa] 48 1E-68 81 XP_002318660.1 RAMOSA2 ortholog/homolog

29 LBD PROTEIN-LIKE [Theobroma cacao] 48 2E-68 81 EOY20851.1 RAMOSA2 ortholog/homolog

30 LBD PROTEIN-LIKE [Prunus persica] 47 3E-67 79 EMJ12162.1 RAMOSA2 ortholog/homolog

31 LBD PROTEIN-LIKE [Fragaria vesca subsp. vesca] 50 1E-67 75 XP_004298984.1 RAMOSA2 ortholog/homolog

32 LBD PROTEIN-LIKE [Solanum lycopersicum] 49 1E-66 77 XP_004236250.1 RAMOSA2 ortholog/homolog

33 LBD PROTEIN-LIKE [Capsella rubella] 57 3E-63 67 EOA15040.1 RAMOSA2 ortholog/homolog

34 ASSYMETRIC LEAVES2-LIKE4 (ASL4) [Arabidopsis thaliana] 43 4E-63 81 NP_201114.1 RAMOSA2 ortholog/homolog

35 LBD PROTEIN-LIKE [Solanum lycopersicum] 45 2E-61 74 XP_004242158.1 RAMOSA2 ortholog/homolog

36 LBD PROTEIN-LIKE [Vitis vinifera] 46 2E-68 82 XP_002281358.1 RAMOSA2 ortholog/homolog

37 LBD PROTEIN25-LIKE [Cucumis sativus] 45 2E-64 79 XP_004169034.1 RAMOSA2 ortholog/homolog

38 LBD PROTEIN25-LIKE [Cucumis sativus] 49 5E-63 75 XP_004156730.1 RAMOSA2 ortholog/homolog

39 LBD PROTEIN-LIKE [Arabidopsis thaliana] 45 2E-57 74 BAB02689.1 RAMOSA2 ortholog/homolog

40 LBD PROTEIN-LIKE [Vitis vinifera] 45 3E-63 76 XP_002273053.1 RAMOSA2 ortholog/homolog

41 LBD PROTEIN-LIKE [Theobroma cacao] 45 2E-66 79 EOY02489.1 RAMOSA2 ortholog/homolog

42 LBD PROTEIN-LIKE [Prunus persica] 45 6E-63 80 EMJ18120.1 RAMOSA2 ortholog/homolog

43 LBD PROTEIN-LIKE [Ricinus communis] 46 1E-64 78 XP_002521459.1 RAMOSA2 ortholog/homolog

44 LBD PROTEIN-LIKE [Populus trichocarpa] 45 2E-65 79 XP_002300051.1 RAMOSA2 ortholog/homolog

45 LBD PROTEIN25-LIKE [Glycine max] 46 1E-62 75 XP_003554815.1 RAMOSA2 ortholog/homolog

46 LBD PROTEIN25-LIKE [Glycine max] 45 2E-65 79 XP_003548568.1 RAMOSA2 ortholog/homolog

47 LBD PROTEIN25 [Glycine max] 45 6E-65 78 XP_003553820.1 RAMOSA2 ortholog/homolog

48 LBD PROTEIN-LIKE [Cucumis sativus] 47 5E-61 74 XP_004157828.1 RAMOSA2 ortholog/homolog

49 LBD PROTEIN-LIKE [Prunus persica] 45 7E-48 66 EMJ13351.1 LBD protein (CLASS I)

50 LBD PROTEIN4-LIKE [Fragaria vesca subsp. vesca] 45 4E-49 66 XP_004293726.1 LBD protein (CLASS I)

51 LBD PROTEIN-LIKE [Populus trichocarpa] 45 2E-47 65 XP_002304584.1 LBD protein (CLASS I)

52 LBD PROTEIN4 isoform 1 [Theobroma cacao] 45 2E-48 66 EOY27472.1 LBD protein (CLASS I)

53 LBD PROTEIN4 [Vitis vinifera] 45 8E-48 64 XP_002270650.1 LBD protein (CLASS I)

54 LBD PROTEIN4-LIKE [Cicer arietinum] 45 2E-47 64 XP_004505337.1 LBD protein (CLASS I)

55 LBD PROTEIN-LIKE - LOC100790989 [Glycine max] 47 6E-48 60 NP_001241363.1 LBD protein (CLASS I)

56 LBD PROTEIN4-LIKE [Solanum lycopersicum] 45 1E-48 65 XP_004250781.1 LBD protein (CLASS I)

57 LBD PROTEIN-LIKE [Ricinus communis] 50 3E-50 60 XP_002513395.1 LBD protein (CLASS I)

58 LBD PROTEIN4 [Theobroma cacao] 46 2E-48 64 EOY13865.1 LBD protein (CLASS I)

59 LBD PROTEIN-LIKE [Medicago truncatula] 45 3E-47 64 XP_003616690.1 LBD protein (CLASS I)

60 LBD PROTEIN-LIKE [Theobroma cacao] 47 3E-49 63 EOY34594.1 LBD protein (CLASS I)

61 LBD PROTEIN-LIKE [Vitis vinifera] 47 4E-48 64 CAN68873.1 LBD protein (CLASS I)

62 LBD PROTEIN-LIKE [Prunus persica] 47 4E-48 64 EMJ07197.1 LBD protein (CLASS I)

63 LBD PROTEIN6-like [Cucumis sativus] 50 8E-55 65 XP_004152887.1 LBD protein (CLASS I)

64 LBD PROTEIN6-like [Glycine max] 61 6E-48 57 XP_003545810.1 LBD protein (CLASS I)

65 LBD PROTEIN-LIKE [Capsella rubella] 51 3E-47 64 EOA35742.1 LBD protein (CLASS I)

66 LBD PROTEIN-LIKE [Populus trichocarpa] 59 5E-49 58 XP_002307250.1 LBD protein (CLASS I)

67 LBD PROTEIN36-like [Cucumis sativus] 51 1E-47 62 XP_004148465.1 LBD protein (CLASS I)

68 LBD PROTEIN-LIKE [Prunus persica] 53 1E-47 61 EMJ04878.1 LBD protein (CLASS I)

69 LBD PROTEIN1-LIKE [Hordeum vulgare] — 8E-57 — Ctg135924_5HL LBD protein (CLASS I)

70 LBD PROTEIN15-LIKE [Hordeum vulgare] — 3E-23 — Ctg1572307_3HL LBD protein (CLASS I)

71 LBD PROTEIN1-LIKE [Hordeum vulgare] — 9E-18 — Ctg62778_4HL LBD protein (CLASS I)

72 LBD PROTEIN11-LIKE [Hordeum vulgare] — 2E-52 — Ctg54974_1H LBD protein (CLASS I)

73 LBD PROTEIN18-LIKE [Hordeum vulgare] — 9E-12 — Ctg1591863_7HS LBD protein (CLASS I)

74 LBD PROTEIN16-LIKE [Hordeum vulgare] — 2E-27 — Ctg46032_6HL LBD protein (CLASS I)

75 LBD PROTEIN24-LIKE [Hordeum vulgare] — 9E-18 — Ctg47723_4HL LBD protein (CLASS I)

76 LBD PROTEIN6-LIKE [Hordeum vulgare] — 2E-76 — Ctg61827_3HL LBD protein (CLASS I)

77 LBD PROTEIN6-LIKE [Hordeum vulgare] — 3E-81 — Ctg45166_1H LBD protein (CLASS I)

78 LBD PROTEIN6-LIKE [Hordeum vulgare] — 2E-76 — Ctg51474_4HS LBD protein (CLASS I)

79 LBD PROTEIN7-LIKE [Hordeum vulgare] — 4E-09 — Ctg98873_3HL LBD protein (CLASS I)

80 LBD PROTEIN-LIKE [Hordeum vulgare] — 4E-10 — Ctg38897_5HL LBD protein (CLASS I)

81 LBD PROTEIN41-LIKE; Class II [Hordeum vulgare] — — Ctg42541_2HL LBD protein (CLASS II)

82 LBD PROTEIN38-LIKE; Class II [Hordeum vulgare] — — Ctg36895 LBD protein (CLASS II)

83 LBD PROTEIN39-LIKE; Class II [Hordeum vulgare] — — Ctg1561909 LBD protein (CLASS II)

84 LBD PROTEIN41-LIKE; Class II [Hordeum vulgare] — — Ctg50476_4HL LBD protein (CLASS II)

References

1. Chen X, et al. (2005) Mutation of the RESURRECTION1 locus of Arabidopsis reveals an association of cuticular wax with

embryo development. Plant Physiol. 139(2):909-919.

2. Forster BP, et al. (2007) The barley phytomer. Ann. Bot. 100(4):725-733.

3. Fukuyama T, Takahashi R, & Hayashi J (1982) Genetic studies on the induced six-rowed mutants in barley. Ber. Ohara

Inst. landw. Biol., Okayama Univ. 18:99-113.

4. Druka A, et al. (2011) Genetic dissection of barley morphology and development. Plant Physiol. 155(2):617-627.

5. Doyle JJ & Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13-15.

6. Komatsuda T, Nakamura I, Takaiwa F, & Oka S (1998) Development of STS markers closely linked to the vrs1 locus in

barley, Hordeum vulgare. Genome 41(5):680-685.

7. Thompson JD, Higgins DG, & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence

alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.

22(22):4673-4680.

8. Shuai B, Reynaga-Pena CG, & Springer PS (2002) The LATERAL ORGAN BOUNDARIES gene defines a novel, plant-

specific gene family. Plant Physiol. 129(2):747-761.

9. Komatsuda T, et al. (2007) Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class

homeobox gene. Proc. Natl. Acad. Sci. USA 104(4):1424-1429.

10. Lundqvist U (1991) Coordinator's report: Ear morphology genes. Barley Genetics Newsletter 20:85.

11. Cuesta-Marcos A, et al. (2010) Genome-wide SNPs and re-sequencing of growth habit and inflorescence genes in barley:

implications for association mapping in germplasm arrays varying in size and structure. BMC Genomics 11:707.

12. Fukuyama T, Hayashi J, & Takahashi R (1975) Genetic and linkage studies of five types of induced six-row mutants.

Barley Genetics Newsletter 5:12-13.

13. Kasha KJ & Walker GWR (1960) Several recent barley mutants and their linkages. Can. J. Genet. Cytol. 2:397-415.

14. Ramsay L, et al. (2011) INTERMEDIUM-C, a modifier of lateral spikelet fertility in barley, is an ortholog of the maize

domestication gene TEOSINTE BRANCHED1. Nat. Genet. 43(2):169-172.