Development of Specific Molecular and Phenotypic Marker-Based Haploid Inducers in Rice

Abstract

Doubled haploid (DH) technology is an efficient strategy for producing completely homozygous lines for breeding programs. Mutations in the MATRILINEAL (MTL) phospholipase trigger intraspecific haploid induction in cereals. Although an in vivo haploid induction system based on OsMTL-edited plants has been established in rice (Oryza sativa), DH technology is still limited by other factors, such as haploid identification, which is one of the essential steps required for DH technology. In the study, we addressed this technical challenge by integrating specific molecular and phenotypic markers into rice haploid inducers. We first generated large fragment insertion or deletion mutations within the OsMTL gene and designed a pair of primers flanking the mutational sites to be used as the specific and universal molecular markers between wild-type and Osmtl plants. Next, we screened for hairy leaf as a single dominant trait and integrated it into specific molecular marker-based haploid inducers using the cross and self-cross method. When crossing cytoplasmic male sterile lines with these haploid inducers, we utilized the specific InDel marker and hairy leaf phenotypic marker to identify putative haploids (or double haploids). These putative haploids were further confirmed through ploidy and phenotypic analysis, demonstrating the high efficiency of haploid identification using these markers. The haploid induction rate (HIR) of the developed specific molecular and phenotypic marker-based haploid inducers ranged from 3.7% to 12.5%. We have achieved successful integration of distinct molecular and phenotypic markers into rice haploid inducers. Our advanced marker-based system has significantly enhanced the accuracy of haploid identification, thereby expediting the adoption of DH technology in rice breeding.