Development of diagnostic marker for functional Lr21 and its validation in pyramiding with Pm36 in bread wheat

Wheat leaf rust, caused by the obligate biotrophic fungus Puccinia triticina ( Pt ), poses a major threat to global wheat production. This makes screening and utilizing effective leaf rust resistance genes key to controlling the disease. Twenty-five wheat cultivars previously reported to carry Lr21 were assayed for the hypersensitive response (HR) to the AvrLr21 protein, and none exhibited HR, indicating that all carry non-functional Lr21 alleles. Sequence comparison further classified these non-functional alleles into two distinct haplotypes, H1 (14 cultivars) and H2 (11 cultivars), and identified six polymorphic sites that distinguish functional from non-functional Lr21 alleles. Based on these sites, a CAPS marker for functionality and four SSP markers for H1/H2 haplotypes were developed, confirming that all twenty-five cultivars carry non-functional haplotypes. Through pedigree tracing of these twenty-five cultivars identified eight core parental germplasm accessions carrying only non-functional Lr21 haplotypes, indicating that functional Lr21 has never been deployed in wheat breeding. To utilize functional Lr21 , TcLr21 was crossed with ZKPm36 to pyramid Lr21 with Pm36 , yielding F 2 progeny with the expected 9:3:3:1 segregation ratio. Resistance assays showed that the pyramided lines exhibited moderate resistance to leaf rust, high resistance to powdery mildew, and moderate resistance to root rot. Collectively, we developed diagnostic markers discriminating functional from non-functional Lr21 haplotypes, and validated their utility in pyramiding Lr21 with Pm36 , providing both tools and genetic materials for marker-assisted selection (MAS) and further improvement of multi-disease resistance in wheat.