AFLP™ markers for DNA fingerprinting in cattle

Abstract

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with EcoRI and MseI is suggested, in mammals the enzyme combination EcoRI/TaqI produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 EcoRI/TaqI primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0.31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.

Pinniped phylogenetic relationships inferred using AFLP markers

“…Briefly, 100-400 ng of genomic DNA was first digested using TaqI (5 U in a 10 ml volume, 65 1C for 2 h) and then with EcoRI (5 U in a 20 ml volume, 37 1C for 2 h). EcoRI and TaqI adapters (Ajmone-Marsan et al, 1997;Madden et al, 2004) were ligated on to the digested DNA using T4 DNA ligase (1 U in a 50 ml volume, 37 1C for 3 h) and then diluted 10 times with low TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 8). For the preamplification, 5 ml of ligation mix was added to a 50 ml PCR reactions containing Tris-HCl (10 mM, pH 8.3), MgCl 2 (1.5 mM), KCl (50 mM), dNTPs (0.2 mM), Taq polymerase (1 U) and 50 ng each of the preamplification primers EcoRI-A and TaqI-C carrying one selective nucleotide.…”

Phylogeny of bovine species based on AFLP fingerprinting

“…AFLP is based on the selective amplification of restriction fragments by primers that bind to adapters and extend at their 3′ ends one to three extra nucleotides beyond the restriction sites. It has been developed as genetic mapping tool for plants, but is now used for genomic typing of several prokaryotic and eukaryotic organisms (Ajmone-Marsan et al, 1997;Heun et al, 1997;Mueller and Wolfenbarger, 1999;Savelkoul et al, 1999). In this study we show that with appropriate precautions AFLP fingerprints are informative for phylogeny and indicate recent speciation events of cattle species better than mitochondrial DNA.…”

“…(Ciulla et al, 1988;Sambrook et al, 1989). AFLP AFLP reactions were basically performed as described (Vos et al, 1995;Ajmone-Marsan et al, 1997). EcoRI and TaqI digested DNA was ligated to adapters 5′-CTCGTAG ACTGCGTACC/3′-CATCTGACGCATGGTTTAA and 5′-GACGATGAGTCCTGAC/3′-TACTCAGGACTGGA.…”

Discrimination between selected lines of pigs using AFLP® markers

“…Of the 1102 observed bands, of length 50-550 bp, 239 were polymorphic AFLP markers (Table 2). Procedures for DNA extraction, AFLP marker analysis, detection and scoring were also described by Ajmone-Marsan et al (1997). The proprietary AFLP marker analysis software (Keygene NV) allowed alignment of multiple gels based on the use of two lanes, containing a size ladder and a standard sample, that were included on every gel-half.…”

“…Amplified fragment length polymorphic (AFLP s ) markers have been used to construct phylogenic trees of cattle (Ajmone-Marsan et al, 1997;Buntjer et al, 2002), pig (SanCristobal et al, 2002) and goat (Ajmone-Marsan et al, 2001) breeds. Rather than being limited to between-breed studies, AFLP markers can provide information at the within-breed level.…”