DETECTION AND PARTIAL SEQUENCE OF PHYTOCHROME GENES IN THE FERNS Anemia phyllitidis (L.)Sw (SCHIZAEACEAE) AND Dryopteris filix‐mas L. (POLYPODIACEAE) BY USING POLYMERASE‐CHAIN REACTION TECHNOLOGY*

Abstract

Phytochrome controls several developmental steps during formation and differentiation of the fern gametophyte, including spore germination, morphogenesis of the gametophyte or differentiation of the sexual cells. To obtain information about the amino acid sequence and the regulation of phytochrome expression at the gene level, two degenerated oligonucleotides against well conserved amino acid regions were designed after an optimal alignment of the known phytochrome sequences. These primers were tested against DNA isolated from Arabidopsis thaliana, and the polymerase-chain reaction (PCR) products were cloned and sequenced. The DNA fragment produced with this method proved to be identical with a phytochrome-A-gene fragment from A. thaliana, and hence this fragment was used in further experiments to prove whether amplified DNA from fern species contains phytochrome-like DNA. With this procedure we successfully detected and cloned gene fragments both from gametophytes of Anemia phyllitidis and Dryopteris filix-mas, cultured for 7 days under vegetative conditions. In addition, poly(A)+ RNA was prepared from 7-day-old gametophytes of A. phyllitidis, induced to differentiate antheridia under generative conditions. This poly(A)+ RNA was transcribed into complementary DNA and used together with both phytochrome specific primers in a PCR experiment. We thereby obtained another DNA fragment. These data strongly suggest that A. phyllitidis has at least two phytochrome genes, and that at least one of them is expressed in light-grown gametophytes.