Background: Identi cation of genetic diversity in heat tolerance and associated traits is of great importance for improving heat tolerance in cool-season grass species. The objectives of this study were to determine genetic variations in heat tolerance associated with phenotypic and physiological traits and to identify molecular markers associated with heat tolerance in a diverse collection of perennial ryegrass (Lolium perenne L.).

Results: Plants of 98 accessions were subjected to heat stress (35/30℃, day/night) or optimal growth temperature (25/20℃) for 24 d in growth chambers. Overall heat tolerance of those accessions was ranked by principal component analysis (PCA) based on eight phenotypic and physiological traits. Among these traits, electrolyte leakage (EL), chlorophyll content (Chl), relative water content (RWC) had high correlation coe cients (− 0.858, 0.769, and 0.764, respectively) with the PCA ranking of heat tolerance. We also found expression levels of four Chl catabolic genes (CCGs), including LpNYC1, LpNOL, LpSGR, and LpPPH, were signi cant higher in heat sensitive ryegrass accessions then heat tolerant ones under heat stress. Furthermore, 66 pairs of simple sequence repeat (SSR) markers were used to perform association analysis based on the PCA result. The population structure of ryegrass can be grouped into three clusters, and accessions in cluster C were relatively more heat tolerant than those in cluster A and B. SSR markers signi cantly associated with above-mentioned traits were identi ed (R2> 0.05, p< 0.01)., including two pairs of markers located on chromosome 4 in association with Chl content and another four pairs of markers in association with EL.

Conclusion: The result not only identi ed useful physiological parameters, including EL, Chl content, and RWC, and their associated SSR markers for heat-tolerance breeding of perennial ryegrass, but also highlighted the involvement of Chl catabolism in ryegrass heat tolerance. Such knowledge is of signi cance for heat-tolerance breeding and heat tolerance mechanisms in perennial ryegrass as well as in other cool-season grass species.