Development and molecular cytogenetic characterization of black-grain wheat derived from wheat-Thinopyrum intermedium hybridization.

Key message: Thirteen black-grain wheat lines with black endosperm and high grain protein were developed through hybridization between wheat cultivars and Thinopyrum intermedium. These lines are disomic chromosome substitution lines designated DS4B/4J^r/St or DS4D/4J^r/St. Thinopyrum intermedium (Host) Barkworth and D. R. Dewey (2n = 6x = 42, StStJ^rJ^rJ^vsJ^vs or StStEEEE) is a valuable genetic resource for wheat improvement. By crossing the common wheat cultivar Kehan 9 and a male-sterile line Taigu with Th. intermedium, we generated 13 wheat lines characterized by black aleurone and endosperm, along with elevated grain protein content. Field trials from 2022 to 2024 demonstrated that these lines exhibited superior yield traits and higher protein content compared to the parental Kehan 9. Sequential multicolor genomic and florescence in situ hybridization analyses revealed that a pair of recombinant J^r and St chromosomes from Th. intermedium replaced wheat chromosomes 4B or 4D, respectively. Single-nucleotide polymorphism (SNP) analysis via RNA-seq further identified this recombinant chromosome from the homoeologous group 4, designated 4J^r/St. These black-grain wheat lines were thus identified as disomic chromosome substitution lines DS4B/4J^r/St or DS4D/4J^r/St. Analysis of an F₂ population derived from a cross between black- and white-grain lines confirmed the association between chromosome 4J^r/St and the black-grain trait. Three anthocyanin-related genes located on chromosome 4J^r/St, and the expression level of Thint.S04G290300 encoding a basic helix-loop-helix (bHLH) DNA-binding superfamily protein, showed a progressive increase during grain development. Black pigmentation was initially detected at 18 days post-anthesis (DPA) in the aleurone layer near the embryo region and in the endosperm at 30 DPA. These lines provide a promising foundation for enhancing wheat nutritional value through the development of colored grain cultivars.