Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

Segregation of random amplified DNA markers in F1 progeny of conifers.

The recently developed approach to deriving genetic markers via amplification of random DNA segments with single primers of arbitrary nucleotide sequence was tested for its utility in genetic linkage mapping studies with conifers. Reaction conditions were optimized to reproducibly yield clean and specific amplification products. Template DNA from several genotypes of Douglas-fir (Pseudotsuga menziesii) and white spruce (Picea glauca) were tested against eight ten-base oligonucleotide primers. Most of the tested primer/parent tree combinations yielded polymorphic PCR products ("RAPD" markers). Selected primers were then used in PCR reactions with template DNA isolated from offspring in Douglas-fir and black spruce diallel crosses among the same parental lines. The diallel study confirmed the appropriate inheritance of RAPD markers in the F1 generation. The value of these dominant RAPD markers for genetic linkage mapping in trees was established from both theoretical and applied perspectives.