Cytogenetics and genomics analysis of cold-hardy perennial wheatgrass: insights into agronomic performance, chromosome composition, and gene expression

Intermedium wheatgrass (Thinopyrum intermedium), a perennial species with extensive root systems and high tolerance to cold, drought, and salinity, is a valuable genetic resource for the development of perennial crops. Over a decade-long selection process, two cold-hardy perennial wheatgrass lines were developed by crossing wheat-Thinopyrum partial amphiploids with Th. intermedium. These lines inherited key traits from Th. intermedium, including plant stature, spike morphology, and postharvest regrowth. Transcriptome-based single-nucleotide polymorphism tracing and sequential multicolor genomic in situ hybridization analyses revealed variations in the chromosome compositions of the perennial wheatgrass lines. The introgression of wheat chromosomes enhanced grain weight and size, while preserving the cold-hardy, perennial characteristics of the wheatgrass lines compared to Th. intermedium. Genome-wide gene expression was generally suppressed in the wheatgrass lines relative to Th. intermedium, particularly in conserved genes. This suppression was especially pronounced in genes involved in cell division and DNA repair pathways. In contrast, genes associated with cold tolerance and the water stress response were upregulated. We identified eight cold-tolerance genes in the Th. intermedium chromosomes and validated three of them, Thint.J05G452200, Thint.J05G452300, and Thint.V05G408900, using qRT-PCR. These genes encode proteins associated with cold tolerance and are potential candidates for further functional validation. Additionally, three chromosomes from homoeologous group 6 were introgressed, carrying six genes potentially associated with superior grain traits. Among them, TraesCS6D02G287800, which encodes a specific protein, exhibited high expression levels in both wheatgrass lines, suggesting its critical role in enhancing grain traits. Our results indicate that the suppression of grass gene expression, likely due to the introgression of wheat chromosomes and the upregulation of pathways related to cold tolerance and overwintering ability, contributes to the adaptive features of the wheatgrass lines. This study provides a genomic foundation for understanding gene expression regulation in distant hybrid progeny and offers valuable insights for designing new breeding strategies for perennial wheat or wheatgrass.