Accumulation of mutations in the AP2 homoeologs causes suppression of anther extrusion with altered spike and culm development in hexaploid wheat

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.