The REDUCED HEIGHT (RHT) dwarfing alleles Rht-B1b and Rht-D1b were essential in the ‘Green Revolution’. The RHT1 gene encodes a DELLA protein, which participates in the gibberellin (GA) growth-stimulating pathway (Peng et al., 1999), and truncations of this protein are responsible for the GA-insensitive semi-dwarf Rht1b alleles (Van De Velde et al., 2021). The growth-repressing effect of Rht1b alleles optimized plant height, reduced lodging and improved harvest index, but also reduced above-ground biomass and coleoptile length, limiting sowing depth and access to deeper soil moisture (Ellis et al., 2004). This has triggered the search for GA-sensitive dwarfing genes with fewer negative pleiotropic effects.

Plant height in grasses is regulated by a complex genetic network, which includes the conserved microRNA172 (miR172)–APETALA2-like (AP2L) module (Patil et al., 2019; Zhu and Helliwell, 2011). In wheat, miR172 expression is induced during the reproductive transition and regulates flowering time, plant height and both spike and floret development by repressing the expression of AP2L genes (Debernardi et al., 2017). Reduction of miR172 activity in the semi-dwarf tetraploid wheat variety ‘Kronos’ (Rht-B1b) using a transgenic target mimicry (MIM172) approach delayed reproductive transition a few days and generated shorter plants with more compact spikes (Debernardi et al., 2017).

Among the four AP2L genes targeted by miR172 in wheat, AP2L2 and AP2L5 regulate flowering transition, stem elongation and spike development (Debernardi et al., 2020). Point mutations in the miR172 target site of the AP2L genes reduce miR172 activity and generate resistant alleles designated hereafter as rAp2l. An rAp2l-A5 allele originated the domestication gene Q and the free-threshing wheats (Debernardi et al., 2017). Additional mutations in the miR172 target site of Q or in the homeolog AP2L-D5 result in plants with reduced height but, unfortunately, with associated spike defects (Greenwood et al., 2017; Zhao et al., 2018). In this study, we explore the effects of chemically induced alleles rAp2l-A2 from tetraploid and rAp2l-B2 from hexaploid wheat (Figure S1a) as well as multiple new CRISPR-induced alleles. All materials and methods are described in the Materials and Methods in Appendix S1.

The rAp2l-A2 EMS-mutation in the semi-dwarf Kronos reduced stem length by 21%, whereas the introgression of the rAp2l-B2 allele into Kronos or Kronos-rAp2l-A2 backgrounds, reduced stem length by 43–45% (Figure S1a–c, Data S1). We next used CRISPR-Cas9 with a gRNA specifically targeting the miR172 target site of AP2L-B2, because AP2L-A2 has a polymorphism that disrupts the gRNA target (Figure 1a, Figure S2a). We generated multiple independent CRISPR T₀ events into Kronos (Rht-B1b) and a near-isogenic tall line (Rht-B1a) (Figure S2, Data S2). Most of the CRISPR mutations were small frameshift indels in the miR172 target site (Figure 1a, Figure S2a), located downstream of the conserved AP2 domains and close to the stop codon (Figure 1a). Both in-frame and frameshift indels resulted in semi-dominant dwarfing effects, suggesting that disruptions of the reading frame at the end of the gene have limited effects on AP2L2 activity. The dominance effect of the dwarfing rAp2l-B2 alleles was similar in the tall Rht-B1a plants (Figure S2b,c) and the semi-dwarf Rht-B1b backgrounds (Figure S2d, Data S2).

Independent T₂ edited lines homozygous for different mutations in both Rht-B1a (Figure 1a–c) and Rht-B1b backgrounds (Figure S3a,b) showed significant effects on plant height that varied depending on the mutations. The strongest rAp2L-B2 alleles in the Rht-B1a background reduced plant height to similar levels as Rht-B1b (Figure 1b,c), suggesting that they can be used to replace the Rht1b alleles.

The rAp2l-B2 plants showed a higher spikelet density (Figure 1d, Figure S3c) as a result of reductions in spike length and slight increases in spikelet number per spike (Data S4). In the Rht-B1a background, the edited lines headed 1.8–2.9 days later, which was comparable to the delay generated by Rht-B1b (Figure 1e). The delay in heading time associated with the rAp2l-B2 alleles was slightly stronger in the Rht-B1b sister lines (4.4 to 5.7 days delay, Figure S3d, Data S4). Finally, plants with and without the rAp2l-B2 mutations showed similar coleoptile and first-leaf lengths in both the Rht-B1a (Figure 1f,g, Data S4) and Rht-B1b backgrounds (Figure S3e,f, Data S4). In summary, these results indicate that the rAp2l-B2 alleles can be used to reduce plant height with limited pleiotropic effects on spike architecture or heading time, and with beneficial effects in coleoptile length relative to the Rht1b alleles.

The highly efficient CRISPR vector makes it possible to rapidly induce different rAp2l2 dwarfing alleles in elite backgrounds without time-consuming crosses. To demonstrate this strategy, we generated semi-dwarf mutants for the triticale cultivar ‘UC-Bopak’ (PVP 202100269). Triticale is an anthropogenic allohexaploid combining wheat and rye (AABBRR genomes), which delivers significantly high biomass and grain yield (Tamagno et al., 2022). However, the taller plant stature of many triticale cultivars combined with their larger and heavier spikes can result in increased lodging. We transformed UC-Bopak using the same gRNA targeting the miR172 binding site in both AP2L-B2 and AP2L-R2 homeologs (Figure 1h). Under greenhouse conditions, we observed significant reductions in plant height in edited lines (Figure 1h,i, Figure S4a,b), which were larger in the lines with mutations in both genomes (Data S5). Plant height was correlated with the predicted effect of the mutations on miR172 binding energy both in lines with mutations in AP2L-R2 (R = −0.94) and in those with mutations in both AP2L-B2 and AP2L-R2 (R = −0.73, Data S5). A combined statistical analysis of the triticale and wheat results (Figure 1a–c and Figure S3a,b) showed that this correlation was highly significant (P = 0.0014, Data S5). By selecting different combinations of rAp2l2 mutations, we were able to fine-tune triticale plant height (Figure 1i, Figure S4b) without affecting coleoptile and first-leaf length or heading time (Figure 1j, Figure S4c,e). The edited plants showed more compact spikes but with the same number of spikelets (Figure S4f, Data S5).

Finally, we evaluated lines with 1-bp deletions in the miR172 target site of AP2L-B2 (B) or AP2L-R2 (R) under field conditions in two consecutive years. In 2023, we used headrows (Figure S5) and in 2024 small yield plots as experimental units (Figure 1k–n). The plants with the 1-bp deletions were 17–18 cm shorter the first year (Figure S5a,b) and 12–14 cm shorter the second year (Figure 1l), indicating some interaction with the environment. The spikes of the edited lines were more compact than the wildtype (Figure S5c), but this was not associated with significant differences in grain yield (Figure 1n, Figure S5d). In the second year, the plots of the wildtype variety suffered significantly more lodging than the edited lines (P < 0.0001, Figure 1k,m). Although the differences in grain yield were not significant (Figure 1n, Figure S5d), the edited lines showed a combined 9.5% increase in grain yield in the second year (P = 0.0528, Data S3), which was likely associated with their superior resistance to lodging.

In summary, we demonstrate that different induced mutations in the miR172 target site of AP2L2 genes can be used to precisely modulate wheat and triticale plant height. Breeders can use this technology to evaluate multiple plant heights in their top lines without lengthy backcrossing programs. Moreover, the rAp2l2 alleles did not reduce coleoptile and first-leaf length, suggesting that they can be a valuable replacement of the gibberellin-insensitive Rht1b alleles.

降低高度 （RHT） 矮化等位基因 Rht-B1b 和 Rht-D1b 在“绿色革命”中至关重要。RHT1 基因编码一种 DELLA 蛋白，该蛋白参与赤霉素 （GA） 生长刺激途径 （Peng et al.， 1999），该蛋白的截断负责 GA 不敏感的半矮小 Rht1b 等位基因 （Van De Velde et al.， 2021）。Rht1b 等位基因的生长抑制作用优化了株高，减少了倒伏，提高了收获指数，但也减少了地上生物量和胚芽鞘长度，限制了播种深度和获得更深的土壤水分（Ellis et al.， 2004）。这引发了对具有较少负多效性效应的 GA 敏感矮化基因的搜索。

禾本科植物中的植物高度受复杂的遗传网络调节，其中包括保守的 microRNA172 （miR172） – APETALA2样 （AP2L） 模块（Patil et al.， 2019;Zhu 和 Helliwell，2011 年）。在小麦中，miR172 表达在生殖过渡期间被诱导，并通过抑制 AP2L 基因的表达来调节开花时间、株高以及穗状花序和小花发育（Debernardi等 人，2017 年）。使用转基因靶模拟物 （MIM172） 方法降低半矮化四倍体小麦品种 'Kronos' （Rht-B1b） 中的 miR172 活性，将生殖过渡延迟了几天，并产生了具有更紧凑穗状花序的更短植株（Debernardi等 人，2017 年）。

在小麦中 miR172 靶向的四个 AP2L 基因中，AP2L2 和 AP2L5 调节开花过渡、茎伸长和穗状花序发育（Debernardi等 人，2020 年）。AP2L 基因的 miR172 靶位点的点突变会降低 miR172 活性并产生以下称为 rAp2l 的耐药等位基因。rAp2l-A5 等位基因起源于驯化基因 Q 和自由脱粒小麦（Debernardi等 人，2017 年）。Q 的 miR172 靶位点或同源 AP2L-D5 中的其他突变导致植物高度降低，但不幸的是，伴有相关的刺突缺陷（Greenwood等 人，2017 年;Zhao et al.， 2018）。在这项研究中，我们探讨了来自四倍体的化学诱导等位基因 rAp2l-A2 和来自六倍体小麦的 rAp2l-B2 的影响（图 S1a）以及多个新的 CRISPR 诱导的等位基因。所有材料和方法均在附录 S1 的材料和方法中进行了描述。

半矮化 Kronos 中的 rAp2l-A2 EMS 突变使茎长减少了 21%，而 rAp2l-B2 等位基因渗入 Kronos 或 Kronos-rAp2l-A2 背景后，茎长减少了 43-45%（图 S1a-c，数据 S1）。接下来，我们使用 CRISPR-Cas9 和特异性靶向 AP2L-B2 的 miR172 靶位点的 gRNA，因为 AP2L-A2 具有破坏 gRNA 靶点的多态性（图 1a、图 S2a）。我们在 Kronos （Rht-B1b） 和一条近等基因高线 （Rht-B1a） 中生成了多个独立的 CRISPR T₀ 事件（图 S2，数据 S2）。大多数 CRISPR 突变是 miR172 靶位点的小移码插入缺失（图 1a、图 S2a），位于保守 AP2 结构域的下游，靠近终止密码子（图 1a）。框内和移码插入缺失都导致半显性矮化效应，表明基因末端阅读框的破坏对 AP2L2 活性的影响有限。矮化 rAp2l-B2 等位基因的显性效应在高大的 Rht-B1a 植物 （图 S2b，c） 和半矮化的 Rht-B1b 背景 （图 S2d，数据 S2） 中相似。

在 Rht-B1a（图 1a-c）和 Rht-B1b 背景（图 S3a，b）中不同突变的独立 T₂ 编辑品系纯合子显示对株高的显着影响，这种影响因突变而异。Rht-B1a 背景中最强的 rAp2L-B2 等位基因将株高降低到与 Rht-B1b 相似的水平（图 1b、c），表明它们可以用来替代 Rht1b 等位基因。

rAp2l-B2 植物显示出更高的小穗密度（图 1d，图 S3c），这是由于穗长的减少和每个穗的小穗数略有增加（数据 S4）。在 Rht-B1a 背景中，编辑后的行在 1.8-2.9 天后航向，这与 Rht-B1b 产生的延迟相当（图 1e）。在 Rht-B1b 姊妹系中，与 rAp2l-B2 等位基因相关的抽穗时间延迟略强（延迟 4.4 至 5.7 天，图 S3d，数据 S4）。最后，有和没有 rAp2l-B2 突变的植物在 Rht-B1a （图 1f，g，数据 S4）和 Rht-B1b 背景 （图 S3e，f，数据 S4） 中显示出相似的胚芽鞘和第一叶长度。总之，这些结果表明 rAp2l-B2 等位基因可用于降低株高，对穗结构或抽穗时间的多效性影响有限，并且相对于 Rht1b 等位基因对胚芽鞘长度产生有益影响。

高效的 CRISPR 载体可以在精英背景中快速诱导不同的 rAp2l2 矮化等位基因，而无需耗时的杂交。为了证明这种策略，我们为黑小麦品种 'UC-Bopak' （PVP 202100269） 生成了半矮化突变体。黑小麦是一种结合小麦和黑麦（AABBRR 基因组）的人为异体六倍体，可提供显着高的生物量和谷物产量（Tamagno et al.， 2022）。然而，许多黑小麦品种较高的植物身材与更大更重的穗状花序相结合，会导致倒伏增加。我们使用靶向 AP2L-B2 和 AP2L-R2 同源体内 miR172 结合位点的相同 gRNA 转化 UC-Bopak（图 1h）。在温室条件下，我们观察到编辑品系中的株高显著降低（图 1h，i，图 S4a，b），在两个基因组均发生突变的品系中更大（数据 S5）。株高与突变对 miR172 结合能的预测影响相关，与 AP2L-R2 突变 （R = -0.94） 和 AP2L-B2 和 AP2L-R2 突变 （R = -0.73，数据 S5） 一致。黑小麦和小麦结果的综合统计分析（图 1a-c 和图 S3a、b）表明这种相关性非常显著（P = 0.0014，数据 S5）。通过选择 rAp2l2 突变的不同组合，我们能够微调黑小麦株高（图 1i，图 S4b），而不会影响胚芽鞘和第一叶长度或抽穗时间（图 1j，图 S4c，e）。编辑后的植物显示出更紧凑的穗状花序，但小穗数量相同（图 S4f，数据 S5）。

最后，我们连续两年在现场条件下评估了 AP2L-B2 （B） 或 AP2L-R2 （R） 的 miR172 靶位点中 1-bp 缺失的细胞系。2023 年，我们使用头行（图 S5），并在 2024 年使用小产量图作为实验单位（图 1k-n）。具有 1 bp 缺失的植物第一年短 17-18 cm（图 S5a，b），第二年短 12-14 cm（图 1l），表明与环境存在一些相互作用。编辑后的品系的尖峰比野生型更紧凑（图 S5c），但这与谷物产量的显着差异无关（图 1n，图 S5d）。在第二年，野生型品种的地块比编辑的品系遭受更多的倒伏（P < 0.0001，图 1k，m）。虽然谷物产量的差异不显著（图 1n，图 S5d），但编辑后的线显示第二年的谷物产量综合增加了 9.5%（P = 0.0528，数据 S3），这可能与它们优异的抗倒伏性有关。

综上所述，我们证明了 AP2L2 基因 miR172 靶位点的不同诱导突变可用于精确调节小麦和黑小麦株高。育种者可以使用这项技术来评估其顶系中的多种植物高度，而无需冗长的回交程序。此外，rAp2l2 等位基因不会减少胚芽鞘和第一叶的长度，这表明它们可以成为赤霉素不敏感的 Rht1b 等位基因的有价值的替代品。