Isolation and characterization of mitochondrial deoxyribonucleic acid (DNA) from cotton

A procedure to isolate mtDNA from cotton seedlings G. hirsutum and S. barbadence was developed. The new protocol, which is different from published procedures for other plant species, ailows for the isolation of cotton mtDNA of high purity, yield and digestibility by restriction endonucleases.

The success of the protocol is based on criticai adjustments in the ionic strengths of the homogenizing medium and washing buffer, the speed of grinding during homogenization, and the methods used for lysis of the mtDNA.

A major problem to be overcome in developing the protocol was the contamination of mtDNA with nuclear DNA. Nuclear DNA was manifested either as degraded DNA or as high molecular weight DNA; the former was detected with gel profiles of undigested DNA. The latter was detected only upon restriction endonuclease digestion followed by gel electrophoresis. A high concentration of NaCI in the homogenizing medium in place of mannitol has enhanced the effectiveness of DNAase treatment in removing the degraded form of nuclear DNA contamination. During CsCI density gradient centrifugation, the high molecular weight nuclear DNA in the form of chromatin which is present in high levels as a result of high speed homogenization, can be readily separated from mtDNA. The ability to use high speed homogenization greatly increases the yield of mtDNA. A second problem to be overcome was the lack of reproducibility in mtDNA restriction fragment patterns. Proteinase K treatment which is standard in many mtDNA isolation protocols was found to be the cause of poor digestibility by restriction endonucleases. Elimination of proteinase K treatment produced cotton mtDNA with reproducible restriction patterns.

The isolated mtDNA was characterized by its (G +C) content, melting point, and its genome size. The melting point is found to be approximately 88°C which gives a (G +C) content in the range of 44 - 45%. Estimations of the genome size of cotton mtDNA were determined by restriction analysis and reassociation kinetics. Molecular weight determination by summation of the fragments resulting from digestion with restriction endonucleases Hind III, Bam HI, and Eco Rl yields an estimate of a genome size in the range of 695 - 712 kb. Kinetic complexities obtained from reassociation kinetic analysis by hydroxyapatite chromatography indicate a mitochondrial genome size of approximately 2000 kb for cotton.