A Large Transposon Insertion in the stiff1 Promoter IncreasesStalk Strength in Maize[OPEN]

Zhihai Zhang,a Xuan Zhang,a Zhelong Lin,a Jian Wang,a Hangqin Liu,a Leina Zhou,a Shuyang Zhong,a Yan Li,a

Can Zhu,a Jinsheng Lai,a Xianran Li,b Jianming Yu,b and Zhongwei Lina,1

a National Maize Improvement Center; Center for Crop Functional Genomics and Molecular Breeding; Joint Laboratory forInternational Cooperation in Crop Molecular Breeding, Ministry of Education; Beijing Key Laboratory of Crop Genetic Improvement,Laboratory of Crop Heterosis and Utilization, China Agricultural University, Beijing 100193, ChinabDepartment of Agronomy, Iowa State University, Ames, Iowa 50011

ORCID IDs: 0000-0001-7870-1112 (Z.Z.); 0000-0002-6635-371X (X.Z.); 0000-0002-3534-2161 (Zhe.L.); 0000-0001-6868-1760 (J.W.);0000-0002-9330-8201 (H.L.); 0000-0001-7558-845X (L.Z.); 0000-0002-4143-9402 (S.Z.); 0000-0002-4247-9967 (Y.L.); 0000-0001-6172-3251 (C.Z.); 0000-0001-9202-9641 (J.L.); 0000-0002-4252-6911 (X.L.); 0000-0001-5326-3099 (J.Y.); 0000-0002-3161-1221(Zho.L.).

Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primarythreat to maize (Zea mays) yield under high-density planting. However, the molecular genetic basis of maize stalk strengthremains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and associationmapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kbtransposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. Thistransposable element insertion repressed the transcription of stiff1, leading to the increased cellulose and lignin contents inthe cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through theCRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealedthat the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case inwhich a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1allele pave the way for efficient improvement of maize stalk strength.

INTRODUCTION

As a typical high-yielding C4 cereal, maize (Zea mays) plays animportant role in global food security, providing more than 30% offood calories for humans (Shiferaw et al., 2011). The utilization ofheterosis has greatly improved maize yield. A key step during theutilization of maize heterosis is the introduction of stiff-stalk elitemaize resources (Tracy and Chandler, 2006). One major heteroticgroup in the United States maize breeding germplasm is the stiff-stalk group, which includes lines with better stalk strength, andimproving stalk strength has been a major breeding objective incommercialhybridsadaptedtohighplantingdensity (Duvick,2005).Improved stalk strength can not only efficiently prevent yield lossfrom stalk lodging but also greatly enhance the tolerance ofovercrowded planting. Notably, stalk lodging can reduce the totalannual maize yield by 5 to 20% worldwide (Flint-Garcia et al.,2003b). The development of the maize stalk is commonly affectedbystalk rot diseasesoriginating frompathogensandenvironmentalstresses such as drought and heat, especially under high-densityplanting conditions (Duvick, 1984; Yang et al., 2010). Specifically,late-seasonstalk lodgingresults inharvestdifficulties,a large lossof

yield, high grain moisture, and low grain quality upon pathogenattack. In contrast, a strong stalk will enhance mechanical grainharvesting. Therefore, highstalk strengthhasbeenpursued inmostmaize breeding programs to improve yield.Stalk strength is related to stalk morphology, including stalk

diameter, rind thickness, and cell wall structure componentscontaining cellulose and lignin (Robertson et al., 2017). Thechallenge in studying maize stalk strength is whether the mea-surement is feasible on a large scale. Several testing methodshavebeendeveloped.Natural stalk lodging rating is oftenaffectedby theweather. Rind penetrometer resistance (RPR)measured bya digital meter is fast and can be applied on a large scale (Flint-Garcia et al., 2003b). However, RPR measurements generally arenot easy to use to differentiate maize lines with moderate andstrong stalks (Pedersen and Toy, 1999). Othermethods, includingthree-point bending tests and x-ray computed tomographyscanning, either involve premature stalk cutting or are time-consuming and expensive (Robertson et al., 2017). Thus, it ischallenging to apply these methods on a large scale. A new easyand cheap method needs to be developed for the measurementsof maize stalk strength on large scales.The improvement of stalk strength is time-consuming in maize

breeding, as exemplified by the decreasing stalk lodging rate from19.6 to13.6%within the IowaStiff StalkSyntheticmaizepopulation,which required more than 50 years of recurrent selection (Lamkey,1992). Several genetic studies have revealed that stalk strength isgenerally controlled by numerous quantitative trait loci (QTLs; Flint-Garcia et al., 2003b; Peiffer et al., 2013; Li et al., 2014). However, the

1 Address correspondence to zlin@cau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Zhongwei Lin (zlin@cau.edu.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00486

The Plant Cell, Vol. 32: 152–165, January 2020, www.plantcell.org ã 2020 ASPB.

molecular genetic mechanism related to stalk strength in maizeremains largely unknown.

Here, we identified the gene underlying a major QTL for stalkbending strength (BS) on chromosome 6 inmaize. The stiff1 (namedafter the term stiff stalk) gene was fine-mapped within a 58-kb ge-nomic region on chromosome 6. Subsequent association mappingrevealed that the insertion of a 27.2-kb transposon in the promoter ofa gene with an F-box domain in this 58-kb fragment was correlatedwith stalk strength. Genetic transformation confirmed that the F-boxgene corresponding to stiff1 controlled stalk strength in maize.Transient expression assays together with in situ hybridizationshowed that this large insertion repressed the expression of stiff1 inmaize stalk vascular bundles. The downregulation of stiff1 led tothicker cell walls and higher stalk cellulose and lignin contents. Ourresults support that transposonsmight play important roles inmaizeimprovement. The identification of stiff1 and our newly edited stiff1allele with the clustered regularly interspaced short palindromic re-peats (CRISPR)/Cas9 system will pave the way for large-scale im-provement in maize stalks with high efficiency.

RESULTS

A Major QTL for Stalk Strength in Maize

To study themolecular geneticmechanismunderlying stalk strengthin maize, we performed QTL analysis of stalk strength ina recombinant inbred line (RIL)populationthatoriginated fromacrossbetween the typical stiff-stalk andnon-stiff-stalk lines (B73andKi11).Maize stalk strength was scored through both RPR and stalk BS,a newmethod that we developed to improve phenotyping efficiency(see Methods; Supplemental Figure 1). QTL mapping identifieda major QTL of stalk strength (Supplemental Figure 2), which ac-countedfor23.8and15.6%ofthetotalphenotypicvariationinBSandRPR, respectively. This QTL was referred to as stiff1, following thecommonly used term, “stiff stalk,” to represent the better stalkstrength. To precisely estimate the stiff1 effect, we compared twonear-isogenic lines (NILs) derived from the mapping population (see“Methods”; Supplemental Figure 3). Compared with the NIL withhomozygousKi11stiff1allele (NIL-Ki11) fromthetropicalparentKi11,theNILwithhomozygousB73stiff1allele (NIL-B73) fromthestiff-stalkparent B73 exhibited significantly enhanced BS, RPR, plant height,and stalk diameter traits as well as higher levels of the two cell wallstructural components (cellulose and lignin; Figures 1A–1J). Theweak stalk of the NIL-Ki11 lodged in the late season (Figures 1D and1E; Supplemental Figure 4).

High-Resolution Mapping of the stiff1 Gene in Maize

To fine-map stiff1, a large population with 11,538 individuals wascreated from twoheterogeneous inbred families (HIFs; Tuinstra et al.,1997) that harbored heterozygous genomic fragments at the stiff1locus but were mostly homozygous at other loci (see “Methods”;Supplemental Figure 3). Fine-mapping with 14 newly developedsimple sequence repeat (SSR)markers in this large population finallynarroweddown thestiff1genewithina58-kbgenomic fragment from96,449,596 to 96,507,664 bp based on the B73 genome sequence(V4; http://www.maizegdb.org), a region that was flanked by P7 and

P10 (Figures 2A and 2B; Supplemental Figure 5). Sequence anno-tation revealed two partial gene fragments, which were derived fromthe twogenes (Zm00001d036652andZm00001d036653), encodingan aluminum-activated malate transporter and an F-box domain,located in this region (Figure 2C). Only two partial gene fragmentswere located in the fine-mapping region because the flankingmarkers P7 and P10 were situated within these two genes.Transcription analysis further revealed that only Zm00001d036653was expressed in the stalk (Supplemental Figure 6). Thus,Zm00001d036653 became the candidate for stiff1.The Zm00001d036653 gene encoding an F-box domain

contained two exons and one intron and had 436 amino acids(Figure 2E).We next sequenced the entire 3.3-kb gene region ofZm00001d036653 between B73 and Ki11, including the 1.5-kbpromoter region, the 1,470-bp open reading frame (ORF), andthe 311-bp 39-untranslated region (39-UTR), using the se-quence from the tropical parent Ki11 as a reference (Figure 2C).Sequence analysis identified several variants, including single-nucleotide polymorphisms (SNPs) and insertions/deletions, inthe 3.3-kb region. Among these variants, two conspicuous27.2-kb and 578-bp insertions were present in the stiff-stalkparentB73at positions21094and2392 in the promoter region(Figure 2C; Supplemental Data Set 1). The 27.2-kb insertionconsisted of four Ty1/Copia transposable elements, as an-notated based on RepeatMasker (http://repeatmasker.org).

Association Mapping Identified a 27.2-kb TransposonInsertion in the stiff1 Promoter Responsible for StalkStrength in Maize

To assess whether the Zm00001d036653 gene is responsible forstalk strength, we conducted sequence analysis of this 3.3-kbfragment across a global maize inbred population with 265 ac-cessions, comprising 34 stiff-stalk, 75 tropical, 137 non-stiff-stalk,and19unknownbackgroundinbred lines(see“Methods”).Fifty-eightvariantswithanallele frequencyover 5%were identified in the3.3-kbfragment based on the Ki11 reference sequence (Figure 2D). As-sociation tests with the mixed linear model detected significantsignals in these two insertions in the promoter (P # 1.72 3 1024),suggesting that the Zm00001d036653 gene corresponding to stiff1was responsible for stalk strength (Figure 2D). The strongest signalwas present at the 27.2-kb insertion (P 5 1.19 3 1025). The nextstrongest signal occurred at the 578-bp insertion (P5 9.683 1025;Figure 2D), which was in high linkage disequilibrium with the largeinsertion (Supplemental Figure 7). This result indicated that both the27.2-kb transposon and 578-bp insertionsweremost likely to be thecausal variants of the stiff1 gene.

Overexpression and CRISPR Editing of stiff1 Confirmed ItsEffect on Stalk Strength

To confirm whether the Zm00001d036653 gene controls stalkstrength in maize, we performed genetic transformations. Aconstruct under the control of the Ubiquitin promoter (Ovstiff1)was first introduced into maize plants, and five independenttransgenic events were obtained (Figure 3A). Compared withcontrol plants, three transgenic plants (T0) with strong stiff1

Maize stiff1 Gene for Stalk Strength 153

Figure 1. stiff1 Phenotypes.

(A) The stiff1 phenotypes between two NILs. Bar 5 28 cm.(B) and (C) TheNIL-B73 plant exhibited a stronger stalk. Under the same horizontal pulling force, bending angle (B)was smaller and stalk thickness (C)washigher in the NIL-B73 plant with homozygous stiff1 than in the NIL-Ki11 (Ki11 type) plant.

154 The Plant Cell

expression were semidwarf and sterile as a result of failures intassel and ear development (Supplemental Figures 8A to 8C). Thestalks were thinner and soft, with low RPR, and lodged later in theseason (Figures 3A to3C). The two remainingT0 transgenic plantsexhibited no obvious phenotypic changes, but the seeds failed togerminate in the next generation.

We then conductedmaize transformation usingCRISPR/Cas9withtwo editing targets in the Zm00001d036653 gene coding sequence(CDS; Cong et al., 2013; Figure 3D). However, only one event wasobtained. This gene knockout plant (Cstiff1) contained two 2-bp de-letions in the CDS of stiff1 and caused a frameshift and then an early-stop translation (Supplemental Figure 8D). Compared with the controlplants, the knockout plants (Cstiff1; T1) harbored no other phenotypicchanges, includingplantheightandstalkdiameter, exceptsignificantlyincreased stalk BS (P 5 8.1 3 1024) and RPR (P 5 1.8 3 1027;Figure 3E; Supplemental Figures 8E to 8G). These transgenic resultssuggested that the Zm00001d036653 gene corresponding to stiff1controlled stalk strength in maize.

The Large Transposon Insertion Repressed stiff1Expression and Led to Better Stalk Strength in Maize

To determine how the large 27.2-kb insertion regulates stiff1 tran-scription,weperformedreal-timeRT-PCR.RT-PCRshowedthat theexpressionofstiff1wasclearly repressed in thestalk fromtheplantofNIL-B73 compared with NIL-Ki11 (Figure 4A). In situ hybridizationfurther revealed that the stiff1 gene was expressed (Figures 4B and4C) in the stalk at the V9 stage (Supplemental Figure 9). The stiff1expression was mainly detected in vascular bundles in the stalk(Figures 4B and 4C). Transient gene expression assays using a lu-ciferase reporter (LUC) were also conducted (Figure 4D). Luciferaseactivity was upregulated in the construct with the longer 1258-bppromoter (Ki11-Pro::LUC)comparedwitha truncatedconstructwitha 733-bp promoter (Ki11-Pro::LUC-T), and luciferase expressionwas also enhanced in the construct with a 461-bp distal end of this1258-bp promoter (Ki11-Pro::LUC-D) compared with Ki11-Pro::LUC, suggesting that a mini-enhancer was located at the distal endof the1258-bppromoter (Figure4D). Luciferaseactivitywasstronglyrepressed when a truncated 2456-bp sequence from the B73 large27.2-kb transposable element (B73-like-Pro::LUC)was inserted intothe 1258-bp Ki11 promoter (Figure 4D). Additionally, no apparentchange in themethylation level was detected in the promoter regionof stiff1 between NIL-B73 and NIL-Ki11 (Supplemental Figure 10).These results indicated that the large27.2-kb insertionmaypush themini-enhancer in thepromoter awayandsubsequently repress stiff1expression in the B73 plant. Therefore, both association mappingandLUC transient assay analysis revealed that the large transposoninsertion was causative to the stiff1 gene.

The sclerenchyma cell wall was thinner in the rind region and thestalk vascular bundles in theNIL-Ki11 plants comparedwith theNIL-B73 plants (Supplemental Figure 11), and these differences weremore apparent between the transgenic (Ovstiff1) and nontransgeniccontrol plants (Figures 4E and 4F). In contrast, the cell walls werethicker in sclerenchyma cells in Cstiff1 plants than in the non-transgenic control plants (Figure 4G). Both NIL-B73 with low ex-pressionandCstiff1plants exhibited significantly increasedcelluloseand lignin levels instalkcellscomparedwith those in theNIL-Ki11andOvstiff1plantswithhighexpression.Theseresultssuggested that thestiff1genecontrolledthedevelopmentof thesclerenchymacellwall inmaizestalks.Asaresult, thepresenceof the large insertion in thestiff1promoter led to increased cell wall thickness in sclerenchyma cellsfrom the rind region and stalk vascular bundles and subsequentlycaused high stalk BS in maize.

RNA-Seq Analysis for the stiff1 Gene

The stiff1 protein contains an F-box domain. Transient expressionassays revealed that STIFF1-GFP was expressed in both the cyto-plasm and nucleus in onion (Allium cepa) epidermal cells, which wasconsistent with the expression observed in maize leaf protoplasts(SupplementalFigure12).To identify thegene regulatorynetwork,weperformed RNA-seq analysis. RNA-seq revealed 2568 differentiallyexpressed (DE) genes between the knockout (Cstiff1) and controlplants. In total, 2061 and 507 DE genes from the Cstiff1 plant wereupregulated and downregulated, respectively, compared with thosefrom the control (Figure 3H). The stiff1gene regulated genetic factorsin several hormone signaling pathways, including gibberellin (GA),auxin, ethylene, andbrassinolide. TheDEgenenumbers from theGAand auxin pathways apparently outweighed those from the ethyleneand brassinolide pathways (Supplemental Data Set 2).

The stiff1 Locus Was under Strong Selection duringMaize Improvement

The global natural population with 265 maize inbred lines was di-videdintostiff-stalk,non-stiff-stalk,andtropical-subtropicalgroupsbasedon randomSNPsacross the entiremaize genome (Figure5A;Flint-Garciaetal.,2005).Mostof the inbred linesbelongingto thestiff-stalkbackgroundweresplit into fourgroupsoriginating fromtheB73,B37, N28, and B14 lines (Liu et al., 2003). B73, B37, and N28 con-tainedthefavorableallelewith the large insertion instiff1,whereas thisfavorable allele was absent in B14. The presence of the favorableB73 stiff1 allele reached 50% in the stiff-stalk group, which wasconsiderably increasedcomparedwith thenon-stiff-stalk (3.6%) andtropical-subtropical (16%) groups; Figure 5A). This result indicatedthat selection of stalk strength resulted in the accumulation of the

Figure 1. (continued).

(D) and (E) The NIL-B73 stalk stood firmly, whereas the NIL-Ki11 stalk lodged in the late season (Supplemental Figure 4).(F) to (H)ComparedwithNIL-Ki11,NIL-B73exhibited significantly enhanced trait values inBS,RPRof the stalk, andstalk diameter (SD;n>100,P<0.0001).(I)and (J)Thecontentsof thecellwall structural componentscellulose (I)and lignin (J)weresignificantly increased in thestalksofNIL-B73overNIL-Ki11 (n56, P < 0.001).Error bars indicate SD.

Maize stiff1 Gene for Stalk Strength 155

Figure 2. High-Resolution Mapping of stiff1.

(A)QTLmapping identified amajorQTL for stalk strength on chromosome6, accounting for 23.8 and15.6%of the total phenotypic variation in stalk BSandRPR, respectively. The blue and green lines represent the BS and RPR traits, respectively. The red dashed line represents the significant logarithm of theodds (LOD) threshold (3.2).

156 The Plant Cell

favorable B73 stiff1 allele in the stiff-stalk group during maizeimprovement.

We further sequencedoneadditional 1.1-kb fragment upstreamofthe large insertion (22500 to21400 bp) and the 1.8-kb ORF and 39-UTR in these maize inbreds and 15 teosinte lines (see “Methods”).Although the inbredmaize lineswith the large 27.2-kb insertionweredistributed in different groups (Figure 5A), theywere clearly clusteredintoasinglecladebasedon the2.9-kbstiff1 fragment (Figure5B).Wenext compared the nucleotide diversity among 29 maize lines in thestiff-stalk subgroup and 15 teosinte lines. DNA diversity may havedecreased in the promoter region, while no clearly changed diversitywas present in the ORF region from all maize lines in the stiff-stalkgroup in comparisonwith teosinte (Figure 5C). The Tajima’sD valueswere 2.73 (P < 0.01; maize stiff-stalk promoter), 20.94 (P > 0.10;teosinte promoter), 0.69 (P > 0.10; maize ORF), and20.57 (P > 0.10;teosinte ORF). Tajima’s D test significantly (P < 0.01) rejected theneutral null model for the promoter region, while no selection signalswere detected in the ORF region (P > 0.1) for these stiff-stalk maizeinbred lines. This result suggested that the stiff1 locus experiencedstrong selection during maize improvement. Strong selection hasswept the promoter region, and no SNPs were present in the pro-moter from21500 to22500bp,excluding the insertions/deletions inthese maize lines with the large insertion (Figure 5C). Two teosintelines split into the maize clade with the large insertion, and the twoteosinte lines contained the large 27.2-kb insertion (Figure 5B). In thisstudy, four non-stiff-stalk lines contained this large transposoninsertion at the stiff1 locus under the non-stiff-stalk genomicbackground. TheB73 stiff1 alleles fromstiff-stalk line genesmightbe introgressed into non-stiff-stalk lines in breeding.

DISCUSSION

Most of the maize genome is composed of transposable ele-ments (Schnable et al., 2009). As oneof themost active factors inthemaize genome evolution, transposons play an important roleinmaize domestication. The key steps frommultiple branches toa single stalk (Studer et al., 2011), seed shattering to non-shattering (Lin et al., 2012), andmultiple tiny ears to a single largeear on plants (Wills et al., 2013) have been achieved through thetransposition of these jumping DNA sequences. However,whether transposable elements reshape key genes for maizeimprovement remains unclear. In this study, we identified theinsertion of Ty1/Copia transposable elements in the stiff1 pro-moter responsible for stalk strength in maize, supporting the

suggestion that transposable elementsmight play a role inmaizeimprovement.Maize stalk strength is a multiple-scale phenomenon that is af-

fected by stalk morphology and chemical composition (Robertsonet al., 2016). StalkRPR testingmainly estimates stalk rind thickness(Robertson et al., 2017). While a three-point bending test and x-raycomputed tomography scanning can precisely investigate stalkstrength andmultiple-scale stalk structure (Robertson et al., 2017),both methods involve premature stalk damage and are time-consuming or expensive, difficult to be used on a large scale.Natural stalk lodging rating, which is often influenced byweather, isstill being applied in current breeding even it has a low selectionefficiency. In this study, we applied stalk BS with a simple device(see “Methods”). Compared with other methods, our stalk BStesting method is fast, cheap, repeatable, and without prematurestalk damage. This measurement of stalk BS can be directly per-formed in the field on a large scale.The knockdown of stiff1 promoted the expression of several

key genes in the GA and auxin pathways based on RNA-seq(Figures 3I and 3J; Supplemental Figure 13; Supplemental Table 1;Supplemental Data Set 2; Peng et al., 1999; Sasaki et al., 2002;Forestan et al., 2012). Then a conserved NAC (SND1)-MYB regu-latory network for secondary cell wall development in plants (Fig-ures3Iand3J;Koetal., 2009, 2014;Zhongetal., 2011;Huangetal.,2015) might be activated, and the transcription of the genes forcellulose, hemicellulose, and lignin synthesis were further pro-moted (Husseyet al., 2011). Therefore, celluloseand lignincontentsare finally increased in the stalk cell wall (Figures 3I and 3J;Supplemental Figure 13).The development of the maize stalk is impacted by numer-

ous internal (genetic) and external (environmental) factors.Improvements in stalk strength remain difficult and inefficientbased on phenotypic selection. Although the allele frequency ofthe large insertion of stiff1 reached 50% in the stiff-stalk sub-population, the allele frequency remained low in other sub-populations frommaize inbred lines for associationmapping. Inthis study, we screened a large global breeding population of1429maize inbred lines, and the favorableallele frequencyof thelarge insertion in stiff1 only reached 19%. These facts sug-gested that this favorable B73 stiff1 allele is notwidely applied inmaize except for the stiff-stalk subgroup. Our cloning of stiff1provides an efficientmethod for improvingmaize stalk ona largescale through marker-assisted selection, and precise geneeditingof stiff1will provideanothermethod forgreatly improvingmaize stalk strength.

Figure 2. (continued).

(B)High-resolutionmappingof stiff1.Only 4of 14markers forfine-mapping (Supplemental Figure 5) arepresented. ThecomparisonofBSbetween twoNILs(St72-56-1 and St72-56-2) originated from a recombination event narrowed down stiff1 to a 58-kb fragment between twomarkers: P7 and P10 (P5 1.03

10210).Orangearrows representmolecularmarkers forfine-mapping,and thepositionsof thesemarkersarepresentedbelowtheseorangearrows.Blueandgreen bars indicate the chromosomal fragments of the parental lines B73 and Ki11. The red flag indicates the target region of stiff1.(C)Sequence analysis of the 58-kb fragment between twoparent lines, B73andKi11. Two insertions of 27.2 kb and578 bpwere present in B73 at positions21094 and2392 in the promoter of the target geneZm00001d036653using theKi11 sequence as a reference. The start codonwas regarded as position 0.The two insertions are highlighted in red and purple. Blue and dark bars represent stiff1 gene exons and introns, respectively. Bar 5 1 kb.(D)Associationmappingwith amixed linearmodel revealed that the large insertion/deletion at position21094 in the stiff1promoterwas strongly correlatedwith stalkBS. The reddashed line is the1%significance threshold afterBonferroni correction for 58 tests. The stiff1genestructure ispresentedon the xaxis.(E) stiff1 protein sequence alignment between B73 and Ki11. The F-box domain is highlighted with the red dashed line.

Maize stiff1 Gene for Stalk Strength 157

Figure 3. Transgenic Analysis.

(A)TheOvstiff1 (strongallele) transgenicplantwithstiff1under thecontrol of ubiquitinwasconsiderably shorter andexhibiteda thinnerandweaker stalk thanthe nontransgenic control plant (CK), and the weak stalk of the Ovstiff1 plant was lodged in the late season.(B)and (C)ThreeOvstiff1plantswith strongexpressionexhibitedaconspicuously smaller stalkdiameter (B)andconsiderably reducedRPR (C) than theCK.*, P < 0.05 and **, P < 0.01.

158 The Plant Cell

METHODS

Plant Materials

The maize (Zea mays) RIL population (F7) between stiff-stalk andtropical parents, B73andKi11, respectively,with 189 linesderived fromone nested association mapping population (Yu et al., 2008) anda global inbred maize population (Flint-Garcia et al., 2005) with 265lines, was planted in a randomized block design with three replicates(Supplemental Data Set 3). Each line of both populations for QTLmapping and associationmappingwith 12 plants was grown in a 50-cm3 300-cm plot in the experimental station of China Agricultural Uni-versity in Beijing. All the plant materials for fine-mapping in this studywere grown in Beijing and Hainan between 2013 and 2017. All maizeplants for the QTL mapping, fine-mapping, and association mappingwere grown in the same field conditions. All these plant materials wereplanted with 50-cm row-to-row distance and 25-cm plant-to-plantdistance. The experimental fields were supplied with 120 kg ha21 N,90 kg ha21 P, and 90 kg ha21 K.

Phenotypic Investigation

To measure stalk strength in maize, we first applied RPR (Flint-Garciaet al., 2003a). RPRscoreswere obtained from themiddle of the first threenodes near the soil for 10 plants from each line from the RIL populationand the transgenic plants with Ovstiff1 using a digital force gauge(ELECALL, YLK-500; Supplemental Figure 1). The average of these RPRscores was calculated to represent the stalk strength of each line.

StalkBSwas then investigatedbypulling the stalk to abendingangleof 20° deviating from a vertical line with a digital force gauge (ELECALL,YLK-500). A simple device was developed to guarantee the same height(50 cm) and angle (20°) of pulling (Supplemental Figure 1) for all thesamples when measuring BS. This BS measurement method is fast andcan be applied for large-scale investigations of stalk strength. Similarly,BS values were collected from 10 plants from each line from both the RILand global natural maize populations, and the mean of these BS valueswas used for further analysis. These BS scores were repeated in 10 plantsfrom each line. Both RPR and BS measurements were performed at theend of the grain-filling stage, which was about 1 month after flowering.For the maize association mapping panel with diverse flowering time,flowering time frommaize diverse inbred lines was first investigated, andRPR and BS were then measured about 1 month after flowering. All thephenotypic comparisons in this study were conducted using Student’st test.

QTL Mapping and Fine-Mapping of stiff1

Themean BS and RPR values for each line from the RIL population withthree replicates were imputed into Cartographer (Silva et al., 2012) for

QTL detection. QTL analysis was performed with composite intervalmapping at a walking speed of 1 cM. The significant logarithm of theodds threshold (3.2) was determined through 1000 permutations.

We fine-mapped the major QTL for stalk strength on chromosome 6(stiff1), which accounted for 23.8 and 15.6% of the total phenotypicvariations in BS and RPR, respectively. A large population with 6190plants was created from two HIFs, Z012E0013 and Z012E0072, witha heterozygous genomic fragment at stiff1 (Supplemental Figure 3).Marker screening of this population with 14 SSRs revealed 18 repre-sentative recombination types. The descendant populations derivedfrom these recombination plants harboring heterozygous/homozy-gous fragments in the stiff1 target region were used for the correlationtest between genotypes and phenotypes. The correlations betweengenotypes and stalk BS were estimated through a linear regressionmodel to test the presence or absence of the target QTL in the re-combination plants. A significant P value (F test) indicated the presenceof the target QTL in the heterozygous fragments. Otherwise, the targetQTL would be categorized among the homozygous fragments. Ac-cording to this modified progeny test (Liu et al., 2015), stiff1was placedbetween two markers: P7 and P10.

Next, sequencing analysis revealed that two insertions of 27.2 kband 578 bp were present in the Zm00001d036653 promoter. To testwhether the Zm00001d036653 promoter region was responsible formaize stalk strength, we next screened another large population (F12)with 5348 individuals derived from SS72-68 (F11) using SSR markers.One recombination plant (St72-56) with a heterozygous fragmentbetween P7 and P10was identified. Further progeny tests between twoNILs (St72-56-1 and St72-56-1) confirmed that stiff1 was locatedbetween P7 and P10 (Supplemental Figure 5).

The two NILs (NIL-B73 and NIL-Ki11; F12) were generated from a HIFfrom the recombination plant (SS72-7-4 [F11]; Supplemental Figure 5A) inthe fine-mapping, which contained heterozygous genotypes in the stiff1locus but was homozygous in the other QTLs on chromosome 10 andmostother chromosomal regions (Supplemental Figure 3). All the primers forthese fine-mapping markers are listed in Supplemental Table 2. TheseNILs were planted in the field for phenotypic comparisons.

Sequence Analysis

The 3.3-kb fragment with a 1.5-kb promoter region, a 1470-bp ORF, anda 311-bp 39-UTRwas sequenced from amaize associationmapping panelwith 265 samples. The variants in the 3.3-kb fragment were applied forassociation mapping. The presence or absence of the large 27.2-kb in-sertion in a total of 1429 maize inbred lines was determined using primerpairs I1, I2, and I3 (Supplemental Figure 14). The sequences of the 1.5-kbpromoter region were not well aligned due to the presence of a lot of in-sertion/deletions. We then sequenced an additional 1.1-kb promoterfragment upstream of the large 27.2-kb transposable element for DNAdiversity analysis. This 2.9-kb sequence containing a 1.1-kb promoter

Figure 3. (continued).

(D)TheCRISPRgene-editingplantwith twocutting targets in theCDSofstiff1exhibitednoadditional apparentphenotypicchanges incontrast toCKexceptthe enhanced stalk BS. Red dashed triangles indicate the 2-bp deletion created by CRISPR.(E)TheBSwassignificantly increased in theCstiff1plant comparedwith that in theCKplant. Thenumberof plantsmeasured is presentedon thebars. **, P<0.001.(F) and (G)Cellulose and lignin levels were significantly increased in theCstiff1 plants but significantly decreased inOvstiff1 plants compared with those inthe CK. **, P < 0.01.(H) to (J)RNA-seqanalysis betweenCstiff1andCK, theDEgenes (H); radar plot of expression levels of keygenes for thegene regulatory networkof stiff1 (I),where thebluenumberson theplot represent theexpression levels (fragmentsper kilobaseofexonpermillion fragmentsmapped); andapotential regulatorynetwork for stiff1 (J).Error bars indicate SD.

Maize stiff1 Gene for Stalk Strength 159

Figure 4. stiff1 Function.

(A) RT-qPCR revealed that stiff1 exhibited increased expression in NIL-Ki11 stalk compared with that in NIL-B73.(B) and (C) In situ hybridization for stiff1 in the stalk of NIL-Ki11 with antisense (B) and sense (C) probes in themiddle of the jointing stage. Bars5 500 mm.

160 The Plant Cell

fragment, a 1.8-kb ORF, and a 39-UTR was amplified for DNA diversityanalysis. All the PCR products were cleaned using the QIAquick PCRPurification Kit (Qiagen) and were subsequently sequenced using an ABI3730 sequencer.

Association Mapping

The association test was performed with R software (http://www.r-project.org) using a simple linear model and the F test as the set statisticaltool given that the stalk BS is a continuous trait. Variants in the 3.3-kbfragment containing a 1.5-kb promoter, a 1470-bp ORF, and a 311-bp 39-UTR with an allele frequency more than 5% were applied for associationmapping. Association mapping was performed with a mixed linear modelusing a genome-wide set of SNPs to calculate population structure andkinship, and the genome-wide SNPs were provided in Tassel software(Bradbury et al., 2007). SNPs for the stiff1 gene were identified fromSanger sequencing in a maize association panel (Supplemental DataSet 3).

The significance threshold was corrected for multiple testing throughBonferroni correction based on the following equation: a9 � a/n5 1.723

1024, where a is the nominal significance threshold (a5 0.01) and n is thenumber of variants (n 5 58; Figure 2D).

DNA Diversity Analysis

Briefly, the 2.9-kb sequences containing the 1.1-kb promoter upstream ofthe large 27.2-kb transposable element, 1.8-kb ORF, and 39-UTR from 15teosinte (Supplemental Table 3) and 29 inbred maize lines in the stiff-stalksubgroup were imported into ClustalW to construct a nucleotide alignmentmatrix, which was further used for nucleotide diversity analysis usingDnaSP V5.10 (Rozas et al., 2003). Tajima’s D test (Tajima, 1989) wasperformed inDnaSPV5.10basedon the 1.1-kb promoter, 1.8-kbORF, and39-UTR sequences.

RNA in Situ Hybridization

RNA in situ hybridization was conducted based on the protocol fromXiaolan Zhang’s lab. The uppermost extending stem tips (2–3 mm) atdifferent developmental stages (V5, V7, V9, and V12) from NIL-Ki11plants were fixed in 3.7% FAA solution (50 mL of ethanol, 5 mL of aceticacid, 10 mL of 37% formaldehyde, and 35 mL of diethyl pyrocarbonate-treated water) on ice (4°C), dehydrated by ethanol from 50 to 100%,infiltrated by Histo-clear (Thermo Fisher Scientific) from 50 to 100%,embedded in Paraplast (Sigma-Aldrich), and stored at 4°C. Fixedsamples were sliced into 8- to 10-mmsections using amicrotome (LeicaRM2145). Sense and antisense probes were amplified based on an;300-bp stiff1 cDNA fragment with gene-specific primers containingSP6 and T7 RNA polymerase binding sites and then labeled with di-goxigenin from a DIG Northern Starter Kit (Roche). RNA in situ hy-bridization was performed with probes on the sections, and slides were

observed and photographed with a microscope (Leica DMR) anda microcolor CCD camera (Apogee Instruments).

Transformation

The CDS of stiff1 amplified from B73 was inserted into the binary vectorpCUNm-eGFP under control of the Ubiquitin promoter. The constructwas then transformed into maize inbred line B73 with Agrobacteriumtumefaciens EHA105, following a reported protocol (Ishida et al., 2007).Cas9 was driven by the rice (Oryza sativa) OsU3 promoter (Trapnell et al.,2010). Two gRNAs targeting two sites in the first exon of the stiff1 genewere designed with CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/)and then introduced into CRISPR/Cas9 binary vector. All these con-structs were introduced into the maize stiff-stalk inbred line B73. FiveOvstiff1 (T0) events were obtained. However, only one homozygousCstiff1 (T0) gene-editing event was created using the CRISPR/Cas9system. The knockout line contained two 2-bp deletions in the stiff1CDS(Supplemental Figure 8D). The T0 Cstiff1 plant was self-crossed togenerate T1 plants for the measurement of the BS of stalks. All these T0overexpressed and T1 CRISPR plants were compared with the controlplants, which contained an empty construct through transformations.Each edited T1 plant with 2-bp deletion was confirmed throughsequencing.

All the T0 and T1 transgenic and nontransgenic control plants weregrownunder14hof light and10hofdark inpots suppliedwith1gN/pot, 1gP/pot, and 1 gK/pot, with 60-cm row-to-row distance and 30-cmplant-to-plant distance, in a greenhouse. BS and RPR measurements were per-formed in the grain-filling stage (1 month after flowering). The transgenicplants showed similar flowering time to the control plants.

Subcellular Localization

The pUbi:stiff1-GFP construct was generated by the fusion of the stiff1CDS with GFP under the control of the maize Ubiquitin promoter. TheUbi:stiff1-GFP construct was then introduced into onion (Allium cepa)epidermal cells and maize leaf protoplasts, and the subcellular locali-zation of GFP signals was examined using a Nikon C1 confocal lasermicroscope.

Bisulfite Sequencing

Genomic DNA was first treated with the EZ DNA Methylation kit (ZymoResearch). After unmethylated cytosines were converted to uracils, PCRwas conducted. All the successful PCR products were further cloned intothe pEASY-T1 vector (Tiangen). Twenty clones from each PCR productwere then sequenced, and the methylation level was determined.

Phylogenetic Analysis

A nucleotide alignment matrix based on 2.9-kb sequences, including the1.1-kb promoter upstream of the large transposable element, 1.8-kb CDS,

Figure 4. (continued).

(D) Luciferase reporter gene transient assay. Four constructs with a 733-bp truncated promoter (Ki11-Pro::LUC-T), a 1258-bp promoter (Ki11-Pro::LUC),and a 461-bp distal end of the 1258-bp promoter (Ki11-Pro::LUC-D) from the tropical parent Ki11 and the 1258-bp promoter with a 2456-bp transposablesequence insertion from the stiff-stalk parent B73 were introduced into maize mesophyll protoplasts. Empty vector was used as a control. The relativeexpression was quantified according to the LUC:REN ratio. Significant P values for the comparisons of relative expression between pairwise constructsusing Student’s t test are presented. The red wedge indicates the insertion of a 2456-bp transposable element from B73. Error bar indicate SE (n 5 6).(E) to (G)Scanning electronmicrographs of transections of stalks fromOvstiff1 (E), nontransgenic control (CK [F]), andCstiff1 (G) plants. A thinner cell wallwas present in the sclerenchyma of the rind region and vascular bundles (V) in the stalk ofOvstiff1, whereas a thicker cell wall occurred inCstiff1 comparedwith that in the CK. Bars 5 100 mm.

Maize stiff1 Gene for Stalk Strength 161

and 39-UTR, was imported into MEGA7 to generate a phylogenetic treeusing a statistical method of maximum likelihood under the Tamura-Neimodel (Kumar et al., 2016). The nucleotide alignment matrix and themachine-readable tree file were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S25304).

RNA-Seq Analysis

RNA samples with three biological replicates were collected from the up-permostextendingstemtips (2–3mm;V9) fromCstiff1andthecontrolplants.

The six DNA-free RNA samples were then sequenced with a Hiseq-2500,

Figure 5. Nucleotide Diversity of stiff1.

(A)Theglobalmaizepopulationwasdivided into threesubgroupsbasedonprincipalcomponentanalysis.Non-stiff-stalk (NSS), stiff-stalk (SS), and tropical-subtropical (TRO) inbredmaize lineswith a large insertion of 27.2 kbare highlighted in red. A set of genome-wide randomSNPs (Supplemental Table 4)wereused to perform the principal component analysis.(B)Phylogenetic tree analysis based on the stiff1gene sequence revealed that these lineswith a large 27.2-kb insertion clustered together (in blue). The twoteosinte lines (T11 and T52) with this large insertion are labeled in orange.(C)Nucleotide diversity of stiff1 in the promoter region andORF and 39-UTR between teosinte andmaize inbred lines in the stiff-stalk subgroup (maize-SS).Error bars indicate SD (n > 15).All the data used in Figure 5 are listed in Supplemental Table 4.

162 The Plant Cell

and 40 Gb of raw sequencing data were obtained. These raw RNA-seqreads were then analyzed following a standard RNA-seq analysis pipeline.Briefly, the raw RNA-seq reads were initially processed to remove theadapter sequences and low-quality bases with Trimmomatic version 0.33(Bolger et al., 2014) in paired end mode with recommended parameters.The virus-like and rRNA-like RNA-seq reads were further removed withfastq_clean (Zhang et al., 2014). Finally, the clean RNA-seq reads weremapped to the reference genomes using STARversion 2.5.0b (Dobin et al.,2013). To improve spliced alignment, STARwasprovidedwith exon junctioncoordinates from the reference annotations. Default alignment parameterswere used, and the outSAMattrIHstart was changed to 0 for compatibilitywith downstream software Cufflinks (Trapnell et al., 2010). DE genes betweenCstiff1 and the control were finally determined by a corrected P value.

Quantitative Real-Time PCR

Total RNAs from the uppermost extending stem tip at the developmentstage of jointing (V9) from the NIL-B73 and NIL-Ki11 plants were isolatedfrom three to five plants using an RNAExtraction Kit (Tiangen). First-strandcDNAwassynthesized from1mgof total RNAusingTransScript-Uni cDNASynthesis SuperMix (TransGen Biotech). qPCR with three technical rep-licates and three biological replicates was performed on an ABI7500thermocycler using the housekeeping geneGADPH as an internal control.The final transcript levels were determined via the relative quantificationmethod DDCT, which is a convenient method for calculating the relativechange in gene expression (Livak and Schmittgen, 2001). The qPCR pri-mers are listed in Supplemental Table 2.

Transient Expression Assays

Briefly, 733- and 1,258-bp promoter sequences (from positions21093 to2360 and21554 to2296, respectively, in the promoter of stiff1 using theKi11 sequence as a reference), the 461-bp distal end of the 1258-bppromoter from the tropical parent Ki11, and the 1258-bp promoter se-quence from Ki11 with a 2456-bp transposable element insertion from thestiff-stalk parent B73 were introduced into the LUC vector (pGreenII 0800-LUC), which harbored a Renilla reporter gene as an internal control drivenby the Cauliflower mosaic virus 35S promoter and a firefly luciferase re-porter gene driven by a custom promoter. These three constructs werefurther introduced into etiolated maize mesophyll protoplasts (Ki11) at theseedling stage. Freshly isolated protoplasts were mixed with 20 mg ofreporter construct in polyethylene glycol transfer solution for 18min on ice.After transformation, the protoplasts were incubated for 18 h at 25°C andthen harvested. The harvested protoplasts were lysed with Passive LysisBuffer (Promega) and assayed using the Dual-Luciferase Reporter AssaySystem (Promega). Six biological replicates were assayed per construct.

Scanning Electron Microscopy

The stems between the second and third nodes close to the ground fromthe NIL-B73 and NIL-Ki11 plants in transgenic and nontransgenic controlplants at the heading stage (V17) were fixed in glutaric dialdehyde solutionovernight. The fixed tissues were then critical-point dried in liquid CO2,sputter-coated with gold and palladium for 60 s, and observed at an ac-celeration voltage using a scanning electron microscope.

Determination of Cellulose and Lignin in Maize Stalks

To determine the cellulose and lignin composition in maize stalks, HPLCand UV spectrophotometry were applied based on National RenewableEnergy Laboratory (NREL/TP-510-42618) procedures (Sluiter et al., 2011).Thedrymassofmaize stalks collected 1month after harvestwas assessedusing this method. Briefly, the cellulose content was determined by HPLC

based on degraded glucose and xylose sugar units, and the amount ofacid-soluble lignin was determined using a UV spectrophotometer.

Accession Numbers

Sequence data from RNA-seq were deposited in the National Center forBiotechnology Information under the Sequence Read Archive accessionnumber PRJNA531708, and the stiff1 gene sequences were deposited inGenBank with the accession numbers MK748607 to MK748986.

Supplemental Data

Supplemental Figure 1. The testing machine for stalk stiffness.

Supplemental Figure 2. QTL mapping of stalk bending strength inB73-Ki11 RIL population.

Supplemental Figure 3. Two HIFs from B73-Ki11 RIL population.

Supplemental Figure 4. Phenotypes of the stiff1 gene.

Supplemental Figure 5. Fine-mapping of stiff1 through a modifiedprogeny test.

Supplemental Figure 6. Transcription analysis for the two genes inthe fine-mapping region in the stalk.

Supplemental Figure 7. Linkage disequilibrium (LD) heat map.

Supplemental Figure 8. Transformation analysis of stiff1.

Supplemental Figure 9. RNA in situ hybridization.

Supplemental Figure 10. DNA methylation analysis for the stiff1.

Supplemental Figure 11. Scanning electron micrographs of trans-ections of stalks from the plants of NIL-B73 and NIL-Ki11.

Supplemental Figure 12. Subcellular localization of the stiff1-GFPfusion protein.

Supplemental Figure 13. Real-time RT-PCR analysis for key genes ofthe regulatory network of stiff1.

Supplemental Figure 14. The identification of the large 27.2-kbinsertion in the stiff1 promoter.

Supplemental Table 1. The key genes from stiff1 gene network.

Supplemental Table 2. Primer list.

Supplemental Table 3. Information about the 15 teosinte lines.

Supplemental Table 4. Maize materials and DNA fragments fordifferent analyses in this study.

Supplemental Data Set 1. The sequence comparison of thestiff1 gene.

Supplemental Data Set 2. The list of differentially expressed (DE)genes based on RNA-seq analysis.

Supplemental Data Set 3. Maize variants for association mappingtesting.

ACKNOWLEDGMENTS

We thank Xiaolan Zhang for technical assistance with in situ hybridization.This work was supported by the National Key Research and DevelopmentProgram of China (grants 2016YFD0101803 and 2016YFD0100303 to Z.L.)and the National Natural Science Foundation of China (grants 91735305and 31871632 to Z.L.).

Maize stiff1 Gene for Stalk Strength 163

AUTHOR CONTRIBUTIONS

Zho.L. designed the study; Z.Z., X.Z., Zhe.L., J.W.,H.L., S.Z., L.Z., Y.L., andC.Z. performed the research; J.L., X.L., and J.Y. contributed to newreagents; Z.Z. and Zho.L. analyzed the data; Z.Z. and Zho.L. wrote thearticle.

Received July 1, 2019; revised October 8, 2019; accepted November 1,2019; published November 4, 2019.

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Maize stiff1 Gene for Stalk Strength 165

DOI 10.1105/tpc.19.00486; originally published online November 4, 2019; 2020;32;152-165Plant Cell

Li, Can Zhu, Jinsheng Lai, Xianran Li, Jianming Yu and Zhongwei LinZhihai Zhang, Xuan Zhang, Zhelong Lin, Jian Wang, Hangqin Liu, Leina Zhou, Shuyang Zhong, Yan

Promoter Increases Stalk Strength in Maizestiff1A Large Transposon Insertion in the

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