CLONING AND SEQUENCING OF RAPD FRAGMENTS AMPLIFIED FROM MITOCHONDRIAL-DNA OF MALE-STERILE AND MALE-FERTILE CYTOPLASM OF SUGAR-BEET (BETA-VULGARIS L)

Mitochondrial DNA fragments of two nearly isogenic lines of sugar beet (Beta vulgaris L.) were amplified by RAPD analysis. A number of fragm ents, most of them unique to either the male-sterile or the male-ferti le cytoplasm, were selected for cloning and sequencing. One fragment w as present in the PCR fingerprint pattern of both cytoplasms, whereas five of the selected fragments were specifically amplified from only o ne type of cytoplasm. The mitochondrial origin of all cloned RAPD frag ments was confirmed by Southern hybridization. One fragment resulted i n a hybridization pattern that suggests its repetitive presence in the mitochondrial genome of sugar beet. Four out of the five cytoplasm-sp ecific RAPD fragments were shown to hybridize specifically to one type of cytoplasm only. One fragment hybridizing with the mtDNA from N-cyt oplasm also revealed hybridization signals with both total and nuclear DNAs of N- as well as S-cytoplasm. Sequence alignments of this clone showed strong homologies with a part of the plastidal ndh C gene of hi gher plants, indicating that the male-fertile-specific mtDNA RAPD frag ment is derived from chloroplast DNA. Sequence analysis of an amplifie d sterile-specific fragment revealed the presence of an open reading f rame of 288 bp. Northern hybridization showed a transcription signal s pecific for the male-sterile cytoplasm. No sequence homology of the op en reading frame to any known sequences was found. The results reveal an extremely high degree of sequence variability between the mtDNA of the N- and S-cytoplasm of Beta vulgaris.