Cloning and sequencing of RAPD fragments amplified from mitochondrial DNA of male-sterile and male-fertile cytoplasm of sugar beet (Beta vulgaris L.)

Abstract

Mitochondrial DNA fragments of two nearly isogenic lines of sugar beet (Beta vulgaris L.) were amplified by RAPD analysis. A number of fragments, most of them unique to either the male-sterile or the male-fertile cytoplasm, were selected for cloning and sequencing. One fragment was present in the PCR fingerprint pattern of both cytoplasms, whereas five of the selected fragments were specifically amplified from only one type of cytoplasm. The mitochondrial origin of all cloned RAPD fragments was confirmed by Southern hybridization. One fragment resulted in a hybridization pattern that suggests its repetitive presence in the mitochondrial genome of sugar beet. Four out of the five cytoplasm-specific RAPD fragments were shown to hybridize specifically to one type of cytoplasm only. One fragment hybridizing with the mtDNA from N-cytoplasm 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 ndhC gene of higher plants, indicating that the male-fertile-specific mtDNA RAPD fragment is derived from chloroplast DNA. Sequence analysis of an amplified sterile-specific fragment revealed the presence of an open reading frame of 288 bp. Northern hybridization showed a transcription signal specific for the male-sterile cytoplasm. No sequence homology of the open 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.