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Variation in Maize Chlorophyll Biosynthesis Alters Plant Architecture12[OPEN]

Chlorophyll is a tetrapyrrole metabolite essential for photosynthesis in plants. The first committed step of chlorophyll biosynthesis is catalyzed by a multimeric enzyme, magnesium chelatase, the subunit I of which is encoded by the oil yellow1 (oy1) gene in maize (Zea mays). A range of chlorophyll contents and net CO2 assimilation rates can be achieved in maize by combining a semidominant mutant allele of oy1 (Oy1-N1989) and a cis-regulatory modifier named very oil yellow1 (vey1) that varies between different inbred lines. We previously demonstrated that these allelic interactions can delay reproductive maturity. In this study, we demonstrate that multiple gross morphological traits respond to a reduction in chlorophyll. We found that stalk width, number of lateral branches (tillers), and branching of the inflorescence decline with a decrease in chlorophyll level. Chlorophyll deficit suppressed tillering in multiple maize mutants, including teosinte branched1, Tillering1, and grassy tillers1. In contrast to these traits, plant height showed a nonlinear response to chlorophyll levels. Weak suppression of Oy1-N1989 by vey1B73 resulted in a significant increase in mutant plant height. By contrast, enhancement of the severity of the Oy1-N1989 phenotype by the vey1Mo17 allele resulted in reduced plant height. We demonstrate that the effects of reduced chlorophyll contents on plant growth and development are complex and depend on the trait being measured. We propose that the lack of chlorophyll exerts growth control via energy balance sensing, which is upstream of the known genetic networks for branching and architecture.

Land plants project their aerial parts into threedimensional space to capture solar radiation and convert it into chemical energy via photosynthesis (Rabinowitch and Govindjee, 1965;Govindjee et al., 2017). Energy is stored as carbon-carbon bonds and primarily transported as sugars from photosynthetically active tissues, such as leaves, to rapidly growing parts of the plant. The addition of biomass to young tissue is achieved by the mobilization of this photosynthetically fixed carbon . Thus, growth of the plant body is achieved by converting sugars from photosynthesis into physical structures and requires an excess of sugar production beyond that required to maintain respiration.
To sustain maximum growth rates, plants must coordinate growth and metabolism to prevent underperformance when resources are plentiful or unsustainably committing to growth when photosynthesis cannot provide sufficient fixed carbon (Lemoine et al., 2013). Unsurprisingly, energy deficits from the manipulation of photosynthesis can result in dramatically decreased growth.
One way to manipulate photosynthetic activity and energy resources in plants is by altering chlorophyll biosynthesis (Walker et al., 2018;Khangura et al., 2019). Magnesium chelatase (MgChl) is a heterooligomeric enzyme complex that catalyzes the first committed step of chlorophyll biosynthesis by the conversion of protoporphyrin IX into magnesium-protoporphyrin IX (Walker and Weinstein, 1991;Gibson et al., 1995). In maize (Zea mays), one of the subunits of the MgChl complex is encoded by the oil yellow1 gene (oy1; B73 v3: GRMZM2G419806; B73 v4: Zm00001d023536). This gene received its name because weak hypomorphic alleles result in a light green to yellow color reminiscent of vegetable oil (Eyster, 1933;Sawers et al., 2006). We previously demonstrated that the semidominant Oy1-N1989 mutant was strongly modified by a quantitative trait locus (QTL) called very oil yellow1 (vey1) that appears to be encoded by cis-acting regulatory polymorphisms at the oy1 gene itself (Khangura et al., 2019). The highly heritable vey1 QTL is a major modifier of chlorophyll contents in heterozygous Oy1-N1989/1 mutants but has no effect on chlorophyll accumulation in wild-type plants (Khangura et al., 2019). The vey1 alleles are detectable as a cis-acting expression quantitative trait locus (eQTL) and show allele-specific expression at oy1 (Li et al., 2013Waters et al., 2017;Khangura et al., 2019Khangura et al., , 2020. The top singlenucleotide polymorphism associated with the vey1 locus was detected ;3.6 kb downstream of the 39 end of the coding sequence of oy1 on chromosome 10 (Khangura et al., 2019). In the B73 3 Mo17 populations, the vey1 allele from B73 (vey1 B73 ) was associated with higher expression of OY1, whereas the vey1 allele from Mo17 (vey1 Mo17 ) was linked with lower expression of OY1. These expression level differences at oy1 are apparent at the mRNA level in both mutants and the wild type, but the impact on chlorophyll content was only visible in the presence of the Oy1-N1989 allele (Khangura et al., 2019). This allelic interaction indicates the value of using mutants to uncover hidden genetic variation in maize.
Using the Oy1-N1989 mutant allele and the two vey1 modifier alleles from B73 and Mo17, we previously developed populations of maize to investigate the effects of this cryptic variant on chlorophyll content (Khangura et al., 2019), net CO 2 assimilation, and reproductive maturity (Khangura et al., 2020). These three phenotypes showed high linear correlations with each other and were consistent with expression of the wild-type oy1 allele. We proposed that the vey1 B73 allele results in greater expression of the wild-type OY1 and suppression of all of these Oy1-N1989/1 mutant phenotypes (Khangura et al., 2019(Khangura et al., , 2020. These results are consistent with the proposal that plants sense carbon assimilation and regulate the vegetative-to-reproductive transition in maize as a response to energy status (Minow et al., 2018).
In this report, we use the allelic combinations at vey1 and oy1 to study the effect of chlorophyll content, and the previously demonstrated effects on net CO 2 assimilation, on plant growth and development. The effect of chlorophyll accumulation on plant architecture and the unexpected opposing effects of the Oy1-N1989 allele and its modifier vey1 on plant height are discussed.

Tillering Is Reduced in Oy1-N1989 Mutants, and This Effect Is Modulated by vey1
Heterozygous Oy1-N1989 plants (Oy1-N1989/1) have their eponymous oil-yellow color due to reduced chlorophyll content, but they are fertile and produce both ears and tassels (Khangura et al., 2019(Khangura et al., , 2020. The heterozygous mutant plants in the B73 genetic background (Oy1-N1989/1:B73) germinate as yellow-green seedlings, but the phenotype is progressively suppressed as they develop and they are difficult to distinguish by color from wild-type plants at maturity (Khangura et al., 2019(Khangura et al., , 2020. We previously described cis-acting expression polymorphisms at oy1 distinguishing the maize inbred lines Mo17 and B73 and proposed that they are the genetic basis of an Oy1-N1989 modifier QTL called vey1 (Khangura et al., 2020). The reduction of chlorophyll content in Oy1-N1989/1 plants decreased CO 2 assimilation and sugar contents in young leaves and increased the time to reproductive maturity (Khangura et al., 2020). When the enhancing vey1 allele from Mo17 was introgressed into the B73 background, Oy1-N1989/1 plants with the vey1 Mo17 allele exhibited lower levels of chlorophyll, decreased CO 2 assimilation, and a longer delay of reproductive maturity than mutants suppressed by the vey1 B73 allele (Khangura et al., 2019(Khangura et al., , 2020. Sugar availability is implicated in axillary bud outgrowth (Barbier et al., 2015). The levels of Suc and other reducing sugars are lower in Oy1-N1989 and further modified by vey1 (Khangura et al., 2020). We tested if Oy1-N1989 modulated tiller outgrowth by crossing the mutant to well-characterized prolific tillering genetic stocks such as (1) the classical dominant tillering mutant Tillering1, (2) a semidominant tb1 allele in the W22 background (Doebley et al., 1995), and (3) the maize inbred P39, which carries the high-tillering allele at tiller number1 (tin1; Zhang et al., 2019). The tillering of the F1 hybrids between Oy1-N1989/1:B73 and all three hightillering lines (Tlr1, tb1, and sweet corn inbred P39) showed suppression in the mutant F1 siblings (Fig. 1, A-C; Supplemental Table S1). To eliminate the effect of hybrid genetic background in these crosses, we further tested the influence of the Oy1-N1989 allele on tillering in tb1 and gt1 mutants in an isogenic B73 background. We used a B73-like near-isogenic line (NIL) that was homozygous for either the vey1 B73 or vey1 Mo17 allele to generate populations that segregated for Oy1-N1989 along with tb1 or gt1 mutants in an otherwise isogenic B73 background. The Oy1-N1989 allele suppressed tillering in both tb1 and gt1 mutants. The effect of Oy1-N1989 on tillering was further modified by vey1 in the tb1;Oy1-N1989/1 and gt1;Oy1-N1989/1 double mutants as compared with tb1 and gt1 mutant siblings ( Fig. 1, D-G). The tb1;Oy1-N1989/1 and gt1;Oy1-N1989/1 plants that inherited vey1 B73 alleles from the NIL had an intermediate number of tillers, whereas those with vey1 Mo17 alleles had little to no lateral tiller outgrowth (Fig. 1, E and G). The gt1 homozygotes were readily distinguishable from gt1 heterozygotes and the wild types due to extended blade tissue from the husk sheath, a previously described recessive phenotype of gt1 (Whipple et al., 2011). Thus, the enhancement and suppression of tiller outgrowth in tb1 and gt1 mutants by the Oy1-N1989 and vey1 alleles displayed the additive and linear relationship previously observed for chlorophyll accumulation and net CO 2 fixation (Khangura et al., 2019(Khangura et al., , 2020. This is consistent with sugar availability controlling bud outgrowth in plants (Mason et al., 2014;Barbier et al., 2015). The possibility that these changes in axillary branch growth in the combinations tin1;Oy1-N1989/1, tb1;Oy1-N1989/1, and gt1;Oy1-N1989/1 are due to a change in photosynthetic capacity suggests strongly that future investigations seeking the identification of relationships between the fundamentals of energy balance sensing and lateral organ growth control are needed.
Tassel Branching Is Reduced in Severe Oy1-N1989 Mutants A recessive mutant allele at oy1 was previously shown to reduce the tassel branch number in maize (Brewbaker, 2015). The availability of NILs segregating for vey1 alleles prompted us to test whether the semidominant Oy1-N1989 allele and its interaction with vey1 alter tassel branching and development. We performed a morphometric analysis of the tassels collected from mutant and wild-type siblings from the F1 families derived from crosses of B73-like and Mo17-like NILs with Oy1-N1989/1:B73. Tassel length traits (measured as the length of the total tassel, branching zone, and central spike) were reduced in Oy1-N1989/1 mutants carrying the enhancing vey1 Mo17 allele ( Fig. 2; Supplemental Table S2). In Oy1-N1989/1 mutants carrying the vey1 B73 allele, tassel length was indistinguishable from wild-type siblings. Mutants derived from crosses of Oy1-N1989/1:B73 with B73-like and Mo17-like NILs carrying vey1 Mo17 introgression displayed reductions in the primary tassel branch number by ;47% and ;35%, respectively. A greater reduction (P , 0.0001) in the number of secondary tassel branches was affected by the combination of Oy1-N1989/1 and vey1 Mo17 . Inbred B73-like NIL F1 mutant plants carrying the vey1 B73 allele displayed lesser reductions (P , 0.05) in branching (;10% and ;20% reduction in primary and secondary branch numbers, respectively), but the hybrid Mo17-like NIL F1 mutant plants carrying the vey1 B73 allele showed no reduction in tassel branching and were indistinguishable from wild-type siblings. These changes in tassel architecture are consistent with the previous report of an ;30% reduction in tassel branching in the oy1-anderson homozygotes in the Hi27 inbred background (Brewbaker, 2015).

Oy1-N1989 Reduces Stalk Width and Is Epistatic to vey1
To determine if the reduced net CO 2 assimilation, sugar content, or chlorophyll reduction affected by variation in oy1 had effects on growth and development, we measured multiple traits in materials with a range of phenotypic severities. We started with F1 crosses of B73 and Mo17 with and without the Oy1-N1989 allele. As reported previously (Khangura et al., 2019), chlorophyll content in leaves was approximated using a hand-held chlorophyll content meter (CCM-200 plus; Opti-Sciences) at two time points (CCMI, ;3-4 weeks after planting; and CCMII, ;6-7 weeks after planting). The CCM values were lower (less chlorophyll Figure 1. The Oy1-N1989 mutant allele suppresses tillering in maize. A to C, Representative F1 hybrid plants from crosses Tlr1/Tlr1;1/1:Hi27 3 1/1;Oy1-N1989/1:B73 (A), tb1/tb1;1/1:W22 3 1/1;Oy1-N1989/1:B73 (B), and 1/1:P39 3 Oy1-N1989/1:B73 (C). D to G, Alleles at vey1 show additive suppression of tillering with Oy1-N1989 in double mutants with both tb1 and gt1. D and E, Representative tb1 (D) and gt1 (E) homozygotes with different allelic combinations at oy1 and vey1 loci in the isogenic B73 background. F and G, Tiller counts (means 6 SD) of these genotypic classes for tb1 (F) and gt1 (G) in the isogenic B73 background. Sample sizes range from 15 to 46 plants for each genotypic class. Different lowercase letters indicate statistically significant differences by Student's t test(P , 0.01). vey1* in D to G indicates that the vey1 alleles (vey1 B73 or vey1 Mo17 ) are of no significance to trait expression in the absence of Oy1-N1989. Bars5 50 cm. content) in mutants in the Mo17 3 B73 F1 background compared with mutants in the B73 background (Supplemental Table S3; Khangura et al., 2019Khangura et al., , 2020. The heterotic Mo17 3 B73 F1 wild-type hybrid plants had greater stalk width (SW) than the B73 plants (Supplemental Table S3). The presence of an Oy1-N1989 allele in the Mo17 3 B73 F1 background eliminated the hybrid advantage producing mutant hybrids with narrower stalks than both wild-type and mutant B73 inbred plants. We hypothesized that the vey1 allele in Mo17 was responsible for enhancing the effect of Oy1-N1989 on SW in the B73 3 Mo17 F1 hybrids. To test this, we measured SW in F1 populations generated by crossing Oy1-N1989/1:B73 to recombinant inbred lines (RILs) from the Intermated B73 3 Mo17 (IBM; Lee et al., 2002) and Syn10 (Hussain et al., 2007) populations. As expected, mutant chlorophyll values showed high positive correlations with mutant SW in both IBM and Syn10 F1 populations (Supplemental Tables S4 and S5). Not surprisingly, as previously reported for chlorophyll content, CO 2 assimilation, and reproductive maturity (Khangura et al., 2019(Khangura et al., , 2020, the vey1 locus modified SW in the Oy1-N1989/1 mutant F1 siblings but not their wild-type counterparts. The vey1 locus was the largest effect QTL for SW in mutant siblings in both mapping populations, explaining ;43% and ;68% of phenotypic variation in IBM and Syn10 F1 populations, respectively ( Fig. 3; Supplemental Tables S6 and S7). Assessment of SW ratio between mutant and wild-type siblings, to control for confounding by RIL genetic background effects on SW, only detected vey1 in IBM, whereas in Syn10, an additional QTL was detected on chromosome 9. The regression using linked markers for this additional QTL (chr09.159.5) and vey1 (chr10.94.5) in Syn10 did not detect any interaction between these two QTLs for the ratio between mutant and wild-type SW. No previously annotated genes with known roles in plant growth control or SW were present within a 400-kb window surrounding the peak maxima on chromosome 9. These results formally extend the epistatic relationship of the Oy1-N1989 allele and vey1 locus to SW. The F1 mutant plants that inherited the suppressor vey1 B73 allele were thicker than the mutant plants harboring the enhancing vey1 Mo17 allele (Figs. 4A and 5). This demonstrated that SW responded similarly to chlorophyll content, reproductive maturity, and CO 2 assimilation. Comparable to chlorophyll content, vey1 was the only QTL detected for mutant SW in both populations.
The SW of wild-type siblings in both populations was also variable and under genetic control. The localization of small-effect QTLs is often imprecise, but candidate genes within a window bound by a 2 2 log of the odds (2-LOD) decrease in the QTL significance were tabulated for each detected QTL (Supplemental Tables S6 and S7). QTL-candidate genes were assessed for roles in growth regulation and marked for eQTL using data from a previous study of the IBM population (supplemental table S3 from Li et al., 2013; please note that the effect direction was reversed due to the correction in Li et al., 2018). Three QTLs, one in the IBM population and two in the Syn10 F1 population, were detected on chromosome 1 for wild-type SW (Supplemental Tables S6 and S7). There was no overlap of the 2-LOD QTL intervals between IBM and Syn10 for SW of the wild-type siblings. Moreover, the Mo17 allele Figure 2. Effect of the vey1 Mo17 allele on tassel morphology of Oy1-N1989 mutants in the B73 and B73 3 Mo17 isogenic backgrounds. The data presented on the schematic of the maize tassel are derived from mutant siblings of B73 and Mo17 NILs crossed to Oy1-N1989/1:B73. Blue colored B73-NIL and Mo17-NIL carry the vey1 B73 allele, whereas gold color represents NILs with the vey1 Mo17 allele. Black crosses and downward-pointing arrows in front of the traits represent no significant reduction and statistically significant reduction in mutants compared with the wild-type siblings, respectively. One arrow and two arrows indicate statistically significant differences at P , 0.05 and P , 0.0001, respectively, as determined using Tukey's honestly significant difference. Full data are provided in Supplemental Table S2.
at the QTL detected in IBM increased wild-type SW, whereas both Syn10 QTLs showed increases in SW with B73 alleles, making an imprecise localization of the same QTL detected in both populations on chromosome 1 unlikely. The QTL in IBM for SW is a moderate-effect QTL with an estimated contribution of 13% of the phenotypic variation (Supplemental Table S6), but such estimates should be regarded as upper estimates (Beavis, 1994). The 2-LOD window at this QTL contains a tandem duplicate pair of candidate genes in the b-expansin family (expb4; B73 v4: Zm00001d033231), which have been fused in the maize B73 v4 assembly but were annotated as separate gene models in the B73 v3 assembly (Supplemental Fig. S1; Jiao et al., 2017). Additional evidence for the tandem duplicate pair of expb4 as separate gene models came from the isolation of full-length cDNA in previous studies (Supplemental Fig. S1; Alexandrov et al., 2009;Soderlund et al., 2009). Rice (Oryza sativa) orthologs of expb4 have been implicated in increased stem elongation in response to gibberellic acid (Lee and Kende, 2001). The abundance of both EXPB4 transcripts was influenced by cis-eQTL (LOD 5 7.01 and 45.82, additive effect 5 20.58 and 25.09, R 2 5 0.16 and 0.7, respectively) and trans-eQTL (chromosome 5 9 and 8, LOD 5 4.81 and 5.67, additive effect 5 20.41 and 2 0.96, R 2 5 0.11 and 0.03, respectively), with B73 alleles decreasing gene expression in all cases. The cis-eQTL at expb4 and the allelic effect of this QTL have the expected effect direction that makes this gene a good candidate for this QTL. A number of coding sequence differences, including a possible N-terminal extension of one of the paralogs in B73, may alternatively encode functional polymorphisms (Supplemental Fig. S1). No other QTL overlapped genes with previously described roles in stem architecture.
Confirmation of the impact of the vey1 locus on SW was provided by crosses between Oy1-N1989/1 mutants and NILs with introgressed vey1 alleles. In all NIL  . SW and plant height of the wild type and Oy1-N1989 mutants with alternative vey1 alleles in isogenic backgrounds. A, Representative cross sections of maize stalks at the middle of the internode above the primary ear at maturity from the wild type and mutants in isogenic B73 (first two pairs) and Mo17 3 B73 (last two pairs) F1 backgrounds. B and C, Representative wild-type and mutant plants at maturity with either a vey1 B73 or vey1 Mo17 allele in B73 NIL 3 Oy1-N1989/1:B73 (B) and Mo17 NIL 3 Oy1-N1989/ 1:B73 (C) crosses. The flag leaf and primary ear node are indicated by gold and white triangles, respectively. Bars 5 2 cm (A) and 50 cm (B and C). F1 crosses to Oy1-N1989/1, the Oy1-N1989/1 mutants were thinner than their wild-type siblings (Figs. 4A and 6,C and F;Supplemental Fig. S2). In both B73-like and Mo17-like NILs, inheritance of the vey1 Mo17 allele enhanced the Oy1-N1989/1 phenotype, leading to thinner plants than isogenic siblings with a suppressing vey1 B73 allele. In all NIL F1 families, the allele at vey1 had no discernible impact on the stem width of the wild-type siblings (Figs. 4A and 6; Supplemental Fig. S2). This is consistent with our previous report demonstrating no impact of vey1 on chlorophyll and reproductive maturity in these NIL populations (Fig. 6, A and D, which include data reanalyzed from Khangura et al., 2019Khangura et al., , 2020. Thus, Oy1-N1989 controlled SW and was epistatic to vey1, as previously found for chlorophyll content, reproductive maturity, and CO 2 assimilation (Khangura et al., 2019(Khangura et al., , 2020.  Table S3). This raised the possibility that the delay in flowering time in Oy1-N1989/1 (Khangura et al., 2020) might also result in a taller plant. However, delays in flowering time did not predict plant height. For example, Mo17 3 B73 F1 hybrid Oy1-N1989/1 mutants showed a reduction in EaHT but not plant height, despite their greater delay in reproductive maturity (Supplemental Table S3).
We measured height traits in the RIL 3 Oy1-N1989/ 1 F1 populations to determine the effect of the vey1 modifier on FlHT in the Oy1-N1989/1 mutants. Chlorophyll values correlated with the height of Oy1-N1989/1 mutants in both IBM and Syn10 F1 populations (Supplemental Tables S4 and S5). In both populations, linkage mapping detected a QTL for FlHT at vey1 for mutant individuals but not wild-type siblings ( Fig. 7; Supplemental Tables S6 and S7). The mutant plants that inherited the vey1 B73 allele were taller than the mutants harboring the vey1 Mo17 allele (Fig. 5, B and E). This effect was clear in both IBM and Syn10 F1 populations. The average FlHT of mutant plants that inherited the vey1 B73 allele was significantly greater than that of the isogenic wild-type siblings. The mutant plants that inherited the vey1 Mo17 allele were shorter than their isogenic wild-type siblings (Fig. 5, B and E). As a result, Oy1-N1989/1 mutants can either be taller or shorter than their wild-type siblings. Therefore, the relationship between the disruption of chlorophyll biosynthesis and final plant height is not simply linear, and Oy1-N1989 can have opposite effects on plant height when modified by alleles at vey1.
In the Syn10 F1 crosses, we also measured the height of the primary ear node (EaHT), which allowed us to obtain the distance between primary ear node and flag leaf (Ea2Fl). Again, linkage mapping detected vey1 as the sole significant locus for mutant EaHT and Ea2Fl (Supplemental Table S7). Similar to the results for FlHT, vey1 B73 resulted in increased EaHT and Ea2Fl in Syn10 F1 plants (Fig. 5E). The average Ea2Fl of vey1 Mo17 mutants was reduced compared with the wild-type counterparts. This allele-specific interaction between vey1 and Oy1-N1989, with opposing effects on mutant plant height by vey1 B73 and vey1 Mo17 relative to the wild-type plants, is inconsistent with the simple interpretation that plant height in the Oy1-N1989 mutants is conditioned solely by reduced growth resulting from less carbon assimilation in the chlorophyll-deficient Oy1-N1989 mutants (reduced height in all cases), nor is it consistent with the delay in reproductive maturity (increased height in all cases).
A number of smaller effect QTLs were also identified for plant height in both IBM and Syn10 F1 populations (Supplemental Tables S6 and S7). Using the same approach described above for SW, candidate genes were sought with a 2-LOD window of QTLs that were not linked to vey1 and affected height. A QTL for wild-type FlHT was detected on chromosome 3 in the Syn10 F1 population (Supplemental Table S7) at a similar position to a previously reported plant height and flowering time QTL in both Syn10 and IBM inbred lines . This QTL overlaps a recently described transcription factor, zmmads69 (B73 v4: Zm00001d042315), that hastens flowering time and reduces plant height in maize-teosinte NILs and in two maize association panels (Liang et al., 2019). Consistent with zmmads69 encoding a QTL segregating between B73 and Mo17, the effect of the QTL on height (Supplemental Table S7) coincides with a strong cis-eQTL with increased accumulation of mRNA from the Mo17 allele of zmmads69 (LOD 5 44.17, additive effect 5 23.84, R 2 5 0.76; Li et al., 2013Li et al., , 2018. In addition to reducing plant height and increasing the accumulation of zmmads69 mRNA, the Mo17 allele (marker chr03.1591) also hastened the flowering of both wild-type (LOD 5 3.04, additive effect 5 0.57, R 2 5 0.05, P 5 0.0002) and mutant (LOD 5 0.86, additive effect 5 1.13, R 2 5 0.02, P 5 0.047) siblings when the data from Khangura et al. (2020) were reanalyzed by single-marker regressions. and Mo17-like NIL 3 Oy1-N1989/1:B73 (D-F) F1 hybrid populations. Averages 6 SD of chlorophyll approximations (CCMI, early-season CCM; CCMII, late-season CCM) and time to reproductive maturity (DTA, days to anthesis; DTS, days to silking) are depicted in A and D (data reanalyzed from Khangura et al., 2019Khangura et al., , 2020; height traits in B and E; and SW in C and F. c.u., Caliper units. Different lowercaseletters indicate statistically significant differences by Tukey's honestly significant difference test (P , 0.05). For each trait measurement, NIL F1s were measured in paired sets of plants, resulting in a sample size of 210 plants each of the wild type and mutant with the vey1 B73 genotype and 120 plants each of the wild type and mutant with vey1 Mo17 alleles in the NILs derived using B73 as the recurrent parent. In the Mo17 recurrent parent NIL F1 population, sample sizes were 30 each for the wild-type and mutant plants with the vey1 B73 allele and 165 each for the wild-type and mutant F1s with the vey1 Mo17 allele.
Multiple QTLs for FlHT were identified on chromosome 9 in both Syn10 and IBM F1 populations (Supplemental Tables S6 and S7). In Syn10 mutant F1 plants, we detected a FlHT QTL, centered at 3.2 Mb, on chromosome 9. A plant height QTL close to this position has been detected in multiple previous studies using B73 3 Mo17 populations (Beavis et al., 1991Austin et al., 2001;Liu et al., 2015). Others have noted that this QTL colocalizes with the dwarf plant3 (d3; B73 v4: Zm00001d045563) gene (Beavis et al., 1991;Wang et al., 2006Wang et al., , 2018 encoding ent-kaurenoic acid oxidase (Winkler and Helentjaris, 1995). There is a strong cis-eQTL in the IBM transcriptomic data that accounts for ;63% of the variation in D3 mRNA accumulation (Li et al., 2013). Recently, it was revealed that this eQTL study of the IBM population (Li et al., 2013) had reversed the signs of the effects on gene expression . As a result of this correction, it is clear that the Mo17 allele at d3 cannot encode this QTL, as it decreases mRNA abundance but is associated with an increase in plant height (Supplemental Table S7). The 2-LOD window around the peak of our QTL localization included the pin1a gene (B73 v4: Zm00001d044812; Forestan et al., 2012) encoding an auxin efflux carrier, but no eQTL was observed for pin1a (Li et al., 2013 and there were no coding sequence polymorphisms between the alleles encoded by B73 and Mo17 (Supplemental Fig. S3). No other candidate gene with a potential role in plant elongation was present. A second QTL on chromosome 9, with a peak at 12 Mb and unlinked to any other QTL, was detected for wild-type Ea2Fl in the Syn10 F1 population. A third QTL on chromosome 9 controlled multiple wild-type and mutant height traits, with peaks at 106 and 118 Mb, respectively. The QTL for mutant FlHT centered at 118 Mb was linked to zmgras45 (B73 v4: Zm00001d047123; Guo et al., 2017), orthologs of which have been previously shown to influence gibberellin-mediated regulation of chlorophyll biosynthesis (Ma et al., 2014). No eQTL was detected for zmgras45 in the previous study of IBM shoot apices (Li et al., 2013. This gene is not annotated in the Mo17 assembly (Sun et al., 2018), but a conserved sequence that contained a singlenucleotide polymorphism changing the canonical start codon from AUG to GUG followed by a frameshift mutation after the first few codons is consistent with Mo17 encoding a loss-of-function allele at this transcription factor (Supplemental Fig. S4).
Of the remaining QTLs, only one had a 2-LOD window that overlapped a gene known to impact plant height. In the Syn10 F1 population, the QTL on chromosome 1 with a peak at 82 Mb was detected for FlHT in both wild-type and mutant siblings (Supplemental Table S7). The 2-LOD window for this locus overlaps a MATE transporter (MATE; B73 v4: Zm00001d029677), which is orthologous to Arabidopsis (Arabidopsis thaliana) genes known to encode dominant alleles affecting hypocotyl elongation  and facilitating abscisic acid transport (Zhang et al., 2014). Again, no cis-eQTL was described for this MATE in the IBM eQTL study (Li et al., 2013, but three coding sequence differences (M67K, A186V, and V239I) between B73 and Mo17 may result in altered protein function. Of these changes, the latter two occur in well-conserved regions of the protein (Supplemental Fig. S5).

Single-Locus Recapitulation and Validation of the Nonlinear Relationship between Plant Height and Chlorophyll Level
To test the single-locus effect of vey1 on plant height of the Oy1-N1989 mutant, we crossed Oy1-N1989/ 1 mutants to NILs in the B73 background with either homozygous vey1 B73 or vey1 Mo17 allele introgressions. The F1 progeny from these crosses segregated for Oy1-N1989 mutants with either vey1 B73 or vey1 Mo17 allele. Supporting a simple explanation of one-locus determination, in the isogenic B73 background the FlHT of Oy1-N1989/1 mutants with a vey1 B73 allele was greater than that of both wild-type siblings and mutants with a vey1 Mo17 allele (Figs. 4B and 6B). Similarly, when vey1 Mo17 enhanced the Oy1-N1989/1 phenotype, the plants were shorter than all other genotypes. Thus, we were able to recapitulate the effect of vey1 on mutant plant height observed in the RIL and doubled-haploid (DH) population using a single-locus introgression in an isogenic inbred background.
Testing this using B73-like NILs removed any effect of hybridity. It seemed possible that some of the nonlinearity of the response of plant height to chlorophyll limitation might be driven by overall plant vigor and hybridity. For instance, in both IBM and Syn10 F1 populations, the vey1 Mo17 allele decreased plant height (Fig. 5, B and E), but the original B73 3 Mo17 F1 materials that also contained the vey1 Mo17 allele did not exhibit a similar decrease in FlHT (Supplemental Table  S3). Each RIL and DH is ;50% Mo17, whereas the Oy1-N1989 mutant is in a B73 inbred background resulting in F1 progeny that were hybrid at a random set of 50% of the loci, contributing to heterosis between B73 and Mo17. To test the effect of vey1 in an isogenic F1 hybrid background, we crossed Oy1-N1989/1:B73 to NILs derived using Mo17 as the recurrent parent with homozygous vey1 B73 introgression. This test maintains hybridity at most loci, unlike the RIL crosses, which retained hybridity for roughly half of the genome. Hybridity did not prevent vey1 B73 from increasing mutant plant height, and these plants were significantly taller than the wild-type siblings (Figs. 4C and 6E), just as observed in the IBM, Syn10, and isogenic B73-like NIL F1 progeny (Figs. 4-6; Supplemental Table S3). However, the mutant F1 progeny derived from Mo17like NILs that inherited the vey1 Mo17 allele showed no discernible difference in FlHT as compared with wildtype siblings, but EaHT decreased and a compensatory increase in the height between the ear and flag leaf was observed (Fig. 6E), just as was observed in the Mo17 3 B73 mutant F1 parental crosses (Supplemental Table  S3). Thus, vey1 B73 consistently increased plant height of the Oy1-N1989/1 plants over wild-type siblings in the isogenic B73 background, across two populations of recombinant inbred lines crossed to B73, and in B73 3 Mo17 F1 NIL hybrids, whereas the vey1 Mo17 allele decreased mutant plant height when compared with wild-type siblings in all materials except the hybrid Mo17 3 B73 F1 background.

Expression Level Variation at oy1 in IBM Correlates with SW and Height in Mutants
We previously proposed that vey1 encodes a cisacting expression polymorphism in the regulatory sequence at oy1 (Khangura et al., 2019(Khangura et al., , 2020. Our previous reanalysis of transcript data derived from shoot apices of IBM (Li et al., 2013, a maize association panel (Kremling et al., 2018), and our allele-specific expression assays in NIL demonstrated that the vey1 B73 allele was linked to higher expression of wildtype OY1 transcript (Khangura et al., 2020). Thus, we also expect SW and height variation in mutant plants to be correlated with OY1 mRNA accumulation. SW and plant height in mutant IBM F1 progeny showed significant linear correlations with OY1 transcript abundance in the shoot apices of the IBM inbred lines (Fig. 8). The OY1 transcript variation in IBM inbreds did not correlate with SW and plant height in the wild-type IBM F1 plants. These results are consistent with vey1 encoding a cis-acting regulatory polymorphism that controls OY1 mRNA accumulation as the genetic basis of SW and height in Oy1-N1989/1 mutants.
If the effect of vey1 on Oy1-N1989/1 mutants is the result of rescuing chlorophyll biosynthesis, rather than some novel function of the protein encoded by Oy1-N1989, then chlorophyll abundance should correlate better with gross morphology than OY1 transcript abundance. Both CCM measures (CCMI and CCMII) vary within each vey1 genotypic class (Supplemental Figs. S6-S10). If the effects on development are mediated by a loss of net CO 2 capture and plant energy status due to a reduction in chlorophyll, then any variation in CCM levels in Oy1-N1989/1 mutants, including the variation other than vey1, should result in a corresponding fluctuation in the chlorophyll-dependent phenotype. To test this hypothesis, we used regression to explore the relationship between chlorophyll abundance variation (measured as CCM) and developmental phenotypes in the mutants. Within each of the vey1 Mo17 and vey1 B73 mutant F1 genotypes, we determined the linear relationships between the reproductive maturity date (Khangura et al., 2020), plant height, and SW (Supplemental Tables S8 and S9) to the mutant CCM values (Khangura et al., 2019). As the effect of vey1 is only visible in the presence of the Oy1-N1989 allele for all traits, we used the difference between congenic wildtype and mutant siblings to study these trait variations with respect to mutant CCM values. Mutant SW was reduced (increase in difference) with a decrease in CCM within both vey1 Mo17 and vey1 B73 genotypes in the IBM and Syn10 F1 populations (Supplemental Fig. S6). Consistent with the effects on net CO 2 capture (Khangura et al., 2020), the slope of the regression line and, therefore, the fit was greater for the mutants with the vey1 Mo17 allele. Thus, the variation in chlorophyll content among individuals within each vey1 allele class was sufficient to alter the SW.
Similarly, the reduction in FlHT was also suppressed in mutants with the vey1 Mo17 allele that accumulated relatively higher chlorophyll in both IBM (R 2 5 0.18, P , 0.0001) and Syn10 (R 2 5 0.09, P 5 0.008) F1 populations (Supplemental Fig. S7). There was no discernible impact of a change in chlorophyll content on plant height in the vey1 B73 mutant F1 siblings (Supplemental Fig. S7). The effect of chlorophyll variation on EaHT was only significant between the later mutant chlorophyll estimates (CCMII) in IBM and the vey1 Mo17 allele and EaHT, although a similar trend was observed for Syn10 F1 progeny inheriting the vey1 Mo17 allele (Supplemental Fig. S8). As expected for a simple relationship, as mutant CCM values increased, the mutant plants matured earlier as determined by days to anthesis in both vey1 classes in both IBM and Syn10 (Supplemental Figs. S9 and S10). Regardless of the reason for these effects, the increase in plant height observed in Oy1-N1989/1 mutants with the vey1 B73 allele remains the outlier for these epistatic relationships.

Seedling Etiolation in Far-Red Light Is Not Altered in Oy1-N1989 Mutants
The nonlinearity of the interaction between Oy1-N1989 and the vey1 alleles on plant height raised the possibility that disrupting subunit I of MgChl alters light signaling in maize. The shared biosynthetic origins of chlorophyll and bilin chromophores required for phytochrome assembly (Frankenberg and Lagarias, 2012) was explored as a possible source of the increase in FlHT observed in Oy1-N1989/1 mutants modified by the vey1 B73 allele. A low red/far-red light ratio is indicative of shade in the plant canopy and elicits a shade-avoidance syndrome via phytochrome signaling (Chen et al., 2004;Pierik and de Wit, 2014; Roig-Villanova and Martínez-García, 2016; Ortiz-Alcaide et al., 2019). Increased plant height and mesocotyl length along with earlier reproductive maturity were reported previously in the elongated mesocotyl1 (elm1) maize mutant, which is defective in chromophore synthesis (Sawers et al., 2002). If a mutation in oy1 perturbs phytochromobilin biosynthesis (e.g. via feedback inhibition), a reduction in the pool of functional phytochrome might underlie the increased plant height observed in the Oy1-N1989/1 mutants carrying the vey1 B73 allele.
We tested whether the range of MgChl defects affected by the interaction between Oy1-N1989 and vey1 alleles influences mesocotyl elongation in a variety of light conditions that stimulate phytochrome-mediated elongation. To obtain segregating vey1 alleles, we grew F1 progeny of two B73-like NILs (one homozygous for vey1 B73 and the other for vey1 Mo17 ) crossed to the Oy1-N1989/1:B73 mutant under four light conditions (continuous dark, white, red, and far-red light) and measured mesocotyl length. The elm1 mutant was used as a positive control for these growth conditions (Sawers et al., 2002(Sawers et al., , 2004. As expected, the elm1 mutants showed significantly longer mesocotyls compared with the wild type in all light conditions, and the stimulatory effect of red light and shade-mimicking far-red light on mesocotyl length in elm1 was evident (Fig. 9). Under all light conditions tested, no statistically significant differences were observed in the mesocotyl length between the wild type and the Oy1-N1989 mutants with either the vey1 Mo17 or vey1 B73 allele. These results indicate no, or very limited, impact of Oy1-N1989 on light-mediated growth responses, consistent with independent regulation of the tetrapyrrole pathway branches leading to chlorophyll and chromophore biosynthesis. DISCUSSION We demonstrate here that modulation of chlorophyll accumulation by Oy1-N1989 was sufficient to alter plant growth and development. Our previous study showed that cis-acting expression polymorphism at the oy1 locus encoded by alleles of vey1 can influence the transcript abundance of wild-type OY1 (Khangura et al., 2019). As a result, a relative increase in wildtype OY1 transcripts by suppressing vey1 alleles was consistent with higher chlorophyll accumulation in Oy1-N1989/1 mutants and vice versa. We further demonstrated that the lower chlorophyll levels in Oy1-N1989 mutants conditioned by vey1 alleles resulted in a lower photosynthetic rate, nonstructural carbohydrate synthesis, and delayed reproductive maturity (Khangura et al., 2020). In this study, we demonstrate that chlorophyll metabolism, and the variation affected by the vey1 locus interactions with the Oy1-N1989 mutant allele, sufficiently disrupt metabolism to alter stem thickness, plant height, tiller production, and tassel branching. In all traits but plant height, Oy1-N1989 mutants had lower trait expression than the wild-type siblings, consistent with the view that loss of growth results from a decrease in photosynthetic output (Long et al., 2006;Evans, 2013). The performance of the mutant plants was further reduced for these traits by the enhancing vey1 Mo17 allele. So far, all phenotypes except plant height show a linear change in trait value with suppressing (e.g. B73) and enhancing (e.g. Mo17) alleles at vey1 (Figs. 4-6). The relationship between developmental phenotypes and chlorophyll was maintained even in the absence of QTLs and mutant segregation. As expected for a mechanistic connection between the metabolism affected by OY1 and these phenotypes, the variation in chlorophyll levels between mutant individuals carrying the same vey1 allele also correlated with consistent changes in plant phenotypes for all traits other than plant height (Supplemental Figs. S6-S10).
The effect of chlorophyll accumulation and vey1 alleles on mutant plant height did not follow a simple linear relationship and could not be explained by a continuous loss of chlorophyll creating a continuum of sugar levels that support differing plant growth. The Oy1-N1989/1 mutants that inherited a vey1 B73 allele had a mild decrease in chlorophyll and reduced net CO 2 assimilation (Khangura et al., 2019(Khangura et al., , 2020) and yet were taller than their wild-type siblings. The Oy1-N1989/1 mutants with the vey1 Mo17 allele that have a further reduction in chlorophyll and net CO 2 assimilation were shorter than their wild-type siblings (Figs. 4-6; Supplemental Table S3). These results are in contrast to those from Arabidopsis, where a 20% decrease in chlorophyll led to a small but significant decrease in hypocotyl elongation in shade and a further decrease in chlorophyll led to greater reduction in hypocotyl length (Ortiz-Alcaide et al., 2019). In our experiments, an ;80% to 90% reduction in chlorophyll at the seedling stage of the Oy1-N1989/1 mutants (Khangura et al., 2020) neither reduced nor increased mesocotyl elongation under continuous white light, darkness, red light, or far-red light. (Fig. 9). This may simply relate to the amount of carbohydrates stored in the maize seed available for mesocotyl elongation. The opposing effects of vey1 alleles on the influence of Oy1-N1989 on plant height strongly argue for additional experiments to distinguish whether a biochemical signal related to tetrapyrrole metabolism or an indirect effect of chlorophyll loss, unrelated to CO 2 assimilation, led to the changes in plant height.
In addition to vey1, multiple QTLs were detected for SW and plant height measurements in both mutant and wild-type siblings. Putative genes underlying these QTLs discussed above could explain the genetics of these traits. One of the QTLs detected on chromosome 3 for FlHT variation in the wild-type siblings in the Syn10 F1 population contained zmmads69 (Supplemental Table S7). QTLs for plant height and flowering time in the IBM and Syn10 inbred populations were previously mapped to a similar position . Recently, zmmads69 was characterized as a positive regulator of flowering time polymorphic between cultivated maize and teosinte and proposed as a component of the ZmRap2.7-ZCN8 regulatory network that allowed the latitudinal adaptation of maize (Liang et al., 2019). Overexpression of zmmads69 led to early flowering time and reduced height (Liang et al., 2019). We detected a strong cis-eQTL for zmmads69 (LOD 5 44.17, R 2 5 0.76) in the previous eQTL study of IBM inbred lines (Li et al., 2013, with the Mo17 allele resulting in greater transcript abundance, corresponding to the QTL effects of reduced plant height and earlier maturity (Supplemental Table S7). Although not detected as a genome-wide significant QTL in our study, when considered without a multiple test correction, the Mo17 allele at zmmads69 decreased days to anthesis in both wild-type and mutant siblings in our materials. Based on these results and allele directions, we predict that previously described QTLs at this position for plant height and flowering time in B73 3 Mo17 mapping populations  are under the genetic control of zmmads69.
Since we only measured tillering and tassel architecture in NIL crosses, it is a formal possibility that a modifier of plant branching is linked to oy1. For example, a tillering modifier that is common to both the gt1 and tb1 responses may be linked to oy1. However, the changes in the developmental phenotypes were contingent on the Oy1-N1989 allele, and it retained the epistatic interaction with vey1 (Fig. 1). These results make it unlikely that a linked QTL differing between B73 and Mo17 underlies this variation in tillering. In the case of tassel branching, our results (Fig. 2) are an independent validation of a previous report of reduced tassel branching by a loss-of-function allele at oy1 (Brewbaker, 2015). In all cases, lateral branch numbers were reduced by the loss of chlorophyll in Oy1-N1989/ 1, although the strength of this effect varied by the modifying allele at vey1 (Fig. 2).
The maize gene tin1 encodes a C2H2-zinc-finger transcription factor that is a dominant factor responsible for increased tiller growth in the Purdue sweetcorn line P51 (Zhang et al., 2019). The high-tillering Purdue sweetcorn line P39 encodes the same tin1 allele as P51 (Zhang et al., 2019), possibly due to their common derivation from cv Golden Bantam (www.ars-grin. gov). The Oy1-N1989 allele suppressed tillering conditioned by the tin1 variant in P39 (Fig. 1C). TIN1 is upstream of GT1 but acts independently of TB1 (Kebrom and Brutnell, 2015;Zhang et al., 2019). The suppression of tillering in gt1 mutants in the Oy1-N1989/1 background (Fig. 1) is consistent with tin1 acting upstream of gt1 (Zhang et al., 2019) to regulate tiller bud outgrowth. Despite the fact that tb1 acts independently of tin1, the Oy1-N1989 allele also suppressed tillering in tb1, suggesting that loss of chlorophyll regulates branching upstream of the branch point between tb1 and tin1-gt1 ( Fig. 1; Supplemental Table S1).
Our data are consonant with previous suggestions that sugar production is a critical determinant of bud outgrowth (Mason et al., 2014;Barbier et al., 2015). The vey1 QTL can modify the photosynthetic capacity and sugar accumulation of Oy1-N1989 mutants (Khangura et al., 2020). Testing in isogenic double mutants of Oy1-N1989 with gt1 and tb1 demonstrated that tiller growth was suppressed in the Oy1-N1989/1 mutants in a vey1-dependent manner (Fig. 1, D-G). A study analyzing young axillary buds of tb1 and gt1 mutants in maize found increased sugar biosynthesis and metabolism to be critical for breaking bud dormancy (Dong et al., 2019). Evidence of a relationship that places tb1 upstream of energy sensing was obtained by the chromatin immunoprecipitation results that show tb1 bound to the promoters of genes encoding enzymes important for energy sensing, such as enzymes involved in trehalose-6-phosphate metabolism (Dong et al., 2019). Analysis of transcript abundance in axillary buds of tin1 variants also found photosynthesis to be the most enriched Gene Ontology term and chlorophyll biosynthesis to be highly upregulated in nondormant tiller buds (Zhang et al., 2019). The expression of genes important for photosynthesis may be a consequence of bud outgrowth and the initiation of photosynthesis in expanding organs. However, it may be that sugar production at the branches is a critical determinant of branch growth. Based on our data that chlorophyll limitation suppresses tillering by tb1, gt1, and tin1 alleles, we predict that Suc concentrations regulate bud outgrowth and that these transcription factors act downstream to promote bud growth. We expect genes important for the patterning and placement of axillary meristems to act upstream of the sensor of carbon status.
Flowering time and plant height displayed a confusing relationship that was not resolved by the idea that later flowering plants are taller. Indeed, as expected based on the correlations with CCM and vey1 but in defiance of a simple explanation, as mutant plants accumulated more chlorophyll, they flowered earlier and were taller in IBM regardless of the vey1 genotype (Supplemental Fig. S11). In the Syn10 F1 population, the plants with the vey1 Mo17 allele behaved in this manner, but the vey1 B73 plants went in the opposite direction, with increasing height and increasing flowering time cooccurring (Supplemental Fig.  S12). Thus, a secondary relationship between plant height and the flowering time delay does not explain the nonlinear change in plant height with respect to chlorophyll levels. Although the plant height in Oy1-N1989 and vey1 segregating materials is extremely reproducible across a range of populations (two RIL populations and both reciprocal NIL populations) and multiple years, the mechanism that explains the complicated relationship between the Oy1-N1989 allele, its modifier vey1, and CCM values to plant height and the paradoxical effect of this epistasis on different morphological traits is elusive to us at this time.
The CCM values that are produced by Oy1-N1989/ 1 mutants with vey1 Mo17 and vey1 B73 alleles do not overlap (compare the range on the x axes in Supplemental Figs. S6-S10). It may be that a Goldilocks value for CCM that maximizes plant height lies between the values we have measured. If so, Oy1-N1989/1 mutants suppressed by a vey1 B73 allele should become taller as CCM decreases. Instead, the vey1 B73 plants show no significant correlations between CCM and plant height, with both the positive and negative slopes of the regression line consistent with the asymptotic relationship between CCM and plant height. If there is a break-even point where decreasing chlorophyll contents increased plant height, it occurs between the CCM values of the two vey1 alleles. Thus far, the relationship between vey1 and plant height and the unusual effect of vey1 B73 on plant height defies any simple explanation. Although not measured by us, a shorter, thinner plant should produce less biomass and utilize less carbohydrate. The plant volume, which is a function of stem thickness and plant height, is built from excess CO 2 assimilation. As a result, it may be that vey1-suppressed Oy1-N1989/ 1 mutants are taller because of the addition of available carbohydrates to thinner stems. Direct measurements of biomass might better estimate this and would also open up the possibility to explore other aspects such as water-and nitrogen-use efficiency.
A similar crossing strategy to that described above for tb1 was used for testing the effect of Oy1-N1989 and vey1 alleles on tillering in gt1 mutants. In the populations segregating gt1, Oy1-N1989, and vey1 alleles, gt1 homozygotes were easily distinguished from heterozygotes and the wild types by the presence of extended blade tissue on the husk sheath of primary and additional ears (Whipple et al., 2011). The phenotypic segregation of the progeny for gt1 was as expected. The Oy1-N1989/1 and wild-type progeny segregated 1:1 (x 2 test, P 5 0.59). The gt1 nonhomozygotes (the wild type and heterozygotes) and gt1 homozygotes segregated 3:1 (x 2 test, P 5 0.69). The genotypes at gt1 in phenotypically selected homozygotes, including gt1/gt1;Oy1-N1989/1 double mutants carrying vey1 B73 or vey1 Mo17 alleles, were confirmed by progeny testing in selfed families. In all cases, all progeny exhibited the gt1 homozygote phenotype, confirming the genotype at gt1 to be homozygous in the progenitor plants.

Location, Experimental Design, and Planting
All the field experiments were performed at the Purdue Agronomy Center for Research and Education in West Lafayette, Indiana. The test cross populations of IBM F1 and Syn10 F1 hybrid progeny were evaluated in 2013 (IBM) and 2014 (Syn10) in a randomized complete block design with one and two replications, respectively. The F1 progeny of Oy1-N1989/1:B73 crossed with B73 and Mo17 were used as parental checks in each range treated as a block. The F1 progeny of Oy1-N1989/1:B73 with NIL and parents (B73 and Mo17) were planted in a randomized complete block design with five replications in 2016. Plots were sown in a 3.84-m-long row with the interrow spacing of 0.79 m and an alley space of 0.79 m. All F1 families were planted as a single plot of 18 seeds (equivalent to a density of 24,017 plants per acre). No irrigation was applied during the entire crop season, as rainfalls were uniformly distributed for satisfactory plant growth. Conventional fertilizer, pest control, and weed control practices for growing field maize in Indiana were followed.

Phenotyping and Data Collection
The plant height and SW were measured in both wild-type and mutant siblings segregating in each family in the F1 populations of Oy1-N1989/1:B73 with the IBM, Syn10, and NIL populations. As described previously (Khangura et al., 2019(Khangura et al., , 2020, genetic backgrounds containing the vey1 B73 allele progressively suppress the mutant phenotype and were tagged at V5 to V7 stage when they were distinguishable from the wild-type siblings, so that traits could be recorded at maturity when the genotypes are difficult to visually distinguish. In each RIL and NIL F1 plot, trait measurements were performed on two to four wild-type and mutant siblings that were picked at random. Therefore, hundreds of mutant and wild-type plants were measured in each genotypic class, in each replicate, of each population. The exact sample size is indicated in the figure legends. Plant height and SW were measured in cm and caliper units, respectively (Chaikam et al., 2011). Briefly, around 3 weeks after reproductive maturity, FlHT was measured as the vertical distance from the soil line to the point of attachment of the flag leaf, and EaHT was measured as the vertical distance from the soil line to the top/primary ear. The EaHT was only measured in Syn10 F1; consequently, any height trait derived from EaHT is only in the Syn10 data. The Ea2Fl was calculated from the difference between EaHT and FlHT. Ratios of mutant to wild-type averages and the difference between mutant and wild-type averages were also determined for these traits. SW measurements were taken with a Vernier caliper (Vernier, 1631) ;3 weeks after reproductive maturity at the middle of the internode above the primary ear using the shortest diameter of each stalk. The SW measurements were recorded as caliper units, where 1 caliper unit 5 1/32 of an inch or ;0.08 cm. The measurements of all the primary traits in the two biparental and NIL populations are provided in Supplemental Tables S8 to S10.
Additional morphological features were quantified as follows. Tassel measurements were performed on wild-type and mutant siblings in the F1 progeny of all B73-like NILs and Mo17-like NILs crossed with Oy1-N1989/1:B73 pollen parents. Tassels were removed from mature plants at the point of flag leaf attachment and saved for measurements. The tassel traits measured included the following: primary tassel branch number, number of primary branches with at least one secondary branch, secondary branch number, length of the tassel from the point of flag leaf attachment to the tip, length of the main rachis of the tassel, length of the branching zone, length of the topmost primary tassel branch, length of the middle primary tassel branch, length of the bottom primary tassel branch, and average of top, middle, and bottom primary tassel branches. Tillering was assessed by counting the number of visible lateral primary branches 1 month after reproductive maturity. Mesocotyl measurements were done on seedlings grown under continuous red light (15-18 mmol m 22 s 21 ), far-red light (6-7 mmol m 22 s 21 ), white light (150 mmol m 22 s 21 ), and dark at 25°C for 7 d at previously described peak wavelengths (Puthiyaveetil, 2008;Macadlo et al., 2020).

Genotypes, Gene Expression, and Sequence Data
The genotypic and gene expression data sets used in this study were described previously in detail (Khangura et al., 2019(Khangura et al., , 2020. Briefly, the public marker data for IBM consist of 2,156 unique markers. The gene expression data set consists of normalized read counts, expressed as reads per kilobase of transcript per million mapped reads, of the maize genes derived from the shoot apices of 14-d-old IBM seedlings (Li et al., 2013, and counts for OY1 were obtained from MaizeGDB (www.maizegdb.org; Sen et al., 2010). The DNA sequences for the B73 and Mo17 alleles of various candidate genes described in this article were obtained from MaizeGDB. The Syn10 genotypes consist of single-nucleotide polymorphisms at 6,611 positions with no missing data . The B73-like and Mo17-like NILs carrying homozygous vey1 introgressions from the reciprocal parent were selected for QTL validation from a previously described B73 3 Mo17 NIL population (Eichten et al., 2011). As a result, the F1 progeny of NIL (ear parent) 3 Oy1-N1989/1:B73 is either heterozygous for the vey1 B73 or vey1 Mo17 allele depending upon the ear parent.

Sequence Alignments
The genome assembly comparison for the syntenic regions between B73 and Mo17 for expb4 was done using the GEvo tool (https://genomevolution.org/ coge/; Lyons and Freeling, 2008). De novo protein translations of cDNA sequences were done using the translate tool in the ExPASy bioinformatics resource portal (https://web.expasy.org). The alignments of DNA and protein sequences were done using the T-coffee web service (http://tcoffee.crg.cat).

Statistical Analyses
Preliminary data exploration and descriptive statistics were calculated using JMP 13.0 (SAS Institute). Trait means for each line were used as phenotypes for QTL analyses. The QTL analyses for all traits were done using single-interval mapping implemented in R/QTL v1.40-8 (Broman et al., 2003). Genome-wide statistical thresholds at a 5 0.05 for each trait were computed by permutation testing using 1,000 iterations (Churchill and Doerge, 1994).

Accession Numbers
The figure legend for Supplemental Figure S1 contains the National Center for Biotechnology Information accession numbers for the sequence data used in this article.

Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Tandem duplicate pairs of expb4 in B73 and Mo17 assembly.
Supplemental Figure S4. The coding sequence and putative protein sequence alignments encoded by the zmgras45 alleles of B73 (B73 v4: Zm00001d047123) and Mo17 (not annotated).
Supplemental Figure S6. Linear regression of CCM measurements in mutants with the difference of SW between congenic mutant and wild-type siblings.
Supplemental Figure S7. Linear regression of CCM measurements in the mutants with the difference of FlHT in congenic mutant and wild-type siblings.
Supplemental Figure S8. Linear regression of CCM measurements in the mutants with the difference of EaHT and Ea2Fl in congenic mutant and wild-type siblings.
Supplemental Figure S9. Linear regression of CCM measurements in the mutants with the difference of days to anthesis and mutant days to anthesis.
Supplemental Figure S10. Linear regression of CCM measurements in the mutants with the difference of days to anthesis and mutant days to anthesis.
Supplemental Figure S11. Linear regression of FlHT of wild-type and mutant siblings with the difference of days to anthesis between congenic mutant and wild-type siblings.
Supplemental Figure S12. Linear regression of the FlHT, EaHT, and Ea2Fl of the wild-type and mutant siblings with the difference of days to anthesis between congenic mutant and wild-type siblings.
Supplemental Table S2. Morphometric analysis of mature tassels in the F1 progeny of the B73 NIL and Mo17 NIL crossed to Oy1-N1989/1:B73.
Supplemental Table S3. Effect of Oy1-N1989 and genetic background on chlorophyll index, SW, and height traits.
Supplemental Table S8. The average trait values of wild-type and mutant F1 hybrid siblings from a cross between Oy1-N1989/1:B73 (as a pollen parent) and IBM-RILs.
Supplemental Table S9. The average trait values of wild-type and mutant F1 hybrid siblings from a cross between Oy1-N1989/1:B73 (as a pollen parent) and Syn10-DH lines.