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. 2019 Nov 4;32(1):152–165. doi: 10.1105/tpc.19.00486

A Large Transposon Insertion in the stiff1 Promoter Increases Stalk 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, Zhongwei Lin a,1
PMCID: PMC6961635  PMID: 31690654

Downregulation of the maize F-box gene stiff1 by a transposon results in thicker stalk cell walls with increased cellulose and lignin, and thus improved stalk strength.

Abstract

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

INTRODUCTION

As a typical high-yielding C4 cereal, maize (Zea mays) plays an important role in global food security, providing more than 30% of food calories for humans (Shiferaw et al., 2011). The utilization of heterosis has greatly improved maize yield. A key step during the utilization of maize heterosis is the introduction of stiff-stalk elite maize resources (Tracy and Chandler, 2006). One major heterotic group in the United States maize breeding germplasm is the stiff-stalk group, which includes lines with better stalk strength, and improving stalk strength has been a major breeding objective in commercial hybrids adapted to high planting density (Duvick, 2005). Improved stalk strength can not only efficiently prevent yield loss from stalk lodging but also greatly enhance the tolerance of overcrowded planting. Notably, stalk lodging can reduce the total annual maize yield by 5 to 20% worldwide (Flint-Garcia et al., 2003b). The development of the maize stalk is commonly affected by stalk rot diseases originating from pathogens and environmental stresses such as drought and heat, especially under high-density planting conditions (Duvick, 1984; Yang et al., 2010). Specifically, late-season stalk lodging results in harvest difficulties, a large loss of yield, high grain moisture, and low grain quality upon pathogen attack. In contrast, a strong stalk will enhance mechanical grain harvesting. Therefore, high stalk strength has been pursued in most maize breeding programs to improve yield.

Stalk strength is related to stalk morphology, including stalk diameter, rind thickness, and cell wall structure components containing cellulose and lignin (Robertson et al., 2017). The challenge in studying maize stalk strength is whether the measurement is feasible on a large scale. Several testing methods have been developed. Natural stalk lodging rating is often affected by the weather. Rind penetrometer resistance (RPR) measured by a digital meter is fast and can be applied on a large scale (Flint-Garcia et al., 2003b). However, RPR measurements generally are not easy to use to differentiate maize lines with moderate and strong stalks (Pedersen and Toy, 1999). Other methods, including three-point bending tests and x-ray computed tomography scanning, either involve premature stalk cutting or are time-consuming and expensive (Robertson et al., 2017). Thus, it is challenging to apply these methods on a large scale. A new easy and cheap method needs to be developed for the measurements of 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 from 19.6 to 13.6% within the Iowa Stiff Stalk Synthetic maize population, which required more than 50 years of recurrent selection (Lamkey, 1992). Several genetic studies have revealed that stalk strength is generally controlled by numerous quantitative trait loci (QTLs; Flint-Garcia et al., 2003b; Peiffer et al., 2013; Li et al., 2014). However, the molecular genetic mechanism related to stalk strength in maize remains largely unknown.

Here, we identified the gene underlying a major QTL for stalk bending strength (BS) on chromosome 6 in maize. The stiff1 (named after the term stiff stalk) gene was fine-mapped within a 58-kb genomic region on chromosome 6. Subsequent association mapping revealed that the insertion of a 27.2-kb transposon in the promoter of a gene with an F-box domain in this 58-kb fragment was correlated with stalk strength. Genetic transformation confirmed that the F-box gene corresponding to stiff1 controlled stalk strength in maize. Transient expression assays together with in situ hybridization showed that this large insertion repressed the expression of stiff1 in maize stalk vascular bundles. The downregulation of stiff1 led to thicker cell walls and higher stalk cellulose and lignin contents. Our results support that transposons might play important roles in maize improvement. The identification of stiff1 and our newly edited stiff1 allele with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system will pave the way for large-scale improvement in maize stalks with high efficiency.

RESULTS

A Major QTL for Stalk Strength in Maize

To study the molecular genetic mechanism underlying stalk strength in maize, we performed QTL analysis of stalk strength in a recombinant inbred line (RIL) population that originated from a cross between the typical stiff-stalk and non-stiff-stalk lines (B73 and Ki11). Maize stalk strength was scored through both RPR and stalk BS, a new method that we developed to improve phenotyping efficiency (see Methods; Supplemental Figure 1). QTL mapping identified a major QTL of stalk strength (Supplemental Figure 2), which accounted for 23.8 and 15.6% of the total phenotypic variation in BS and RPR, respectively. This QTL was referred to as stiff1, following the commonly used term, “stiff stalk,” to represent the better stalk strength. To precisely estimate the stiff1 effect, we compared two near-isogenic lines (NILs) derived from the mapping population (see “Methods”; Supplemental Figure 3). Compared with the NIL with homozygous Ki11 stiff1 allele (NIL-Ki11) from the tropical parent Ki11, the NIL with homozygous B73 stiff1 allele (NIL-B73) from the stiff-stalk parent B73 exhibited significantly enhanced BS, RPR, plant height, and stalk diameter traits as well as higher levels of the two cell wall structural components (cellulose and lignin; Figures 1A–1J). The weak stalk of the NIL-Ki11 lodged in the late season (Figures 1D and 1E; Supplemental Figure 4).

Figure 1.

Figure 1.

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stiff1 Phenotypes.

(A) The stiff1 phenotypes between two NILs. Bar = 28 cm.

(B) and (C) The NIL-B73 plant exhibited a stronger stalk. Under the same horizontal pulling force, bending angle (B) was smaller and stalk thickness (C) was higher in the NIL-B73 plant with homozygous stiff1 than in the NIL-Ki11 (Ki11 type) plant.

(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) Compared with NIL-Ki11, NIL-B73 exhibited significantly enhanced trait values in BS, RPR of the stalk, and stalk diameter (SD; n > 100, P < 0.0001).

(I) and (J) The contents of the cell wall structural components cellulose (I) and lignin (J) were significantly increased in the stalks of NIL-B73 over NIL-Ki11 (n = 6, P < 0.001).

Error bars indicate sd.

High-Resolution Mapping of the stiff1 Gene in Maize

To fine-map stiff1, a large population with 11,538 individuals was created from two heterogeneous inbred families (HIFs; Tuinstra et al., 1997) that harbored heterozygous genomic fragments at the stiff1 locus but were mostly homozygous at other loci (see “Methods”; Supplemental Figure 3). Fine-mapping with 14 newly developed simple sequence repeat (SSR) markers in this large population finally narrowed down the stiff1 gene within a 58-kb genomic fragment from 96,449,596 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 annotation revealed two partial gene fragments, which were derived from the two genes (Zm00001d036652 and Zm00001d036653), encoding an aluminum-activated malate transporter and an F-box domain, located in this region (Figure 2C). Only two partial gene fragments were located in the fine-mapping region because the flanking markers P7 and P10 were situated within these two genes. Transcription analysis further revealed that only Zm00001d036653 was expressed in the stalk (Supplemental Figure 6). Thus, Zm00001d036653 became the candidate for stiff1.

Figure 2.

Figure 2.

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High-Resolution Mapping of stiff1.

(A) QTL mapping identified a major QTL for stalk strength on chromosome 6, accounting for 23.8 and 15.6% of the total phenotypic variation in stalk BS and RPR, respectively. The blue and green lines represent the BS and RPR traits, respectively. The red dashed line represents the significant logarithm of the odds (LOD) threshold (3.2).

(B) High-resolution mapping of stiff1. Only 4 of 14 markers for fine-mapping (Supplemental Figure 5) are presented. The comparison of BS between two NILs (St72-56-1 and St72-56-2) originated from a recombination event narrowed down stiff1 to a 58-kb fragment between two markers: P7 and P10 (P = 1.0 × 10−10). Orange arrows represent molecular markers for fine-mapping, and the positions of these markers are presented below these orange arrows. Blue and green 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 two parent lines, B73 and Ki11. Two insertions of 27.2 kb and 578 bp were present in B73 at positions −1094 and −392 in the promoter of the target gene Zm00001d036653 using the Ki11 sequence as a reference. The start codon was 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 = 1 kb.

(D) Association mapping with a mixed linear model revealed that the large insertion/deletion at position −1094 in the stiff1 promoter was strongly correlated with stalk BS. The red dashed line is the 1% significance threshold after Bonferroni correction for 58 tests. The stiff1 gene structure is presented on the x axis.

(E) stiff1 protein sequence alignment between B73 and Ki11. The F-box domain is highlighted with the red dashed line.

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 of Zm00001d036653 between B73 and Ki11, including the 1.5-kb promoter region, the 1,470-bp open reading frame (ORF), and the 311-bp 3′-untranslated region (3′-UTR), using the sequence from the tropical parent Ki11 as a reference (Figure 2C). Sequence analysis identified several variants, including single-nucleotide polymorphisms (SNPs) and insertions/deletions, in the 3.3-kb region. Among these variants, two conspicuous 27.2-kb and 578-bp insertions were present in the stiff-stalk parent B73 at positions −1094 and −392 in the promoter region (Figure 2C; Supplemental Data Set 1). The 27.2-kb insertion consisted of four Ty1/Copia transposable elements, as annotated based on RepeatMasker (http://repeatmasker.org).

Association Mapping Identified a 27.2-kb Transposon Insertion in the stiff1 Promoter Responsible for Stalk Strength in Maize

To assess whether the Zm00001d036653 gene is responsible for stalk strength, we conducted sequence analysis of this 3.3-kb fragment across a global maize inbred population with 265 accessions, comprising 34 stiff-stalk, 75 tropical, 137 non-stiff-stalk, and 19 unknown background inbred lines (see “Methods”). Fifty-eight variants with an allele frequency over 5% were identified in the 3.3-kb fragment based on the Ki11 reference sequence (Figure 2D). Association tests with the mixed linear model detected significant signals in these two insertions in the promoter (P ≤ 1.72 × 10−4), suggesting that the Zm00001d036653 gene corresponding to stiff1 was responsible for stalk strength (Figure 2D). The strongest signal was present at the 27.2-kb insertion (P = 1.19 × 10−5). The next strongest signal occurred at the 578-bp insertion (P = 9.68 × 10−5; Figure 2D), which was in high linkage disequilibrium with the large insertion (Supplemental Figure 7). This result indicated that both the 27.2-kb transposon and 578-bp insertions were most likely to be the causal variants of the stiff1 gene.

Overexpression and CRISPR Editing of stiff1 Confirmed Its Effect on Stalk Strength

To confirm whether the Zm00001d036653 gene controls stalk strength in maize, we performed genetic transformations. A construct under the control of the Ubiquitin promoter (Ovstiff1) was first introduced into maize plants, and five independent transgenic events were obtained (Figure 3A). Compared with control plants, three transgenic plants (T0) with strong stiff1 expression were semidwarf and sterile as a result of failures in tassel and ear development (Supplemental Figures 8A to 8C). The stalks were thinner and soft, with low RPR, and lodged later in the season (Figures 3A to 3C). The two remaining T0 transgenic plants exhibited no obvious phenotypic changes, but the seeds failed to germinate in the next generation.

Figure 3.

Figure 3.

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Transgenic Analysis.

(A) The Ovstiff1 (strong allele) transgenic plant with stiff1 under the control of ubiquitin was considerably shorter and exhibited a thinner and weaker stalk than the nontransgenic control plant (CK), and the weak stalk of the Ovstiff1 plant was lodged in the late season.

(B) and (C) Three Ovstiff1 plants with strong expression exhibited a conspicuously smaller stalk diameter (B) and considerably reduced RPR (C) than the CK. *, P < 0.05 and **, P < 0.01.

(D) The CRISPR gene-editing plant with two cutting targets in the CDS of stiff1 exhibited no additional apparent phenotypic changes in contrast to CK except the enhanced stalk BS. Red dashed triangles indicate the 2-bp deletion created by CRISPR.

(E) The BS was significantly increased in the Cstiff1 plant compared with that in the CK plant. The number of plants measured is presented on the bars. **, P < 0.001.

(F) and (G) Cellulose and lignin levels were significantly increased in the Cstiff1 plants but significantly decreased in Ovstiff1 plants compared with those in the CK. **, P < 0.01.

(H) to (J) RNA-seq analysis between Cstiff1 and CK, the DE genes (H); radar plot of expression levels of key genes for the gene regulatory network of stiff1 (I), where the blue numbers on the plot represent the expression levels (fragments per kilobase of exon per million fragments mapped); and a potential regulatory network for stiff1 (J).

Error bars indicate sd.

We then conducted maize transformation using CRISPR/Cas9 with two editing targets in the Zm00001d036653 gene coding sequence (CDS; Cong et al., 2013; Figure 3D). However, only one event was obtained. This gene knockout plant (Cstiff1) contained two 2-bp deletions in the CDS of stiff1 and caused a frameshift and then an early-stop translation (Supplemental Figure 8D). Compared with the control plants, the knockout plants (Cstiff1; T1) harbored no other phenotypic changes, including plant height and stalk diameter, except significantly increased stalk BS (P = 8.1 × 10−4) and RPR (P = 1.8 × 10−7; Figure 3E; Supplemental Figures 8E to 8G). These transgenic results suggested that the Zm00001d036653 gene corresponding to stiff1 controlled stalk strength in maize.

The Large Transposon Insertion Repressed stiff1 Expression and Led to Better Stalk Strength in Maize

To determine how the large 27.2-kb insertion regulates stiff1 transcription, we performed real-time RT-PCR. RT-PCR showed that the expression of stiff1 was clearly repressed in the stalk from the plant of NIL-B73 compared with NIL-Ki11 (Figure 4A). In situ hybridization further revealed that the stiff1 gene was expressed (Figures 4B and 4C) in the stalk at the V9 stage (Supplemental Figure 9). The stiff1 expression was mainly detected in vascular bundles in the stalk (Figures 4B and 4C). Transient gene expression assays using a luciferase reporter (LUC) were also conducted (Figure 4D). Luciferase activity was upregulated in the construct with the longer 1258-bp promoter (Ki11-Pro::LUC) compared with a truncated construct with a 733-bp promoter (Ki11-Pro::LUC-T), and luciferase expression was also enhanced in the construct with a 461-bp distal end of this 1258-bp promoter (Ki11-Pro::LUC-D) compared with Ki11-Pro::LUC, suggesting that a mini-enhancer was located at the distal end of the 1258-bp promoter (Figure 4D). Luciferase activity was strongly repressed when a truncated 2456-bp sequence from the B73 large 27.2-kb transposable element (B73-like-Pro::LUC) was inserted into the 1258-bp Ki11 promoter (Figure 4D). Additionally, no apparent change in the methylation level was detected in the promoter region of stiff1 between NIL-B73 and NIL-Ki11 (Supplemental Figure 10). These results indicated that the large 27.2-kb insertion may push the mini-enhancer in the promoter away and subsequently repress stiff1 expression in the B73 plant. Therefore, both association mapping and LUC transient assay analysis revealed that the large transposon insertion was causative to the stiff1 gene.

Figure 4.

Figure 4.

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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 the middle of the jointing stage. Bars = 500 μm.

(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 transposable sequence insertion from the stiff-stalk parent B73 were introduced into maize mesophyll protoplasts. Empty vector was used as a control. The relative expression was quantified according to the LUC:REN ratio. Significant P values for the comparisons of relative expression between pairwise constructs using 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 = 6).

(E) to (G) Scanning electron micrographs of transections of stalks from Ovstiff1 (E), nontransgenic control (CK [F]), and Cstiff1 (G) plants. A thinner cell wall was present in the sclerenchyma of the rind region and vascular bundles (V) in the stalk of Ovstiff1, whereas a thicker cell wall occurred in Cstiff1 compared with that in the CK. Bars = 100 μm.

The sclerenchyma cell wall was thinner in the rind region and the stalk vascular bundles in the NIL-Ki11 plants compared with the NIL-B73 plants (Supplemental Figure 11), and these differences were more apparent between the transgenic (Ovstiff1) and nontransgenic control plants (Figures 4E and 4F). In contrast, the cell walls were thicker in sclerenchyma cells in Cstiff1 plants than in the nontransgenic control plants (Figure 4G). Both NIL-B73 with low expression and Cstiff1 plants exhibited significantly increased cellulose and lignin levels in stalk cells compared with those in the NIL-Ki11 and Ovstiff1 plants with high expression. These results suggested that the stiff1 gene controlled the development of the sclerenchyma cell wall in maize stalks. As a result, the presence of the large insertion in the stiff1 promoter led to increased cell wall thickness in sclerenchyma cells from the rind region and stalk vascular bundles and subsequently caused high stalk BS in maize.

RNA-Seq Analysis for the stiff1 Gene

The stiff1 protein contains an F-box domain. Transient expression assays revealed that STIFF1-GFP was expressed in both the cytoplasm and nucleus in onion (Allium cepa) epidermal cells, which was consistent with the expression observed in maize leaf protoplasts (Supplemental Figure 12). To identify the gene regulatory network, we performed RNA-seq analysis. RNA-seq revealed 2568 differentially expressed (DE) genes between the knockout (Cstiff1) and control plants. In total, 2061 and 507 DE genes from the Cstiff1 plant were upregulated and downregulated, respectively, compared with those from the control (Figure 3H). The stiff1 gene regulated genetic factors in several hormone signaling pathways, including gibberellin (GA), auxin, ethylene, and brassinolide. The DE gene numbers from the GA and auxin pathways apparently outweighed those from the ethylene and brassinolide pathways (Supplemental Data Set 2).

The stiff1 Locus Was under Strong Selection during Maize Improvement

The global natural population with 265 maize inbred lines was divided into stiff-stalk, non-stiff-stalk, and tropical-subtropical groups based on random SNPs across the entire maize genome (Figure 5A; Flint-Garcia et al., 2005). Most of the inbred lines belonging to the stiff-stalk background were split into four groups originating from the B73, B37, N28, and B14 lines (Liu et al., 2003). B73, B37, and N28 contained the favorable allele with the large insertion in stiff1, whereas this favorable allele was absent in B14. The presence of the favorable B73 stiff1 allele reached 50% in the stiff-stalk group, which was considerably increased compared with the non-stiff-stalk (3.6%) and tropical-subtropical (16%) groups; Figure 5A). This result indicated that selection of stalk strength resulted in the accumulation of the favorable B73 stiff1 allele in the stiff-stalk group during maize improvement.

Figure 5.

Figure 5.

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Nucleotide Diversity of stiff1.

(A) The global maize population was divided into three subgroups based on principal component analysis. Non-stiff-stalk (NSS), stiff-stalk (SS), and tropical-subtropical (TRO) inbred maize lines with a large insertion of 27.2 kb are highlighted in red. A set of genome-wide random SNPs (Supplemental Table 4) were used to perform the principal component analysis.

(B) Phylogenetic tree analysis based on the stiff1 gene sequence revealed that these lines with a large 27.2-kb insertion clustered together (in blue). The two teosinte lines (T11 and T52) with this large insertion are labeled in orange.

(C) Nucleotide diversity of stiff1 in the promoter region and ORF and 3′-UTR between teosinte and maize 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.

We further sequenced one additional 1.1-kb fragment upstream of the large insertion (−2500 to −1400 bp) and the 1.8-kb ORF and 3′-UTR in these maize inbreds and 15 teosinte lines (see “Methods”). Although the inbred maize lines with the large 27.2-kb insertion were distributed in different groups (Figure 5A), they were clearly clustered into a single clade based on the 2.9-kb stiff1 fragment (Figure 5B). We next compared the nucleotide diversity among 29 maize lines in the stiff-stalk subgroup and 15 teosinte lines. DNA diversity may have decreased in the promoter region, while no clearly changed diversity was present in the ORF region from all maize lines in the stiff-stalk group in comparison with teosinte (Figure 5C). The Tajima’s D values were 2.73 (P < 0.01; maize stiff-stalk promoter), −0.94 (P > 0.10; teosinte promoter), 0.69 (P > 0.10; maize ORF), and −0.57 (P > 0.10; teosinte ORF). Tajima’s D test significantly (P < 0.01) rejected the neutral null model for the promoter region, while no selection signals were detected in the ORF region (P > 0.1) for these stiff-stalk maize inbred lines. This result suggested that the stiff1 locus experienced strong selection during maize improvement. Strong selection has swept the promoter region, and no SNPs were present in the promoter from −1500 to −2500 bp, excluding the insertions/deletions in these maize lines with the large insertion (Figure 5C). Two teosinte lines split into the maize clade with the large insertion, and the two teosinte lines contained the large 27.2-kb insertion (Figure 5B). In this study, four non-stiff-stalk lines contained this large transposon insertion at the stiff1 locus under the non-stiff-stalk genomic background. The B73 stiff1 alleles from stiff-stalk line genes might be introgressed into non-stiff-stalk lines in breeding.

DISCUSSION

Most of the maize genome is composed of transposable elements (Schnable et al., 2009). As one of the most active factors in the maize genome evolution, transposons play an important role in maize domestication. The key steps from multiple branches to a single stalk (Studer et al., 2011), seed shattering to nonshattering (Lin et al., 2012), and multiple tiny ears to a single large ear on plants (Wills et al., 2013) have been achieved through the transposition of these jumping DNA sequences. However, whether transposable elements reshape key genes for maize improvement remains unclear. In this study, we identified the insertion of Ty1/Copia transposable elements in the stiff1 promoter responsible for stalk strength in maize, supporting the suggestion that transposable elements might play a role in maize improvement.

Maize stalk strength is a multiple-scale phenomenon that is affected by stalk morphology and chemical composition (Robertson et al., 2016). Stalk RPR testing mainly estimates stalk rind thickness (Robertson et al., 2017). While a three-point bending test and x-ray computed tomography scanning can precisely investigate stalk strength and multiple-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 by weather, is still being applied in current breeding even it has a low selection efficiency. In this study, we applied stalk BS with a simple device (see “Methods”). Compared with other methods, our stalk BS testing method is fast, cheap, repeatable, and without premature stalk damage. This measurement of stalk BS can be directly performed 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 regulatory network for secondary cell wall development in plants (Figures 3I and 3J; Ko et al., 2009, 2014; Zhong et al., 2011; Huang et al., 2015) might be activated, and the transcription of the genes for cellulose, hemicellulose, and lignin synthesis were further promoted (Hussey et al., 2011). Therefore, cellulose and lignin contents are finally increased in the stalk cell wall (Figures 3I and 3J; Supplemental Figure 13).

The development of the maize stalk is impacted by numerous internal (genetic) and external (environmental) factors. Improvements in stalk strength remain difficult and inefficient based on phenotypic selection. Although the allele frequency of the large insertion of stiff1 reached 50% in the stiff-stalk subpopulation, the allele frequency remained low in other subpopulations from maize inbred lines for association mapping. In this study, we screened a large global breeding population of 1429 maize inbred lines, and the favorable allele frequency of the large insertion in stiff1 only reached 19%. These facts suggested that this favorable B73 stiff1 allele is not widely applied in maize except for the stiff-stalk subgroup. Our cloning of stiff1 provides an efficient method for improving maize stalk on a large scale through marker-assisted selection, and precise gene editing of stiff1 will provide another method for greatly improving maize stalk strength.

METHODS

Plant Materials

The maize (Zea mays) RIL population (F7) between stiff-stalk and tropical parents, B73 and Ki11, respectively, with 189 lines derived from one nested association mapping population (Yu et al., 2008) and a global inbred maize population (Flint-Garcia et al., 2005) with 265 lines, was planted in a randomized block design with three replicates (Supplemental Data Set 3). Each line of both populations for QTL mapping and association mapping with 12 plants was grown in a 50-cm × 300-cm plot in the experimental station of China Agricultural University in Beijing. All the plant materials for fine-mapping in this study were grown in Beijing and Hainan between 2013 and 2017. All maize plants for the QTL mapping, fine-mapping, and association mapping were grown in the same field conditions. All these plant materials were planted with 50-cm row-to-row distance and 25-cm plant-to-plant distance. The experimental fields were supplied with 120 kg ha−1 N, 90 kg ha−1 P, and 90 kg ha−1 K.

Phenotypic Investigation

To measure stalk strength in maize, we first applied RPR (Flint-Garcia et al., 2003a). RPR scores were obtained from the middle of the first three nodes near the soil for 10 plants from each line from the RIL population and the transgenic plants with Ovstiff1 using a digital force gauge (ELECALL, YLK-500; Supplemental Figure 1). The average of these RPR scores was calculated to represent the stalk strength of each line.

Stalk BS was then investigated by pulling the stalk to a bending angle of 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 the samples when measuring BS. This BS measurement method is fast and can be applied for large-scale investigations of stalk strength. Similarly, BS values were collected from 10 plants from each line from both the RIL and global natural maize populations, and the mean of these BS values was used for further analysis. These BS scores were repeated in 10 plants from each line. Both RPR and BS measurements were performed at the end of the grain-filling stage, which was about 1 month after flowering. For the maize association mapping panel with diverse flowering time, flowering time from maize diverse inbred lines was first investigated, and RPR and BS were then measured about 1 month after flowering. All the phenotypic comparisons in this study were conducted using Student’s t test.

QTL Mapping and Fine-Mapping of stiff1

The mean BS and RPR values for each line from the RIL population with three replicates were imputed into Cartographer (Silva et al., 2012) for QTL detection. QTL analysis was performed with composite interval mapping at a walking speed of 1 cM. The significant logarithm of the odds 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 phenotypic variations in BS and RPR, respectively. A large population with 6190 plants was created from two HIFs, Z012E0013 and Z012E0072, with a heterozygous genomic fragment at stiff1 (Supplemental Figure 3). Marker screening of this population with 14 SSRs revealed 18 representative recombination types. The descendant populations derived from these recombination plants harboring heterozygous/homozygous fragments in the stiff1 target region were used for the correlation test between genotypes and phenotypes. The correlations between genotypes and stalk BS were estimated through a linear regression model to test the presence or absence of the target QTL in the recombination plants. A significant P value (F test) indicated the presence of the target QTL in the heterozygous fragments. Otherwise, the target QTL would be categorized among the homozygous fragments. According to this modified progeny test (Liu et al., 2015), stiff1 was placed between two markers: P7 and P10.

Next, sequencing analysis revealed that two insertions of 27.2 kb and 578 bp were present in the Zm00001d036653 promoter. To test whether the Zm00001d036653 promoter region was responsible for maize 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 fragment between P7 and P10 was identified. Further progeny tests between two NILs (St72-56-1 and St72-56-1) confirmed that stiff1 was located between P7 and P10 (Supplemental Figure 5).

The two NILs (NIL-B73 and NIL-Ki11; F12) were generated from a HIF from the recombination plant (SS72-7-4 [F11]; Supplemental Figure 5A) in the fine-mapping, which contained heterozygous genotypes in the stiff1 locus but was homozygous in the other QTLs on chromosome 10 and most other chromosomal regions (Supplemental Figure 3). All the primers for these fine-mapping markers are listed in Supplemental Table 2. These NILs 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, and a 311-bp 3′-UTR was sequenced from a maize association mapping panel with 265 samples. The variants in the 3.3-kb fragment were applied for association mapping. The presence or absence of the large 27.2-kb insertion in a total of 1429 maize inbred lines was determined using primer pairs I1, I2, and I3 (Supplemental Figure 14). The sequences of the 1.5-kb promoter region were not well aligned due to the presence of a lot of insertion/deletions. We then sequenced an additional 1.1-kb promoter fragment upstream of the large 27.2-kb transposable element for DNA diversity analysis. This 2.9-kb sequence containing a 1.1-kb promoter fragment, a 1.8-kb ORF, and a 3′-UTR was amplified for DNA diversity analysis. All the PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen) and were subsequently sequenced using an ABI 3730 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 statistical tool given that the stalk BS is a continuous trait. Variants in the 3.3-kb fragment containing a 1.5-kb promoter, a 1470-bp ORF, and a 311-bp 3′-UTR with an allele frequency more than 5% were applied for association mapping. Association mapping was performed with a mixed linear model using a genome-wide set of SNPs to calculate population structure and kinship, and the genome-wide SNPs were provided in Tassel software (Bradbury et al., 2007). SNPs for the stiff1 gene were identified from Sanger sequencing in a maize association panel (Supplemental Data Set 3).

The significance threshold was corrected for multiple testing through Bonferroni correction based on the following equation: α′ ≈ α/n = 1.72 × 10−4, where α is the nominal significance threshold (α = 0.01) and n is the number of variants (n = 58; Figure 2D).

DNA Diversity Analysis

Briefly, the 2.9-kb sequences containing the 1.1-kb promoter upstream of the large 27.2-kb transposable element, 1.8-kb ORF, and 3′-UTR from 15 teosinte (Supplemental Table 3) and 29 inbred maize lines in the stiff-stalk subgroup were imported into ClustalW to construct a nucleotide alignment matrix, which was further used for nucleotide diversity analysis using DnaSP V5.10 (Rozas et al., 2003). Tajima’s D test (Tajima, 1989) was performed in DnaSP V5.10 based on the 1.1-kb promoter, 1.8-kb ORF, and 3′-UTR sequences.

RNA in Situ Hybridization

RNA in situ hybridization was conducted based on the protocol from Xiaolan Zhang’s lab. The uppermost extending stem tips (2–3 mm) at different developmental stages (V5, V7, V9, and V12) from NIL-Ki11 plants were fixed in 3.7% FAA solution (50 mL of ethanol, 5 mL of acetic acid, 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. Fixed samples were sliced into 8- to 10-μm sections using a microtome (Leica RM2145). Sense and antisense probes were amplified based on an ∼300-bp stiff1 cDNA fragment with gene-specific primers containing SP6 and T7 RNA polymerase binding sites and then labeled with digoxigenin from a DIG Northern Starter Kit (Roche). RNA in situ hybridization was performed with probes on the sections, and slides were observed and photographed with a microscope (Leica DMR) and a microcolor CCD camera (Apogee Instruments).

Transformation

The CDS of stiff1 amplified from B73 was inserted into the binary vector pCUNm-eGFP under control of the Ubiquitin promoter. The construct was then transformed into maize inbred line B73 with Agrobacterium tumefaciens 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 gene were designed with CRISPR‐P (http://crispr.hzau.edu.cn/CRISPR2/) and then introduced into CRISPR/Cas9 binary vector. All these constructs were introduced into the maize stiff-stalk inbred line B73. Five Ovstiff1 (T0) events were obtained. However, only one homozygous Cstiff1 (T0) gene-editing event was created using the CRISPR/Cas9 system. The knockout line contained two 2-bp deletions in the stiff1 CDS (Supplemental Figure 8D). The T0 Cstiff1 plant was self-crossed to generate T1 plants for the measurement of the BS of stalks. All these T0 overexpressed and T1 CRISPR plants were compared with the control plants, which contained an empty construct through transformations. Each edited T1 plant with 2-bp deletion was confirmed through sequencing.

All the T0 and T1 transgenic and nontransgenic control plants were grown under 14 h of light and 10 h of dark in pots supplied with 1 g N/pot, 1 g P/pot, and 1 g K/pot, with 60-cm row-to-row distance and 30-cm plant-to-plant distance, in a greenhouse. BS and RPR measurements were performed in the grain-filling stage (1 month after flowering). The transgenic plants showed similar flowering time to the control plants.

Subcellular Localization

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

Bisulfite Sequencing

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

Phylogenetic Analysis

A nucleotide alignment matrix based on 2.9-kb sequences, including the 1.1-kb promoter upstream of the large transposable element, 1.8-kb CDS, and 3′-UTR, was imported into MEGA7 to generate a phylogenetic tree using a statistical method of maximum likelihood under the Tamura-Nei model (Kumar et al., 2016). The nucleotide alignment matrix and the machine-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 uppermost extending stem tips (2–3 mm; V9) from Cstiff1 and the control plants. The six DNA-free RNA samples were then sequenced with a Hiseq-2500, and 40 Gb of raw sequencing data were obtained. These raw RNA-seq reads were then analyzed following a standard RNA-seq analysis pipeline. Briefly, the raw RNA-seq reads were initially processed to remove the adapter 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 with fastq_clean (Zhang et al., 2014). Finally, the clean RNA-seq reads were mapped to the reference genomes using STAR version 2.5.0b (Dobin et al., 2013). To improve spliced alignment, STAR was provided with exon junction coordinates from the reference annotations. Default alignment parameters were used, and the outSAMattrIHstart was changed to 0 for compatibility with downstream software Cufflinks (Trapnell et al., 2010). DE genes between Cstiff1 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 development stage of jointing (V9) from the NIL-B73 and NIL-Ki11 plants were isolated from three to five plants using an RNA Extraction Kit (Tiangen). First-strand cDNA was synthesized from 1 μg of total RNA using TransScript-Uni cDNA Synthesis SuperMix (TransGen Biotech). qPCR with three technical replicates and three biological replicates was performed on an ABI7500 thermocycler using the housekeeping gene GADPH as an internal control. The final transcript levels were determined via the relative quantification method ΔΔCT, which is a convenient method for calculating the relative change in gene expression (Livak and Schmittgen, 2001). The qPCR primers are listed in Supplemental Table 2.

Transient Expression Assays

Briefly, 733- and 1,258-bp promoter sequences (from positions −1093 to −360 and −1554 to −296, respectively, in the promoter of stiff1 using the Ki11 sequence as a reference), the 461-bp distal end of the 1258-bp promoter from the tropical parent Ki11, and the 1258-bp promoter sequence from Ki11 with a 2456-bp transposable element insertion from the stiff-stalk parent B73 were introduced into the LUC vector (pGreenII 0800-LUC), which harbored a Renilla reporter gene as an internal control driven by the Cauliflower mosaic virus 35S promoter and a firefly luciferase reporter gene driven by a custom promoter. These three constructs were further introduced into etiolated maize mesophyll protoplasts (Ki11) at the seedling stage. Freshly isolated protoplasts were mixed with 20 μg of reporter construct in polyethylene glycol transfer solution for 18 min on ice. After transformation, the protoplasts were incubated for 18 h at 25°C and then harvested. The harvested protoplasts were lysed with Passive Lysis Buffer (Promega) and assayed using the Dual-Luciferase Reporter Assay System (Promega). Six biological replicates were assayed per construct.

Scanning Electron Microscopy

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

Determination of Cellulose and Lignin in Maize Stalks

To determine the cellulose and lignin composition in maize stalks, HPLC and UV spectrophotometry were applied based on National Renewable Energy Laboratory (NREL/TP-510-42618) procedures (Sluiter et al., 2011). The dry mass of maize stalks collected 1 month after harvest was assessed using this method. Briefly, the cellulose content was determined by HPLC based on degraded glucose and xylose sugar units, and the amount of acid-soluble lignin was determined using a UV spectrophotometer.

Accession Numbers

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

Supplemental Data

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

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

AUTHOR CONTRIBUTIONS

Zho.L. designed the study; Z.Z., X.Z., Zhe.L., J.W., H.L., S.Z., L.Z., Y.L., and C.Z. performed the research; J.L., X.L., and J.Y. contributed to new reagents; Z.Z. and Zho.L. analyzed the data; Z.Z. and Zho.L. wrote the article.

Footnotes

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