MICRODISSECTION AND AMPLIFICATION OF CODING SEQUENCES FROM A CHROMOSOME FRAGMENT RESTORING MALE-FERTILITY IN ALLOPLASMIC MALE-STERILE TOBACCO

An alloplasmic male-sterile line of tobacco, containing the nucleus of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, is restored to fertility by introgression of an alien chromosome fragment obtaine d from the cytoplasm donor. To isolate the restorer gene(s), the alien chromosome fragment was microdissected from metaphase plates of the r estored line. The microdissected chromosomes represented only 0.1 pg o f DNA, which was amplified using a degenerate oligonucleotide-primed P CR method (DOP-PCR), from which a chromosome fragment specific library was created. Compared with previous strategies used for microcloning, a modified and improved method was developed by the subsequent isolat ion of expressed sequences. The library was screened with cDNA probes synthesized by reverse transcription and DOP-PCR amplification (RT/DOP -PCR), of total RNAs isolated from early developing restored and male- sterile flower buds. By this strategy, transcribed DNA sequences speci fic for the restored line were cloned.