Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies

The phytochrome photoreceptors play important roles in the photoperiodiccontrol of vegetative bud set, growth cessation, dormancy induction, andcold-hardiness in trees. Interestingly, ecotypic differences inphotoperiodic responses are observed in many temperate- zone tree species.Northern and southern ecotypes of black cottonwood (Populus trichocarpaTorr. & Gray), for example, exhibit marked differences in the timing ofshort-day-induced bud set and growth cessation, and these responses arecontrolled by phytochrome. Therefore, as a first step toward determiningthe molecular genetic basis of photoperiodic ecotypes in trees, wecharacterized the phytochrome gene (PHY) family in black cottonwood. Werecovered fragments of one PHYA and two PHYB using PCR-based cloning and byscreening a genomic library. Results from Southern analyses confirmed thatblack cottonwood has one PHYA locus and two PHYB loci, which we arbitrarilydesignated PHYB1 and PHYB2. Phylogenetic analyses which included PHY fromblack cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum)suggest that the PHYB/D duplications in these species occurredindependently. When Southern blots were probed with PHYC, PHYE, and PHYEheterologous probes, the strongest bands that we detected were those ofblack cottonwood PHYA and/or PHYB. These results suggest that blackcottonwood lacks members of the PHYC/F and PHYE subfamilies. Although blackcottonwood could contain additional PHY that are distantly related to knownangiosperm PHY, our results imply that the PHY family of black cottonwoodis less complex than that of other well-characterized dicot species such asArabidopsis and tomato. Based on Southern analyses of five black cottonwoodgenotypes representing three photoperiodic ecotypes, substantialpolymorphism was detected for at least one of the PHYB loci but not for thePHYA locus. The novel character of the PHY family in black cottonwood, aswell as the differences in polymorphism we observed between the PHYA andPHYB subfamilies, indicates that a number of fundamental macro- andmicroevolutionary questions remain to be answered about the PHY family indicots.