Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non-actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high-molecular-weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)^?1, with a ratio A₂₀₀/A₂₈ of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al-2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI-2 vs AI-2), Betula papyrifera (BP-4 vs BP-8) and subclones of Ainus glutinosa AG-1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.