Cleistogamy or closed flowering is a widely used trait in barley (Hordeum vulgare) breeding because it reduces the risk of fungal infection in florets at anthesis. Cleistogamy in barley is caused by a point mutation within the microRNA172 (miR172) target site of the Cly1 gene, which encodes the Apetala2 (AP2) transcription factor. Because cleistogamy is not apparent in cultivars of hexaploid wheat (Triticum aestivum), a strategy to develop cleistogamous wheat was proposed by inducing point mutations in all three AP2 homoeologs, which are the wheat orthologs of barley Cly1. In this study, we investigated the effects of miR172 target site mutations on wheat cleistogamy using double mutants by combining three previously obtained mutant alleles (AP2-A1, D1 and D2) in a near-isogenic background. The AP2-D2 allele had the greatest effect on reducing the anther extrusion rate and lodicule size compared with the other two mutant alleles. The double mutant containing the AP2-A1 and AP2-D2 alleles had a much greater suppression of anther extrusion by reducing the lodicule size than the single AP2-D2 mutant, suggesting cumulative effects of the two mutant alleles. In addition, both single and double mutants exhibited compact spikes and shorter plant heights due to reduced rachis and culm internodes in the upper parts. The presence or absence of the wild-type AP2-B homoeolog had no significant effect on phenotype. This study provides insights into the cumulative effects of mutant AP2 alleles in suppressing open flowering and provides a basis for further research on the development of complete cleistogamy in hexaploid wheat.

闭锁花或闭花是大麦（ Hordeum vulgare ）育种中广泛使用的性状，因为它可以降低花期小花真菌感染的风险。大麦中的闭锁受精是由Cly1基因的 microRNA172 (miR172) 靶位点内的点突变引起的，该基因编码 Apetala2 (AP2) 转录因子。由于闭锁受精在六倍体小麦（ Triticum aestivum ）品种中并不明显，因此提出了通过在所有三个AP2同源物（大麦Cly1的小麦直系同源物）中诱导点突变来开发闭锁受精小麦的策略。在这项研究中，我们通过在近等基因背景下组合三个先前获得的突变等位基因（AP2-A1、D1和D2 ），使用双突变体研究了miR172靶位点突变对小麦闭花受精的影响。与其他两个突变等位基因相比，AP2-D2 等位基因对降低花药挤出率和浆片大小的影响最大。与单一AP2- D2突变体相比，含有AP2-A1和AP2 -D2等位基因的双突变体通过减小浆片大小对花药挤出具有更大的抑制作用，表明两个突变体等位基因的累积效应。此外，由于上部的轴和秆节间减少，单突变体和双突变体均表现出紧凑的穗状花序和较短的株高。野生型AP2-B同源物的存在或不存在对表型没有显着影响。这项研究提供了突变AP2等位基因在抑制开放开花方面的累积效应的见解，并为进一步研究六倍体小麦完全闭锁受精的发展提供了基础。