Cultivated bread wheat (Triticum aestivum L.) is a predominantly autogamous crop with low rates of natural outcrossing in the absence of environmentally or chemically-induced male sterility. This reproductive system has limited the range of breeding approaches applied to wheat. Labor requirements and low seed yield of traditional crosses have limited rapid cycling recurrent selection as a breeding strategy and present logistical barriers to production of large numbers of backcross progeny for complex gene pyramids. The Ms3 dominant male sterility trait has been deployed in some programs, and has been proposed as a strategy for long-term recurrent selection and for utilization of exotic germplasm. Seedling identification, or seed identification, of Ms3/ms3 progeny from fertilization of an Ms3/ms3 spike would improve the efficiency of Ms3-facilitated breeding strategies. Our experience with Ms3-crossing is that crossing efficiency is substantially reduced by initiating pollination after visible evidence of sterility. Therefore, we have relied heavily on second-spike crossing, which has reduced efficiency of greenhouse operations, decreased available tillers for pollination, and prevented implementation of whole-plant crossing blocks. Early marker selection for sterile genotypes will transform the management of Ms3-based breeding programs.

Ms3 was previously localized to the centromeric region of chromosome 5A with an RFLP marker, which was not sequenced and is no longer available. The centromeric location of Ms3 limits the prospects for utilizing existing SNP-based marker sets, which are derived principally from gene-rich regions with higher recombination. The benefit of the centromeric location is that, once identified, a broadly informative marker would provide a reliable predictor of the malesterile phenotype.

A set of 429 hybrids incorporating Ms3 were constructed, both within hard winter wheat adapted to the Great Plains of the United States, and between these winter wheats and Asian spring wheats. Hybrid plants were visually evaluated for the male-sterile phenotype, both at anthesis and at final harvest. DNA extracted from these plants was sequenced using restriction enzyme-based reduced representation sequencing. Polymorphisms among the sequence tags were identified by a reference-based SNP calling pipeline. Association of polymorphic tags localized to chromosome 5A with the malesterile phenotype was tested using case-control association analysis. Two highly significant (LOD>30) SNP-trait associations were obtained, both with absolute association of a specific tag sequence with the male-sterile phenotype. These SNP-trait associations provided the foundation for development of a highly sensitive, reliable SNP marker for the Ms3-associated male-sterile phenotype that can be incorporated into routine marker selection regimes in wheat breeding programs designed to utilize Ms3 as an enabling technology for new breeding approaches.