Comparative karyology, DNA methylation and restriction pattern analysis of male and female plants of the dioecious alga Chara tomentosa (Charophyceae)

Abstract

Comparison of karyology, restriction patterns and DNA methylation levels (as gender separation could be related to some epigenetic mechanism, e.g. DNA methylation) has been performed between male and female individuals of the dioecious species, Chara tomentosa. In contrast to angiosperms, sex determination systems in Charophyceae have not been studied. High-performance liquid chromatography (HPLC) showed that the cytosine methylation level in female plants of Chara tomentosa reached 5·9%, while in male plants it reached 9·1%. Karyological analysis did not show any differences in metaphase chromosome number or morphology between sexes. Cytophotometric measurements of DNA content revealed differences in 1C DNA content : 7·0 pg in females and 7·4 pg in males (p < 0·001). Computer image analysis showed that in the interphase nuclei condensed chromatin consists of 34·3% of the total chromatin in female plants, while in males it was 29·6% of total chromatin (p < 0·001). Digestion of isolated DNAs with 12 enzymes showed poor enzymatic cleavage (for example with SacI) or restriction patterns which differed between the sexes (TaqI, Bst0I). The restriction endonucleases RsaI and BamHI revealed repetitive DNA families in males. BamHI generated bands of over 3000 bp while RsaI revealed populations of fragments 650–2000 bp long. DNA isolated from male individuals of C. tomentosa was less susceptible than DNA isolated from female plants to digestion with enzymes that are inhibited by methylation of dC residues (HpaII, HhaI).