USE OF COMPETITIVE PCR TO ASSAY COPY NUMBER OF REPETITIVE ELEMENTS INBANANA

Banana is one of the most important subtropical fruit crops. Genetic i mprovement by traditional breeding strategies is difficult and better knowledge of genomic structure is needed. Repeated sequences are power ful markers for genetic fingerprinting. The method proposed here to de termine the copy number of nuclear repetitive elements is based on com petitive reverse transcription-polymerase chain reaction and can also be used for quantifying cytosolic sequences. The reliability of this m ethod was investigated on crude preparations of total DNA. Variations due to the heterogeneity of crude DNA extracts showed that a single lo cus reference is needed for accurate quantification. A mapped microsat ellite locus was used to normalize copy number measurements. Copy numb er assay of repetitive elements using this method clearly distinguishe s between the two banana subspecies investigated: Musa acuminata spp. banskii and M. acuminata spp. malaccensis. Two repetitive sequence fam ilies, pMaCIR1115 and pA9-26, were assayed that cover up to 1% of the M. acuminata genome. Their copy number varied up to six fold between t he two subspecies. Furthermore, sequence quantification showed that mi tochondrial genomes are present in crude leaf-extracted banana DNA at up to 40 copies per cell.