Molecular genetic basis of pod corn (Tunicate maize)Luzie U. Wingen1,2, Thomas Münster1, Wolfram Faigl, Wim Deleu3, Hans Sommer, Heinz Saedler, and Günter Theißen4,5

Department of Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany

Edited by John F. Doebley, University of Wisconsin, Madison, WI, and approved March 19, 2012 (received for review July 21, 2011)

Pod corn is a classic morphological mutant of maize in which themature kernels of the cob are covered by glumes, in contrast togenerally grown maize varieties in which kernels are naked. Podcorn, known since pre-Columbian times, is the result of a dominantgain-of-function mutation at the Tunicate (Tu) locus. Some classicarticles of 20th century maize genetics reported that the mutantTu locus is complex, but molecular details remained elusive. Here,we show that pod corn is caused by a cis-regulatory mutation andduplication of the ZMM19MADS-box gene. Although the WT locuscontains a single-copy gene that is expressed in vegetative organsonly, mutation and duplication of ZMM19 in Tu lead to ectopicexpression of the gene in the inflorescences, thus conferring veg-etative traits to reproductive organs.

compound locus | morphological novelty | domestication |transcription factor | copy number variation

Maize (Zea mays ssp. mays) is an important cereal crop witha history of at least 8,000–9,000 y of human domestication,

which has resulted in maize being an important resource of 21stcentury agriculture. During this long history, some traits mayhave been chosen for different reasons than favorable agricul-tural properties, as is most likely the case for pod corn. Pod cornplants are mutant at the Tunicate (Tu) locus and show a strikingphenotype. The predominant phenotypic feature of Tu maize isa foliaceous elongation of the glumes, which cover the kernels inthe ears (1–4), different from other maize varieties in whichglumes are not present or are invisible in the mature ear (Fig. 1).Depending on gene dosage, the Tu mutation may also causeother strong phenotypic features in male (tassel) and female(ear) inflorescences of maize, including branching and de-velopment of lower florets in the ears and development of seedsin the tassel (5). Because of its bizarre phenotype, pod corn hasbeen of religious significance for certain native tribes of Amer-ican Indians since pre-Columbian times, who believed it to havemagical and curative properties (5–7). This presumably led to thepropagation of a mutant by medicine men that might otherwisehave been discarded as worthless (7). Pod corn was described as“Zea Mais var. tunicata” almost 2 centuries ago by the Frenchnaturalist Saint-Hilaire, who proposed that pod corn representsthe natural state of maize (8). This raised a considerable, long-lasting scientific interest in pod corn, as is evident from the ex-tensive literature on the topic (1–11). Despite this, the molecularmechanism responsible for the Tu phenotype remained elusive.MADS-box genes encode transcription factors involved in

plant development. One subgroup, formed by the MIKCc-typegenes, is best known for its important role in the control offlower development (reviewed in 12). Some MIKCc-type MADS-box genes, however, have functions outside of flower de-velopment and are mainly expressed in vegetative organs, forexample, the STMADS11-like genes (13–15). MADS-box genefunctions are frequently conserved between plant species andconfer comparable organ identities in different species (16). Inother cases, the role of MADS-box genes has diversified duringevolution and changes in gene functions have given rise tomorphological novelties (16).We studied MADS-box genes in maize to identify the roles of

these genes in development and evolution of a grass species.Maize is a long-standing genetic model plant with a rich resource

of classic morphological mutants. We argued that knowing thechromosomal location of maize MADS-box genes would openthe possibility to link individual genes to classic morphologicalphenotypes (17). We mapped the STMADS11-like MADS-boxgene ZMM19 close to the Tu locus, which thus became a candi-date gene for this locus (17).The present study provides several lines of evidence indicating

that the bizarre Tu phenotype is attributable to ectopic expres-sion of the developmental control gene ZMM19 in the maize ear,a gene that is normally expressed only in vegetative tissue.Moreover, we show that a change in the 5′-upstream region ofthe gene and gene duplication are the most likely causes for themutant phenotype of Tu.

ResultsRestriction Fragment Length Polymorphism Mapping of ZMM19. Toestablish the closeness of linkage between ZMM19 and the Tulocus, we performed a restriction fragment length polymorphism(RFLP) analysis of a population of 93 individuals, segregating formutant (Tu/+) and WT plants (+/+) using a ZMM19 hybridiza-tion probe. No recombination between the Tu phenotype and theZMM19 locus was found (Table S1), which suggests a mappingdistance of 0 cM (95% confidence interval: 0–3.8 cM). This tightlinkage between the ZMM19 gene and the Tu locus supports thehypothesis that ZMM19 represents the Tu locus itself.

Expression Patterns of ZMM19 in WT and Tu Plants. Inspired by dataabout homeobox genes from maize, such as Knotted1 (18),Gnarley1 (19), and Rough Sheath1 (20), as well as Hooded frombarley (21), we reason that the codominance (2, 11), cell au-tonomy (22), and gain-of-function characteristics (4) of Tu couldbe based on the ectopic expression of a transcription factorencoded by ZMM19 in those organs of maize that show a mutantphenotype. We tested this hypothesis by RNA gel blot analysesof our segregating population and found that ZMM19 is ex-pressed in vegetative leaf blades and husk leaves in WT plants(Fig. 2). In all Tu mutant plants investigated (15 of 30 individ-uals), expression of ZMM19 in vegetative and husk leaves wasabout the same as in WT plants, indicating that the Tu mutationdoes not significantly change the expression of ZMM19 in veg-etative organs (Fig. 2 and Fig. S1). In addition, however, in allmutant plants, a strong ectopic expression in male inflorescences

Author contributions: T.M., H. Saedler, and G.T. designed research; L.U.W., T.M., W.F., W.D.,and H. Sommer performed research; and L.U.W., T.M., and G.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the Euro-pean Molecular Biology Laboratory database (accession nos. AJ850298–AJ850303 andHE657274–HE657295).1L.U.W. and T.M. contributed equally to this work.2Present address: Department of Crop Genetics, John Innes Centre, Norwich NR4 7UH,United Kingdom.

3Present address: Ramiro Arnedo S.A., E-04716 Las Norias de Daza, Almería, Spain.4Present address: Department of Genetics, Friedrich Schiller University Jena, D-07743Jena, Germany.

5To whom correspondence should be addressed. E-mail: guenter.theissen@uni-jena.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1111670109/-/DCSupplemental.

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and a very strong ectopic expression in female inflorescenceswere found (Fig. 2 and Fig. S1). Thus, ZMM19 is ectopicallyexpressed in exactly those structures that show morphologicalchanges in the mutant. We corroborated this finding by in situhybridization studies, which revealed a somewhat patchy patternof ZMM19 expression throughout spikelets of mutant (Tu/+)ears from early to late stages of development (Fig. 3, row A). Theexpression is initially stronger in lateral regions, which give riseto the glumes; however, in later stages, it also involves largerparts of lower and upper spikelet primordia. The ZMM19 ex-pression signal differs considerably in both space and time fromthat of GAPDH (Fig. 3, row C) and the homeobox geneKNOTTED1 (Fig. 3, row D), thus documenting the specificity ofthe ZMM19 signal. ZMM19 expression appears to be completelyabsent in WT inflorescences comprising spikelets (Fig. 2). Fe-male inflorescences show a stronger phenotype in Tu/+ plantsthan male inflorescences. The strength of phenotype and ex-pression of ZMM19 are thus positively correlated, underpinningthe hypothesis of the identity of ZMM19 and Tu. There are atleast four different STMADS11-like genes in the maize genome(12). However, no strong ectopic expression in Tu mutants wasdetected for the genes of this subfamily that we tested: ZMM20and ZMM26 (Fig. 2). This makes a mutation of a cis-regulatoryelement of ZMM19 a likely candidate for the cause of the ectopicexpression of ZMM19.

Sequence Analysis of Different ZMM19 Alleles.We next investigatedthe nature of this putative mutation and sequenced ZMM19alleles from Tu/+ (pod corn) and +/+ (WT) plants. We detecteda rearrangement over at least 1.5 kb in the putative promoterregion of ZMM19 (Fig. S2), which might cause or contribute tothe ectopic expression of the gene, and thus to the pod cornphenotype. The deviant upstream sequence shows similarity to

mudrA, encoding the transposase of a MuDR-like transposableelement. This aberrant putative promoter region was present inZMM19 alleles of all 11 pod corn accessions tested, as revealedby DNA blot hybridization (Fig. S3) and sequence analysis (Figs.S2 and S4). In contrast, no such deviant promoter was found inany WT maize accession (13 tested), including a number ofprimitive maize lines (Fig. S5). This survey on the allelic varia-tion in all available pod corn accessions and several WT acces-sions supports the identity of Tu and ZMM19. Although theperfect correlation between an aberrant allele structure anda mutant phenotype in all accessions tested represents consid-erable evidence in itself that mutant structure and phenotype arecausally linked (21), these findings also imply that there are noindependent alleles that could be used to corroborate further ourconclusions of ZMM19 being Tu.

DNA Gel Blot Analysis Reveals a Compound ZMM19 Locus in Tu Plants.Previous genetic analyses suggested that the Tu locus is com-pound (1–3). Two lines (“half tunicates”; Tu-md and Tu-l)exhibiting similar but weaker phenotypes than Tu mutants wereisolated several times independently, and could be recombinedagain subsequently to reconstitute the “full” Tu (2). However,another recombinant version of Tu was isolated and called Tu-d(2). To disclose the structure of the Tu locus, we isolated andsequenced genomic clones containing the ZMM19 locus fromWT plants and the different mutants. By means of comparative

Fig. 1. Morphology of an ear from pod corn (Lower) and WT (Upper) maize.The pod corn ear is from a heterozygous Tu/+ plant; homozygous plantshave an even stronger phenotype.

wild type

L H T E

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ZMM20

rRNA

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rRNA

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Fig. 2. Hybridization signals of ZMM19-, ZMM20-, and ZMM26-specificprobes to an RNA gel blot containing total RNA from vegetative leaf blades(L), husk leaves (H), tassels (T), and ears (E) of WT plants (+/+) and hetero-zygous Tu mutant plants (Tu/+). ZMM20 and ZMM26 are two of the otherSTMADS11-like genes in the maize genome (17). The rRNA signals representloading controls.

D

C

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AHu

SpPSpP

loGl

uFP

Fig. 3. Expression patterns of ZMM19 in longitudinal sections through earsof heterozygous Tu mutant (Tu/+) plants as revealed by in situ hybridization.Early (Left), intermediate (Center), and late (Right) stages, respectively, ofspikelet development. (Row A) Utilization of a ZMM19 antisense probereveals strong ectopic expression of ZMM19 in a somewhat patchy patternthroughout the spikelets of mutant (Tu/+) ears. (Row B) Negative controlsusing a ZMM19 sense probe. (Row C) Positive controls using an antisenseprobe of the cytosolic GAPDH gene. (Row D) Positive controls using an an-tisense probe of the maize homeobox gene KNOTTED1 (18). GAPDH andKNOTTED1 signals are distinct, and hence gene-specific. Hu, husk leaf; loGl,lower glume; SpP, spikelet pair primordium; uFP, upper floret primordium.(Scale bar = 100 μm.)

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sequence analyses, we identified conceptual RFLPs that differ-entiate between the WT (wt ZMM19) and two different putativemutant copies of the ZMM19 locus, which we named ZMM19/Tu-A and ZMM19/Tu-B. The two mutant copies differ by aninsertion of a Jare transposable element into the first intron ofZMM19/Tu-A and a number of point mutations (Fig. S2). DNAgel blot hybridization using suitable restriction enzymes revealedbands specific for each wt ZMM19, ZMM19/Tu-A and ZMM19/Tu-B, in Tu/+ plants (Fig. 4B). This indicates that ZMM19 isduplicated in the case of the mutant Tu allele, comprising copiesZMM19/Tu-A and ZMM19/Tu-B, as predicted by the classic ge-netic analyses (2). We have determined 5.8 kb of DNA sequenceupstream of the translation start site of both ZMM19/Tu-A andZMM19/Tu-B (published in sequences AJ850302 and AJ850303,respectively) without finding homology to any of the ZMM19region, and thus conclude that the two copies must be at least5.8 kb apart. DNA gel blot analysis (Fig. 4B) revealed that Tu-md, Tu-l, and Tu-d have indeed lost one of the mutant copies,either ZMM19/Tu-A (Tu-d and Tu-l) or ZMM19/Tu-B (Tu-md).

Mutant Phenotype, Number of Mutant Components, and Strength ofEctopic Expression Are Correlated. Classic genetic analyses dem-onstrated a strong correlation between the number of mutantcomponents of Tu (mutant “gene dosage”) and the expression ofthe phenotype (1). We thus analyzed the correlation between the

amount of ectopic expression of ZMM19 in ears (Fig. 4C) and thestrength of mutant phenotype (1) (Fig. 4A), and found a goodagreement. The correlation of these features to the number ofmutant copies of ZMM19 (Fig. 4B) was less strict, presumablyattributable to differences in the genetic backgrounds of the mu-tant lines. Ears of heterozygotes Tu-d/+ and Tu-l/+, having onlyone mutant gene copy per diploid genome, showed only a mildphenotype compared with heterozygote Tu/+ with two mutantgene copies (1) (Fig. 4A). In line with this, the level of ectopicexpression of ZMM19 in ears of Tu-d and Tu-l plants was signif-icantly reduced compared with that of Tu (Fig. 4C). Tu-md/+plants, however, despite also having only one mutant gene copy,showed quite a strong phenotype under our growth conditions anda level of ectopic expression of ZMM19 very similar to that ofTu/+ plants (Fig. 4C).

Leaf Characteristics Are Promoted Under ZMM19 Ectopic Expression.ZMM19 is mainly expressed in vegetative tissue (17), and wehypothesized that ectopic expression could promote vegetativecharacteristics. To test this, we conducted morphological analysisof glumes from Tu/+ and WT plants and found the glumes fromTu/+ plants highly populated with trichomes, a leaf sheath-likefeature, in comparison to glumes from WT plants, which hadnearly no trichomes (Fig. 5). Moreover, on heterologous ex-pression of ZMM19 cDNA under control of the cauliflowermosaic virus (CaMV) 35S promoter in Arabidopsis, sepals areconsiderably enlarged and develop vegetative characteristics(Fig. 6). The comparative morphological analyses of WT, mu-tant, and transgenic plants suggest that ZMM19 expressionpromotes the development of leaf features, thus providing a clueconcerning the function of ZMM19 in WT maize.

DiscussionEvidence That Tu Is a Mutant Allele of ZMM19. The agreementsdocumented here between molecular changes at the ZMM19locus, changes in ZMM19 expression in both space and intensity,and the pod corn (Tu) phenotype in quantitative terms providecompelling evidence that Tu is a mutant allele of ZMM19.Moreover, even though we cannot provide an analysis of multipleindependent mutants, we argue that the fact that several partialphenotypic revertants of the pod corn phenotype (Tu-d/+,Tu-l/+, and Tu-md/+ plants) all show loss of one of the duplicatemutant copies (Fig. 4B) comes quite close to such kind ofevidence.The difference in the expression pattern between ZMM19 and

the closely related paralogs suggests that a mutation of a cis-regulatory element of ZMM19 is responsible for the ectopic ex-pression of ZMM19 in Tu mutants. RFLP and sequence analysisof genomic DNA from different Tu accessions from severalmaize collections show nearly identical aberrant promoterstructures for the ZMM19 gene (Figs. S3 and S4). We thussuggest that pod corn has originated only once so far in thehistory of maize. In line with this, a spontaneous origin of podcorn has never been reported, despite the millions of acres ofcorn that are grown each year.

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Fig. 4. Correlation of mutant gene dosage with the amount of ectopicexpression of ZMM19 in ears and with the strength of mutant phenotype.(A) Phenotypes of cobs of a WT (Left), Tu/+ (Center), and Tu-d/+ (Right) plant,respectively. (B) Number of ZMM19 copies in WT, Tu/+, Tu-d/+, Tu-md/+, andTu-l/+ plants as revealed by DNA blot analysis. The positions of the bandsrepresenting the different ZMM19 genes are indicated. In addition to oneWT allele of ZMM19, Tu/+ plants have two mutant gene copies in cis(ZMM19/Tu-A and ZMM19/Tu-B), whereas Tu-d/+, Tu-l/+, and Tu-md/+ plantshave only one mutant gene copy (either ZMM19/Tu-A or ZMM19/Tu-B).Numbers on the left side indicate band lengths (kb). (C) RT-PCR analysis ofZMM19 expression in female inflorescences (ears). Columns show normal-ized expression levels, with the expression levels of ZMM19 in Tu/+ plants setto 1. Error bars represent SD.

Fig. 5. Abaxial epidermal surface structure of glumes from WT (Left) andTu/+ plants (Center) compared with the surface structure of leaf sheathsfrom WT plants (Right). The images were obtained by SEM. C, carpel; loGl,lower glume; T, trichome; upGl, upper glume. (Scale bar = 100 μm.)

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Structural Evolution of the Tu Locus. We detected a rearrangementat least 1.5 kb long, and possibly longer than 5.3 kb, of the 5′-upstream promoter region of the ZMM19 allele from a Tu plant(Fig. S2). The deviant upstream sequence shows similarity tomudrA (Fig. S2), suggesting that illegitimate recombination fa-cilitated by a transposon-derived sequence was involved in thepromoter rearrangement. Similar DNA rearrangements havebeen described for other regulatory loci in maize encoding di-verse transcription factors (23, 24). The requirement for a com-plex change in the promoter region may explain why noindependent alleles of Tu ever occurred.DNA gel blot analyses (Fig. 4B) revealed that the Tu locus is

composed of two ZMM19 copies ZMM19/Tu-A and ZMM19/Tu-B.A compound locus was already predicted by the classic geneticanalyses almost 50 years ago (2). However, ZMM19 appears to be

a single-copy gene rather than a compound locus in WT maize(Fig. 4B), in contrast to previous assumptions (2).Comparison of sequences of ZMM19/Tu-A and ZMM19/Tu-B

to that of the WT copy of ZMM19 allowed us to retrace thecomplex mutational history of the Tu locus, first involvinga rearrangement in the promoter region and then gene dupli-cation, followed by sequence divergence of both copies, in-cluding the insertion of a Jare element (Fig. 7).Three half tunicate lines (Tu-md, Tu-d, and Tu-l) exhibiting

similar but weaker phenotypes than full Tu mutants were iso-lated after recombination occurred within the Tu locus (1, 2, 4).Genetic separation of Tu into three predicted components (m, d,and l), however, was never achieved (1, 4). This is now explainedby our molecular data, which reveal just two copies of ZMM19 atthe mutant Tu locus.The origin of half tunicates and the reconstitution of full

tunicates from half tunicates by recombination events at consid-erable high frequencies were striking observations of the classicgenetic experiments (1, 2, 4), which the DNA gel blot hybrid-ization experiment (Fig. 4B) now helps us to understand better.In the case of Tu-d and Tu-l, the band representing the WT copyof ZMM19 is about twice as intense as that of the mutant copy(ZMM19/Tu-B), whereas in the case of Tu-md, the bands of themutant (ZMM19/Tu-A) and WT copy are of equal intensity (Fig.4B). This observation suggests that in the process leading to halftunicates, recombination occurred between the single WT copyon one chromatid and the duplicate mutant copies on the other

wild type 35S::ZMM19

Fig. 6. Phenotype of an Arabidopsis flower in which the ZMM19 cDNA isexpressed under control of the 35S promoter of the CaMV (35S::ZMM19),compared with a WT flower. Note the enlarged, leaf-like sepals with tri-chomes in the transgenic plant.

Fig. 7. Presumed mutational history of the Tu locus. The origin of the mutant Tu locus, causing pod corn, by promoter rearrangement, gene duplication, andsequence diversification is schematically depicted. ZMM19 exons, labeled with E plus a number, are shown as boxes, with open boxes representing 5′- and3′-UTRs and filled boxes representing the coding region. Upstream, downstream, and intron sequences are depicted as lines. Breaks in the lines indicate anomission of sequence; red flashes indicate the positions of major molecular events, question marks and the red zig-zag line indicate the foreign DNA in theZMM19/Tu promoters, and the open triangle highlights the insertion locus of a “Jare” transposon (blue line) into the first intron of the ZMM19/Tu-A gene.

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chromatid during meiosis in Tu/+ plants. This would have cre-ated two gamete types, either with single mutant copies or withchimeric duplicate loci comprising a wt ZMM19 and a mutantcopy (ZMM19/Tu-B in observed cases) in cis. Fertilization of WTgametes may then have generated plants with two WT copies ofZMM19 (in trans) and a mutant copy (ZMM19/Tu-B) in the caseof Tu-d and Tu-l, and a WT copy and mutant copy (ZMM19/Tu-A) in the case of Tu-md. It is quite likely that reconstitution ofa full tunicate from half tunicates may have involved re-combination between chimeric duplicate loci composed of WTand mutant copies of ZMM19.

ZMM19 Expression May Promote the Development of Leaf Features.Analysis of trichome formation, texture, stomatal development,vascular system, and bundle structure revealed that the glumes ofTu plants have all the characteristics of typical leaf sheaths ratherthan of WT glumes (3) (Fig. 5). Because ZMM19 is stronglyexpressed in leaves in WT plants and ectopically expressed inglumes in pod corn, ZMM19 expression may promote the de-velopment of leaf features. On heterologous expression ofZMM19 cDNA under control of the CaMV 35S promoter inArabidopsis, sepals are considerably enlarged and develop vege-tative characteristics (25) (Fig. 6), indicating that inflationarygrowth of leaf-like organs (sepals and glumes) surrounding re-productive organs is a conserved response to ectopic expression ofZMM19. Similarly, ectopic expression of closely related homologs(STMADS11-like genes) of ZMM19 from Arabidopsis (SVP), po-tato (STMADS16), and rice (OSMADS22 and OsMADS47) in theflowers of Arabidopsis or tobacco, respectively, yields very similarreactions (14, 26–28), and heterotopic expression of the ZMM19ortholog MPF2 brings about the “inflated-calyx-syndrome” of the“Chinese lantern” of Physalis (27), all suggesting that ZMM19-likegenes promote vegetative development. Detailed morphologicalstudies even indicated that the sepals of tobacco flowers express-ing STMADS16 are transformed into vegetative leaves (14).Three STMAD11-like genes are present in rice. They seem to

encode negative regulators of brassinosteroid responses and in-duce vegetative growth via this pathway (29–31). Most relevantly,ectopic expression of the putative ZMM19 ortholog OsMADS22of rice in transgenic rice plants leads to the elongation of glumes(31), somewhat mimicking the Tu phenotype and thus corrobo-rating that ZMM19 is Tu.

Pod Corn Is Not an Ancestral Form of Maize. Based on archaeo-logical data, pod corn was long suspected to represent the nat-ural state of maize (8) and the Tu locus was suspected to beresponsible for controlling the switch from hard to soft glumesduring maize domestication (5, 6, 9). However, the hypothesisthat Tu was involved in maize evolution has been refuted bychromosomal mapping data (10). In line with this, we did notfind the deviant promoter structure that is typical for Tu allelesof ZMM19 in diverse close relatives of maize collectively termed“teosintes,” among which is found the putative direct ancestor ofmaize, Zea mays ssp. parviglumis (Fig. S5).These findings stronglysupport the view that pod corn does not represent an ancestralform of maize but traces back to a unique mutational event in thepromoter of ZMM19 that probably occurred only once after thedomestication of maize. Except for maize, whose glumes, palea,and lemma are so reduced that the mature grain emerges nakedabove them, almost all other grasses (including all cereals) havetheir kernels enclosed in at least one kind of these organs. It isclear from our data, however, that pod corn just phenocopies thistrait rather than representing an ancestral state. In line with this,it has been shown that changes at the TEOSINTE GLUMEARCHITECTURE1 locus, encoding an SBP-domain transcrip-tion factor, and thus not mutations at the Tu locus, controlledthe critical changes in glume structure during the domesticationof Zea mays (32).

Copy Number Variation as an Underestimated Source of GeneticDiversity in Domestication. Little is known about the role of tan-dem gene duplications and loss of duplicate copies in domesti-cation processes, but our findings concerning the Tu locus arenot unparalleled. Strikingly similar cases for copy number vari-ation are provided by the Agouti loci controlling coat color insheep and goats (33, 34). The typical coat color of WT sheep isdark-bodied with a pale belly, but artificial selection for whitefibers during domestication led to a high frequency of the whitecoat phenotype in certain breeds of sheep (33). Recently, it couldbe determined that a 190-kb tandem duplication, including theovine agouti signaling protein gene (ASIP), brought a secondcopy of the ASIP gene under the control of a nearby promoter,which led to high levels of deregulated expression of ASIP and,consequently, a dominant white color phenotype (33). Moreover,reciprocal deletions generating mutant single-copy loci of ASIPwere observed, which very much reminds us of the selective lossof one of the duplicate copies at the Tu locus during the origin ofhalf tunicates (Fig. 4B). Quite similar to what we suggest for theTu locus, a process involving recombination between duplicatedcopies has also been suggested as a likely route of mutant copyloss at the Agouti locus (33).There is evidence that a similar mechanism to that of sheep is

also responsible for the white coat color in goats (34). The com-bination of gene duplication, promoter rearrangement and de-regulated expression, and duplicate mutant copy loss, as exem-plified by pod corn (this work) and white sheep and goats (33, 34),may be of more general importance in domestication, and possiblyalso in natural evolution, than has previously been assumed.

Materials and MethodsPlant Material and Growth Conditions. A description of all maize and teosinteaccessions used is given in Table S2. Different accessions from the MaizeGenetics Stock Center with Tu phenotype (e.g., Tu, Tu-md, Tu-d, Tu-laccessions) were used to compare expression levels and morphological fea-tures; however, they were in undefined genetic backgrounds. A populationsegregating for the Tu phenotype comprised 93 individuals and was gen-erated by crossing a heterozygous Tu/+ plant with a WT (+/+) plant. Thirty ofthese plants were used for the RNA blot analyses. All plants were cultivatedunder standard greenhouse conditions.

DNA and RNA Blot Analysis. Maize genomic DNA was extracted from youngleaf material using a diethyldithiocarbamate sodium-based protocol (35).DNA blot analysis was performed following described methods (36).Hybridizations with radioactive-labeled DNA probes were always performedunder conditions of high stringency [hybridization: 68 °C in 5× SSC, 5×Denhardt’s solution, 0.5% SDS, 1 mg/mL herring sperm DNA; washing: 68 °Cin 0.1× SSPE (3.0 M sodium chloride, 0.2 M sodium hydrogen phosphate,0.02 M EDTA, pH 7.4), 0.1% SDS].

DNA restricted with enzyme BstF5I and a probe derived from the region−11 to −327 upstream of the ATG codon of the ZMM19 gene of WT maizeline T232 were used for DNA blot analysis distinguishing between differentZMM19 alleles. The same probe was used with SacI-digested DNA to screensix additional pod corn accessions and for the RFLP analysis of the 93 plantssegregating for mutant (Tu/+) and WT (+/+).

Total RNA was isolated, separated by electrophoresis, and transferred topositively charged nylon membranes (Pall) as described (35). The followingtissues were used for the RNA preparation: leaf blades of 6-wk-old plants,husk leaves covering 0.3- to 2.5-cm immature ears, tassels of 0.5–2.5 cm, andimmature ears 0.3–2.5 cm in length. Hybridization under stringent con-ditions [68 °C, DIG Easy Hybridization Solution (Roche)] was performed usingdigoxigenin-labeled riboprobes representing the C-regions and the 3′-UTRsof ZMM19, ZMM20, and ZMM26 cDNA sequences, respectively.

Construction of Genomic DNA Libraries. Genomic libraries of heterozygote Tu/+,Tu-d/+, Tu-md/+, and Tu-l/+ plants were produced in Lambda EMBL3 phages[primary titers were about 1.6 × 106 pfu/mg (phage arms)]. The genomic DNAwas prepared as described, but an additional CsCl-gradient purification stepwas carried out (37). BamHI-digested phage arms and Gigapack III XL packagingextracts were supplied by Stratagene. The cloning, packaging, and ream-plification procedures followed the manufacturer’s instructions. In addition, the

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Sau3a-digested DNA fragments were size-fractionated in a sucrose gradient(36). A maize T232 genomic library cloned in Lambda DASH2 had been madepreviously in a similar way.

Cloning and Sequencing of ZMM19 Genomic Loci. Radioactive-labeled probesrepresenting different regions of the ZMM19 cDNA were used to screen thegenomic libraries. Plaque hybridizations were performed under stringent con-ditions to avoid cross-hybridization with the closely related ZMM26 locus (hy-bridization: 65 °C in 5× SSC, 5× Denhardt’s solution, 0.5% SDS, 1 mg/mL herringsperm DNA; washing: 65 °C in 0.1× SSPE, 0.1% SDS). The following probes wereused: (i) a probe representing the complete ZMM19 cDNA to screen the T232library; (ii) a probe representing a small part of the 5′-UTR, the MADS-box, andabout 300 bp of the first intron of the ZMM19 T232 allele to screen the Tu-,Tu-d, Tu-md, and Tu-l libraries (each made of the DNA of heterozygous plants);and (iii) a probe representing the region between the seventh exon and a re-gion immediately downstream of the 3′-UTR of the ZMM19 T232 allele for anadditional screen of the Tu library. The isolated clones were preliminarily char-acterized by means of PCR assay using different pairs of ZMM19-specific primers,and selected clones were sequenced via primer walking.

Promoter Isolation by RAGE. Promoter analyses were done using the GenomeWalker Kit (Stratagene), following the instructions in the manual, and se-quencing. Sequences of ZMM19-specific primers are available on request.

In Situ Hybridization Studies. In situ hybridization studies were done asgenerally described with digoxigenin-labeled riboprobes (35) using ZMM19antisense cDNA. Hybridization with a ZMM19 sense probe is shown asa negative control. Hybridizations with antisense probes of GAPDH andKNOTTED1 are shown as positive controls.

Generation and Analysis of Transgenic Arabidopsis Plants. Plants of Arabi-dopsis thaliana ecotype Columbia were transformed using the plant binary

vector pBAR-A harboring the ZMM19 coding sequence under control of theconstitutive CaMV 35S promoter. Transformation and characterization ofthe transgenic plants were carried out as described by He and Saedler (27).

SEM. SEMof glumes and husk leaves of young cobs (1 cm long) of Tu/+ andWTplants was done essentially as described (37).

RT-PCR Analysis. For RT-PCR analysis, single-stranded cDNA of equal amountsof total RNA samples of the tissuematerials described above were generated.Pairs of ZMM19- and actin-specific oligonucleotide primers were used for theamplification of DNA fragments of 297 and 422 bp, respectively. Sampleswere taken after 28, 30, and 32 cycles and separated on agarose gels. Theintensities of the obtained signals were quantified using the ImageQuantsoftware package (Molecular Dynamics). Quantities of ZMM19 amplificationproducts were first standardized for the signals of actin controls and then tothe respective expression in ears of Tu/+ plants.

Statistics. Mapping distance and the conservative estimate of a 95% confi-dence interval were calculated as described (38).

ACKNOWLEDGMENTS. We thank Susanne Werth for skillful technicalassistance, the late Zsuzsanna Schwarz-Sommer for help with the SEM,and Maret Kalda for help with photography work. We also thank DavidJackson and Sarah Hake for providing a KNOTTED1 cDNA and Bill Martin forproviding a GAPDH cDNA from maize; the Maize Genetics Stock Center,Centro Internacional de Mejoramiento de Maíz y Trigo, Cornell University,and the Leibniz Institute of Plant Genetics and Crop Plant Research Gate-rsleben for providing seed material; and two anonymous reviewers and thePNAS editor for many helpful comments on a previous version of the man-uscript. This work was partly supported by Bundesministerium für Bildungund Forschung Grant “Entwicklungskontrollgene zum gezielten Designvon Nutzpflanzen.”

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