Project description:It is crucial for north temperate tree species like white spruce to control timing of growth cessation and bud set in order to survive winter conditions. While much work has examined both endogenous and exogenous mechanisms influencing this transition within angiosperms, the mechanisms underlying this transition in conifers; including photoperiod perception, growth cessation and development of endodormancy, have been less well-characterized. The objective of this research was to compare the genetic responses of spruce genotypes exhibiting an early transition from active growth to bud initiation with those that delay the transition, to tease apart the molecular mechanisms underlying these complex traits. In order to gain a deeper understanding of how genomic architecture influences bud phenology and related regulatory pathways, transcript levels were quantified using a 25,045 element spruce oligonucleotide microarray. Individuals exhibiting early versus late time to bud set were selected from a large unrelated full-sib family that had been phenotyped multiple years under natural and controlled environment conditions, and harvested at an early and mid stage of bud formation. Progeny designated as early time to bud set were more likely to undergo second flush, particularly under long day (16 h) and constant temperature (20°C) conditions. Approximately 5400 genes were differentially expressed in either or both of the early and late time to bud set phenotypic classes between time points, while ca. 1500 genes were differentially expressed between phenotypic classes at either or both time points. These analyses revealed about 400 genes with putative regulatory roles. The comparison between early and late time to bud set trees revealed a large number of specifically differentially expressed genes in the early developmental stage for bud formation (957 genes, including 53 putative regulators) relative to the later developmental stage (81 genes, including 4 putative regulators). Genes implicated in the circadian cycle, photoperiodic pathway and chromatin remodeling figured prominently in these lists, providing new insight into mechanisms that may regulate bud formation in determinate species such as white spruce.

Project description:It is crucial for north temperate tree species like white spruce to control timing of growth cessation and bud set in order to survive winter conditions. While much work has examined both endogenous and exogenous mechanisms influencing this transition within angiosperms, the mechanisms underlying this transition in conifers; including photoperiod perception, growth cessation and development of endodormancy, have been less well-characterized. The objective of this research was to compare the genetic responses of spruce genotypes exhibiting an early transition from active growth to bud initiation with those that delay the transition, to tease apart the molecular mechanisms underlying these complex traits. In order to gain a deeper understanding of how genomic architecture influences bud phenology and related regulatory pathways, transcript levels were quantified using a 25,045 element spruce oligonucleotide microarray. Individuals exhibiting early versus late time to bud set were selected from a large unrelated full-sib family that had been phenotyped multiple years under natural and controlled environment conditions, and harvested at an early and mid stage of bud formation. Progeny designated as early time to bud set were more likely to undergo second flush, particularly under long day (16 h) and constant temperature (20M-BM-0C) conditions. Approximately 5400 genes were differentially expressed in either or both of the early and late time to bud set phenotypic classes between time points, while ca. 1500 genes were differentially expressed between phenotypic classes at either or both time points. These analyses revealed about 400 genes with putative regulatory roles. The comparison between early and late time to bud set trees revealed a large number of specifically differentially expressed genes in the early developmental stage for bud formation (957 genes, including 53 putative regulators) relative to the later developmental stage (81 genes, including 4 putative regulators). Genes implicated in the circadian cycle, photoperiodic pathway and chromatin remodeling figured prominently in these lists, providing new insight into mechanisms that may regulate bud formation in determinate species such as white spruce. A clonally replicated QTL mapping population (cross C94-1-2516, M-bM-^YM-^@77111 M-CM-^W M-bM-^YM-^B2388), previously characterized by Pelgas et al. (2011), was used for this study. Phenotypic data collected between 2006-2008 was used to identify extremes for time to bud set within the QTL population. Genotypes falling within the lower quartile for time to bud set for at least three of six experiments were designated as early time to bud set, while genotypes falling within the upper quartile for time to bud set for at least three of six experiments were designated as late time to bud set. Shoot tips from four ramets of each genotype were harvested for molecular analysis. Two ramets were harvested at the first sign of bud formation (Time 1) and the remaining two ramets were harvested six weeks later at which time the majority of seedlings were at the mid stage of bud set (Time 2). Microarray hybridizations were conducted to make the following comparisons: a comparison of bud development (Time 1 v. Time 2) in early time to bud set seedlings, a comparison of bud development (Time 1 v. Time 2) in late time to bud set seedlings, and a comparison of early and late time to bud set seedlings within early stages of bud formation or mid stage of bud formation. Each of two biological replicates for each time point and each phenotypic class was hybridized.

Project description:Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce (Picea glauca [Moench] Voss.), a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next season's growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for ABA, cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce. Shoot tips (terminal buds), needles, and secondary stems were collected from two-year-old white spruce plants over a 10-week time course of 0, 1, 3, 7, 14, 28, and 70 days after switching from 6 to 8 weeks of long daylight photoperiods (LD; 16 hours of light and 8 hours of dark) to short daylight photoperiods (SD; 8 hours of daylight and 16 hours of dark). Remaining plants were kept in short days for an additional 8-15 weeks, and then transferred to low temperature (LT; 2°C to 4°C) for 3 to 4 weeks with continuing SD prior to harvest. Another set of plants was grown and harvested under the same conditions as described above, but remained in LD at all times. Four sets of dye-swap design microarray experiments were conducted. The first set of experiments (samples 1-7) studied the SD time course of buds development. Terminal buds from each time point (1d, 3d, 7d, 14d, 28d, and 70d) and LT were co-hybridized with actively growing shoot tips (0d). The same time point comparison (without LT) of shoot tips from LD-treated trees was carried out as the second set of experiments (samples 8-13). The third experiment (samples 14-20) denoted a separate LD/SD comparison at seven different time points (0d, 1d, 3d, 7d, 14d, 28d, and 70d), and the last experiment (samples 21-26) compared SD shoot tips, needles, and secondary stems with each of the other tissues at 14d and 70d. In each experiment, four biological replicates were used, with two replicates representing the dye-swaps.

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