Identification and Development of Functional Markers Associated with Triple Pistil Phenotype in Wheat

Triple pistil (TP) wheat is a historical genetic resource capable of producing up to three grains in a single floret and bolstering the current stagnant grain yield potential. TP phenotype is speculated to be the result of a spontaneous mutation; however, the exact underling genetic mechanism remains elusive, with lack of functional markers for early generation trait selection in hybrid wheat breeding programs. Here, scanning electron microscopy highlighted clear developmental differences between single and triple pistil plants started to arise during 1-2 cm long young spike stages. Using a forward genetics approach, we identified consistent TP-associated mutations in two genes ( TraesCS2D02G490900 and TraesCS2D02G491600 ) exhibiting a nearly complete co-segregation with TP phenotype and developed functional markers for early generation trait selection. CRISPR-Cas9 mediated gene-editing of TraesCS2D02G490900 shifted grain set toward single-grain florets in one edited line in transgenic wheat plants. Furthermore, grain yield evaluation exhibited a significant increase in grains per spike, with no statistically significant reduction in grain weight per spike. Hybrids between common and TP wheat exhibited relatively higher yields, highlighting TP wheat as a significant donor to fortify grain yield potential. This study provides co-dominant functional markers for early generation TP trait selection and valuable targets for hybrid wheat breeding programs.