Freezing tolerance and expression of candidate genes in timothy(Phleum pratens L.).

Abstract

Low temperature is the major abiotic stress limiting the productivity and the geographical distribution of many important crops, including forage grasses. To gain a better understanding of low temperature stress responses in timothy (Phleum pratense L.), we investigated variation in freezing tolerance among 30 genotypes of timothy and gene expression of two frost tolerant candidate genes, sucrose: fructan 6-fructosyltransferase (6-SFT) and QM in response to low temperature treatment. Plant materials were established, cold acclimated at 1°C for two weeks, and freezing tolerance was assayed at -10 °C for 24 h by visual scoring of regrowth after 2 weeks using a scale ranging from 0 (completely dead) to 9 (re-growth without damage). An average survival score was determined and high and low frost tolerant genotypes were identified. The relative gene expression of the two candidate genes was measured for two high and two low frost tolerant genotypes using the comparative cycle threshold (CCT) method. The results indicate that there is significant variation among some of the genotypes for freezing tolerance. Increased transcript levels for both candidate genes in response to cold stress were observed in all the genotypes except for 6-SFT in the high frost tolerant genotype AP31 (from Russia) which was unaffected or slightly down-regulated at all time points studied. Although not conclusively shown, the reason for this result could be lack of primer binding due to sequence variation in the binding sites. Generally, an increase in transcript levels was observed for both genes until day 7 after commencement of cold acclimation and declined thereafter. Variation for freezing tolerance exists among genotypes of timothy that can be utilized for improvement for frost tolerance in this species. Both genes studied might possibly be good candidate genes for further investigations and selection for freezing tolerance of timothy.