low binding affinity for DRL1 atUPA2releases
LG1 to activateZmRAVL1transcription, which
further up-regulatesbrd1expression, resulting
in increased brassinosteroid and leaf angle.
TeosinteUPA2and editingZmRAVL1
enhance maize yields
To examine the distribution of S2 atUPA2in
the maize natural population, we genotyped S2 in
508 maize inbred lines ( 21 ) and 50 maize land-
races (table S2). None of these maize accessions
carried the TG allele at S2 (table S3). We further
genotyped S2 in 45 teosinte accessions (table S4).
Only two (4.4%) teosinte accessions carried the
TG allele at S2 (table S3). These results suggested
that S2 is a rare variant in teosinte that was lost
during maize domestication.
Although the TG allele at S2 that reduces leaf
angle was lost during maize domestication, this
allele could be exploited for generating upright
leaf architecture for dense planting in modern
breeding. To test this possibility, we planted
UPA2-NILW22andUPA2-NIL^8759 under five dif-
ferent planting densities in two environments
(Tieling and Sanya, both in China) in 2017. In
both field trials,UPA2-NIL^8759 with upright leaf
angle out-yieldedUPA2-NILW22under high plant-
ing densities (>67,500 and 105,000 plants per hect-
are in Sanya and Tieling, respectively) (Fig. 6, A
and B, and fig. S23, A to F). Consistent with this
result,UPA2-NIL^8759 exhibited a lower rate of
decrease of grain yield per plant thanUPA2-
NILW22as planting density increased (Fig. 6A
and fig. S23C). To evaluate the value ofUPA2in
hybrids, we introgressed the teosinteUPA2allele
conferring upright leaf angle, through repeated
backcrossing and molecular marker assisted
selection, into the two parents of Nongda108
(HuangC × Xu178), an elite maize hybrid widely
planted in China. We analyzed yield in field
trials of the improved Nongda108 and original
Nongda108 hybrids under three different plant-
ing densities in Sanya, China, in 2018 (Fig. 6, C
and D, and fig. S23, G and H). Under planting
densities of 45,000 and 75,000 plants per hect-
are, the original Nongda108 and the improved
Nongda108 exhibited similar grain yield (Fig. 6,
C and D). However, as planting density increased
to 105,000 plants per hectare, the improved
Nongda108 showed higher grain yield than the
original Nongda108 (Fig. 6, C and D). These
results suggested that the teosinteUPA2allele
can both alter the architecture of modern maize
plants and improve yield potentials under dense
planting.
ZmRAVL1edited lines also displayed reduced
leaf angle. We evaluated the grain yield ofZmRAVL1-
KO#1 (Cas9-free) under five different plantingdensities in two locations (Tieling and Sanya) in- Yield fromZmRAVL1-KO#1 was greater
than from wild-type plants under high planting
densities in both locations (Fig. 6, E and F, and
fig. S24). In theZmRAVL1-KO#1T1family,Cas9-
positive plants were selected and crossed with six
elite maize inbreds. In the resulting F 1 plants, the
wildZmRAVL1allele introduced by inbreds was
edited (fig. S25). As a result, the F 1 hybrids showed
reduced leaf angle compared with the control
hybrids (fig. S27). We also crossedZmRAVL1-
RNAi#1 with the six maize inbreds. The resulting
F 1 hybrids exhibited decreasedZmRAVL1expres-
sion compared with the control hybrids (fig. S26),
leading to reduced leaf angle (fig. S27). Therefore,
manipulatingZmRAVL1can generate upright leaf
architecture for increased planting density.
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Fig. 6. TeosinteUPA2allele andZmRAVL1edited allele enhanced
maize grain yield under high planting densities.(AandB)Compar-
ison of grain yield per plant (A) and grain yield per hectare (B)
betweenUPA2-NILW22andUPA2-NIL^8759 under different planting
densities in the field trial in Sanya, China, in 2017. (CandD)Comparison
of grain yield per plant (C) and grain yield per hectare (D) between
the original Nongda108 and the improvedNongda108 carrying the
8759 UPA2allele under different planting densities in the field trial
in Sanya, China, in 2018. (EandF)Comparisonofgrainyield
per plant (E) and grain yield per hectare (F) betweenZmRAVL1-KO#1
and wild-type under different planting densities in the field trial in
Tieling, China, in 2018. Values are means ± SD (n= 3 replications).
Different letters denote significant differences (P<0.05)from
Duncan’s multiple-range tests. ha, hectare.RESEARCH | RESEARCH ARTICLE