 PERGAMON Phytochemistry 49 "0888# 808Ð813 9920!8311:88 ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII]S9920!8311"87#99517!0 Customisation of AFLP analysis for cassava varietal identi_cation Hui!Lee Wong\ Hock!Hin Yeoh\ Saw!Hoon Lim Department of Biological Sciences\ National University of Singapore\ Kent Ridge\ Singapore 008519\ Singapore Received 8 April 0887^ revised 01 November 0887 Abstract Ampli_ed fragment length polymorphism "AFLP# markers were used in the characterization of eight cassava varieties[ This non! radioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 2? end of primers[ EcoRI:MseI and HindIII:MseI fragments generally gave monomorphic pro_les while ApaI:TaqI fragments produced polymorphic pro_les suggesting a genome with high G¦C content[ It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer[ For cassava\ the AFLP system provided a higher number of loci detected per run when compared to RAPD[ The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Manihot esculenta^ Euphorbiaceae^ AFLP markers^ Selective nucleotides^ Varietal identi_cation^ Cyanogenic potential 0[ Introduction Cassava "Manihot esculenta Crantz# provides dietary carbohydrate to more than _ve hundred million people in developing countries "Cock\ 0874#[ Cassava food safety\ however\ is compromised by the varying levels of cyano! genic potential "mainly attributed to linamarin content# in the storage roots "Yeoh + Truong\ 0882#[ Thus\ the assessment of genetic variation in this crop not only facili! tates e.cient conservation and utilization of germ plasm resources\ it also assists in breeding strategies for safe consumption[ Determination of genetic variation can be conducted through a range of molecular markers "Paul + Yeoh\ 0878^ Yeoh + Paul\ 0880^ Fregene et al[\ 0883^ Marmey\ Beeching\ Hamon\ + Charrier\ 0883^ Yeoh + Joseph\ 0884#[ Previous work which employed the use of RAPD in detecting DNA polymorphism between varieties showed that primers with higher G and C contents pro! duced better DNA ampli_cation patterns "Wong\ Yeoh\ Lim\ + Looi\ 0886#[ However\ RAPD analyses do not always produce a very high number of reproducible frag! ments per run and this may limit its usefulness in varietal identi_cation[ This study thus aims to customize another PCR based technique\ ampli_ed fragment length poly! morphisms "AFLP#[ This method is based on the selective ampli_cation of restriction fragments from total genomic  Corresponding author[ DNA with di}erent primer pairs "Vos et al[\ 0884#[ We _rst focused on the restriction enzymes used for AFLP analysis and tested three combinations namely ApaI: TaqI\ EcoRI:MseI and HindIII:MseI[ We then added di}erent selective nucleotides to the 2? ends of the primers used and observed the banding pattern obtained[ Two criteria were considered^ the ability of the restriction enzyme and primer set to distinguish among genotypes and the number of loci:bands detected per experiment[ 1[ Results and discussion Three sets of restriction enzyme combinations were tested in the AFLP analysis of cassava varieties[ Eco! RI:MseI is generally useful for G¦C!poor genomes\ Hin! dIII:MseI for genomes with G¦C contents of 39Ð49 mol) and Apa0:TaqI for G¦C!rich genomes "Janssen et al[\ 0885#[ In our work\ we found that the EcoRI:MseI and HindIII:MseI combinations generally produced monomorphic AFLP pro_les "Figure 0a# when ampli_ed with a selection of primers "Table 0#[ However\ the ApaI: TaqI template\ provided strong polymorphic banding patterns "Figure 0b# when ampli_ed with all the primers tested[ This suggests that the cassava genome is high in G¦C content as enzymes that recognise high G¦C stretches\ like ApaI\ are able to restrict the cassava DNA[ This agrees well with the earlier observation that primers with high G¦C content showed better RAPD patterns