Genetic variation within and between two cultivars of red clover (Trifolium pratense L.), Essi from Europe and Ottawa from Canada was estimated using morphological, isozyme and random amplified polymorphic DNA (RAPD) markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Nine 10-mer primers were used to assay 20 individuals from each cultivar for RAPD markers. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. The mode of inheritance of seven isozyme loci: Aat-2, Amy-1, Est-4, Est-7, Pgd-1, Pgd-2 and Skd-1, in red clover was verified. The genetic basis of banding patterns for 16 other isozyme loci: Aat-3, Adh-1, Dia-1, Dia-2, Dia-3, Est-1, Est-2, Gpi-2, Idh-1, Mdh-1, Mdh-2, Mdh-3, Mdh-4, Me-1, Me-2 and Pgm-2, was also postulated, based on the segregation patterns observed within cultivars. Two pairs of linked enzyme-coding loci, Est-4/Est-7 and Pgd-2/Skd-1, were found with joint segregation analysis. Estimates of genetic variability of 15 red clover cultivars from three different origins indicated that within-cultivar variation was much higher than between-cultivar variation. Allele frequencies of these isozymes could discriminate the five North American cultivars assayed, but they could not differentiate cultivars from Europe and Japan. The use of RAPD markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. Twenty was found to be an appropriate number of red clover individuals per bulk for homogenizing genetic variation within cultivars. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origin

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