The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints.
OBJECTIVE
Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used.


METHODS
AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used.


RESULTS
Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid.


CONCLUSIONS
We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.
Fay
Michael F
MF
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK. m.fay@kew.org
Cowan
Robyn S
RS
Leitch
Ilia J
IJ
D002467:Cell Nucleus / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods*; D018744:DNA, Plant / Q000032:analysis* / Q000235:genetics; D018745:Genome, Plant; D019684:Magnoliopsida / Q000235:genetics*; D011003:Ploidies; D012150:Polymorphism, Restriction Fragment Length; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D012641:Selection, Genetic; D013045:Species Specificity
D018744:DNA, Plant