Amplified Fragment Length Polymorphism (AFLP) : A Review of The Procedure and Its Applications

Journal of Industrial Microbiology & Biotechnology (1998) 21, 99–114
 1998 Society for Industrial Microbiology 1367-5435/98/$12.00

http://www.stockton-press.co.uk/jim
Amplified fragment length polymorphism (AFLP): a review of

the procedure and its applications
MJ Blears1,2, SA De Grandis2, H Lee1 and JT Trevors1
1
Department of Environmental Biology; 2Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada
N1G 2W1
Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to
DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded
nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers
complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as
selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence
of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant
DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. How-
ever, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships
and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the
molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms.
Keywords: AFLP; molecular markers; genetic mapping; PCR; polymorphism; DNA fingerprinting
Introduction Netherlands). With AFLP, molecular genetic polymor-

phisms are identified by the presence or absence of DNA
Amplified fragment length polymorphism (AFLP) is a new
fragments following restriction and amplification of gen-
molecular technique for fingerprinting DNAs of any origin
omic DNA. Figure 1 outlines the four steps of the AFLP
and complexity. It has a number of potential applications
technique: DNA digestion, ligation, amplification and gel
such as monitoring inheritance of agronomic traits in plant
analysis. Genomic DNA is first digested by two restriction
and animal breeding, diagnostics of genetically inherited
enzymes. Double-stranded oligonucleotide adapters, hom-
diseases, pedigree analysis, forensic typing, parentage
ologous to one 5′- or 3′-end generated during restriction
analysis, screening of DNA markers linked to genetic traits
digestion, are ligated to the DNA fragments. The ligated
and microbial typing. The AFLP technique has several
DNA fragments are amplified by PCR using primers comp-
advantages over other DNA fingerprinting systems. The
lementary to the adapter and restriction site sequence with
most important of these are the capacity to inspect an entire
additional selective nucleotides at their 3′-end. The use of
genome for polymorphism and its reproducibility. AFLP
selective primers reduces the complexity of the mixture.
can be applied to any DNA samples including human, ani-
Only those fragments with complementary nucleotides
mal, plant and microbial DNAs, giving it the potential to
become a universal DNA fingerprinting system. The objec-
tives of this review are to discuss, in detail, the AFLP pro-
cedure and its applications.
Principle of the method

Amplified fragment length polymorphism (AFLP) is the
selective amplification of restriction fragments from a
digest of total genomic DNA using the polymerase chain
reaction (PCR). The technique, developed by Zabeau and
Vos [46], is patented by Keygene NV (Wageningen, The
Correspndence: JT Trevors and H Lee, Department of Environmental

Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Abbreviations: A, adenine; AFLP, amplified fragment length polymor-
phism; ATP, adenosine triphosphate; bp, base pair(s); C, cytosine; cM,
centimorgan(s); dCTP, deoxycytidine triphosphate; DNA, deoxyribonu- Figure 1 A schematic displaying the four basic steps of AFLP: diges-
cleic acid; G, guanine; HG, hybridization group; ISTR, inverse sequence- tion, ligation, amplification and gel analysis. Genomic DNA is digested
tagged repeat; kbp, kilobase pair(s); Mb, megabases; P, phosphorus; PCR, by restriction enzymes and adapters are ligated to the restriction fragments.
polymerase chain reaction; PFGE, pulsed-field gel electrophoresis; RAPD, A subset of the ligated fragments are amplified by PCR, using primers
randomly amplified polymorphic DNA; RFLP, restriction fragment length with selective nucleotides at the 3′-end. Polymorphism is revealed by run-
polymorphism; T, thymine ning the amplified products of various samples on a denaturing polyacryla-
Received 19 December 1997; accepted 3 June 1998 mide gel.
Amplified fragment length polymorphism
MJ Blears et al
100
extending beyond the restriction site will be amplified by
the selective primers under stringent annealing conditions.
Polymorphisms are revealed by analysis of amplified frag-
ments on a denaturing polyacrylamide gel, and comparison
of the patterns generated for each sample.
Digestion of genomic DNA

Restriction fragments are generated using two restriction
endonucleases, a ‘rare’-cutting enzyme with 6- to 8-base
recognition, in combination with a ‘frequent’-cutting
enzyme of 4-base recognition. The high degree of speci-
ficity of restriction enzymes results in production of a
reproducible set of DNA fragments. The complexity of the
genome and factors such as the methylation status of the
DNA influence the choice of enzymes [1]. The use of two
different enzymes allows the researcher to manipulate the
number of fragments generated for amplification and pro-
duce fingerprint patterns of desired complexity. In addition, Figure 2 A schematic outlining the ligation of adapters to the ends of
a restriction fragment. Genomic DNA is first restricted by EcoRI and MseI.
when the fragments are cut using two enzymes, a large Double-stranded adapters, complementary to the short single-strand exten-
number of different fingerprints can be produced using the sion generated by the restriction enzymes, are ligated to the DNA frag-
combinations of only a few primers. Examples of rare-cut- ment. The EcoRI and MseI recognition sites are not restored by ligation
ting enzymes employed in AFLP include EcoRI, AseI, Hin- because of a base change in the adapter sequence (shown in lower case).
dIII, ApaI and PstI. MseI and TaqI are the commonly used
frequent-cutters. However, the DNA of most eukaryotes is
AT-rich making MseI (recognition sequence TTAA) the In addition, adapter-to-adapter ligation is prevented by
preferred frequent-cutter for AFLP [45]. The frequent-cut- using nonphosphorylated adapters. Both of these features
ter generates small fragments within the desired size range ensure that adapters are ligated to virtually all restriction
of 100–1000 base pairs required for efficient PCR amplifi- fragments.
cation and separation on denaturing polyacrylamide gels The complexity of the DNA mixture can be reduced prior
[1]. to amplification using biotin-labeled adapters complemen-
Three types of restriction fragments are generated fol- tary to the rare-cutting restriction half-site (eg biotin-lab-
lowing digestion: (i) fragments cut by the rare-cutting eled EcoRI adapters). Biotinylated restriction fragments cut
enzyme on both ends; (ii) fragments cut with the frequent- by the rare-cutting enzyme are selected using streptavidin-
cutting enzyme on both ends; and (iii) fragments that have coated magnetic beads as described by Zabeau and Vos
been cut by both the rare-cutter and frequent-cutter. Using [46]. The use of biotin-labeled adapters separates fragments
EcoRI and MseI restriction enzymes as examples, EcoRI- having at least one rare-cutting restriction site from the
EcoRI, MseI-MseI and EcoRI-MseI fragments would be large number of fragments having two frequent-cutting
produced during restriction enzyme digestion. More than restriction sites (eg EcoRI-EcoRI and EcoRI-MseI frag-
90% of the fragments are expected to have frequent-cutter ments are separated from MseI-MseI fragments). This step
sites on both ends (eg MseI-MseI fragments). is not essential but may be useful for decreasing back-
ground smears on gels.
Ligation of adapters
Selective amplification
AFLP is not dependent on prior sequence knowledge. Dou-
ble-stranded nucleotide adapters (10–30 base pairs long), AFLP primers for selective amplification contain three
complementary to the sticky ends of the corresponding types of DNA sequence: the 5′ region complementary to
restriction site, are ligated to the restriction fragments using the adapter, the restriction site sequence and the 3′ selective
T4 DNA ligase (Figure 2). The sequence of the adapters nucleotides (Figure 3). Two AFLP primers are used; one
and the adjacent restriction half-site serve as primer binding primer is complementary to the adapter and adjacent rare-
sites for subsequent PCR amplification. Adapters are com- cutter restriction site sequence with one to three additional
posed of two synthetic oligonucleotides that are in part selective nucleotides at the 3′-end (eg EcoRI primer 3′-
complementary to each other and form a double-stranded XXX, where X denotes the selective nucleotides), and the
structure in solution under appropriate conditions. Ligation second primer is complementary to the adapter and fre-
does not restore the original restriction enzyme site because quent-cutter recognition site sequence with an additional
of a base change incorporated into the adapter sequence selective one- to three-base extension (eg MseI primer 3′-
(Figure 2). This change in the recognition site prevents XXX).
restriction from taking place after ligation has occurred, Following the restriction-ligation reaction, a limited
enabling restriction and ligation reactions to be performed number of ligated restriction fragments are selectively
in the same tube. With these reactions occurring simul- amplified by the AFLP primers (Figure 3). Only a subset
taneously, any fragment-to-fragment product is restricted. of the template fragments, with complementary nucleotides
MJ Blears et al
101
Figure 4 A schematic illustrating the selectivity of AFLP primers. The

first example shows successful extension of a primer with three selective
nucleotides matching the template sequence. The arrow indicates the direc-
Figure 3 A schematic depicting the PCR amplification of a ligated tion of DNA synthesis. In the second example, there is a mismatch
restriction fragment. The AFLP primers have three regions, the 5′-end between the three selective bases and the restriction fragment preventing
complementary to the oligonucleotide adapter sequence, the restriction site extension of the primer. The final example illustrates the mismatch ampli-
sequence and the 3′ selective nucleotides. The horizontal arrows indicate fication that can occur with primers having four selective bases. D = G,
the direction of DNA synthesis. With each PCR cycle, the amount of DNA A or T.
is doubled.
the PCR primers. There is an almost linear correlation

extending beyond the restriction site, will be amplified between the number of amplified fragments and the genome
under stringent annealing conditions (Figure 4). The nucle- size [45]. As the number of selective nucleotides is
otide extensions on the 3′-end of the primers serve two increased, the complexity of the DNA fingerprint decreases.
purposes: (i) they allow a variety of restriction fragment The number of amplified fragments is reduced approxi-
subsets to be amplified; and (ii) they provide additional mately four-fold with each additional selective base,
possibilities for polymorphisms to be detected beyond the assuming random base distribution [45]. Although there is
restriction site itself. no standard method for chosing selective nucleotides,
Although fragments cut only by the frequent-cutter (eg nucleotide extensions composed of rare di- or tri-nucleo-
MseI-MseI fragments) are the predominant species tides will greatly reduce the number of amplified fragments.
(⬎90%), fragments cut by both enzymes (eg EcoRI-MseI The length and nature of the base extension on the 3′-end
fragments) are preferentially amplified. There are two of the primers can be manipulated to generate fingerprints
reasons for the efficiency: (i) the primer complementary to of desired complexity.
the rare-cutting restriction site and adapter (eg EcoRI For small genomes of 106–107 base pairs (bp), one to
primer) has a higher annealing temperature than the primer two selective nucleotides on the 3′-end of each primer may
of the frequent-cutter (eg MseI primer); and (ii) the frag- be sufficient to reveal polymorphism. More complex gen-
ments cut by both enzymes (eg EcoRI-MseI fragments) are omes ranging from 108–109 bp will require additional selec-
amplified using two primers (eg MseI and EcoRI primers) tive nucleotides to yield the desired number of amplified
preventing the formation of an inverted repeat at the ends fragments. Typically, the ideal number of amplified restric-
[45]. Base-pairing of the ends of the fragment forms a stem- tion fragments ranges from 50–100 [45].
loop structure which competes with primer annealing. The Although the complexity is reduced with each additional
PCR conditions are an important feature of the AFLP tech- selective nucleotide, the selectivity is maintained with
nique. Most restriction digestion techniques generate such nucleotide additions to a maximum of three selective bases.
complex patterns that differences cannot be discerned. The fingerprints generated are merely a subset of the pre-
However, by increasing the efficiency of the primer comp- vious pattern. However, selectivity is lost with a 4-base
lementary to the rare-cutter, only a subset of the fragments extension. This loss of selectivity was demonstrated by Vos
(⬍10%) are efficiently amplified (eg EcoRI-MseI et al [45] when additional bands were amplified using four
fragments) to allow polymorphism to be revealed. selective bases, which had not been detected in the corre-
The number of amplified fragments is determined by the sponding fingerprint with primers having three selective
complexity of the genomic DNA, the choice of enzymes, bases. This indicates a tolerance of mismatches during
as well as the number and type of selective nucleotides in amplification by primers with 4-base extensions (Figure 4).
MJ Blears et al
102 Table 1 Exprimental protocols of studies utilizing the AFLP technique

Gel analysis
for analysis of genetic diversity in eukaryotes
Prior to loading the amplified products into polyacrylamide
gels, the DNA samples are denatured by heating at 90– Reference Organism Preselective Selective No. of
95°C for 3–5 min. To prevent the occurrence of double studied primers primers primer
pairs
bands or ‘doublets’ on the gel due to unequal mobility of tested
the two strands of an amplified fragment, only one strand
is labeled. Two labeling techniques, using either a radioac- [8] Pisum sativum L. MseI 3′-1 MseI 3′-3 N/A
tive label or a fluorescent dye are employed. Typically, the (pea) EcoRI 3′-1 EcoRI 3′-3ab
primer corresponding to the rare-cutting restriction enzyme [9] Oryza sativa Not utilized TaqI 3′-3 1
is labeled by phosphorylating the 5′-end with [␥-32P or (rice) japonica PstI 3′-2bc
and indica
-33P]ATP using T4 polynucleotide kinase. A relatively new cultivars
labeling technique involves the use of fluorescent dyes as [12] Globodera MseI 3′-0 MseI 3′-2 12
described below. rostochiensis and EcoRI 3′-0 EcoRI 3′-2a
Following electrophoretic separation of the amplified G. pallida (potato
fragments on a 4.5% or 5% polyacrylamide gel, typical cyst nematodes)
[13] Miscanthus spp MseI 3′-1 MseI 3′-3 6
AFLP patterns are visualized by autoradiography. The (perennial grass) EcoRI 3′-1 EcoRI 3′-3a
resulting banding patterns may be visualized by eye, or cap- [15] Lactuca spp MseI 3′-1 MseI 3′-3 3(4)
tured by high-resolution densitometric scanning for com- (lettuce) EcoRI 3′-1 EcoRI 3′-3a
puter-assisted analysis (eg Gel Compar 3.1 software, [28] Symbiotic fungi Not utilized PstI 3′-2 6 primers
of fungus-
Applied Maths, Kortrijk, Belgium). growing ant
Chalhoub et al [8] evaluated silver staining for the vis- Cyphomyrmex
ualization of AFLP products in polyacrylamide gels minutus
(Table 1). Comparison of AFLP fingerprints visualized by [32] Camellia sinensis MseI 3′-1 MseI 3′-3 5
autoradiography and silver staining revealed similar sensi- (L.) O. Kuntze EcoRI 3′-1 EcoRI 3′-3a
(Indian and
tivity and resolution. The silver staining procedure does not Kenyan tea)
include labeled primers, thus both strands of amplified frag- [35] Glycine max and Not utilized TaqI 3′-2/3a 6
ments are visualized. Regardless of the presence of double Glycine soja PstI 3′-2/3
bands, no complexities in the banding patterns were (soybean) HindIII 3′-2/3
[36] Hordeum vulgare MseI 3′-1 12 MseI 3′-3 96
observed. An advantage of the silver staining technique is (barley) EcoRI 3′-1 8 EcoRI 3′-3a
the possible recovery of fragments from the dried gels at a [38] Vitis vinifera L. MseI 3′-1 MseI 3′-3 8
later date by simple rehydration and direct transfer to a Sangiovese and EcoRI 3′-1 EcoRI 3′-3a
PCR tube for amplification. This is particularly useful in Colorino
cases where further characterization of specific fragments (grapevine
cultivars)
is required such as cloning, sequencing, preparation of [39] Lens (lentil) Not utilized MseI 3′-2/3 4
probes or development of sequence-characterized ampli- PstI 3′-2/3a
fied regions. [41] Astragalus Mse 3′-1 MseI 3′-3 9
Similar results were obtained by Cho et al [9] in a study cremnophylax var EcoRI 3′-1 EcoRI 3′-3a
cremnophylax
evaluating the cloning and mapping of AFLP fragments (endangered
from silver-stained polyacrylamide gels (Table 1). Similar sentry milk-vetch)
banding patterns were obtained using 32P and silver stain-
ing. However, the resolution of silver-stained gels was N/A = information not available.
found to be greater than that of 32P-labeled gels particularly a
5′-end of primer labeled using [␥-32P or ␥-33P]ATP.
b
in the region of the gel containing fragments greater than Selective primers not labeled for silver staining.
c
5′-end of primer labeled using [32P]dCTP.
300 bp. The silver staining method is advantageous in that
it does not involve the use of radioactive isotopes. In
addition, the high resolution and ability to excise bands Preamplification reduces the overall complexity of the mix-
directly from the gel make silver staining a more useful ture up to 16-fold, allowing the target sequence to become
and versatile detection method for AFLP amplification pro- the predominant species. The inclusion of preamplification
ducts. reduces background smears in the fingerprint pattern
resulting from mismatched amplification, that is tolerated
Inclusion of PCR preamplification for complex at a low level, by primers with three selective bases
genomes (Figure 4).
A two-step amplification strategy was developed for com-

AFLPTM kits available
plex genomes (108-109 bp) requiring three selective nucleo-
tides on one or both primers. The first PCR amplification, AFLPTM Plant Mapping Kit
called preamplification, utilizes primers having a single or PE Applied Biosystems (Foster City, CA, USA) has
no selective nucleotide. PCR products from preamplifi- developed an AFLPTM Plant Mapping Kit based on the
cation are diluted and used as templates for a second ampli- AFLP procedure patented by Keygene NV (Wageningen,
fication reaction using primers with full base extensions. The Netherlands). Two modules are available depending on
MJ Blears et al
103
the genome size. The Small Plant Genome Kit is used for software currently available to conduct cluster analysis
genomes ranging from 50–500 megabases, and the Regular using GeneScan files directly, it is possible to construct
Plant Genome Kit is for genomes of 500–5000 megabases. dendrograms using software packages such as TREECON
Restriction fragments are generated using EcoRI and MseI [44] if the AFLP data are first converted to a binary file.
restriction enzymes. For preamplification, both preselective Instead of manually scoring the presence or absence of
primers in the Regular Plant Genome Kit have an additional fragments to generate a binary file, AFLPapp software [4]
selective nucleotide at the 3′-end. However, only the MseI can be used to convert GeneScan data directly to a
preselective primer has a selective base in the Small Plant binary file.
Genome Kit.
There are 16 selective primers available, eight MseI pri-
Factors affecting reproducibility
mers and eight EcoRI primers. The MseI primers have a
total of three selective nucleotides at the 3′-end. The EcoRI Genomic DNA of high purity is required for AFLP to
primers have two selective nucleotides (for the Small Plant ensure complete digestion by the restriction endonucleases.
Genome Kit) or three selective nucleotides (for the Regular Incomplete restriction of DNA generates partial fragments,
Plant Genome Kit) at the 3′-end. Any combination of one predominantly of high molecular weight. Amplification of
EcoRI and one MseI primer can be used, providing 64 poss- fragments that are not fully digested generates an altered
ible primer pair combinations. banding pattern, and may be misinterpreted as false poly-
morphisms [45].
AFLPTM Microbial Fingerprinting Kit Although the AFLP procedure is affected by DNA qual-
A similar AFLP kit using EcoRI and MseI restriction ity, it is insensitive to the template DNA concentration. The
enzymes is offered by PE Applied Biosystems for microbial protocol is optimized such that the amplification reaction
fingerprinting. For the less complex genomes of micro- ceases when the labeled primer is consumed [45]. This
organisms, preselective amplification is performed using ensures that fingerprints of equal intensity are produced
primers composed of only the core sequence (adapter and despite variations in template concentration. However, at
restriction site sequence) without the addition of selective very high template dilutions (picogram quantities), the
nucleotides. Selective nucleotides are introduced in the nucleotide sequences flanking the restriction site will no
second amplification step. Eight MseI and eight EcoRI longer be random for a small pool of restriction fragments
selective primers are available, four of each type have one and variations in the banding patterns may be observed.
selective nucleotide and four have two selective nucleotides Typically, 0.05 ␮g of genomic DNA is required for small
at the 3′-end. genomes ranging from 50–500 megabases and 0.5 ␮g for
An unique feature of the AFLPTM Plant Mapping and genomes of 500–5000 megabases.
AFLPTM Microbial Fingerprinting Kits is the 5′-end label
on the EcoRI selective primers. Instead of a 5′-radioactive
Advantages of AFLP
label, the EcoRI primers have a fluorescent dye label at the
5′-end. The fluorescent dyes are excited by laser radiation The AFLP technique can be used for DNA samples of any
and are visualized by their characteristic absorption-emis- origin or complexity. Small sequence variations can be
sion frequencies. Thus, only the fragments containing an detected using only small quantities of genomic DNA
EcoRI restriction site are detected using systems such as (0.05–0.5 ␮g). The capacity to reveal many polymorphic
ALFexpressTM DNA Sequencer (Pharmacia Biotech, bands in one lane is a major advantage of AFLP markers.
Uppsala, Sweden) and ABI PRISMTM DNA Sequencer The numerous bands on a gel are analyzed simultaneously
(Perkin Elmer Corporation, Foster City, CA, USA). making AFLP an extremely efficient technique. AFLP has
Three types of fluorescent dyes are used to label the the capacity to inspect a much greater number of loci for
EcoRI selective primers, carboxyfluorescein (FAM), car- polymorphism than other currently available PCR-based
boxytetramethyrhodamine (TAMRA), and carboxy-4′,5′- techniques, such that the number of polymorphisms
dichloro-2′,7′-dimethoxyfluorescein (JOE). A fourth fluor- detected per reaction are much higher. AFLP is superior in
escent dye, carboxy-X-rhodamine (ROX), is loaded along terms of the number of sequences amplified per reaction
with each sample to serve as an internal size standard. The and their reproducibility. The markers produced are reliable
standard ensures that all amplification fragments are accu- and reproducible within and between laboratories, and are
rately sized. For high throughput, PCR products from up relatively easy and inexpensive to generate. A virtually
to three reactions, labeled with different coloured dyes, can unlimited number of markers can be generated by simply
be loaded into a single lane because each dye label has varying the restriction enzymes, and the nature and number
its characteristic absorption-emission frequency [29]. The of selective nucleotides.
fluorescent dye labels are detected with high sensitivity by
the DNA sequencers. Results are analyzed with GeneScan
Comparison to other molecular-based techniques
Analysis software (PE Applied Biosystems) and displayed
in any combination of electropherograms and tabular data. AFLP has advantages over other molecular-based tech-
GeneScan Analysis software can be used to prepare data for niques for DNA fingerprinting including restriction frag-
further analysis using GenoTyperTM software (PE Applied ment length polymorphism (RFLP) and random amplified
Biosystems). GenoTyper converts data into the format polymorphic DNA (RAPD). The technique of RFLP
required by downstream applications, such as linkage includes digestion of genomic DNA with restriction
analysis, databases, or spreadsheets. Although there is no enzymes. The pattern of restriction fragments generated for
MJ Blears et al
104
each sample is compared, following Southern hybridiz- Generation of high-resolution genetic maps in plants
ation, to reveal polymorphism. In a relatively well-characterized mapping population of
The major difference between AFLP and the traditional potato, van Eck et al [43] studied the localization of AFLP
RFLP technique is that PCR amplification is used for the markers relative to a mapping population of 197 RFLP,
detection of restriction fragments in AFLP analysis. How- nine isoenzyme, and 11 morphological markers. Six primer
ever, in a modified RFLP technique, called PCR-RFLP, combinations generated 264 segregating AFLP amplifi-
PCR amplification is followed by restriction digestion. Fur- cation products in a diploid backcross population from non-
thermore, AFLP fragments are run on a denaturing polyac- inbred potato parents. More details on experimental proto-
rylamide gel to detect the presence or absence of restriction col are outlined in Table 2. The segregating patterns of the
fragments as opposed to RFLP which displays length dif- AFLP amplification products observed in the offspring
ferences of restriction fragments on agarose or polyacryla- were used to evaluate the inheritance of putative AFLP loci.
mide gels following hybridization. Due to the nature of the AFLP mapping generated more than double the number of
RFLP technique, only the restriction site is scanned for dif- genetic markers when compared to RFLP. Athough the
ferences in DNA sequence. The selective nucleotides AFLP markers were randomly distributed, they targeted
included in AFLP provide additional possibilities for poly- regions already mapped by RFLP markers such that the
morphisms to be detected beyond the restriction site itself. total map length increased only 5% from 1120 to 1170 cen-
AFLP has the capacity to detect more point mutations than timorgans (cM). van Eck et al [43] concluded that AFLP
RFLP [3]. Insertions and deletions are detected at approxi- may substitute for other marker systems. The AFLP ampli-
mately the same frequency [3]. In a single hybridization fication products mapped by van Eck et al [43] will enable
experiment, RFLP can detect, at most, a few genetic loci the chromosomal identity of linkage groups to be estab-
compared to 100–200 loci detected using AFLP [26]. In lished in future mapping studies because of the locus speci-
addition to a greater number of polymorphisms per reac- ficity of AFLP markers. In addition, the identification and
tion, AFLP is also superior in terms of efficiency as it does mapping of comigrating AFLP fragments from other gen-
not require template DNA sequencing. Fingerprints are pro- omes will be facilitated.
duced without prior sequence knowledge. In a similar study on barley, Becker et al [3] generated
118 AFLP markers using 16 primer combinations (Table
RAPD is a PCR-based technique similar to AFLP. How-
2). Two of the linked AFLP markers could not be assigned
ever, AFLP uses primers specific to the adapter and restric-
to one of seven linkage groups. The remaining 116 markers
tion site sequence, whereas RAPD utilizes arbitrary pri-
were mapped to an existing barley RFLP map including
mers. The arbitrary primers, having no known homology
five microsatellite and four protein marker loci. The AFLP
to the target sequence, are used to randomly amplify seg-
markers mapped to all regions of the genome including
ments of the target DNA. Fragments of various sizes are three gaps, 2L, 4L and 6, in which no RFLP loci had been
generated during PCR. To allow the primer to anneal to mapped. Addition of markers at the chromosome tips, and
several locations on the two strands of target DNA, the bridging the three gaps in the original RFLP map, increased
amplification is performed at low stringency (annealing at the barley map of 1096 cM to a length of 1873 cM. A 58%
36–45°C) [33]. RAPD analysis is easier to perform than increase in the barley map length was determined and is
AFLP [3]. However, the RAPD technique is very sensitive quite dramatic in comparison to the 5% increase observed
to the reaction conditions, template DNA concentration and by van Eck et al [43] in potato. The results of Becker et
purity, and PCR temperature profiles, limiting its appli- al [3] indicate that AFLP is very useful for marker enrich-
cation. AFLP analysis uses stringent annealing conditions
which guarantee a better reproducibility [12].
For PCR-based techniques, such as RAPD and PCR- Table 2 Experimental protocols employed in studies utilizing the AFLP
RFLP, the term AFLP is sometimes used in a general sense technique for generation of high-resolution maps
to refer to a unique pattern of amplifed products separated
by gel electrophoresis. However, these techniques are not Reference Organism Preselective Selective No. of
based on the patented procedure of Zabeau and Vos [46], studied primers primers primer
and do not employ adapters or selective nucleotides. pairs
tested
[3] Hordeum vulgare MseI 3′-1 MseI 3′-3 16

L. (barley) EcoRI 3′-1 EcoRI 3′-3a
Applications [25] Oryza sativa MseI 3′-0/1 MseI 3′-3 18
(rice) japonica EcoRI 3′-0 EcoRI 3′-3/2a
AFLP has practical applications for DNA fingerprinting of and indica
cultivars
prokaryotes and eukaryotes. It has been used for marker- [30,31] Rattus MseI 3′-1 MseI 3′-3 4
assisted plant breeding, the construction of high-density norvegicus (rat) SseI 3′-1 SseI 3′-2a
molecular linkage maps, and for the positional cloning of [37] Beta vulgaris L. MseI 3′-1 MseI 3′-3 4
genes of interest. AFLP also has important applications for (sugar beet) EcoRI 3′-1 EcoRI 3′-3a
[43] Solanum MseI 3′-1 MseI 3′-3 6
strain identification and characterization of bacteria and tuberosum EcoRI 3′-1 EcoRI 3′-3a
fungi, as well as eukaryotic pathogens of plants and ani- (potato)
mals. In a modified procedure, AFLP has also been used
to generate RNA fingerprints using cDNA as template. a
MJ Blears et al
Table 3 Experimental protocols employed in studies utilizing the AFLP 105

ment and in some cases may extend the length of existing
linkage maps. technique for positional cloning of genes of interest
In a study on rice, Mackill et al [25] compared the levels
Reference Organism Preselective Selective No. of
of polymorphism for AFLP, RAPD, and microsatellite mar- studied primers primers primer
kers on 12 japonica and two indica rice cultivars. Using pairs
18 AFLP primer pairs, 143 of a total of 539 generated tested
bands were polymorphic (Table 2). Twenty-one RAPD pri-
mers produced 103 bands, 43 of which were polymorphic. [2] Solanum Mse 3′-1 MseI 3′-3 144
The number of alleles detected using 14 microsatellite pri- [26] tuberosum HindIII3′-1 HindIII 3′-3a 108
(potato)
mers ranged from one to six. Although the rice accessions [5] Solanum Not utilized MseI 3′-3 216
were classified into the same subspecies using all marker tuberosum PstI 3′-2a
types, the level of polymorphism varied considerably. (potato)
Within japonica cultivars, the average percent polymor- [6,7] Populus spp MseI 3′-1 MseI 3′-3 144
phism between any two accessions was 22% for AFLP, (poplar tree) EcoRI 3′-1 EcoRI 3′-3a
[10] Arabidopsis MseI 3′-0 MseI 3′-3 112
24% for RAPD and 36% for microsatellite markers thaliana EcoRI 3′-0 EcoRI 3′-2a
(monomorphic bands excluded). The average percent poly- SacI 3′-0 MseI 3′-2 192
morphism between indica and japonica accessions was SacI 3′-2a
highest for microsatellite (76%) and AFLP (65%) markers [40] Lycopersicon Not utilized MseI 3′-3 728
esculentum (Cf-9) PstI 3′-2a
as compared with RAPD (35%) markers. Although × L. pennellii
microsatellite markers produced the highest level of poly- (tomato)
morphism, the total number of polymorphic bands was
much higher for AFLPs with an average of eight per gel. 5′-end of primer labeled using [␥-32P or ␥-33P]ATP.
a
Only one locus per gel can be scored for microsatellite

analysis unless multiplexing is employed. To evaluate the
genetic mapping of AFLP markers, 80 F2 plants of an 3000–3300 informative loci were analyzed. Three RFLP,
indica × japonica cross were analyzed using seven primer one RAPD and two AFLP markers were linked to the Gro1
combinations. An existing RFLP-RAPD map containing locus. The RFLP and RAPD markers were inseparable from
115 RFLP, 10 RAPD markers and one morphological trait the Gro1 locus, while the two AFLP markers flanked Gro1
was used to map 50 of 54 scored AFLP bands to specific at genetic distances of 0.6 cM and 0.8 cM (where
chromosomes. The markers were distributed throughout the 1 cM = 100 kilobase pairs (kbp)). The closely linked AFLP
rice genome revealing the potential of AFLP markers for markers identified will facilitate molecular cloning of the
mapping important genes in rice. Gro1 gene based on map position. Cloning the Gro1 gene
AFLP and RFLP markers were used by Meksem et al may contribute to the elucidation of the mechanism of
[26] to construct a high-resolution genetic map of the R1 resistance, and the Gro1 gene can be directly transformed
locus on chromosome V of potato. The R1 allele confers, into susceptible potato cultivars that are agronomically
on potato, a race-specific resistance to Phytophthora sound.
infestans, a fungal pathogen. Bulked segregant analysis and Using bulked segregant pools from F2 progeny, of the
AFLP markers were used to select markers mapping less cross Lycopersicon esculentum (Cf-9) × L. pennellii, and
than 1 cM from the R1 gene. Of 3200 informative AFLP AFLP, Thomas et al [40] screened approximately 42 000
loci, 29 displayed linkage to the R1 locus. These 29 frag- AFLP loci for tight linkage to the tomato Cf-9 gene. The
ments were amplified by 21 of a total of 108 primer combi- Cf-9 gene, on chromosome 1 of tomato, codes for resistance
nations tested. The AFLP protocol used by Meksem et al to the leaf mould pathogen, Cladosporium fulvum. A total
[26] is outlined in Table 3. Genotypic analysis of 461 F1 of 728 primer combinations were tested using 13 PstI and
plants revealed that eight loci mapped within a genetic dis- 56 MseI primers. Additional details of the experimental
tance of 6 cM as defined by two marker loci, GP21 and protocol are outlined in Table 3. Of the 42 000 AFLP loci
GP179, flanking the R1 locus. Only six were linked in inspected for polymorphism, approximately 14 000 were
coupling with the resistance allele R1. The DNA markers potentially informative. This equates to one AFLP marker
generated in the study by Meksem et al [26] will aid in the every 70 kbp if the markers are distributed at random over
further characterization of the R1 gene and may help to the tomato genome estimated at 109 bp. Analysis of F2
elucidate the mechanism of race-specific resistance to P. recombinants identified three markers co-segregating with
infestans. Cf-9. Further analysis of plasmid clones containing the gene
A high-resolution map of the Gro1 locus on potato chro- allowed the position of the markers to be determined. Two
mosome VII was constructed by Ballvora et al [2] using of the markers were found to be located on opposite sides
RFLP, RAPD and AFLP markers. The dominant allele, of the Cf-9 gene, separated by 15.5 kbp.
Gro1, confers resistance to a root cyst nematode, Globo- The AFLP technique was used by Cnops et al [10], in
dera rostochiensis. The AFLP protocol of Meksem et al a chromosome landing strategy, for the isolation of the
[26] was employed for this study (Table 3). AFLP patterns TRN1 gene. The tornado1 (trn1) mutation was identified in
were generated using 24 primers for a total of 144 primer a T-DNA transgenic line of Arabidopsis (ecotype C24) and
combinations tested. Bulked segregant analysis was used to is located 5 cM from a T-DNA insertion. Visible markers
select RAPD and AFLP markers closely linked to Gro1 and RFLP markers were used to map the TRN1 locus to
in a segregating population of 1105 plants. Approximately the bottom half of chromosome 5. The AFLP technique
MJ Blears et al
106
was applied to recombinant classes, within a 3-cM region primer combinations identified three markers linked to and
around the TRN1 locus, to build a high-resolution map in on either side of Rysto. Four additional AFLP markers were
this region. Two different restriction enzymes, SacI and identified in a population of 360 segregating progeny of a
EcoRI, were used in combination with MseI, to generate potato cross between a resistant (Rysto) and a susceptible
template DNA for amplification (Table 3). Approximately parent. Two markers co-segregated with Rysto and the other
26 000 AFLP fragments were generated using 192 SacI- two were located on either side of Rysto, separated by one
MseI primer combinations and 112 EcoRI-MseI primer recombination event. AFLP analysis was successfully used
combinations. Seventeen AFLP markers were identified, to map the Rysto gene, however the closest flanking markers
four of which were so closely linked to the TRN1 locus were estimated to be 0.6 cM apart (where 1 cM = 1 Mb).
that no recombination event was detected. Three AFLP Additional markers around the resistance gene are neces-
fragments were purified from the dried gel and cloned: two sary for positional cloning.
trn1 co-segregating markers and one marker located The linkage map of sugar beet (Beta vulgaris L.) was
immediately below trn1. To facilitate the recovery of the extended by Schondelmaier et al [37] using the AFLP tech-
fragments, a simplified banding pattern (with 16-fold fewer nique on an F2 population consisting of 94 individuals. A
bands) was generated using SacI and MseI primers with total of 120 AFLP markers, generated using four primer
an additional selective nucleotide on the 3′-ends. The three combinations (Table 2) were integrated into an existing
tightly linked and co-segregating AFLP markers were used linkage map based on 207 RFLP loci. The AFLP markers
as probes to identify two yeast artificial chromosomes span- were evenly distributed over all nine linkage groups with
ning the TRN1 locus. Chromosome landing of the TRN1 the exception of linkage group V. The low number of AFLP
gene in Arabidopsis was proven to be successful using an loci on linkage group V was thought to be due to the low
AFLP-based strategy. Isolation and characterization of the number of primer combinations tested. Integration of AFLP
TRN1 gene will allow the process leading to dwarfism and loci served to extend the sugar beet map from 544 cM to
twisting to be investigated on a molecular level. 557 cM. The increase in genetic length was brought about
Using Populus spp as a model tree, Cervera et al [6] through the addition of AFLP markers at the ends of chro-
showed and discussed three possible applications of mosomes II, IV and VII, as well as AFLP loci filling some
AFLPTM in forest tree breeding. The first application, which gaps in the RFLP map. AFLP technology proved to be a
is described in more detail by Cervera et al [7], involves the promising tool for estimating the genetic diversity of sugar
use of AFLP to identify molecular markers tightly linked to beet breeding lines because of the even distribution of
the Mer locus conferring resistance against three races (E1, AFLP markers throughout the genome.
E2 and E3) of the leaf rust fungus, Melampsora larici-pop-
ulina. This disease causes premature defoliation, leaving Analysis of genetic diversity in plants
the tree susceptible to secondary pathogen infection and The variation in AFLP patterns within barley species was
environmental stress, and can reduce growth by more than investigated by Qi and Lindhout [36]. Two barley lines,
20%. The study was carried out using a hybrid progeny L94 and Vada, were used to screen 96 primer combinations
from a cross between a resistant P. deltoides female and a (Table 1). Forty-eight of the best primer pairs were selected
susceptible P. nigra male. Data suggested that resistance is and used to generate AFLP profiles for 16 representative
determined by a single dominant marker. AFLP analysis barley lines, including L94 and Vada. The data from 24
using 144 primer combinations (Table 3) and bulked seg- primer combinations were evaluated to study the polymor-
regant analysis, identified three AFLP markers tightly phism rates. Of 2188 clearly visible bands within the range
linked to the Mer locus. These markers can be used in cur- of 80–510 bp, 55% showed some degree of polymorphism
rent breeding programs and for future cloning of the resist- among the 16 lines. The large number of bands and the
ance gene. The second application involves the generation high rate of polymorphism indicate the efficiency of the
of genomic fingerprints for each species of Populus, using AFLP technique for marker generation in barley. Compari-
AFLP, for identification and taxonomic analysis. The son of 48 AFLP profiles of parent lines revealed more than
results for the study on the diversity among Populus spp 100 common markers (possibly locus-specific) among
have not yet been reported. The final application involves populations or crosses. These markers will greatly facilitate
the generation of high-density linkage maps of P. deltoides, the merging of existing data into one integrated genetic map
P. nigra and P. trichocarpa using the ‘two-way pseudo- of barley.
testcross’ strategy in combination with AFLP analysis. The The genetic diversity within and among populations of
‘two-way pseudo-testcross’ mapping strategy is based on the endangered sentry milk-vetch, Astragalus cremnophy-
linkage analysis of those markers that are heterozygous in lax var cremnophylax, was assessed by Travis et al [41]
one parent and null in the other parent. These results have using AFLP. Three populations in the Grand Canyon
also not yet been published. The multiple studies on Pop- National Park (Arizona, USA) comprise all known individ-
ulus spp using AFLP reveals the versatility of the technique uals remaining in the wild. The oldest population on the
for a variety of applications. South Rim (site 1) consists of approximately 500 individ-
Brigneti et al [5] combined AFLP and bulked segregant uals. A second population on the South Rim (site 2) con-
analysis of an F1 tetraploid potato population to identify sists of only two individuals. The third population, located
AFLP markers tightly linked to the Rysto gene conferring on the North Rim, is estimated to consist of 1000 individ-
resistance to all strains of the potato virus Y. Rysto is a uals who are distributed into four distinct subpopulations
dominant gene and was mapped to chromosome XI of the (A, B, C and D). A total of 220 polymorphic fragments
potato genome. Preliminary AFLP analysis using 216 were scored using nine primer combinations (Table 1).
MJ Blears et al
107
Diversity was measured within each population and subpo- ratio. The expected heterozygosity and multiplex ratio were
pulation on the basis of average heterozygosity and the pro- multiplied to generate a single parameter, termed marker
portion of polymorphic genes. Genetic diversity within index, used to evaluate the marker system’s overall
each population varied directly with population size, with efficiency in detecting polymorphism. Although the
the largest population having the greatest diversity. There expected heterozygosity was among the lowest for AFLP,
was no significant difference in the diversity among the the marker index for AFLP markers was almost an order
subpopulations at the North Rim. Comparisons of AFLP of magnitude higher than the other assays because of the
patterns revealed greater similarity among plants collected contribution of the large effective multiplex ratio. Compari-
from the same population than among plants collected from son of genetic relationships involving both cultivated (G.
separate populations. The genetic relationship among the max) and wild (G. soja) accessions revealed a high corre-
three populations, as revealed by the AFLP results, will be lation of RFLP, AFLP and microsatellite analyses. How-
very important for conservation strategies. The information ever, correlations of RAPD data were lower because
can be used to determine the potential of individuals to RAPDs are less effective in resolving interspecific simi-
adapt to other locations if wild populations are to be aug- larities. The correlations between marker systems were sig-
mented. nificantly lower when the comparisons involved only the
Sensi et al [38] compared AFLP and inverse sequence- 10 G. max accessions. Within G. max, RAPD and AFLP
tagged repeat (ISTR) analyses in their potential to reveal similarity estimates were closely correlated as were AFLP
polymorphisms in a group of 19 Vitis vinifera L. and RFLP similarities. The microsatellite similarities were
accessions, including 13 Sangiovese and six Colorino uncorrelated for G. max comparisons. The AFLP assay was
grapevines. ISTR analysis is based on the selective PCR determined to have the highest marker index value, indicat-
amplification of genomic DNA using primers derived from ing its superiority in detecting polymorphism in soybean
copia-like repetitive elements. It is similar to AFLP in genotypes.
terms of the number of loci detected and percentage of AFLP analysis was employed by Paul et al [32] to detect
polymorphic bands, but it has the added advantage of not genetic variation among 32 genotypes of tea (Camellia
requiring any manipulation of DNA following isolation. A sinensis (L.) O. Kuntze) collected from India and Kenya.
total of 264 polymorphic DNA fragments (57.6% AFLP analysis, using five primer combinations, discrimi-
polymorphism) were generated by AFLP using eight primer nated all of the genotypes including those that could not
combinations (Table 1). Five ISTR primer combinations be distinguished by morphological or phenotypic traits. The
generated 249 polymorphic markers (71% polymorphism). AFLP results were used to construct a dendrogram by clus-
The AFLP and ISTR results were used to construct dendro- ter analysis. The dendrogram separated the tea populations
grams based on cluster analysis. Comparable results were
into China type (sinensis), Assam type (assamica) and
obtained by AFLP and ISTR. The Colorino accessions were
Cambod type (assamica spp lasiocalyx). The grouping of
found to be genetically distinct from Sangiovese, and the
populations in the dendrogram was, in most cases, consist-
Colorino americano ecotype significantly diverged from
ent with the taxonomy, known pedigree of some of the
both groups. However, a higher proportion of polymor-
genotypes, and their geographical origin. However, six of
phism was detected within the Sangiovese group by ISTR
analysis. The genetic variability within and among cultivars the 19 Assam genotypes (two Indian and four Kenyan) have
was explained by the putative polyclonal origin and the been previously classified as China type using morphologi-
variation in selection pressures in different locations. Both cal traits, which may be subject to substantial environmen-
techniques were useful for investigating genetic diversity tal changes. In addition, the dendrogram grouping of the
among Vitis vinifera ecotypes and indicated the potential four Assam genotypes from Kenya has been supported by
application for clonal differentiation and/or identification. RAPD analysis. For the three Cambod genotypes, one was
Genetic relationships among 12 soybean genotypes, previously reported as a China type by RAPD analysis. No
including ten cultivated (Gycine max) and two wild explanation was offered for this discrepancy. Similar
soybean (G. soja) accessions, were determined by Powell grouping of the genotypes into Assam, China and Cambod
et al [35] using RFLP, RAPD, AFLP and microsatellite types was achieved by principal coordinate analysis. The
markers. The AFLP protocol employed is outlined in Table Assam clones from India and Kenya clustered closely on
1. The four marker assays were compared using three fea- the principal coordinate analysis plot showing common
tures: (1) information content (expected heterozygosity); ancestry. This provides support for the belief that Kenyan
(2) number of loci (or bands) simultaneously analyzed per teas originated from India. The China types were more dis-
experiment (multiplex ratio); and (3) effectiveness in persed on the plot indicating greater genetic variation.
assessing relationships among accessions. The expected However, clones collected from the same region exhibited
heterozygosity is a function of a marker system’s ability to less overall genetic variation because of similar selection
distinguish among genotypes. The highest levels of poly- pressures. Calculations on genetic diversity revealed that
morphism (expected heterozygosity) were detected with 79% was detected within populations and 21% between
microsatellite analysis and the lowest with RAPD and Indian and Kenyan populations. Estimates of diversity
AFLP (not significantly different). However, AFLP mark- within populations showed that the China-type teas were
ers generated the highest effective multiplex ratio. The the most variable and the Cambod population exhibited the
AFLP assay provides a high degree of flexibility because lowest variability. However, the Cambod population size
the restriction enzymes, and nature and number of selective was the smallest. Successful discrimination of genotypes
nucleotides can be manipulated to adjust the multiplex revealed that AFLP analysis can be used as an additional
MJ Blears et al
108
molecular marker assay for studying genetic diversity and analysis in that the genetic identity between spp orientalis
population genetics in tea. and macrosperma was comparable with microsperma sug-
The phylogenetic relationships among 44 morphologi- gesting a near simultaneous evolution. Among the wild
cally diverse lines of cultivated lettuce, Lactuca sativa, and taxa, the most closely related species/subspecies were L.
13 accessions of the wild species L. serriola, L. saligna, L. nigricans and L. ervoides. There was additional inconsist-
virosa, L. perennis, and L. indica were investigated by Hill ency between RAPD and AFLP analysis with the phylogen-
et al [15] using AFLP markers (Table 1). The same geno- etic placement of the wild population, L. odemensis.
types (excluding cv ‘Lakeland’) were analyzed by RFLP Although the two methods provided similar conclusions for
markers in a previous study. The AFLP data were used to the phylogeny of Lens, RAPD analysis was unable to dis-
construct a dendrogram by cluster analysis that was found criminate among several genotypes. However, using the
to be consistent with known taxonomic relationships, and 148 AFLP markers, it was possible to differentiate among
was similar to the phenetic tree constructed with RFLP all the genotypes. AFLP analysis was shown to provide
data. Although the dendrograms were similar, the overall a higher degree of resolution in discerning the phylogeny
genetic distance among taxa was genetically higher with of Lens.
RFLP markers. The difference was explained by the The genetic diversity of 48 samples of European Miscan-
method used to select probes for RFLP analysis, in that thus species (perennial grass), including 11 clones of M.
probes known to detect polymorphism are chosen. AFLP sinensis, two clones of M. sacchariflorus, 31 accessions of
data revealed that accessions of L. serriola L. had the high- M. × giganteus and four hybrids created by crossing M.
est mean intra-specific distance and clustered on a sister sinensis and M. sacchariflorus clones was analyzed by
branch of the L. sativa complex in the dendrogram. The Greef et al [13] using the AFLP technique. Approximately
close relationship provides additional support that L. serri- 250 polymorphic markers were generated using six primer
ola is the likely progenitor species of cultivated lettuce. combinations (Table 1). Genetic variation was calculated
With the exception of L. serriola and L. sativa, all species using cluster analysis and principal coordinate analysis.
formed their own major branch with a distance value AFLP revealed two main groups represented by M. sinensis
greater than 1.0. The 44 accessions of L. sativa were further and M. sacchariflorus clones. The M. × giganteus
subdivided as discrete branches according to their plant accessions were clustered under the M. sacchariflorus
type: butterhead, crisphead, romaine, and looseleaf. AFLP group with a relatively high distance of about 0.3 units.
and RFLP distance matrices were compared to test the The divisions, based on AFLP analysis, were found to agree
ability of the marker systems to distinguish the L. sativa with taxonomic classification. The genetic diversity was
accessions. It was determined that AFLP analysis provided highest among the M. sinensis pool and low in the
a more definitive grouping of the accessions. AFLP was M. × giganteus pool. The M. × giganteus accessions were
clearly shown to be a reliable technique for studying gen- divided into two clusters with a high genetic similarity.
etic relationships, both at the species and cultivar level. Only three of the 31 accessions could be differentiated from
AFLP was used by Sharma et al [39] to analyze the gen- the rest. No polymorphism was detected between micro-
etic diversity and phylogeny of 54 lentil accessions includ- and rhizome-propagated M. × giganteus accessions. How-
ing 26 cultivated genotypes of Lens culinaris (13 each of ever, more polymorphism may be detected among
var. macrosperma and microsperma), and seven genotypes M. × giganteus accessions using additional primer combi-
of the wild taxa, L. culinaris spp orientalis, L. odemensis, nations. The AFLP technique was also useful for classify-
L. nigricans and L. ervoides. Results of AFLP analysis ing many samples that were incorrectly named by botanical
were compared with RAPD results previously obtained gardens as M. saccariflorus clones, the majority belonging
using the same material. A total of 148 AFLP markers were to the M. × giganteus pool and one clone clustered in the
generated using four primer pairs with an average of 37 M. sinensis group. In addition to evaluating genetic vari-
informative bands per primer combination. RAPD analysis ation, and detecting incorrect classification, AFLP was used
using 100 10-mer primers produced 10-fold fewer informa- to determine self-fertilization of the M. sinensis clones in
tive bands per primer. The 23 AFLP markers produced by the hybridization of M. sinensis and M. sacchariflorus
one primer pair were used to generate a dendrogram of the material.
six Lens populations and a second dendrogram of the 54
genotypes. Similar results were obtained with the three Generation of high-resolution genetic maps in
other primer combinations. Information was also provided animals
by principal coordinate analysis and construction of an AFLP analysis by Otsen et al [30] contributed a total of
unrooted phylogenetic tree using the 23 AFLP markers. A 18 AFLP markers to the linkage map of the rat, and showed
second unrooted tree was prepared using all of the 148 mar- the potential of AFLP markers for the detection of quanti-
kers to provide further discrimination at the varietal or sub- tative trait loci. Details of the experimental protocol are
species level. The greatest intra-specific genetic variability outlined in Table 2. The 112 progeny of a
was detected for the L. nigricans accessions and the least (BN × ACI)F1 × ACI backcross were subjected to AFLP
diverse group was var microsperma. Subspecies orientalis analysis, revealing eight polymorphic markers. A genetic
and var macrosperma showed the greatest inter-specific linkage map constructed in the backcross experiment, using
similarity. A high degree of genetic identity was also exhib- 12 biochemical, two immunological, one RFLP and 55 sim-
ited between spp orientalis and var microsperma providing ple sequence length polymorphism markers, was successful
strong support that spp orientalis is the progenitor species in localizing seven of the eight AFLP markers to a specific
of cultivated lentils. RAPD results differed from AFLP chromosome. A panel of 34 H × B/B × H recombinant
MJ Blears et al
Table 4 Experimental protocols of studies utilizing the AFLP technique 109

inbred strains was also subjected to AFLP analysis. Eleven
of 15 identified polymorphic bands could be assigned to for the genotypic analysis of bacteria
specific chromosomes by comparison with 450 loci of the
Reference Organism Preselective Selective No. of
recombinant inbred strain linkage map. Genotypes of the studied primers primers primer
11 AFLP markers were tested for correlation with blood pairs
pressure data available for 32 of the recombinant inbred tested
strains. A suggestive correlation was observed between the
mean arterial pressure and two closely linked AFLP mark- [14] Paenibacillus larvae Not utilized MseI 3′-1 1
ers on chromosome 20. The three AFLP markers assigned and P. pulvifaciens EcoRI 3′-1a
(honeybee pathogens)
to chromosome 20 may facilitate the mapping of the puta- [16] Aeromonas N/A N/Aa N/A
tive blood pressure regulatory gene to a more restricted [17,18] Aeromonas Not utilized TaqI 3′-1 1
region. ApaI 3′-1a
[19] Xanthomonas Not utilized TaqI 3′-1/2 14
Characterization of mammalian genotypes Stenotrophomonas ApaI 3′-1a
Aeromonas MseI 3′-1
The AFLP technique was used by Otsen et al [31] for gen- Clostridium EcoRI 3′-0/1a
etic characterization of 12 rat inbred strains. AFLP analysis, Bacillus HindIII 3′-1a
using four primer combinations (Table 2), revealed con- Acinetobacter
siderable variation among strains. However, identical band- Pseudomonas
Vibrio
ing patterns were generated for animals of the same strain [21] Bacillus anthracis MseI 3′-0 MseI 3′-1 16
using the four primer pairs. AFLP profiles were used to EcoRI 3′-0 EcoRI 3′-1a
estimate the genetic relationship among the 12 strains. A [22] Aeromonas Not utilized TaqI 3′-1 1
dendrogram was constructed, based on cluster analysis, [23] ApaI 3′-1a 3
using the AFLP data. Interpretation of strain relationship [24] Escherichia coli and MseI 3′-0 MseI 3′-1 N/A
Agrobacterium EcoRI 3′-0 EcoRI 3′-1a
was not attempted because the chromosomal location of the tumefaciens
AFLP markers was unknown. However, the linkage map
constructed by Otsen et al [30] may assist in this matter. N/A = information not available.
Nevertheless, the high degree of inter-strain variation a
detected by AFLP analysis indicates its utility for charac-
terizing rat inbred strains used in biomedical research.
hybridization group. Analysis of the banding patterns
Genotypic analysis of bacteria revealed 13 AFLP clusters. In many cases, the fingerprints
The AFLP technique was evaluated by Huys et al [16] for enabled isolates within a hybridization group to be differen-
its usefulness in the genotypic classification of aeromonads. tiated at the strain level. The AFLP protocol of Huys et al
A total of 249 presumptive aeromonads were isolated from [17] included only one primer pair (Table 4). Performing
water samples collected from drinking water production a second AFLP reaction with other primer combinations
plants in Flanders, Belgium. Gas-liquid chromatographic may differentiate those isolates producing very similar pat-
analysis of total cellular fatty acids classified 228 isolates terns. The high level of correlation between AFLP results
in the genus Aeromonas using a commercial database. Huys and previously published DNA hybridization data showed
et al [16] established an extended database, based on fatty AFLP to be a valuable technique for classification of Aero-
acid analysis, using 70 Aeromonas reference and type monas species.
strains representing the 13 known hybridization groups The AFLP patterns produced by Huys et al [17] were
(genomic species). All but 42 isolates were identified into entered as representative library entries to generate a new
phenotypically defined groups, also called phenospecies, hybridization group-specific genotypic database called
using the extended database. Of the 186 identified isolates, AERO94. This database was employed in a subsequent
125 were further allocated to an existing hybridization study by Huys et al [18] to determine the genotypic diver-
group. AFLP analysis was performed by Huys et al [16] sity among 168 Aeromonas isolates obtained from drinking
using the protocol of Zabeau and Vos [46] (Table 4). The water production plants in Flanders, Belgium. Of the 168
resulting banding patterns enabled the 125 Aeromonas isolates, 86% (144) could be allocated to one of the 14
strains to be characterized to one particular hybridization recognized DNA hybridization groups. A fatty acid analysis
group. Even strains within one hybridization group could database constructed using the same collection of type and
be differentiated from each other using the fingerprint pat- reference strains used to generate the AFLP library,
terns. Huys et al [16] found that the AFLP data confirmed AERO94, was only able to classify 55 of the 144 Aero-
the results of fatty acid analysis. Thus the study revealed monas strains. The results of Huys et al [18] clearly indi-
the usefulness of AFLP for the classification of Aero- cate the higher potential of AFLP for taxonomic identifi-
monas isolates. cation and classification of unknown aeromonads compared
In a subsequent study, Huys et al [17] investigated the to chemotaxonomic methods.
ability of AFLP to differentiate the 14 defined DNA To evaluate the potential of AFLP to characterize bac-
hybridization groups in the genus Aeromonas. A total of terial strains at the subgeneric level, 36 Xanthomonas
98 Aeromonas type and reference strains representing the strains (including 29 pathovars), one Stenotrophomonas
14 hybridization groups were included in the study, as well strain, and 90 Aeromonas strains (representing the 14
as four phenospecies not yet allocated to a particular hybridization groups) were subjected to AFLP by Janssen
MJ Blears et al
110
et al [19]. DNA template for AFLP was prepared using ation groups, and whether the production of virulence fac-
TaqI and ApaI restriction endonucleases (Table 4). The tors is prevalent among human isolates. Isolates of the
high discriminatory power of AFLP enabled individual phenotype BD-2 and hybridization group 1 predominated
strains within the pathovars of Xanthomonas species to be in patients. In addition, these isolates produced relatively
differentiated based on their AFLP banding patterns. AFLP high levels of virulence factors suggesting that the
analysis of the Aeromonas isolates revealed similar results. HG1/BD-2 type may represent a true human pathogenic
Strains of the same hybridization group clustered together Aeromonas.
and most strains within a given hybridization group could In a second study by Kuhn et al [23], the AFLP tech-
be differentiated from each other. In both cases, there was nique was used to study the diversity and persistence of
good correlation between the AFLP data and existing taxo- coliforms and Aeromonas isolated from a Swedish drinking
nomic data. To determine if the use of other restriction water well. A total of 40 water samples were collected over
enzymes would influence the AFLP-based grouping of bac- a 4-year study period. When available, 32 bacterial colonies
terial strains, AFLP analysis of the 37 Xanthomonas and were isolated from each water sample. Preliminary bio-
12 Aeromonas strains (representing hybridization groups 1, chemical characterization of the isolates was performed
2 and 3) was repeated with DNA fragments generated using using the PhenePlate fingerprinting system. A total of 170
EcoRI and MseI restriction endonucleases (Table 4). For different phenotypes were identified among 1143 studied
both Xanthomonas and Aeromonas, the generation of fewer isolates. Isolates that were suspected of representing pre-
restriction fragments resulted in unevenly distributed band- dominant clones in the well water were further charac-
ing patterns with a higher concentration of high molecular terized using the API 20E system, cellular fatty acid analy-
weight fragments. This was attributed to the lower cleavage sis, and AFLP. Most phenotypes were only represented by a
frequency of EcoRI and MseI with G+C-rich Xanthomonas few isolates and (or) were restricted to one or two sampling
and Aeromonas DNA. Although the grouping of Xantho- occasions. Only one phenotype, identified as A. hydrophi-
monas strains was highly similar to the clusters achieved lia, was identified in more than three water samples. Thirty-
with Apa1-Taq1 templates, the linkage levels were 5–10% nine percent of the isolates were found to belong to this
lower among the various clusters and among the individual phenotype and were present in 28 samples distributed over
strains. For the Aeromonas strains, the linkage levels of the whole study period. Eleven representative A. hydrophi-
the three hybridization groups dropped considerably with lia isolates from 11 different sampling occasions were sub-
hybridization groups 1 and 2 grouping together in one clus- jected to AFLP analysis. Nearly identical banding patterns
ter. This clearly shows that the choice of restriction were generated for each of the three different ApaI-TaqI
enzymes can affect the accuracy of the AFLP analysis. The selective primer pairs (Table 4). Cluster analysis of the
best combination of endonucleases should be determined banding patterns revealed that all 11 A. hydrophilia isolates
empirically for each organism to ensure accurate results. grouped within the same hybridization group, HG3, indicat-
In the same study, Janssen et al [19] tested the general ing that they were most probably of the same clonal origin.
applicability of the AFLP technique in bacterial taxonomy Although most strains were only transient inhabitants of
using four strains belonging to the genera Clostridium, Aci- the well, the AFLP results suggest that the A. hydrophilia
netobacter, Bacillus, Pseudomonas and Vibrio. Of the four phenotype represents a genetically stable Aeromonas clone
strains investigated for each genera, three belonged to the which persisted in the well water for the entire 4-year study.
same species, or were highly related species as with B. Keim et al [21] used AFLP to analyze 79 Bacillus
cereus and B. thuringiensis. In all cases, the four isolates anthracis isolates and isolates of six related Bacillus spec-
could be discriminated. However, the three related strains ies (B. cereus, B. thuringiensis, B. mycoides, B. subtilis, B.
had very similar banding patterns with the fourth strain hav- polymyxa and B. stearothermophilus) for molecular vari-
ing a much different pattern. Use of the AFLP technique ation (Table 4). B. anthracis has two large plasmids, pX01
for DNA of any source and complexity was demonstrated and pX02, that carry essential genes for pathogenesis. To
in this comprehensive study. The genomes of the genera focus on chromosome-based relationships, purified plasmid
investigated vary both in size and base composition, yet the DNA of B. anthracis was analyzed to exclude plasmid-
superior discriminative power of the technique for differen- specific AFLP fragments from the fingerprints. Great AFLP
tiating highly related strains was maintained. diversity was observed among the Bacillus taxa. Cladistic
To gain a better understanding of the mechanism of viru- and phenetic analysis of the banding patterns revealed the
lence of Aeromonas spp, Kuhn et al [22] analyzed 120 iso- phylogenetic relationships among the related Bacillus spec-
lates including 80 fecal isolates from patients in Bangladesh ies, and among the B. anthracis isolates. B. cereus and B.
(69 from patients with diarrhea and 11 from healthy thuringiensis were determined to be the closest taxa to B.
controls), and 40 environmental isolates from surface anthracis with B. mycoides being slightly more distant. An
water. All isolates were phenotyped using a high-resolution extremely low level of molecular variation was detected
biochemical fingerprinting system (the PhenePlate system). among the isolates of B. anthracis, which is thought to be
Most (106) were assigned to hybridization groups by fatty one of the most genetically uniform bacterial species. Of a
acid profiles. However, for 29 isolates requiring further total of 1221 amplified fragments, 97% (1184) were mono-
characterization, or of special interest, AFLP analysis was morphic. In contrast, only 40% of the fragments were simi-
employed (Table 4). In addition, all isolates were assayed lar between B. anthracis and its closest relatives, B. cereus
for the production of hemolysin and cytotoxin. The aim of and B. thuringiensis. Although there was little variation,
the study was to determine whether pathogenicity can be cluster analysis identified two well-defined groups of B.
associated with certain phenospecies and/or DNA hybridiz- anthracis isolates. AFLP proved to be an effective tech-
MJ Blears et al
111
nique for molecular typing of B. anthracis, a relatively strains suggests that each cluster has a common clonal ori-
monomorphic bacterial species, because of its capacity to gin.
inspect a large percentage of the genome for genetic vari- AFLP was evaluated by Janssen et al [20] for its useful-
ation. ness in the epidemiological typing of 25 Acinetobacter
Lin et al [24] used AFLP to analyze different strains of strains isolated during five hospital outbreaks in three coun-
Escherichia coli and Agrobacterium tumefaciens. The E. tries (Table 5). A dendrogram, constructed by cluster analy-
coli strains tested included BL21, BL21F′IQ, DH5␣, sis of AFLP data, revealed five clusters with a minimum
DH5␣F′IQ, HB101 and W. The AFLP technique employed of 94% similarity. Each cluster comprised strains from one
(Table 4) was successful in differentiating the E. coli particular outbreak and shared identical banding patterns.
strains. Polymorphic bands found between BL21F′IQ and The AFLP data were verified by earlier published typing
BL21 as well as between DH5␣F′IQ and DH5␣ demon- data including antibiogram typing, biotyping, cell envelope
strated that AFLP is able to differentiate fragments as small protein electrophoretic profiling and ribotyping. AFLP
as 100 kb, since the F′IQ is the only difference between proved to be a valuable alternative in epidemiological
the strains. The A. tumefaciens strains tested included the typing.
octopine-like strain LBA4404 and napoline-like strains C58 Characterization of water and human isolates of Myco-
and EHA101. AFLP was shown to be a superior molecular bacterium kansasii using a variety of typing methods,
typing system for analysis of A. tumefaciens than traditional including RFLP analysis with the major tandem repeat
phenotypic ribotyping. This further demonstrates the gen- probe and the IS1652 probe, pulsed-field gel electro-
eral applicability of AFLP for determining genotypic phoresis (PFGE), PCR restriction analysis of the hsp-65
relationships in bacterial genera. gene and AFLP analysis, allowed Picardeau et al [34] to
establish an epidemiological relationship between environ-
Epidemiological typing of bacteria mental and clinical isolates. The isolates investigated
AFLP analysis, in addition to ribotyping, biotyping, cell included the type strain and 62 M. kansasii isolates includ-
envelope protein electrophoretic typing and antibiogram ing 38 clinical strains (14 from AIDS patients) and 24
typing, were used by Dijkshoorn et al [11] to compare 31 strains from tap water samples, all recovered in France.
Acinetobacter baumannii strains. Fourteen strains from AFLP analysis involved the use of only one restriction
nosocomical outbreaks in different northwestern European endonuclease, PstI and amplified products (3–8 products of
cities, and 17 sporadic strains apparently not associated 0.5–2.5 kb) were separated by gel electrophoresis using a
with outbreaks were investigated. The AFLP procedure 2% agarose gel (Table 5). Although the five methods reveal
employed is provided in Table 5. A dendrogram, based on independent polymorphisms and provide different levels of
AFLP data, revealed 18 clusters and single strains at a strain characterization, the results indicated five homo-
delineation level of 89.0% similarity. Twelve of the 14 out- geneous clusters suggesting the existence of five distinct
break strains were grouped into two clusters. The first of subspecies within M. kansasii. The PFGE and AFLP data
these included nine outbreak strains with 89.9% similarity were particularly informative because they revealed poly-
and the second cluster comprised three outbreak strains and morphisms within each cluster. Mycobacterial infections
one sporadic strain linked at 92.8%. The AFLP patterns of are considered to be acquired from the environment and M.
the two clusters differed by only one or two bands. Combi- kansasii has been almost exclusively recovered from tap
nation of all typing results linked 18 of the 31 strains into water. All clusters contained clinical strains (both from
four groups. Only two outbreak strains and eleven nonout- AIDS and non-AIDS patients) and environmental strains
break strains failed to cluster. The strains in AFLP clusters providing further evidence for the involvement of tap water
one and two comprised Groups I and II, respectively. The in human infections.
uniformity of typing characters of the two sets of outbreak The AFLP technique was evaluated by Valsangiacomo et
al [42] for the molecular typing of 28 strains of Legionella
pneumophilia including clinical and environmental strains
Table 5 Experimental protocols of studies utilizing the AFLP technique
from different regions of Switzerland. A single restriction
for epidemiological typing of bacteria endonuclease, PstI, was used to generate restriction frag-
ments. Following selective amplification, the fragments (5–
Reference Organism Preselective Selective No. of 10) were separated by gel electrophoresis on a 1.5% aga-
studied primers primers primer rose gel (Table 5). AFLP analysis of the 10 clinical strains
pairs isolated from patients and their living environments (from
tested three cases of legionellosis) made it possible to identify the
origin of infection. The banding patterns of the 18 environ-
[11] Acinetobacter Not utilized TaqI 3′-2 1
baumannii HindIII 3′-1a mental isolates revealed genetic heterogeneity. The poten-
[20] Acinetobacter Not utilized TaqI 3′-2 1 tial of AFLP for epidemiological studies was revealed in
HindIII 3′-1a this study.
[34] Mycobacterium Not utilized PstI 3′-2 N/A
kansasii PstI 3′-5 Genotypic classification of fungi
[42] Legionella Not utilized PstI 3′-1 16
pneumophilia PstI 3′-2 A simplified AFLP protocol, using only PstI restriction
endonuclease, was used by Mueller et al [28] to detect gen-
N/A = information not available. etic differences among 14 symbiotic fungi of the fungus-
a
5’-end of primer labeled using [␥-32P or ␥-33P]ATP. growing ant, Cyphomyrmex minutus (Table 1). The isolates
MJ Blears et al
112
were grouped into four distinct fingerprint ‘types’ by each lations. Further testing with additional primer combinations
of six PstI primers. The fungi within each type showed may permit the clear clustering of G. pallida populations.
identical banding patterns. It was suggested that each fungal
type may represent a distinct clone propagated vegetatively RNA fingerprinting using cDNA-AFLP
by the ant, or may correspond to a free-living species of A novel study by Bachem et al [1] combined an RNA
fungus acquired by the ant. The genetic differences among fingerprinting technique based on AFLP, called cDNA-
the 14 isolates predicted by vegetative-compatibility assays AFLP, with a highly synchronous in vitro potato tuberiz-
corresponded to the fungal types revealed by AFLP analy- ation system to analyze transcriptional changes at and
sis, suggesting that vegetative compatibility can be used as around the time of tuberization. The cDNA-AFLP tech-
a crude indicator of genetic differences among dikaryotic nique uses the standard AFLP protocol on a cDNA template
fungi. (Table 6). The expression of two genes expressed during
tuberization was analyzed. One gene codes for the major
potato storage protein, patatin, and a second codes for
Characterization and classification of pathogens ADP-glucose pyrophosphorylase, a key enzyme in the
AFLP analysis was used by Heyndrickx et al [14], in starch biosynthetic pathway. The kinetics of expression
addition to other morphological and biochemical tests, a revealed by cDNA-AFLP were comparable to those found
variety of chemotaxonomic and genomic fingerprints, and in Northern analysis, the traditional fingerprinting method.
DNA relatedness measurements, to support the reclassi- The verification of band identity is typically a difficult pro-
fication of the honeybee pathogens Paenibacillus larvae cedure in RNA fingerprinting. However, by using selective
and P. pulvifaciens as P. larvae subsp larvae and P. larvae primers with nucleotide extensions of three bases in the
subsp pulvifaciens. Paenibacillus larvae is an obligate cDNA-AFLP procedure (Table 6), it was possible to elim-
pathogen of honeybee (Apis mellifera) larvae causing inate virtually all non-target bands. The results of Bachem
American foulbrood disease. P. pulvifaciens causes a rare et al [1] revealed the use of cDNA-AFLP for identifying
disease called powdery scale characterized by a powdery developmentally regulated genes. The technique may allow
decay of the larvae. A total of eight strains (four of each the detailed characterization of gene expression in a wide
species) were analyzed by the AFLP procedure outlined in range of biological processes.
Table 4. The AFLP patterns of P. larvae and P. pulvifaciens Money et al [27] investigated the use of AFLP to gener-
strains grouped into two distinct clusters, indicating the ate mRNA fingerprints in hexaploid wheat and one of its
existence of two subgroups. The AFLP technique proved deletion mutants. Messenger RNA was extracted from
to be very useful for Heyndrickx et al [14] for the charac- wheat cv Chinese Spring and a mutant of this variety with
terization of bacteria at the subgeneric level and allowed deletions on chromosomes 3A and 3B. The standard AFLP
differentiation within the range of biovar to genomic procedure was performed using double-stranded cDNA
species. synthesized from the extracted mRNA (Table 6). Compari-
Folkertsma et al [12] used AFLP to characterize a total son of the AFLP fingerprints revealed 16 polymorphic frag-
of 24 potato root cyst nematode populations including nine ments. These fragments were excised and reamplified. Pur-
Globodera rostochiensis and 15 G. pallida populations. A ified reamplified products were labeled and hybridized with
total of 987 marker-loci were screened using 12 primer various genomic DNA digests from Chinese Spring and the
combinations. More details on experimental protocol are deletion mutant. Five products with similar hybridization
outlined in Table 1. The polymorphic DNA fragments for patterns were found to cross-hybridize with the 18S-5.8S-
the nine G. rostochiensis populations were separated into 28S rRNA genes of wheat. The remaining 11 probes
two subsets based on presence or absence polymorphisms hybridized to single or low copy sequences. Two of these
and band intensity polymorphisms. There was close agree- fragments, present in Chinese Spring, were found to be
ment between the dendrograms generated using both AFLP located on chromosome 3A. Generation of mRNA finger-
data sets. The nine populations branched into three similar prints using AFLP proved to be useful for isolating
groups containing three, five and one population(s), respect- sequences mapping to deleted chromosome segments in
ively. The pathotype classification of the nine G. rostochi- hexaploid wheat.
ensis populations corresponded to the dendrograms gener-
ated using AFLP. Although all G. pallida populations were Table 6 Experimental protocols of studies utilizing the cDNA-AFLP
differentiated using AFLP, band intensity polymorphisms technique for RNA fingerprinting
could not be identified. The banding patterns generated for
the 15 G. pallida populations were very complex, with the Reference Organism Preselective Selective No. of
studied primers primers primer
primer combinations used providing only qualitative data. pairs
Unlike the G. rostochiensis populations, the clustering of tested
G. pallida populations by AFLP, did not resemble their
pathotype classification. The possible explanations pro- [1] Solanum TaqI 3′-0 TaqI 3′-2 N/A
vided by Folkertsma et al [12] included the inadequacy of tuberosum AseI 3′-0 AseI 3′-2a
the pathotype scheme for G. pallida and the structure of (potato)
[27] Triticum MseI 3′-0 MseI 3′-2/3 49
the genetic variation among G. pallida populations (ie the (wheat) PstI 3′-0 PstI 3′-0a
distribution of the polymorphic DNA fragments). A large
proportion of the polymorphic DNA fragments were scat- N/A = information not available.
tered, hindering the classification of the G. pallida popu- a
MJ Blears et al
voort, A Schots, FJ Gommers, J Helder and J Bakker. 1996. Gene 113

Future perspectives
pool similarities of potato cyst nematode populations assessed by
The AFLP technique can be applied to DNA of any source AFLP analysis. Mol Plant Microbe Interact 9: 47–54.
or origin. There is an extensive number of applications for 13 Greef JM, M Deuter, C Jung and J Schondelmaier. 1997. Genetic
diversity of European Miscanthus species revealed by AFLP finger-
this virtually universal marker system. Since its develop- printing. Genet Res Crop Evol 44: 185–195.
ment in 1993, it has been widely used in the field of plants. 14 Heyndrickx M, K Vandemeulebroecke, B Hoste, P Janssen, K Ker-
Its application for other eukaryotes and prokaryotes is sters, P de Vos, NA Logan, N Ali and RCW Berkeley. 1996. Reclassi-
expected to increase. Although all publications to date util- fication of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura
ize radioactive isotopes for the 5′-end labeling of one 1984) Ash et al 1994, a later subjective synonym of Paenibacillus
(formerly Bacillus) larvae (White 1906) Ash et al 1994, as a subspec-
primer of the pair, the use of fluorescent dye-labeled pri- ies of P. larvae, with emended descriptions of P. larvae as P. larvae
mers will become more prevalent. The many advantages of subsp larvae and P. larvae subsp pulvifaciens. Int J Syst Bacteriol 46:
the technique, primarily its reproducibility and number of 270–279.
loci detected per reaction, will enhance its overall appli- 15 Hill M, H Witsenboer, M Zabeau, P Vos, R Kesseli and R Michelmore.
cation. 1996. PCR-based fingerprinting using AFLPs as a tool for studying
genetic relationships in Lactuca spp. Theor Appl Genet 93: 1202–
1210.
16 Huys G, R Coopman, M Vancanneyt, I Kersters, W Verstraete, K Ker-
Acknowledgements sters and P Janssen. 1993. High resolution differentiation of aeromon-
ads. Med Microbiol Lett 2: 248–255.
The authors would like to thank Drs Shu Chen and Deena 17 Huys G, R Coopman, P Janssen and K Kersters. 1996. High-resolution
Errampalli for critical reading of the manuscript. MJ Blears genotypic analysis of the genus Aeromonas by AFLP fingerprinting.
was the recipient of University of Guelph Graduate Student Int J Syst Bacteriol 46: 572–580.
Scholarships in Fall 1996, Winter and Fall 1997. This 18 Huys G, I Kersters, R Coopman, P Janssen and K Kersters. 1996.
research was supported by a group Strategic grant from the Genotypic diversity among Aeromonas isolates recovered from drink-
ing water production plants as revealed by AFLPTM analysis. System
Natural Sciences and Engineering Research Council of Appl Microbiol 19: 428–435.
Canada and an operating grant from Ontario Ministry of 19 Janssen P and L Dijkshoorn. 1996. High resolution DNA fingerprinting
Agriculture, Food and Rural Affairs. of Acinetobacter outbreak strains. FEMS Microbiol Lett 142: 191–194.
20 Janssen P, R Coopman, G Huys, J Swings, M Bleeker, P Vos, M
Zabeau and K Kersters. 1996. Evaluation of the DNA fingerprinting
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Amplified Fragment Length Polymorphism (AFLP) : A Review of The Procedure and Its Applications

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Journal of Industrial Microbiology & Biotechnology (1998) 21, 99–114

 1998 Society for Industrial Microbiology 1367-5435/98/$12.00

Amplified fragment length polymorphism (AFLP): a review of

Introduction Netherlands). With AFLP, molecular genetic polymor-

Principle of the method

Correspndence: JT Trevors and H Lee, Department of Environmental

Digestion of genomic DNA

Figure 4 A schematic illustrating the selectivity of AFLP primers. The

the PCR primers. There is an almost linear correlation

102 Table 1 Exprimental protocols of studies utilizing the AFLP technique

A two-step amplification strategy was developed for com-

[3] Hordeum vulgare MseI 3′-1 MseI 3′-3 16

Table 3 Experimental protocols employed in studies utilizing the AFLP 105

Only one locus per gel can be scored for microsatellite

Table 4 Experimental protocols of studies utilizing the AFLP technique 109

voort, A Schots, FJ Gommers, J Helder and J Bakker. 1996. Gene 113

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