Molecular genetics of freeze tolerance among natural populations of Populus balsamifera across the active growing season



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Boreal forest trees such as Populus balsamifera. L exhibit high levels of population differentiation in quantitative traits of ecological importance. Climate change is likely to have severe effects on high latitude species. On one hand, due to long warm spells in winter the chilling requirement of some species is not fulfilled which is likely to influence the timing growth initiation in spring and on the other hand due to increased frequency of sporadic freeze events during the active growing season. Although ample study has been conducted to understand winter survival strategies in plants, we currently lack an understanding of how species survive through freeze events during the active growing season. Despite the role of freezing temperatures on determining plant species distribution, very few studies have integrated intraspecific phenotypic variation in freeze tolerance to nucleotide diversity and gene expression patterns at the associated loci. This is the first study to incorporate these into providing an understanding of latitudinal and seasonal dynamics of freeze tolerance in Populus balsamifera (Balsam poplar). Intraspecific variation in the freezing point and freeze damage (LT50) of balsam poplar leaves, across the growing season, was assessed using both the electrolyte leakage assay (EL) and supercooling assay. Simultaneously, changes in leaf thickness were also studied. LT50 was much lower than the freezing point, indicating that this species is likely to tolerate freezing during the growing season. Freezing tolerance showed clinal and temporal variation across the latitude of genotype origin and growing season months respectively. We observed only a moderate latitudinal cline in freezing tolerance during June and July; however, in August the latitudinal cline increased dramatically with the northern populations developing much high levels of freeze tolerance. We compared phenotypic variation in freeze tolerance to genetic variation in the coding regions of putative genes and to patterns of differential gene expression. The CBF gene family in Populus has been shown to influence freeze tolerance, hence we surveyed nucleotide variation in 6 members of the poplar CBF gene family to understand the molecular basis of freeze tolerance variation at the coding region of CBF genes. CBF2 showed strong genetic differentiation (FST=0.083) and a clinal pattern of change in minor allele frequency that was greater than the expected pattern of neutral genome wide differentiation observed in P. balsamifera. Although all 6CBF genes were cold inducible, they exhibit varying patterns of expression during the growing season, with a majority being differentially expressed across latitude only in August and September. Overall, the length of growing season was as an important determinant of foliar cold tolerance, which resulted in a strong latitudinal difference in freeze tolerance and gene expression pattern in August and September. The presence of strong latitudinal cline in freeze tolerance appears to be a threshold phenomenon and suggests an interaction between shortening of day length and lowering of temperatures in freeze induction. Hence, our results indicate that photoperiod cues might play an important role in freeze tolerance induction for woody perennials like Populus balsamifera.



Population genetics, Populus balsamifera, CBF genes, Cold tolerance