MADS-box encoding gene Tunicate1 positively controls maize yield by increasing leaf number above the ear

Li, Yan; Wang, Jian; Zhong, Shuyang; Huo, Qiang; Wang, Qun; Shi, Yunlu; Liu, Hangqin; Liu, Jiacheng; Song, Yang; Fang, Xiaojian; Lin, Zhongwei

doi:10.1038/s41467-024-54148-7

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Published: 12 November 2024

MADS-box encoding gene Tunicate1 positively controls maize yield by increasing leaf number above the ear

Nature Communications volume 15, Article number: 9799 (2024) Cite this article

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Abstract

The leaves above the ear serve as a major source of carbohydrates for grain filling in maize. However, increasing the number of leaves above the ear to strengthen the source and improve maize yield remains challenging in modern maize breeding. Here, we clone the causative gene of the quantitative trait locus (QTL) associated with the number of leaves above the ear. The causative gene is the previously reported MADS-box domain-encoding gene Tunicate1 (Tu1), which is responsible for the phenotype of pod corn or Tunicate maize. We show that Tu1 can substantially increase the leaf number above the ear while maintaining the source‒sink balance. A distal upstream 5-base pair (bp) insertion of Tu1 originating from a popcorn landrace enhances its transcription, coregulates its plastochron activators and repressors, and increases the number of leaves above the ear. Field tests demonstrate that the 5-bp insertion of Tu1 can increase grain yields by 11.4% and 9.5% under regular and dense planting conditions, respectively. The discovery of this favorable Tu1 allele from landraces suggests that landraces represent a valuable resource for high-yield breeding of maize.

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Introduction

The optimization of crop yield from limited land areas is crucial in addressing the growing global demand for food¹. Enhancing the photosynthetic capacity (source) is an effective measure to improve yield per unit area. The photosynthetic capacity of maize is closely linked to the number of leaves and stage of plant development. Leaves located above the main ear are relatively younger and more metabolically active compared to those below the ear, which are susceptible to shading and senescence^2,3. Additionally, the leaves above the ear primarily contribute to the provision of carbohydrates for grain filling of the ear, whereas the leaves below the ear primarily sustain the stem and roots⁴. Therefore, the number of leaves above the ear in maize is an important factor in determining the source strength, source‒sink balance and overall yield of the plant. A proper increase in the number of leaves above the ear can increase the source strength, leading to improved photosynthetic capacity and greater accumulation of assimilates in the ear. This, in turn, leads to a larger ear size and increased grain yield.

Maize has undergone significant changes in source strength depending on the number of leaves above the ear during the course of its domestication and improvement (Fig. 1a). Teosinte generally produces ears on most leaf axils. Compared with their wild ancestor, maize landraces typically present a more limited number of leaves above the ear. Conversely, maize inbred lines are often characterized by more leaves situated above the ear. Hence, maize inbred lines typically possess a stronger source than teosinte and landraces. However, the genetic basis underlying this reshaping of leaf number above the ear to increase the source strength during maize domestication and improvement remains poorly understood.

The shoot apical meristem (SAM) initiates early during embryogenesis to form a series of leaves through sequential initiation from the leaf primordia until floral transition occurs^5,6. The number of leaves produced is determined by both the onset or rate of leaf initiation (plastochron) and the duration of the vegetative phase, which is controlled by the timing of floral transition⁷. A number of genes involved in these processes have been identified. For example, paused (psd) mutants in Arabidopsis exhibit a delay in leaf initiation, leading to fewer leaves than those of the wild type⁸. Conversely, the Knotted1 (Kn1) gene suppresses leaf initiation and preserves meristem identity in maize⁹. The PLASTOCHRON1 (PLA1)^10,11, PLA2¹², and PLA3¹³ genes in rice, TERMINAL EAR1 (TE1)¹⁴, WEE1¹⁵ and VIVIPAROUS8¹⁶ in maize, ALTERED MERISTEM PROGRAM1 (AMP1)¹⁷ in Arabidopsis, and MANY-NODED DWARF genes in barley regulate the plastochron, and loss of function leads to an increase in leaf number^18,19,20,21. The SBP-box gene unbranched 2 (ub2) regulates the rate of leaf initiation, and loss of function results in an increase in leaf number²². Similarly, Big embryo1 (Bige1) is involved in lateral organ initiation, and loss-of-function mutations of the gene can accelerate leaf initiation in maize²³. Furthermore, the MADS-box transcription factors ZmMADS1 and ZmMADS3^24,25, the auxin efflux transporter PIN1a²⁶, and the meristem size regulator Thick Tassel Dwarf1 (TD1) have all been linked to maize leaf number²⁷.

As the timing of floral transition is a determining factor in the duration of the vegetative phase, leaf number is often used as an indicator of flowering time in maize. However, flowering time is a critical factor in crop adaptation and can influence numerous other agronomic traits, making it an unsuitable approach to improve leaf number for increased grain yield while also delaying flowering time. To date, no exploitable natural loci that enhance maize grain yield by increasing the leaf number above the ear without influencing flowering time have been identified.

In this study, we clone the gene underlying a major QTL associated with leaf number above the ear. We show that the causative gene (Tunicate1) (Tu1) encodes a MADS-box protein²⁸. An upstream 5-bp (AGAAG) insertion in the promoter of Tu1 enhances its transcription, regulating plastochron activators such as TD1, PIN1a and yabby1 and repressors such as ub2, Bige1, Kn1, ZmMADS1, ZmMADS3 and WEE1 to moderately increase the number of leaves above the ear, while keeping the majority of other traits unchanged. The favorable 5-bp insertion allele is originally derived from teosinte. Its ability to improve maize yield while maintaining the source‒sink balance indicates its application value in maize breeding.

Results

Lna1 is a major QTL associated with leaf number above the ear in maize

To discover the molecular genetic basis underlying the leaf number above the ear in maize, a landrace called Strawberry Popcorn (Popcorn hereafter) was crossed with a maize nested association mapping population (NAM) parental inbred line, MS71, to construct an F₂ population (Fig. 1). Popcorn produced only 3–4 leaves above the ear, whereas MS71 produced 5 or 6 leaves (Fig. 1b). Genome-wide QTL analysis revealed six QTLs for leaf number above the ear located on chromosomes 1, 2, 3, 4, 5 and 7 in this F₂ population. Among these identified QTLs, the one located on the long arm of chromosome 4, which was named Leaf number above the ear 1 (Lna1), was found to be the only QTL with a genetic effect originating from the Popcorn parent, accounting for 9.3% of the total phenotypic variation (Fig. 1j).

To precisely quantify the genetic effect of Lna1, a pair of near-isogenic lines (NILs) were then generated (see “Methods”). Compared with the NIL carrying the homozygous MS71 Lna1 allele (NIL-MS71), the NIL with the homozygous Popcorn Lna1 allele (NIL-Popcorn) had more leaves above the ear (Fig. 1 c, d). The total leaf number of NIL-Popcorn was greater than that of NIL-MS71, with no significant difference in the leaf number below the ear (Fig. 1e, f). This increase in the leaf number above the ear resulted in an increase in the total leaf number in NIL-Popcorn. Although leaf number is typically strongly correlated with flowering time in maize, our results revealed that there was no significant difference in flowering time between NIL-MS71 and NIL-Popcorn (Fig. 1g). Similarly, the plant height and ear height did not differ significantly between the two NILs (Fig. 1h, i). Furthermore, the length of each internode above the ear was measured, and it was found that the 1st−4th internodes (from top to bottom) of NIL-Popcorn were shorter than those of NIL-MS71, whereas the 5th internode was similar in length between the two NILs (Supplementary Fig. 1). These results suggested that Lna1 can significantly increase the number of leaves above the ear while keeping most other traits unchanged.

High-resolution mapping of the Lna1 locus in maize

Genetic linkage analysis of the F_2:3 population revealed that Lna1 was located between two markers, M1 and M2, on the long arm of chromosome 4 (Fig. 2a). To further fine map the Lna1 locus, a large population with 11,914 individuals was constructed from 17 representative F₇ recombinant plant lines, and a modified progeny testing strategy was employed (see Methods and Supplementary Fig. 2)²⁹. Using 12 simple sequence repeat (SSR) and insertion/deletion (Indel) markers, the causal gene of Lna1 was finally narrowed down within a 195-kilo base pair (kb) genomic fragment from 181,861,347 to 182,056,761 bp on the basis of the B73 genome sequence (V4; http://www.maizegdb.org), which was flanked by the markers M9 and M11 (Fig. 2b, Supplementary Fig. 2). Sequence annotation revealed that only the first exon of one gene (Zm00001d052180) was present in this region (Fig. 2c). Zm00001d052180 is known to be responsible for the phenotype of the Tunicate1 (Tu1) mutant, i.e., pod corn or Tunicate maize³⁰. Thus, Zm00001d052180 was considered the candidate causative gene of Lna1. To avoid confusion, we keep the previous name Tunicate1 (Tu1) in this study.

**Fig. 2: High-resolution mapping of the *Lna1* locus.**

Tu1 contains eight exons and seven introns (Fig. 2c), encoding a MADS-box protein with 228 amino acid (aa) residues. Phylogenetic analysis revealed that this MADS-domain protein clusters within the MIKC-type MADS group and is specifically associated with the Arabidopsis SHORT VEGETATIVE PHASE (SVP) protein³¹ (Supplementary Fig. 3). A sequence comparison between MS71 and Popcorn revealed that only one exon of Tu1 in the fine-mapped interval had no variation, but many single nucleotide polymorphisms (SNPs) and insertions/deletions between the two parental lines were detected upstream of the gene within this 195-kb interval. To determine which variant was causative of leaf number above the ear, we performed association mapping in the NAM populations, which consisted of approximately 5000 individuals^32,33. A total of 475 variants were identified in the 195-kb fragment on the basis of whole-genome sequencing data from 26 NAM parents (https://nam-genomes.org)³⁴. Association mapping revealed that the strongest signal correlated with the number of leaves above the ear (P = 1.8 × 10⁻⁵), which was located at a 5-bp insertion (AGAAG) in the distal region positioned −194 kb upstream of Tu1 (Fig. 2d). This region was also reported as a chromatin accessibility landscape as determined by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) data from a public repository (Supplementary Fig. 4). In addition, this region was identified to have a H3K27me3-HiChIP loop for long-distance interaction with Tu1, and H3K27me3 functions as a transcriptional silencing element (Supplementary Fig. 5). As other variants had signals below the threshold (P = 1.0 × 10⁻⁴), the 5-bp insertion with the strongest signal in Popcorn compared with MS71 was then identified as the causal variant of Lna1. These results suggest that Tu1 is the causal gene for the number of leaves above the ear in maize.

The effect of the Tu1 gene on the number of leaves above the ear was confirmed through transgenic analysis

To determine the role of the Tu1 (Zm00001d052180) gene, which corresponds to Lna1, in maize leaf number above the ear, genetic transformations were conducted (Fig. 3). A construct controlled by the Ubiquitin promoter (Tu1-OE) was introduced into the maize inbred line LH244, resulting in three independent transgenic events (Fig. 3a). Compared with those of the control plants, the leaf number above the ear and the total number of leaves significantly (P < 2.5 × 10⁻⁵) increased in the transgenic plants that overexpressed the Tu1 gene from the three events (Fig. 3d–f). Like the natural Tu1 allele, ectopic overexpression of this MADS-box gene also induced large spikelets on the tassel and long glumes covering grain on ear in this study (Supplementary Fig. 6). Maize transformation via CRISPR/Cas9 was subsequently performed with a single cutting target in the coding sequence (CDS) of the Zm00001d052180 gene. Three independent edited events (Tu1-CR1, Tu1-CR2, and Tu1-CR3) with loss of function in the Tu1 gene were obtained (Fig. 3b). The edited plants carried deletions in the first exon of Tu1, which introduced a frameshift mutation and resulted in early termination of translation (Supplementary Fig. 7). These edited plants were semidwarf and had a significantly (P < 2 × 10⁻³⁰) lower number of leaves above the ear, averaging only 3–5 leaves, than the control plants, with an average of 6.5 leaves above the ear (Fig. 3b, i–m). These transgenic results confirm that the Tu1 gene, corresponding to Lna1, controls the leaf number above the ear in maize.

We investigated the plastochron number in the SAMs of both overexpressing and knockout-edited maize plants by scanning electron microscopy (SEM) and light microscopy. At 30 days after planting (DAP), the overexpression plants presented 19 plastochrons (Supplementary Fig. 8a, e, f), whereas the knockout-edited plants presented 15 plastochrons (Supplementary Fig. 8b, g, h). By 35 DAP, the plastochron number had increased to a maximum of 21 in the overexpression plants and 16 in the knockout-edited plants. Inflorescence primordia had emerged on the SAMs of the overexpression plants by this time, whereas they had not yet appeared in the knockout-edited plants (Supplementary Fig. 8c, d). These findings indicate that the number of leaves in maize is determined by the plastochron number before the emergence of floral primordia in the SAM, and the SAMs in the overexpression plants remained more active than those in the knockout-edited plants.

A 5-bp insertion moderately enhances Tu1 expression and slightly increases the number of leaves above the ear

To determine how the 5-bp insertion in the distal region upstream of Tu1 regulates its transcription, we performed real-time RT‒PCR in SAMs from NIL-Ms71 and NIL-Popcorn at stages V3-V5. RT‒PCR revealed that the expression level of Tu1 was obviously greater in NIL-Popcorn with the 5-bp insertion than in NIL-MS71 (Fig. 4a). To further test the effect of this 5-bp insertion on Tu1 expression, we performed a dual-luciferase (LUC) transcriptional activation assay in maize protoplasts (Fig. 4b). The constructs harboring the LUC reporter gene driven by the distal promoter segments of MS71 and Popcorn were subsequently transformed into protoplasts. Compared with that in the MS71 fragment, luciferase activity in the Popcorn segment was greater. Furthermore, when the 5-bp insertion was deleted from the Popcorn segment, the luciferase activity was clearly repressed (Fig. 4b). These results indicated that the 5-bp insertion increased Tu1 expression in the Popcorn plants. Therefore, both association mapping and LUC transient assay analysis consistently revealed that the 5-bp insertion (AGAAG) in the distal promoter region was causative to the Tu1 gene.

**Fig. 4: Gene function analysis of *Tu1* in maize.**

To elucidate the regulatory role of Tu1 in determining leaf number above the ear, a comprehensive investigation into leaf number-related traits, including leaf numbers above/below the ear, date to pollen shedding (DTP), and the node of ear emergence (equivalent to leaf number below the ear), is imperative (Fig. 1d–i, and Fig. 3e–l). A comparison of these traits across the overexpression and edited transgenic plants with those of the nontransgenic controls revealed robust patterns. The transgenic overexpression plants presented a significant increase in both the number of leaves above and below the ear, leading to a considerable increase in the total leaf number (Fig. 3e–g). The average number of nodes associated with ear emergence in these overexpression plants showed an increase by one internode in comparison to the control plants (Fig. 3g). Additionally, the average DTP in the overexpression plants was significantly delayed in contrast to that in the control plants (Fig. 3h).

Conversely, the edited transgenic plants presented a marked decrease in leaf number above/below the ear, resulting in a significant reduction in total leaf number (Fig. 3i–k). Compared with those in the control plants, the average number of nodes associated with ear emergence in the edited plants decreased by nearly one internode (Fig. 3k). However, the average DTP in the edited plants was significantly delayed (Fig. 3l), resembling that in the overexpression plants. These findings underscore the role of Tu1 in controlling leaf number above and below the ear, influencing both the node of ear emergence and DTP in the transgenic plants.

In contrast, the plant with a 5-bp insertion in the promoter of Tu1, which induced moderately increased expression, regulated the total leaf number without influencing the nodes associated with ear emergence and DTP (Fig. 1f, g). Thus, the number of leaves below the ear of the plant with the 5-bp insertion did not differ, and the number of leaves above the ear was controlled (Fig. 1d, f). Both the overexpression and edited knockout plants (overrepression), along with the plants featuring the 5-bp insertion, consistently demonstrated that Tu1 regulates the total leaf number without conflicts. Unlike the overexpression and overrepression plants, those with the 5-bp insertion and moderately enhanced expression of Tu1 did not alter the node where the ear occurs or flowering time, indicating that changes in these factors depend on Tu1’s expression levels. Therefore, the control exerted by Tu1 on leaf number above the ear is attributed to the unaltered node of ear emergence and flowering time, a result of the moderately increased expression in the maize plant with the 5-bp insertion in the promoter of Tu1.

TU1 might function as a transcriptional repressor

The expression levels of the Tu1 gene were low in the leaves, stem nodes, internodes, roots, and 1-cm or 2-mm ears but were significantly greater in the SAM (Fig. 4a). The accumulation of Tu1 transcripts in the SAM decreased gradually from the V3 to V5 developmental phases, with higher transcript abundance in the NIL-Popcorn than in the NIL-MS71, regardless of developmental stage (Fig. 4a).

The TU1 protein is a MADS-box transcription factor (Supplementary Fig. 3). To determine the subcellular localization of TU1, a construct encoding a TU1-GFP fusion protein was introduced into maize leaf protoplasts, and the fluorescent signals for TU1-GFP were detected only in the nuclei, in contrast to free GFP (Fig. 4c). To examine the self-activating activity of TU1, a yeast two-hybrid assay was performed, which revealed that the TU1 and BD (GAL4 DNA-binding domain) fusion proteins did not activate the expression of the reporter gene, suggesting that TU1 lacks transcriptional activation activity (Supplementary Fig. 9). To confirm this, transcriptional activity assays were conducted by generating chimeric proteins in which TU1 was fused with the DNA-binding domain from the yeast GAL4 transcription factor (GAL4-DB) and the activation domain from the herpes simplex virus protein 16 (VP16) (Fig. 4d). The reporter constructed for these assays consisted of the luciferase reporter gene driven by a synthetic promoter that comprises five copies of the GAL4 upstream activating sequence (UAS) and a TATA box (Fig. 4d). While GAL4BD-VP16 strongly activated luciferase expression (as indicated by high luciferase activity), the GAL4BD-VP16- TU1 chimeric protein dramatically repressed luciferase activity from the same reporter (Fig. 4e). These results indicate that TU1 might act as a transcriptional repressor of its downstream genes.

The gene regulatory network of TU1 in terms of leaf number in maize

To understand how TU1 controls downstream genes involved in leaf number, RNA-seq was performed on the SAMs of a transgenic gene-edited plant (Tu1-CR1) with a 2-bp deletion mutation inducing the loss of function of TU1 and a nontransgenic control plant (LH244). The analysis revealed 1,715 and 2,574 differentially expressed genes (DEGs) whose expression was upregulated and downregulated, respectively, in the edited plants compared with the control plants (Fig. 5a, Supplementary Data 1). We subsequently conducted a gene ontology (GO) analysis on the DEGs identified through RNA-seq (http://systemsbiology.cau.edu.cn/agriGOv2/)³⁵. The analysis highlighted five of the top 20 enriched GO terms associated with biological processes, including organonitrogen compound metabolic process, organonitrogen compound biosynthetic process, nitrogen compound metabolic process, cellular nitrogen compound biosynthetic process, and cellular nitrogen compound metabolic process (Supplementary Data 2). These results suggest that TU1 governs organonitrogen and nitrogen biosynthesis, as well as metabolic processes, in the development of SAM. Leaves sequentially grow out from the leaf primordium in the SAM until floral transition. The leaf number in maize is determined by both the plastochron and the time of floral primordia emergence. A careful search of the RNA-seq data revealed 9 DEGs, namely, TD1²⁷, ub2²², Bige1²³, WEE1¹⁵, yabby1, PIN1a²⁶ and Kn1⁹, which are related primarily to plastochron, meristem development and the timing of floral primordia emergence (Supplementary Data 3). The MADS-box genes ZmMADS1 and ZmMADS3 function in floral activation and leaf development^24,25. TU1 repressed the expression of WEE1 and ZmMADS1, whereas it upregulated the transcription of TD1, ub2, Bige1, ub2, yabby1, PIN1a and Kn1. The relative expression levels of all of these DEGs were confirmed by real-time qPCR (Supplementary Fig. 10). As the function of yabby1 has not been identified in maize, we generated plants with a loss-of-function mutation in the yabby1 gene via the CRISPR/Cas9 editing system. Two edited events carried deletions in the CDS, introducing frameshift mutations and resulting in early translation stops (Supplementary Fig. 11). Compared with the nontransgenic control plants, the edited plants from these two events presented fewer leaves above the ear (Fig. 3c, n).

**Fig. 5: Gene regulatory network of TU1.**

We reanalyzed a previously reported maize single-cell RNA-seq dataset, focusing on the SAM and following the reported method³⁶. Tu1 is expressed across 20 distinct cell types, with obvious expression observed in leaf primordia cells (Supplementary Fig. 12), which is consistent with the transcriptional pattern based on in situ hybridization (Supplementary Fig. 8i). Among the nine chosen downstream target genes, excluding ZmMADS3, which was undetected in this single-cell RNA-seq dataset, we identified correlated expression patterns in these leaf primordium cells, providing robust evidence of their association with TU1 in the regulation of leaf number.

We next performed DAP-seq for TU1 (Supplementary Data 4). This analysis revealed 52,806 TU1 binding peaks in the regulatory regions of 20,821 genes, with a GG(A/T)₆CC motif being predominantly enriched in these binding sites (Fig. 5b). This motif was found to be congruent with the binding site of previously identified MADS-box transcription factors^37,38. The ZmMADS1 gene contains the TU1 binding site “GG(A/T)₆CC” in its promoter region (Fig. 5c). The expression level of the ZmMADS1 gene was suppressed by TU1. Additionally, the region with the GG(A/T)₆CC motif in the ZmMADS1 promoter showed high chromatin accessibility (Supplementary Fig. 13). Luciferase activity assays and electrophoretic mobility shift assays (EMSAs) confirmed that TU1 can directly bind to the motifs in the ZmMADS1 promoter and subsequently repress its expression (Fig. 5d-f).

These results indicated that TU1 can directly bind to the ZmMADS1 promoter, leading to direct repression of its expression. Moreover, TU1 indirectly upregulated TD1, ZmMADS3, PIN1a, Kn1, Bige1, ub2 and yabby1 and indirectly downregulated WEE1 (Fig. 5g). The genes TD1 (Supplementary Fig. 14), PIN1a, and yabby1 have a positive influence on the number of leaves, whereas the genes WEE1, ZmMADS1, ZmMADS3, Kn1, ub2, and Bige1 have a negative effect on this characteristic (Fig. 5g). As a result, TU1 modulates the effects of these genes and mildly enhances the total leaf number in maize.

The Lna1 locus was under selection in landraces during maize diversification

To investigate whether the Lna1 locus was under selection in maize, we performed large-scale sequencing of a 1.4-kb promoter fragment harboring a 5-bp insertion/deletion from 136 accessions, including 42 teosinte lines, 37 landraces and 57 common maize inbred lines (Supplementary Data 5). The majority of the accessions did not possess the 5-bp insertion, with the exception of 4.76% (2 lines) of teosinte, 48.65% (18 lines) of landraces, and 5.26% (3 lines) of maize inbred lines (Fig. 6a). This result suggested that the Popcorn Lna1 allele accumulated during the domestication of maize from teosinte to landrace but was gradually lost during the improvement of maize. Furthermore, phylogenetic analysis was performed on all of the accessions (23 lines) with the 5-bp insertion, and the results revealed that the teosinte lines and maize lines with the 5-bp insertion (AGAAG) shared a single common ancestor (Fig. 6b). We then compared the nucleotide diversity among teosinte lines, landraces, and maize inbred lines. The teosinte lines presented the greatest degree of DNA diversity, which decreased in the maize inbred lines, whereas the landraces presented the lowest level of diversity. The 1.4-kb fragment was divided into three segments (1–600 bp, 601–1000 bp, and 1001–1400 bp). We subsequently performed Tajima’s D tests on the three segments from these accessions. Tajima’s D test significantly rejected the neutral null hypothesis (Tajima’s D = −1.8303, P < 0.05) only for the second segment (601–1000 bp), with a 5-bp insertion in the landraces, whereas no significant Tajima’s D signals were detected in the teosinte or maize inbred lines in any sequenced region (Fig. 6c). These findings indicate that the Lna1 locus underwent selection in landraces during maize diversification.

**Fig. 6: The *Lna1* locus was under selection.**

Popcorn Lna1 allele enhances maize yield by increasing the number of leaves above the ear

The results of the sequence analysis revealed that only two out of 42 teosinte lines and 21 out of 94 maize lines carried the 5-bp insertion. The majority of the maize lines with the insertion are landraces. These findings indicate that the 5-bp insertion is a rare variant in teosinte that mostly accumulated in landraces, but has been largely lost during maize improvement.

Despite being almost completely lost during maize improvement, the 5-bp variant in the Lna1 locus, which is known to increase the number of leaves above the ear, has the potential to be exploited for optimizing leaf architecture, particularly in densely planted modern breeding programs. To test this possibility, we conducted a yield test from three sets of NIL-MS71 and NIL-Popcorn lines planted at normal (8000 plants ha⁻¹, Fig. 7a–c) and high (16,000 plants ha⁻¹, Fig. 7d–f) densities (see “Methods”). The results at a regular planting density revealed that the single ear weight, grain weight per ear, and hundred-grain weight improved by an average of 10.2%, 11.4%, and 2.6%, respectively, in NIL-Popcorn relative to NIL-MS71. When the NILs were planted at double the density, the single ear weight, grain weight per ear, and hundred-grain weight improved by an average of 10.8%, 9.5%, and 5.5%, respectively, in NIL-Popcorn compared with NIL-MS71. These findings indicate that the Popcorn Lna1 allele can increase both the number of leaves above the ear and the yield potential in both normal- and high-density fields (Fig. 7g, h). As such, the Popcorn Lna1 allele can be utilized to improve plant architecture and increase maize yield in both regular and densely planted fields.

**Fig. 7: Yield test between NIL-MS71 and NIL-popcorn.**

Discussion

The source strength and balance between the source and sink play critical roles in determining the final yield of crops. The term “source” refers to the photosynthetic capacity of the leaves, which serves as the main source of carbohydrates for the ear. In maize, leaves located above the ear are more metabolically active, contributing a greater amount of carbohydrates to the ear compared to leaves located below the ear. A proper increase in the number of leaves above the ear strengthens the source strength of maize plants, thereby improving the final yield. However, an excessive number of leaves above the ear can disrupt the source‒sink balance, leading to greater plant stature, delayed flowering and grain filling, and lower yields in maize. How to increase the source strength while maintaining the source‒sink balance remains a challenge in maize breeding.

In this study, we identified a favorable allele with a 5-bp insertion in the Lna1 locus from a landrace called Strawberry Popcorn. This favorable allele of Lna1 has a positive effect on the number of leaves above the ear, with no effect on flowering time or plant stature (Fig. 1). Our field studies have shown that, compared with maize plants without the 5-bp insertion in the Lna1 locus, maize plants with this favorable allele of Lna1 significantly improved the final yield at regular and doubled high planting densities. On the one hand, TU1 regulates plastochron activators such as TD1, PIN1a and yabby1 and repressors such as WEE1, ub2, Bige1, and Kn1. The overall effect of this regulation slightly favors positive impacts on leaf number, leading to a limited increase in the number of leaves above the ear. On the other hand, TU1 modulates the expression of floral genes, including ZmMADS1 and ZmMADS3 (Supplementary Data 3). ZmMADS1 functions as a flowering activator. The flowering time gene ZmMADS1 was predicted to bind to the site close to this 5-bp insertion through PlantRegMap (http://plantregmap.gao-lab.org/index.php). In addition, ZmMADS1 is directly repressed by TU1. ZmMADS1 and TU1 might mutually repress each other in terms of transcription. Thus, the complex regulatory network involving TU1 may fine-tune the timing of flowering. Hence, the Tu1 gene can strengthen the source strength while maintaining the source‒sink balance, resulting in an increase in maize yield. This finding suggests that the favorable allele of the Lna1 locus from Popcorn has high potential for improving maize yield in maize breeding.

In this study, NIL plants carrying a 5-bp insertion in the Tu1 promoter, which led to moderately increased expression of Tu1, did not show changes in either the node where the ear formed or the flowering time. However, these plants presented an increased total leaf number compared with the NIL plants lacking the insertion. As a consequence, the number of leaves above the ear increased. The number of leaves below the ear is generally strongly correlated with flowering time in maize³⁹, suggesting that flowering time determines the node of ear formation. While TU1 regulates flowering time-related genes such as ZmMADS1 and ZmMADS3, the moderate changes in their expression levels induced by TU1 were insufficient to alter the flowering time. Thus, both the flowering time and the ear node remained consistent between NIL plants with and without the 5-bp insertion in Tu1. Moreover, TU1 moderately regulates genes associated with plastochron activators and repressors, including yabby1, WEE1, PIN1a, ub2, TD1, Kn1, and Bige1, leading to a slight increase in plastochron number. Consequently, the NIL plants with the 5-bp insertion in Tu1 showed an increased leaf number above the ear.

MADS genes are classified into types I and II, with plant type II MADS genes playing pivotal roles in various plant developmental processes⁴⁰. Plant type II MADS proteins, also known as MIKC-type MADS proteins, encompass the MADS (M), Intervening (I), Keratin-like (K), and C-terminal (C) domains. Phylogenetic tree analysis revealed that TU1 clustered with the group of Arabidopsis MIKC-type SVP proteins (Supplementary Fig. 3a). Additionally, MIKC domains were identified in seven TU1 orthologous proteins from maize, rice, wheat, barley, sorghum, foxtail millet, and Arabidopsis (Supplementary Fig. 3b). The MADS and K domains displayed high conservation across these seven species, whereas lower conservation was observed in the I- and C-terminal domains (Supplementary Fig. 3b). Our transcriptional assays in this study indicated that TU1 functions as a transcriptional repressor (Fig. 4e and Supplementary Fig. 9). Consistent with our findings, ChIP-seq data demonstrated that Arabidopsis SVP also functions as a transcriptional repressor⁴¹. New ChIP-seq studies support that MIKC-type proteins, such as TU1, can act as transcriptional repressors as well as activators.

Maize was initially domesticated from its wild ancestor, teosinte, and then underwent diversification into landraces and further development into improved inbred lines. During this process, the diversity of maize gradually decreased. Teosinte exhibits a high level of DNA diversity, and maize landraces retain a substantial amount of diversity from teosinte. In contrast, the maize inbred lines exhibit a sharp reduction in diversity. Compared with other crop species, maize landraces exhibit lower differentiation from their progenitor teosinte⁴². The narrow diversity of maize inbred lines has become a major obstacle to further enhancing maize yield from hybrids. In this study, the favorable allele of the 5-bp insertion in the Lna1 locus was found to have originated from teosinte and was significantly enriched in maize landraces, whereas it was nearly absent in maize inbred lines. Teosinte harbors a wealth of advantageous alleles in the wild, and the domestication process may have led to the loss of many of these alleles in maize inbred lines. However, these alleles are largely preserved in maize landraces. Given the lower prevalence of unfavorable traits in maize landraces than in teosinte, maize landraces offer a readily usable resource in breeding programs to increase the diversity of maize inbred lines with limited genetic diversity. Consequently, maize landraces represent a highly valuable resource for future maize breeding to improve maize yield.

Methods

Plant materials

The maize landrace (Strawberry Popcorn; Ames 14282) was crossed with the maize inbred line (MS71; PI 587137) from the NAM population to generate an F₂ population comprising 240 individuals. QTL mapping was conducted in 2015 on both the F₂ and F₃ populations, which were planted during summer and winter in Beijing (40°08′ N, 116°11′ E) and Hainan (18°09′ N, 108°56′ E), respectively. Between 2015 and 2020, plants from the F₂ and F₃ populations, as well as the plant materials used for fine mapping, were grown at a spacing of 25 cm from neighboring plants with a row-to-row distance of 50 cm at the experimental stations of China Agricultural University in Hainan and Beijing. To construct near-isogenic lines, we selfed a heterogeneous inbred family (HIF, F₇) that contained heterozygous genotypes at the Lna1 locus and homozygous fragments at other loci, resulting in the generation of NIL-Popcorn and NIL-MS71 (F₈). To identify whether Lna1 enhances maize yield, three sets of NIL plants were planted at normal (25-cm plant-to-plant distance and 50-cm row-to-row distance) and high-density (12.5-cm plant-to-plant distance and 50-cm row-to-row distance) locations in Hainan in 2019. Each NIL was planted in 8 rows with 15/30 plants in each row. The fields were supplied with 120 kg ha⁻¹ N, 90 kg ha⁻¹ P, and 90 kg ha⁻¹ K.

QTL mapping

The F₂ population comprising 240 lines was genotyped using 218 SSR markers, which were evenly distributed across the 10 maize chromosomes. We subsequently constructed a genetic map, which spanned 1614.69 centimorgans (cMs) and had an average genetic distance of 7.41 cM between pairs of neighboring markers. The phenotypic data and genetic map were then input into R/qtl⁴³ for QTL detection using a multiple-QTL mapping method. We initiated simple interval mapping using the Haley–Knott regression method with the R/qtl function scanone, and a significance threshold of P = 0.05 was established for each trait via 1,000 permutations. We subsequently refined the positions of the QTLs with logarithm of odds (LOD) scores above the threshold via the R/qtl function refineqtl. Next, we scanned additional QTLs with the function addqtl on the basis of the refined QTLs. Upon detecting an additional significant QTL with an LOD score above the threshold, we added it to the model and refined the positions of all of the QTLs again. These steps were repeated until no significant QTL was added. Once all QTL positions had been refined, we finally assessed the genetic effect and significance of each QTL using drop-one-QTL analysis in the full model.

Fine mapping of the Lna1 locus

To fine map the major QTL responsible for leaf number above the ear, Lna1, a large population consisting of approximately 12,000 F₇ individuals (Supplementary Fig. 2), was developed. This population was derived from a single residual heterozygous line (RHL, F₅), which carried a heterozygous genomic fragment at the Lna1 locus and homozygous genotypes at other loci. The population was screened with 12 markers to identify 17 representative recombination types (Supplementary Fig. 2). Descendant populations from the selfing of these recombinant plants, all carrying heterozygous/homozygous fragments within the Lna1 target region, were used to examine the correlation between genotypes and leaf number above the ear. A linear regression model was used to determine the correlation, and a significant P-value indicated the presence of Lna1 in the heterozygous segments, whereas Lna1 was mapped to the homozygous segments in the presence of a nonsignificant P-value. Using this modified progeny test, the Lna1 locus was narrowed down to the region between two markers, M9 and M11. The sequences of the primers used in the fine-mapping process are listed in Supplementary Data 6.

Plant transformation

The CDS of Tu1 of MS71 was inserted into the binary vector pBECXUN under the control of the ubiquitin promoter. The resulting construct was then transformed into the maize inbred line LH244 via a protocol based on HiII⁴⁴. Cas9 is driven by the OsU3 promoter from rice, and a gRNA targeting a site in the first exon of the Tu1 gene was designed using CRISPR-P software and subsequently introduced into the CRISPR/Cas9 binary vector. All of these constructs were then introduced into the maize inbred line LH244. Three Tu1-overexpressing (T₀) events and three homozygous Tu1-CRISPR (T₀) gene-editing events were obtained. The T₀ transgenic plants were self-crossed to create homozygous T₁ plants for the investigation of leaf number above the ear, with LH244 used as the control.

Association mapping

To conduct association mapping analysis for leaf number above the ear in NAM populations consisting of approximately 5,000 individuals^32,33, we identified 475 variants from a 195-kb upstream fragment of the Tu1 gene on the basis of the genomes of 26 NAM parents. We performed association mapping testing using a mixed linear model implemented in TASSLE5⁴⁵. To correct for multiple testing, we used the Bonferroni correction. The significance threshold (α′ = 0.0001) was calculated via the following equation:

$$\alpha ^{\prime} \approx \alpha /n$$

(1)

where α is the nominal significance threshold (α = 0.05) and n is the number of variants (n = 475).

RNA sequencing

Tu1-CRISPR and control plants at the v5 stage with similar growth vigor were simultaneously collected. Shoot apices were dissected at the base of P2 or P3 leaves using a stereomicroscope and rapidly frozen in liquid nitrogen. Approximately 20 shoot apices were pooled per biological replicate, and three biological replicates were collected for each genotype. Total RNA samples were extracted from the shoot apices and sequenced using a HiSeq-2500 System (Illumina), resulting in 50 Gb of raw sequencing data. The raw RNA-seq reads were analyzed using a standard RNA-seq pipeline⁴⁶. Specifically, the raw reads were trimmed using Trimmomatic⁴⁷, cleaned by fastq_clean⁴⁸, and aligned to the maize B73 reference genome (v4) using STAR⁴⁹. Gene expression was then calculated using the fragments per kilobase of exon per million fragments mapped (FPKM) method with Cufflinks and Cuffdiff2⁵⁰. Differentially expressed (DE) genes between the transgenic and control groups were identified on the basis of their corrected P values (q values).

DNA affinity purification sequencing

A genomic DNA library was created by modifying a previously reported protocol^51,52. In brief, genomic DNA was extracted from V5 shoot apices and fragmented into 200 bp fragments. The resulting fragments were ligated with a truncated Illumina TruSeq adaptor to generate the library. The Tu1 coding sequence was fused with a HaloTag and expressed in the wheat germ extract system in two independent experiments. The resulting HaloTagged TU1 was immobilized onto Magne HaloTag beads, which were then incubated with the genomic DNA library (300 ng) for 1 h and washed. The washed beads, which contained bound genomic DNA fragments, were tagged with dual-indexed multiplexing barcodes through 15 cycles of PCR amplification. The resulting libraries from the two independent replicates were pooled and sequenced on the Illumina NovoSeq 6000 platform. Input DNA libraries were also prepared using the same protocol to control the background noise.

The raw reads from each replicate and input DNA library were processed by trimming adapter sequences and low-quality bases using fastp⁵³. The resulting clean reads were mapped to the maize B73 reference genome (v4) using Bowtie2 v2.35⁵⁴. The mapped reads were then filtered with SAMtools 1.9⁵⁵ to restrict the reads that aligned to multiple positions with the following parameters: -h -q 30 -F 4 -F 256. Peak calling was conducted using MACS2 v2.2.7.1⁵⁶ with a cutoff q value of 0.05, using the input DNA library as the control. The final list of candidate peaks was generated by identifying significant overlapping peaks from the two replicates. The bam files were converted to bigwig files and visualized in the Integrative Genome Browser. The most enriched motif for these overlapping peaks was determined using the MEME suite v5.5.1⁵⁷.

Subcellular localization

The CDS of Tu1 was merged with green fluorescent protein (GFP) to create the 35Spro::TU1-GFP construct, which was regulated by the cauliflower mosaic virus (CaMV) 35S promoter. The 35Spro::TU1-GFP construct was subsequently transferred into maize MS71 leaf protoplasts, and the subcellular GFP signal was analyzed using an Olympus FV1000 laser scanning microscope with a 488 nm laser line.

Protoplast transient expression assay

Transient assays were conducted in maize leaf protoplasts to assess the impact of different parental sequences in the distal regulatory regions of Tu1 on gene expression. The ~500-bp fragments containing the 5-bp (AGAAG) insertion/deletion variation from MS71 (MS71-Pro::LUC) and Popcorn (Popcorn-Pro::LUC) were amplified, and site-specific mutations targeting this 5-bp insertion were introduced using specific primers through standard oligonucleotide-directed mutagenesis techniques. These segments were integrated into the LUC vector (pGreenII 0800-LUC), which consisted of a Renilla reniformis reporter gene (REN) controlled by the cauliflower mosaic virus (CaMV) 35S promoter and a firefly luciferase reporter gene (LUC) controlled by a customized promoter. These constructs were then introduced into etiolated maize MS71 mesophyll protoplasts at the seedling stage. Approximately 10 µg of the reporter construct was mixed with the newly isolated protoplasts in PEG transfer solution for 18 min at room temperature before being returned to WI medium. After incubation at 25 °C for 17 h, the transformed protoplasts were harvested by centrifugation, lysed in Passive Lysis Buffer (PLB, Promega), and analyzed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Three to six biological replicates of each construct were carried out, and all of the assays were replicated three times.

To examine the impact of TU1 on the expression of two potential target genes, we then introduced the promoter fragment upstream of ZmMADS1 (1,786 bp) with a 35S mini-promoter into the pGreenll-0800-LUC vector to construct another set of reporters. The complete coding sequence of Tu1 introduced into the pGreenll 62-SK vector under the control of the 35S CaMV promoter was constructed as the effector construct. The appropriate sets of reporter and effector constructs were cointroduced into maize leaf protoplasts, and a relative reporter with the empty effector pGreenll 62-SK was used as the control. Luciferase activity was assessed via the same method described above.

Real-time PCR

Total RNAs were extracted from various tissues, including the tassel, ear (2 mm, 1 cm), bud, leaf, root, node, root, and SAM (v3, v4, v5) of the NIL plants, using an RNA Extraction Kit (Aidlab). First-strand cDNA was synthesized from 1 μg of total RNA via TransScript-Uni cDNA Synthesis SuperMix (TransGen Biotech). Quantitative PCR (qPCR) was performed using TB GreenTM Premix Ex TaqTM II (Takara), using the maize housekeeping gene GADPH1 as the internal control. qPCR was carried out on a CFX ConnectTM Real-time System (Bio-Rad) with three technical replicates and three biological replicates. The final relative transcript levels were determined using the ΔΔCT (DDCT) relative quantification method⁵⁸.

Transcriptional activity assay

To evaluate the transcriptional activity of the TU1 protein, we conducted a transcriptional activity assay using the Matchmaker GAL4 Two-Hybrid System 3 (Clontech). The full-length and two truncated coding sequences of Tu1 were subsequently cloned and inserted into the pGBKT7 vector to fuse TU1 with the DNA-binding domain of GAL4 (GAL4-BD). As a positive control, the transcription factor ZmCCT was fused with GAL4-BD. The resulting constructs were subsequently transformed into the yeast strain AH109 according to the manufacturer’s instructions. The colonies were then spotted onto yeast synthetic drop-out media that lacked Trp or Trp, Ade, and His.

To further evaluate the transcriptional activity of the TU1 protein, we conducted a dual-luciferase transient expression assay in maize leaf protoplasts. The Tu1 coding sequence was cloned and inserted into a vector to fuse TU1 with GAL4-DB and VP16, constructing the effector construct GAL4DB-VP16-TU1. To generate the reporter construct, we introduced a promoter with a 5×GAL4 UAS sequence and a TATA box into pGreenII 0800-LUC. We cotransformed the reporter and effector constructs into maize leaf protoplasts and used an empty effector construct as a control.

DNA diversity analysis

To analyze DNA diversity, we amplified two segments flanking the 5-bp deletion/insertion break point, each containing a forward 814-bp sequence and a downstream 612-bp sequence, from a global maize population consisting of 57 inbred lines, 37 landraces, and 42 teosinte accessions (Supplementary Data 5). The resulting PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and then sequenced on an ABI 3730 sequencer. The obtained sequences were aligned using ClustalW to construct a nucleotide alignment matrix. This matrix was imported into DnaSPv5.1 to calculate nucleotide diversity (π) with a sliding window of 100 bp and a step size of 25 bp⁵⁹, with sites containing gaps being excluded. Additionally, Tajima’s D tests were calculated using DnaSPv5.1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq and DAP-seq data were deposited at the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA960713. Source data are provided with this paper.

Change history

29 January 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41467-025-56464-y

References

Tilman, D., Balzer, C., Hill, J. & Befort, B. L. Global food demand and the sustainable intensification of agriculture. Proc. Natl. Acad. Sci. USA 108, 20260–20264 (2011).
ADS CAS PubMed PubMed Central Google Scholar
Shaver, D. L. Genetics and breeding of maize with extra leaves above the ear. In Proceedings of 38th Annual Corn and Sorghum Research Conference (Chicago, IL: American Seed Trade Association), 161-180 (1983).
Stewart, D. W., Dwyer, L. M., Andrews, C. J. & Dugas, J. A. Modeling carbohydrate production, storage, and export in leafy and normal maize (Zea mays L.). Crop Sci. 37, 1228–1236 (1997).
Palmer, A., Heichel, G. H. & Musgrave, R. B. Patterns of translocation, respiratory loss, and redistribution of 14c in maize labeled after flowering. Crop Sci. 13, 371–376 (1973).
CAS Google Scholar
Kaplan, D. R. Fundamental concepts of leaf morphology and morphogenesis: A contribution to the interpretation of molecular genetic mutants. Int. J. Plant Sci. 162, 465–474 (2001).
CAS MATH Google Scholar
Langdale, J. A. The then and now of maize leaf development. Maydica 50, 459–467 (2005).
Google Scholar
Colasanti, J. & Muszynski, M. The maize floral transition. Handbook of Maize Its Biology, 41-55 (2009).
Telfer, A., Bollman, K. M. & Poethig, R. S. Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 124, 645–654 (1997).
CAS PubMed Google Scholar
Vollbrecht, E., Veit, B., Sinha, N. & Hake, S. The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature 350, 241–243 (1991).
ADS CAS PubMed MATH Google Scholar
Itoh, J. I., Hasegawa, A., Kitano, H. & Nagato, Y. A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice. Plant Cell 10, 1511–1522 (1998).
CAS PubMed PubMed Central Google Scholar
Miyoshi, K. et al. PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450. Proc. Natl. Acad. Sci. USA 101, 875–880 (2004).
ADS CAS PubMed PubMed Central MATH Google Scholar
Kawakatsu, T. et al. PLASTOCHRON2 regulates leaf initiation and maturation in rice. Plant Cell 18, 612–625 (2006).
CAS PubMed PubMed Central MATH Google Scholar
Kawakatsu, T. et al. PLASTOCHRON3/GOLIATH encodes a glutamate carboxypeptidase required for proper development in rice. Plant J. 58, 1028–1040 (2009).
CAS PubMed MATH Google Scholar
Veit, B., Briggs, S. P., Schmidt, R. J., Yanofsky, M. F. & Hake, S. Regulation of leaf initiation by the terminal ear 1 gene of maize. Nature 393, 166–168 (1998).
ADS CAS PubMed Google Scholar
Wang, F. et al. ZmTE1 promotes plant height by regulating intercalary meristem formation and internode cell elongation in maize. Plant Biotechnol. J. 20, 526–537 (2022).
CAS PubMed MATH Google Scholar
Suzuki, M. et al. The Maize Viviparous8 locus, encoding a putative ALTERED MERISTEM PROGRAM1-like peptidase, regulates abscisic acid accumulation and coordinates embryo and endosperm development. Plant Physiol. 146, 1193–1206 (2008).
CAS PubMed PubMed Central Google Scholar
Helliwell, C. A. et al. The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase. Plant Cell 13, 2115–2125 (2001).
CAS PubMed PubMed Central MATH Google Scholar
HARLAN, H. V. & POPE, M. N. Many-noded dwarf barley. J. Heredity 13, 269–273 (1922).
Google Scholar
Mascher, M. et al. Mapping-by-sequencing accelerates forward genetics in barley. Genome Biol. 15, R78 (2014).
PubMed PubMed Central MATH Google Scholar
Walla, A. et al. An Acyl-CoA N-Acyltransferase regulates meristem phase change and plant architecture in barley. Plant Physiol. 183, 1088–1109 (2020).
ADS CAS PubMed PubMed Central MATH Google Scholar
Hibara, K. I. et al. Regulation of the plastochron by three many-noded dwarf genes in barley. PLoS Genet 17, e1009292 (2021).
CAS PubMed PubMed Central Google Scholar
Chuck, G. S., Brown, P. J., Meeley, R. & Hake, S. Maize SBP-box transcription factors unbranched2 and unbranched3 affect yield traits by regulating the rate of lateral primordia initiation. Proc. Natl Acad. Sci. USA 111, 18775–18780 (2014).
Suzuki, M., Sato, Y., Wu, S., Kang, B. H. & McCarty, D. R. Conserved functions of the MATE transporter BIG EMBRYO1 in Regulation of lateral organ size and initiation rate. Plant Cell 27, 2288–2300 (2015).
CAS PubMed PubMed Central Google Scholar
Alter, P. et al. Flowering time-regulated genes in maize include the transcription factor ZmMADS1. Plant Physiol. 172, 389–404 (2016).
CAS PubMed PubMed Central MATH Google Scholar
Heuer, S. et al. The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis. Plant physiol. 127, 33–45 (2001).
CAS PubMed PubMed Central MATH Google Scholar
Gallavotti, A. et al. The relationship between auxin transport and maize branching. Plant Physiol. 147, 1913–1923 (2008).
Bommert, P. et al. thick tassel dwarf1 encodes a putative maize ortholog of the Arabidopsis CLAVATA1 leucine-rich repeat receptor-like kinase. Development 132, 1235–1245 (2005).
CAS PubMed Google Scholar
Wingen, L. U. et al. Molecular genetic basis of pod corn (Tunicate maize). Proc. Natl Acad. Sci. USA 109, 7115–7120 (2012).
ADS CAS PubMed PubMed Central MATH Google Scholar
Wang, J., Zhang X. & Lin Z. QTL mapping in a maize F2 population using genotyping-by-sequencing and a modified fine-mapping strategy. Plant Sci. 276, 171–180 (2018).
Han, J.-J., Jackson, D. & Martienssen, R. Pod corn is caused by rearrangement at the Tunicate1 locus. Plant Cell 24, 2733–2744 (2012).
CAS PubMed PubMed Central MATH Google Scholar
Hartmann, U. et al. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J. 21, 351–360 (2000).
CAS PubMed MATH Google Scholar
Yu, J., Holland, J. B., McMullen, M. D. & Buckler, E. S. Genetic design and statistical power of nested association mapping in maize. Genetics 178, 539–551 (2008).
PubMed PubMed Central MATH Google Scholar
Buckler, E. S. et al. The genetic architecture of maize flowering time. Science 325, 714–718 (2009).
ADS CAS PubMed MATH Google Scholar
Hufford, M. B. et al. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science 373, 655–662 (2021).
ADS CAS PubMed PubMed Central MATH Google Scholar
Tian, T. et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 45, W122–W129 (2017).
CAS PubMed PubMed Central Google Scholar
Satterlee, J. W. et al. A Wox3-patterning module organizes planar growth in grass leaves and ligules. Nat. Plants 9, 720–732 (2023).
CAS PubMed PubMed Central Google Scholar
Pollock, R. & Treisman, R. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 5, 2327–2341 (1991).
CAS PubMed Google Scholar
Shore, P. & Sharrocks, A. D. The MADS-box family of transcription factors. Eur. J. Biochem. 229, 1–13 (1995).
CAS PubMed MATH Google Scholar
Li, D. et al. The genetic architecture of leaf number and its genetic relationship to flowering time in maize. N. Phytol. 210, 256–268 (2016).
CAS MATH Google Scholar
Ng, M. & Yanofsky, M. F. Function and evolution of the plant MADS-box gene family. Nat. Rev. Genet. 2, 186–195 (2001).
CAS PubMed MATH Google Scholar
Gregis, V. et al. Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis. Genome Biol. 14, R56 (2013).
PubMed PubMed Central MATH Google Scholar
Hufford, M. B. et al. Comparative population genomics of maize domestication and improvement. Nat. Genet. 44, 808–811 (2012).
CAS PubMed PubMed Central MATH Google Scholar
Broman, K. W., Hao, W., Śaunak, S. & Churchill, G. A. R/qtl: QTL mapping in experimental crosses. Bioinformatics, 19, 889–890 (2003).
Vega, J. M., Yu, W., Kennon, A. R., Chen, X. & Zhang, Z. J. Improvement of agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep. 27, 297–305 (2008).
CAS PubMed Google Scholar
Bradbury, P. J., Zhang, Z., Kroon, D. E., Casstevens, T. M. & Buckler, E. S. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).
CAS PubMed MATH Google Scholar
Zhang, X. et al. The tin1 gene retains the function of promoting tillering in maize. Nat. Commun. 10, 5608 (2019).
ADS CAS PubMed PubMed Central MATH Google Scholar
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
CAS PubMed PubMed Central Google Scholar
Mi, Z., Sun, H., Fei, Z., Feng, Z. & Shan, G. Fastq_clean: An optimized pipeline to clean the Illumina sequencing data with quality control. 2014 IEEE International Conference on Bioinformatics and Biomedicine (BIBM). 2015.
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
CAS PubMed Google Scholar
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
CAS PubMed PubMed Central MATH Google Scholar
Bartlett, A. et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat. Protoc. 12, 1659–1672 (2017).
CAS PubMed PubMed Central MATH Google Scholar
O’Malley, R. C. et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell 165, 1280–1292 (2016).
PubMed PubMed Central MATH Google Scholar
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).
PubMed PubMed Central MATH Google Scholar
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
CAS PubMed PubMed Central MATH Google Scholar
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
PubMed PubMed Central MATH Google Scholar
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
PubMed PubMed Central MATH Google Scholar
Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).
CAS PubMed PubMed Central MATH Google Scholar
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402 (2001).
CAS PubMed MATH Google Scholar
Librado, P. & Rozas, J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452 (2009).
CAS PubMed MATH Google Scholar

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Acknowledgements

This work was supported by the National Key R&D Program of China (2022YFF1003400 to Z.L.) and the National Natural Science Foundation of China (32272069 to Z.L.).

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State Key Laboratory of Maize Bio-Breeding; National Maize Improvement Center; Department of Crop Genetics and Breeding; China Agricultural University, 100193, Beijing, China
Yan Li, Jian Wang, Shuyang Zhong, Qiang Huo, Qun Wang, Hangqin Liu, Jiacheng Liu, Yang Song, Xiaojian Fang & Zhongwei Lin
Center for Crop Functional Genomics and Molecular Breeding; China Agricultural University, 100193, Beijing, China
Yunlu Shi
Sanya Institute of China Agricultural University, Sanya, 572025, China
Zhongwei Lin

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Contributions

Z.L. designed the study. Y. L., J. W., S. Z., Q. H., Q. W., Y. S., H. L., J. L., Y. S. and X. F. performed the research. Y.L. and Z.L. analyzed the data. Y.L. and Z.L. wrote the manuscript.

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Correspondence to Zhongwei Lin.

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Li, Y., Wang, J., Zhong, S. et al. MADS-box encoding gene Tunicate1 positively controls maize yield by increasing leaf number above the ear. Nat Commun 15, 9799 (2024). https://doi.org/10.1038/s41467-024-54148-7

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Received: 30 May 2023
Accepted: 01 November 2024
Published: 12 November 2024
DOI: https://doi.org/10.1038/s41467-024-54148-7