Use of Amplified Fragment Length Polymorphism (AFLP) Markers in Surveys of Vertebrate Diversity

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[10] Use of Amplified Fragment Length Polymorphism (AFLP) Markers in Surveys of Vertebrate Diversity By Aure´lie Bonin, Franc¸ois Pompanon, and Pierre Taberlet Abstract

The amplified fragment length polymorphism (AFLP) technique is one of the most informative and cost-effective fingerprinting methods. It produces polymerase chain reaction (PCR)–based multi-locus genotypes helpful in many areas of population genetics. This chapter focuses on technical laboratory information to successfully develop the AFLP technique for vertebrates. Several AFLP protocols are described, as well as recommendations about important factors of the procedure such as the choice of enzyme and primer combinations, the choice and scoring of markers, the influence of the genome size on the AFLP procedure, and the control and estimation of genotyping errors. Finally, this chapter proposes a troubleshooting guide to help resolve the main technical difficulties encountered during the AFLP procedure. Introduction

Recently developed by Vos et al. (1995), the amplified fragment length polymorphism (AFLP) technique has become one of the most reliable and promising DNA fingerprinting methods, producing hundreds of informative polymerase chain reaction (PCR)–based genetic markers to provide a wide multi-locus screening of any genome. The AFLP analysis has been largely documented in the literature (Blears et al., 1998; Jones et al., 1997; Mueller et al., 1999; Savelkoul et al., 1999); here, we emphasize one of its more overlooked aspects—technical information. We discuss the important factors of the procedure (enzyme, primer, and marker choice; influence of genome size; genotyping errors) and give several recommendations and protocols to successfully develop AFLP markers for vertebrates.

AFLP Features and Applications

The essence of the AFLP procedure lies in the combined use of two basic tools in molecular biology: restriction, which reduces the total genomic DNA into a pool of fragments, and PCR, which amplifies a subset of

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these restriction fragments thanks to primers with arbitrary selective extensions (Mueller et al., 1999; Savelkoul et al., 1999). Three kinds of AFLP polymorphisms can then be observed: a mutation in the restriction site, a mutation in the sequence adjacent to the restriction site and complementary to the primer extensions, or a deletion/insertion within the amplified fragment (Ajmone-Marsan et al., 2001; Matthes et al., 1998). Polymorphisms are revealed by the presence of a fragment of a given size in some AFLP profiles versus its absence from other profiles. AFLP fingerprinting has been of great interest in population genetics because of several advantageous characteristics. First, it is the method of choice for studies of non-model organisms (Blears et al., 1998; Vos et al., 1995). Theoretically, it can be performed on any genome, regardless of its complexity and structure and without any prior sequence knowledge, in contrast to other kinds of molecular markers like microsatellites that require taxon-specific primers (Dogson et al., 1997). Practically, commercial AFLP primer sets are available that work on most organisms. Second, large numbers (up to several hundreds) of AFLP markers can be typed quickly and at low cost, offering fine-scale genome coverage (Blears et al., 1998; Mueller et al., 1999), although several studies have reported AFLP clustering in centromeric regions (Lindner et al., 2000; Young et al., 1998). AFLP markers are also largely independent, because 90% of them reflect point mutations in enzyme restriction site (Buntjer et al., 2002) that remove the fragments from the AFLP profile rather than change its size (Albertson et al., 1999). Third, AFLP markers usually reveal a greater amount of diversity compared to simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs) (Archak et al., 2003; Barker et al., 1999) and provide valuable fingerprints of organisms like birds in which microsatellite markers are difficult to obtain (Dogson et al., 1997; Knorr et al., 1999). Fourth, thanks to stringent hybridization conditions and relative insensitivity to template DNA concentration, the AFLP fingerprint is highly reproducible and reliable (Ajmone-Marsan et al., 1997; Bagley et al., 2001; Jones et al., 1997). As a result, it can be standardized, reproduced easily between different technicians and laboratories, and computer-scored for subsequent comparisons (Hong and Chuah, 2003). This makes it particularly well-adapted for large-scale studies involving several research centers (Jones et al., 1997). Fifth, only small amounts of genomic DNA are necessary to generate several informative AFLP profiles with different primer combinations (Blears et al., 1998; Savelkoul et al., 1999; Vos et al., 1995). Finally, AFLP markers have been shown to follow mendelian inheritance in plants (Blears et al., 1998; Savelkoul et al., 1999), as well as in animals (Ajmone-Marsan et al., 1997; Otsen et al., 1996).

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Despite its attractiveness, the AFLP method has some detrimental aspects. First, AFLP markers should be considered as dominant biallelic markers: fragment presence versus absence, with the fragment presence allele dominant over the absence allele (Ajmone-Marsan et al., 2001; Mueller et al., 1999). It is indeed difficult to distinguish between heterozygous individuals and individuals homozygous for the presence allele because of differential efficiencies between distinct PCR amplifications, unless exact genotypes can be inferred by means of pedigree studies (Van Haeringen et al., 2002). AFLP data are thus of poor information contents in analyses requiring precise estimations of heterozygosity. Nonetheless, several studies have managed to score up to 65% of the markers in a codominant way by rigorous standardization of profile intensities (Ajmone-Marsan et al., 1997), and new protocols have been developed to investigate AFLP-like codominant markers (Bradeen and Simon, 1998; Hakki and Akkaya, 2000). Second, fragments originating from distinct loci may have the same length by chance (homoplasy of size) (O’Hanlon and Peakall, 2000; Vekemans et al., 2002). Such fragments display exactly the same electrophoretic mobility and thus overlap on the AFLP profile, introducing an undesirable source of artifacts. However, comigration of distinct fragments has proven to be a rare event (Mechanda et al., 2003; Rosendahl and Taylor, 1997). Third, the AFLP procedure is particularly sensitive to contamination by exogenous DNA; even low and unobtrusive levels of bacterial or fungal contaminants, for example, may alter the AFLP profiles (Dyer and Leonard, 2000; Savelkoul et al., 1999). When working with organisms prone to such kinds of contaminations, one should take special precautions to ensure the reliability of the results. Originally worked out for plants and microorganisms, the AFLP analysis now finds more and more applications within the animal kingdom, especially in vertebrate species. Because their resolution power extends from the individual to the species level, AFLP markers have proven to be valuable tools in individual identification (Ovilo et al., 2000), sex determination (Griffiths and Orr, 1999; Questiau et al., 2000), parentage analysis (Questiau et al., 1999), genetic diversity assessment (Ajmone-Marsan et al., 2001, 2002; Mickett et al., 2003; Mock et al., 2002), population assignments (Campbell et al., 2003), investigations of population structure and estimations of gene flow (Dearborn et al., 2003; Jorde et al., 1999), hybridization studies (Bensch et al., 2002b; Nijman et al., 2003), and taxonomic and phylogenetic inferences (Albertson et al., 1999; Buntjer et al., 2002; Giannasi et al., 2001; Ogden and Thorpe, 2002). For higher taxonomic levels (e.g., infrageneric), the multi-locus fingerprint becomes too variable, increasing the risk of size homoplasy for the fragments generated

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(Vekemans et al., 2002) and rendering the analysis of AFLP profiles too complex and largely meaningless. In addition, AFLP markers have encountered considerable success in production of high-resolution genetic and quantitative trait loci (QTL) maps, in fish (Lindner et al., 2000; Liu et al., 2003; Naruse et al., 2000; Ransom and Zon, 1999; Young et al., 1998), amphibians (Kochan et al., 2003; Voss et al., 2001), birds (Groenen et al., 2000; Herbergs et al., 1999; Knorr et al., 1999), and mammals (Otsen et al., 1996; Van Haeringen et al., 2002). The AFLP technique has found a new and productive application in the search for informative single nucleotide polymorphisms (SNPs) in nonmodel vertebrates (Bensch et al., 2002a; Meksem et al., 2001; Nicod and Largiader, 2003).

AFLP Basic Steps for a Complex Genome

Genomic DNA Extraction and Preparation Any DNA extraction method is suitable to isolate total genomic DNA for subsequent AFLP analysis as long as it provides good quality DNA (no or limited degradation). For example, the DNeasy Tissue Kit (Qiagen) usually gives good results. After the extraction, an extra purification step might be necessary for DNA extracts containing restriction or PCR inhibitors. Genomic DNA Digestion The objective of restriction digestion is to reduce the big genomic DNA molecules into a mixture of fragments enabling posterior amplification and electrophoretic detection. The DNA amount needed for the AFLP procedure depends mainly on the genome size and structure, as well as on the DNA quality (see below for further details). The DNA concentration should be standardized among samples to yield comparable and homogeneous fingerprints. Restriction fragments of genomic DNA are generated using two restriction enzymes, a rare cutter like EcoRI (6-bp restriction site) and a frequent cutter like MseI (4-bp restriction site). The enzyme choice is discussed further in this chapter. After digestion, three categories of fragments exist in the mixture: fragments with EcoRI cuts at both ends (longer ones on average), fragments with MseI cuts at both ends (smaller ones on average), and fragments with an EcoRI cut at one end and a MseI cut at the other end. The AFLP protocol is designed to amplify and preferentially detect this last kind of fragment.

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Ligation of Oligonucleotide Adapters Using a T4 ligase enzyme, restriction fragments are ligated to doublestranded adapters specific to one particular restriction enzyme. The adapter structure is composed of a core sequence followed by an enzyme-specific sequence. These adapters are conceived so that the restriction site is not recreated after ligation, which eventually allows simultaneous digestion and ligation if restriction enzymes and T4 ligase are active at the same temperature. If ligation is performed after digestion, restriction enzymes should not be denatured after digestion, to prevent the formation of adapter concatenates. Once the ligation reaction is achieved, only one strand of the adapters is ligated to the restriction fragments, as the T4 ligase enzyme lacks a 30 to 50 activity. Preselective Amplification Preselective amplification aims to decrease the complexity of the initial fragment mixture by amplifying only a subset of fragments. It is conducted with a set of primers whose structure consists of a core sequence, an enzymespecific sequence, and a selective single-base extension at the 30 end. As a result, the adapter sequences offer primer binding sites and the selective base will recognize the fragments having the matching nucleotide after the restriction site. Fragment amplification can occur only if two primers can bind perfectly at both ends of the fragment, so statistically, 1 fragment out of 16 (4  4) originally present in the mixture will be amplified. Before the preselective amplification, the preselective mix undergoes an initial incubation at 72 , taking advantage of the 30 to 50 ligase activity of the DNA polymerase to complete the ligation of the adapters to the restriction fragments before first denaturation. Selective Amplification The selective amplification is based on the same principle as the preselective one. Using primers identical to the preselective primers, plus one or two extra selective bases at the 30 end, a second complexity reduction is performed on the pool of preselective fragments. Selective primers thus have two or three selective bases, depending on the genome complexity. Finally, after two rounds of amplification, the mixture complexity is divided by a 256 (44) factor (selective primers both carrying 2 selective bases) or by a 4096 (46) factor (selective primers both carrying 3 selective bases). During this step, hybridization conditions are very stringent: a ‘‘touchdown’’ PCR (i.e., with a particularly high annealing temperature decreasing progressively on the first cycles) ensures highly specific amplification and thus good reproducibility of the technique.

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EcoRI selective primers are specially designed to have a higher annealing temperature than MseI selective primers. As a result, the ‘‘touchdown’’ PCR allows a preferential amplification of EcoRI/MseI versus MseI/MseI fragments. In addition, because only EcoRI primers are fluorescently or radio-labeled, the AFLP patterns display EcoRI/MseI fragments exclusively in a 50–500 bp detection range, with EcoRI/EcoRI fragments being statistically longer. Finally, after the complexity reduction and the preferential amplification and detection of EcoRI/MseI fragments, 20–150 discrete bands can be visualized on a typical AFLP profile. Electrophoresis and Analysis of the AFLP Profiles After selective amplification, the restriction fragments are denatured, separated by electrophoresis, and visualized by either fluorescence or radioactivity (autoradiography) detection. Autoradiography may enable easier characterization of codominant markers, given that the detected radioactive signal is known to increase linearly with the number of labeled fragments (Hawkins et al., 1992). Automated sequencers are now more exploited for the separation and detection of fluorescent AFLP fragments. Multi-locus fingerprints can then be visualized using software packages like GeneScan Analysis 3.1 (PerkinElmer). This program determines the size of amplified restriction fragments with the help of an internal size standard (Rox 500, Perkin-Elmer), then it classifies them according to their size with single-base resolution. The fragments (bands) can thus be scored as present/absent for a given size, producing a binary matrix. Some software packages can construct this binary matrix semiautomatically, on which subsequent analyses can be performed (Genographer, available at http://hordeum.oscs.montana.edu/genographer/) (Papa et al., 2005). Capillary electrophoresis with an automated sequencer has been shown to increase the AFLP data throughput and reliability (Papa et al., 2005). Certain parameters should then be optimized according to the manufacturer’s instructions for high-quality fingerprints. For example, longer injection times improve the peak intensities for longer fragments, which tend to be loaded with more difficulty during the electrokinetic process. Important Factors in the AFLP Procedure

Choice of Enzyme and Primer Combinations Restriction enzymes and primer pairs are key parameters in the AFLP procedure, influencing the number of amplified fragments, the level of polymorphism detected, and the possibility of comparing AFLP profiles from different studies. In theory, any restriction enzyme can be used in an

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AFLP protocol. However, some of them cut too often, generating comigrating non-homologous fragments, or too rarely, reducing the probability of polymorphism detection. Thus, the choice of enzyme combinations for the AFLP protocol consists of a compromise between adequate levels of polymorphism and readable AFLP profiles. Restriction patterns for several enzyme combinations can be prescreened on agarose gel, and the range of fragment sizes (ideally between 50 and 500 bp) provides an insight into the complexity of the mixture produced. The EcoRI/MseI enzyme combination originally published by Vos et al. (1995) is traditionally used for AFLP analysis, although several studies of vertebrate diversity are based on other enzyme combinations, for example, NotI/HpaII (Voss et al., 2001), SseI/ MseI (Van Haeringen et al., 2002), or EcoRI/MspI (Knorr et al., 1999). It appears that the EcoRI/TaqI combination is particularly efficient in generating high-quality AFLP profiles for avian and mammalian genomes (Ajmone-Marsan et al., 1997; Knorr et al., 1999). This may be because the TaqI restriction site contains a CpG dinucleotide, which is a hot spot for mutations in the genome of hot-blooded animals (Ajmone-Marsan et al., 1997; Gardiner-Garden and Frommer, 1987). When selecting enzymes for an AFLP use, one should exclude enzymes sensitive to DNA methylation, which is a phenomenon known to be tissue- and age-specific in plants and in animals (Bird, 2002), unless AFLPs are being used to investigate DNA methylation patterns (Cervera et al., 2002). The length and bases composition of primer pairs appropriate for the AFLP procedure are also important, because the fine-tuning of polymorphisms detected relies mainly on the addition or suppression of 30 selective bases. As a result, multiple combinations of different selective primers give access to hundreds of polymorphic markers. Vos et al. (1995) have established that selectivity is good for selective primers with elongations constituted of one or two extra bases. With three or more selective bases, nonspecific annealing may occur at the first selective base after enzymespecific sequence, leading to artifactual AFLP bands. As for the choice of appropriate primer pairs to use in organisms never analyzed with AFLP markers, there are unfortunately no general rules except that in most cases, an extensive screening of different primer combinations is necessary. Trying all pairwise combinations is a reasonable and effective approach, but it can rapidly become expensive and time-consuming. If nucleotide frequencies are available for the organism under consideration, an alternative strategy is to test in priority primer pairs with similar base frequencies. In practice, the choice of primer pairs should be performed on four to eight individuals representing the widest possible range of genetic diversity. Testing two or three dozen different primer pairs then is generally sufficient to select three to five clear primer combinations at the end.

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Influence of Genome Size and Complexity The size and complexity of the studied genome can have a tremendous influence on the AFLP analysis in three ways. First, they will condition the total quantity of DNA required for the initial restriction. Indeed, a general rule in the AFLP procedure is that the larger the genome is, the larger the required amount of DNA is. It is indeed essential to ensure a sufficient number of AFLP loci copies, even if this number may vary according to the proportion of repeated sequences in the genome. Although the AFLP protocol is relatively insensitive to the template concentration (Jones et al., 1997; Vos et al., 1995), stochastic amplifications can occur if DNA is limiting, so we recommend using DNA slightly in excess, with appropriate concentrations of reactants and enzymes. For example, 400 ng of DNA is necessary to produce clear, intense AFLP patterns for the common frog (Rana temporaria, genome size: 4.3  109 bp) with the protocol mentioned below. Genome size for many vertebrate species can be checked in the Animal Genome Size Database (http://www.genomesize.com). Second, the genome size and complexity will determine the number of selective bases used for the preselective and selective amplifications. Traditionally, preselective primers count one selective base, whereas the selective primers count three selective bases. However, more complex genomes may require the use of additional selective bases, that is, two selective bases in the first amplification step and four in the second one. For some highcomplexity genomes, it is impossible to achieve clear AFLP profiles only by varying the enzyme choice or the number of selective bases. In this case, a variant of the AFLP procedure can be considered: the three endonuclease (TE)-AFLP technique (Van der Wurff et al., 2000). Compared to traditional AFLP, this technique, based on the use of three restriction enzymes, has an extra reduction step due to a selective ligation. Finally, the ploidy level is an important factor to consider when developing AFLP markers for vertebrates, because differences in ploidy levels can occur within natural populations and species in fish, amphibians, and reptiles, although this phenomenon is less common than in the plant kingdom (Otto and Whitton, 2000). It is, therefore, a potential source of bias for AFLP pattern comparisons and allele frequencies estimates and should remain a major concern when working with individuals of unknown karyotypes. Choice and Scoring of Markers The choice and scoring of markers are perhaps the most critical points of the AFLP procedure—or at least the most exacting steps in terms of experience and rigor. They largely determine the reproducibility and reliability of the technique. A good marker has to fulfill several requirements. First, it has to be polymorphic enough to be informative. A peak appearing

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for only one individual must raise suspicion, because contamination and technical artifacts can never be totally ruled out. Second, it must be clearly distinct (at least 1 bp) from other peaks on the profiles, so that it can be scored properly. Overlapping peaks should be discarded as early as the pilot study for primer choice, because dealing with more individuals usually adds more confusion to the profiles. Third, as many individuals as possible should be scored unambiguously for the chosen marker. AFLP profiles usually display many potential markers, but most of them may not be helpful for subsequent analysis, when their scoring is doubtful. When it comes to the scoring of the markers, several protocols can be adopted. One can choose to score very high and very low peaks the same way. Nevertheless, this method is uncertain and not conservative enough, as a considerable number of low peaks can be attributed to background noise. An absolute or relative intensity threshold can also be set up, under which a peak is considered to be absent. To determine a scoring threshold, we personally scrutinize a drop in intensity among the peaks corresponding to the marker under consideration. A clear discontinuity indicates the frontier between non-selective (i.e. background) and selective amplifications and it usually shows up around 10% of the highest peak’s intensity. Whatever the scoring protocol established, it appears essential to follow it strictly, and because of the amount of subjectivity entering in this process, the same person should be charged to score all the data, to ensure consistency of the results. Normalization of the profiles obtained from different runs and double reading of the data by two experimenters are also efficient measures to take to facilitate and confirm the scorings. AFLP and Genotyping Errors Even if several studies have reported high levels of reproducibility of the AFLP technique (Ajmone-Marsan et al., 1997; Bagley et al., 2001; Jones et al., 1997), the problem of genotyping errors should not be overlooked, because every AFLP data set includes typing errors that might greatly bias the final results. Methods to track and monitor such errors are not the purpose of this chapter, so we will mention only major recommendations to fulfill this objective. First, when developing AFLP markers for a new organism, a systematic pilot study should be carried out before any extensive investigation, providing the opportunity to acquire experience with the AFLP technique and to achieve reproducibility. Second, including blind samples throughout the procedure provides a reliable way to (1) estimate the error rate, (2) detect contamination, tube mixings, or biochemical anomalies, and (3) eliminate unreliable markers (i.e., makers that are unstable or difficult to score). Third, automation, capillary electrophoresis, and semiautomatic scoring have been proven to limit the overall experimental

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error (Papa et al., 2005). Like every other genotyping method, the AFLP procedure is subject to common errors (chance, human factors, technical artifacts), but the vast majority of errors are specific to the AFLP technique. Indeed, differences in peak intensities and comigration of non-homologous fragments cause most of them, whereas contamination, for example, counts for a marginal number (Bonin et al., 2004). In the literature, reproducibility of AFLP data in vertebrates is usually higher than 95% (Ajmone-Marsan et al., 1997, 2001; Bagley et al., 2001; Ovilo et al., 2000).

Methods: AFLP Protocols for Vertebrates

Method 1. AFLP Procedure with TaqI/EcoR1 Enzymes Combination (Birds, Mammals) Volumes are indicated for one sample. 1. Digestion of total DNA. Digest genomic DNA (50–500 ng) in a 25-l reaction containing DNA, 2.5 l of 10 TaqI buffer (New England Biolabs [NEB]), 5 units of restriction endonuclease TaqI (NEB) and q.s.p ultrahigh-quality (UHQ) water. Incubate up to 2 h at 65 , then add 1.5 l of 10 EcoRI buffer (NEB), 5 units of restriction endonuclease EcoRI (NEB), and make up to 40 l with UHQ water. Incubate up to 2 h at 37 . 2. Preparation of 10 M double-stranded adapters. Prepare 10 M double-stranded adapters by mixing equal volumes of 10 M individual synthetic oligonucleotides. Denature by heating 5 min at 65 in a hot block and cool slowly down to room temperature. Store adapters at 20 . When being aliquoted for subsequent ligation, adaptors should be kept in a refrigerated rack to avoid denaturation. 3. Ligation of adapters to restriction fragments. Ligate adaptors to 40 l of the digested genomic DNA by adding 1 l of 10 M EcoRI adapter, 5 l of 10 M TaqI adapter, 1 l of 10 mM ATP, 0.5 l of 1 mg/l bovine serum albumin (BSA), 1 l of 10 T4 ligase buffer (NEB), 100 units of T4 DNA ligase (NEB) and UHQ water to 50 l. Incubate another 3 h at 37 . 4. Preparation of template DNA for preselective amplification. Dilute the ligation reaction mixture 5–10 times with UHQ water. Store diluted DNA at 20 . 5. Preselective amplification. Prepare the preselective mix with the following components: 3 l of diluted template DNA, 2.5 of l 10 Amplitaq buffer (Applied Biosystems), 1.5 l of 25 mM MgCl2, 2 l of

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10 mM dNTPs, 0.5 l of 10 M EcoRI preselective primer, 0.5 l of 10 M TaqI preselective primer, 1 unit of Amplitaq DNA polymerase, and UHQ water to 25 l. Preamplify using the following program: initial incubation 2 min at 72 ; 25–30 cycles of 30 s at 94 , 30 s at 56 , and 2 min at 72 ; final extension 10 min at 72 ; storage at 4 . For extended periods (few days to several months), preselective product should be stored at 20 . After amplification, the preselective PCR product can be monitored on a 2% agarose gel and usually gives a faint smear in the 100–1000 bp range. 6. Preparation of template DNA for selective amplification. Dilute the preselective product 20 times with UHQ water. Store diluted DNA at 20 . 7. Selective amplification. Prepare the selective mix with following components: 5 l of diluted preselective product, 2.5 l of 10 Amplitaq buffer, 2.5 l of 25 mM MgCl2, 2 l of 10 mM dNTPs, 0.5 l of 10 M labeled EcoRI selective primer, 0.5 l of 10 M TaqI selective primer, 1 unit of Amplitaq Gold DNA polymerase, UHQ water to a final volume of 25 l. Amplify using the following program: initial incubation 10 min at 95 ; 13 cycles of 30 s at 94 , 1 min at 65 (first cycle, then decrease of 0.7 for the 12 last cycles) and 1 min at 72 ; 23 cycles of 30 s at 94 , 1 min at 56 , and 1 min at 72 ; final extension 10 min at 72 ; storage at 4 . Selective product can be stored several days at 20 . On a 2% agarose gel, the selective PCR product gives a clearly distinguishable smear in the 100–500 bp range. 8. Electrophoresis of AFLP products. AFLP products can be separated, detected, and sized with any automated DNA sequencer. Refer to the manufacturer for further instructions. Method 2. AFLP Procedure with MseI/EcoR1 Enzymes Combination (All Vertebrates except Mammals) 1. Preparation of 10 M double-stranded adapters. See Method 1, step 2. 2. Simultaneous digestion and ligation of total DNA. Digest and ligate total genomic DNA (50–500 ng) in an 11-l reaction containing DNA, 1.1 l of 10 T4 ligase buffer (NEB), 1.1 l of 0.5 M NaCl, 0.55 l of 1 mg/l1 BSA, 1 l of 10 M MseI adapter, 1 l of 10 M EcoRI adapter, 5 units of restriction endonuclease MseI (NEB), 5 units of restriction endonuclease EcoRI (NEB), 5 units of T4 DNA ligase (NEB), and UHQ water to 11 l. Incubate 2 h at 37 . 3. For the followings steps, see Method 1, steps 4–8.

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comparing macromolecules TABLE I Technical Problems Encountered during AFLP Procedure, Their Possible Causes, and Solutions Problem

Possible causes

Solutions

 No DNA restriction and/or no amplification

 DNA extract contains restriction or PCR inhibitor  Restriction enzymes or T4 DNA ligase or adapters are limited  Adapters are denatured

 Dilute the DNA extract  Purify the DNA extract before the AFLP procedure  Check that restriction enzymes, T4 DNA ligase and adapters are in excess   Heat the adapters 5 min at 65 and let cool down to room temperature; store in the freezer before use

 No amplification after the preselective PCR

 Ligation of the adapters to the restriction fragments is not completely achieved before first denaturation

 Check that the preselective PCR program includes an  initial 2-min incubation at 72

 No amplification after the selective PCR

 Amplitaq Gold DNA polymerase has not been activated before amplification

 Check that the selective PCR program includes an  initial 10-min activation at 95

 No or few polymorphism

 The chosen enzymes do not detect polymorphic sites

 Test other enzyme combinations

 Profiles with not enough peaks

 Only a few peaks can be amplified

 Discard some of the selective bases

 Profiles with not enough peaks for a large genome

 Enzymes cut within repeated sequences

 Test other enzyme combinations

 Profiles with too many peaks

 Too many peaks can be amplified

 Add some selective bases

 Profiles with too many peaks, even with three selective bases

 Genome too large

 Test preselective primers with two selective bases and selective primers with four selective bases  Test TE-AFLP (continued)

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AFLP markers for vertebrates TABLE I (continued) Problem

 No reproducibility

 Parasite peaks

Possible causes  Too much/too few DNA leading to stochastic amplification  Degraded DNA  ‘‘Star activity’’ of the restriction enzymes, which cut nonspecifically  Contamination during one or several steps of the procedure

Solutions  Use less/more DNA for the whole procedure  Check for DNA quality  Shorten the digestion time or modify the enzyme buffer composition

 Use disposable tips and tubes throughout the procedure  Monitor contamination using negative controls at each step of the protocol and sample references

 Weak profiles

 Too little DNA

 Low fluorescence/ radioactivity levels

 Try the AFLP procedure with more DNA  Load more AFLP products for the electrophoresis  Check the quality of primer labeling

 Presence of peak doublets

 Incomplete addition by the Taq polymerase of additional adenine residues at the 30 end of amplified fragments

 Difficulties when comparing different profiles

 Profiles come from different  Normalize the intensities runs and display different in the Genographer levels of intensities software  Use some samples as internal standards

 Add a final 10-min  extension at 72 after amplification

Troubleshooting Guide

Table I lists the solutions to the principal technical problems encountered when developing AFLP markers for vertebrates.

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[11] Use of AFLP Markers in Surveys of Arthropod Diversity By Tamra C. Mendelson and Kerry L. Shaw Abstract

Arthropods comprise the most diverse group of animals on earth and as such have been the subject of considerable evolutionary research. For example, much of our understanding of the genetic basis of evolutionary change is derived from the insect genus Drosophila, one of the most wellstudied organisms in biology. Arthropods are also of tremendous economic importance as both providers and chief destroyers of food for human consumption. Thus, the genetic diversity of arthropods is of interest from both a pure research perspective and for practical economic reasons.The amplified fragment length polymorphism (AFLP) method of genetic analysis, developed in the early and mid-1990s (Vos et al., 1995; Zabeau, 1992; Zabeau and Vos, 1993), offers a relatively new method for assessing genetic diversity and has been increasingly applied in studies of arthropods. Originally coined selective restriction fragment amplification (SRFA) (Zabeau and Vos, 1993), the method was renamed (Vos et al., 1995) presumably to reflect its similarity to restriction fragment length polymorphism (RFLP). Since then, AFLPs have become a popular tool in both population genetics to estimate population parameters such as heterozygosity, F-statistics, migration rates, and genetic distances, as well as phylogenetics, to infer relationships among closely related taxa. In arthropods, AFLPs have been used to assess genetic variation both within and between

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