Download The use of amplified fragment length polymorphism (AFLP) in the

Molecular Ecology (1999) 8, 671Ð674
S H O RT C O M M U N I C AT I O N
The use of amplified fragment length polymorphism
(AFLP) in the isolation of sex-specific markers
R I C H A R D G R I F F I T H S a n d K AT E O R R
Molecular Laboratory, DEEB, Graham Kerr Building, Glasgow University, Glasgow G12 8QQ, UK
Abstract
Sex identification is a problem in research and conservation. It can often be solved using a
DNA test but this is only an option if a sex-specific marker is available. Such markers can
be identified using the amplified fragment length polymorphism (AFLP) technique. This
is usually a taxonomic method, as it produces a DNA fingerprint of 50Ð100 PCR bands.
However, if male and female AFLP products are compared, sex-specific markers are
confined to the heterogametic sex and can rapidly be identified. Once a marker is found,
AFLP can be used to sex organisms directly or the marker can be sequenced and a
standard PCR test designed.
Keywords: AFLP, avian, Phalacrocorax aristotelis, sex identification, Struthio camelus, W chromosome
Received 24 August 1998; revision received 19 October 1998; accepted 19 October 1998
Introduction
Sexual identification can be achieved in many species using
a DNA-based test. Despite its strengths, a DNA test can
only be used if a sex-linked marker has been identified.
The availability of sex-specific DNA is dictated by the
relative contribution of genotype and environment in sex
determination (Charlesworth 1991). Even if this has
moved to the point where a sex-specific Y chromosome
exists (ÔYÕ refers to Y or W chromosomes unless stated otherwise), much of the DNA on this chromosome is not
unique. This is because copies of mobile genetic elements,
repeat sequences and a pseudoautosomal region can
occur elsewhere in the genome (Charlesworth 1991).
Accordingly, sex-linked markers are difficult to isolate.
We present a method that uses the nonacronymous
amplified fragment length polymorphism (AFLP) technique (Vos et al. 1995) to identify sex-specific markers. The
technique is used on DNA from known males and females
where AFLP amplifies three types of marker. Most are
monomorphic and common to all samples. The second
type of markers are polymorphic and therefore differ
between individuals irrespective of sex. However, the
third type are confined to a single sex. These Y-specific
markers can either be used to identify gender using AFLP
alone or be sequenced and used to design primers for a
standard PCR-based sexing technique.
Correspondence: R. Griffiths. Fax: +44 (0) 141 3571132; E-mail:
R. [email protected]
© 1999 Blackwell Science Ltd
We demonstrate AFLP on the ostrich (Struthio camelus)
whose W and Z sex chromosomes have diverged so little,
that few sex-linked markers are available (female ZW;
male ZZ; Takagi et al. 1972; Ogawa et al. 1998). We also
include the shag (Phalacrocorax aristotelis) which resists
any attempts at sex identification using the apparently
ubiquitous avian CHD test (F. Daunt, personal communication; Griffiths & Tiwari 1995; Griffiths et al. 1998).
Materials and methods
AFLP
The AFLP procedure can be taken from the study by Vos
et al. (1995) but we used the ÔAFLP analysis system 1Õ kit
(BRL, cat. no. 10544Ð013). Twelve ostriches were sexed by
plumage and eight shags were sexed by behaviour.
Genomic DNA (250 ng) (Milligan 1998; protocol 9) from
three male and three female samples was processed individually as follows.
Step 1: Restriction of genomic DNA with MseI (TTAA)
and EcoRI (GAATTC) in 10 mM Tris (pH 7.5), 10 mM MgAc
and 50 mM KAc. Digestion was confirmed by electrophoresis of samples on a 0.8% agarose gel.
Step 2: Ligation of 100 ng of the two double-stranded
adapters to the MseI and EcoRI sites in adapter/ligation
solution with 1 unit of T4 DNA ligase (20 ¡C/2 h).
Step 3: Preselective PCR amplification with primers
specific to the two adapters added in Step 2. The primers
are complementary to the adapters except that each has
672
R. GRIFFITHS AND K. ORR
an additional 3' preselective nucleotide that extends into
the genomic DNA (E-n and M-n).
Step 4: Selective PCR amplification with E-nnn and Mnnn primers. These are similar to the preselective primers
but two further 3' selective nucleotides are added and the
E-primer is end-labelled with [γ33P]-dATP. The amplification was carried out in 20 mM Tris (pH 8.4), 1.5 mM MgCl2,
50 mM KCl with dNTPs and Taq DNA polymerase
(Promega). The thermal conditions were 94 ¡C/30 s,
65 ¡C/30 s 72 ¡C/60 s where 0.7 ¡C was lost from the
annealing temperature over 13 cycles in a touchdown to
56 ¡C. Once at 56 ¡C a total of 23 standard cycles was performed.
Step 5: The results were displayed by mixing the PCR
products with an equal volume of formamide dye (98%
formamide, 10 mM EDTA, 0.025% xylene cyanol). The
mixture was heated to 90 ¡C/3 min and put on ice before
being run on a 6% denaturing acrylamide gel (Sequagel)
for 2.5Ð3 h/50 W. The gel was dried and exposed to
Kodak BioMax film overnight.
Design of a PCR test
Sex-linked AFLP markers were cut from the dried gel and
DNA removed by the Ôcrush and soakÕ method in 100 µL
of TE (10 mM Tris (pH 7.6), 1 mM EDTA) (Sambrook et al.
1989). We performed PCR on 5 µL of the isolated DNA.
The selective primers were those in the original AFLP
reaction and 20 thermal cycles were required: 94 ¡C/30 s,
56 ¡C/30 s 72 ¡C/60 s. The product was purified with a
Qiaquick kit (Qiagen) and 10 µL was phosphorylated,
extracted with phenolÐchloroform and cloned into SmaIcut pUC18 (Pharmacia). Sequencing of 50Ð100 ng of plasmid template was carried out by the MBS Unit, Glasgow
University, using an ABI 373A sequencer.
PCR primers were designed to two cloned ostrich
sequences: ScW1F 5'-GAATTCAGGACCTTGGTGAA-3',
ScW1R 5'-ACAAGATGTTTTGGAAAGAAGAG-3' and
ScW2F
5'-CAATAAACAACATGTGAAAGAACA-3',
ScW2R 5'-ACATGAGGACAATGTGTAAGGA-3' (Rychlik
& Rhoads 1989). These primers amplified 50Ð250 ng of
genomic DNA in the presence of positive control primers
designed to the CHD gene P8 5'-CTCCCAAGGATGAGRAAYTG-3' (Griffiths et al. 1998) and P18 5'-GAGATGGAGTCACTATCAGATCC-3'.
The
final
reaction
conditions were: 50 mM KCl; 10 mM Tris pH 9 (25 ¡C);
1.5 mM MgCl2; 0.1% Triton X-100; 200 µM each dNTP;
100 ng of each primer and 0.75 units of Taq DNA polymerase (Promega). An UnoII thermal cycler (Biometra)
denatured the reaction at 95 ¡C for 1.5 min then performed 30 cycles of 52 ¡C for 20 s, 72 ¡C for 20 s and 94 ¡C
for 30 s with a final run of 48 ¡C for 1 min and 72 ¡C for 5
min. PCR products were viewed by electrophoresis in a
3% agarose gel.
Results
The AFLP technique successfully isolated three W-chromosome-linked markers from the ostrich and two from
the shag.
The Gibco BRL kit was used to carry out AFLP. It provides eight E selective primers and eight different M
primers, potentially giving 64 selective PCRs. The AFLP
of three male and three female ostriches used a single E
primer (E-AGG) against each of the eight M primers.
Every primer pair amplified an array of ≈ 120 DNA
bands between 100 and 400 bp in length (Fig. 1). The EAGG/M-CAG primer combination provided two W
chromosome markers (Figs 1, 2) and E-AGG/M-CAA a
third marker. These two primer pairs were tested on a
total of eight birds and identified the sex correctly in
each (n = 8, P = 0.004).
In the shag, eight primer pairs were chosen to check
whether primers other than E-AGG identified sex-specific
markers. Two W chromosome markers were found (EAAG/M-CAA; E-ACT/M-CAC; Fig. 2).
Two of the ostrich markers were cloned and
sequenced. After the AFLP E 5'-CGACTGCGTACCAATTC-3' and M 5'-GATGAGTCCTGAGTAA-3' primer
sequences were removed, there were no significant alignments to the sequences on the GenBank database
(BLASTN; Altschul et al. 1997).
Primer pairs ScW1F/ScW1R and ScW2F/ScW2R were
designed to amplify each of the ostrich sex-specific markers. However, a reliable sex identification technique must
also have a positive internal control for PCR amplification. In both cases the coamplification of a larger CHD
fragment with the P8/P18 CHD primers was incorporated. The two tests were employed to sex DNA samples
from 12 ostriches whose sex was confirmed using
plumage characteristics (n = 12, P = 2.4 × 10Ð4; Fig. 3).
Discussion
If a DNA sex identification test is required, a Y-specific
marker needs to be isolated in an inexpensive, effective
manner. The best option was to use random amplified
polymorphic DNA (RAPD; Griffiths & Tiwari 1993;
Lessells & Mateman 1998). Similar to AFLP, RAPD provides a means to identify sex-linked markers by comparing male and female PCR products. The RAPD
protocol is simple. It uses short primers in a low-stringency PCR to generate randomly primed products
which are compared on an agarose gel. Due to its
simplicity, RAPD outperforms AFLP because it does not
employ acrylamide gels, radioactive markers or highpressure liquid chromatography (HPLC) purified
primers. Consequently, the safety requirements, cost
and preparation time are low.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 671Ð674
AFLP AND SEX-SPECIFIC MARKERS
Fig. 1 AFLP profiles generated from four male and four female
ostriches with the E-AGG/M-CAG selective primers.
Approximately 120 markers are visible, two are W-linked and
unique to the female (W), some are polymorphic markers (e.g. P),
but most are monomorphic (e.g. M).
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 671Ð674
673
Despite this, AFLP has two advantages. First, whilst a
RAPD produces 5Ð10 bands (Griffiths & Tiwari 1993) AFLP
is 10-times as powerful, producing 50Ð100 bands (Fig. 1;
Vos et al. 1995). Second, RAPDs are criticised for variation
in the number and concentration of the products due to
small changes in reaction conditions (Bielawski et al. 1995;
Lessells & Mateman 1998). In contrast, Vos et al. (1995) have
demonstrated that the AFLP reaction is stable over a 1000fold difference in template quantity. Moreover, the preselective/selective AFLP amplifications are carried out
with targets that have been ligated in place. Any selection is
due to one or two, nonligated 3' nucleotides. Consequently,
when the AFLP primers do not match, no amplification
occurs. As a result the annealing temperature is standardized and the AFLP bands tend not to vary in intensity.
If AFLP is used to sex organisms directly the consistency of the technique is beneficial. However, a reference band that is less intense than the Y-specific band
should be used as a positive control. This will guarantee
that the gender is correctly scored even if band intensity
is reduced because of a poor AFLP reaction or a short
exposure time.
The main threat to the accuracy of sex identification
using AFLPs is the possible occurrence of null alleles,
where the Y chromosome is present but the marker does
not amplify. Large numbers of known-sex individuals of
the heterogametic sex are needed to achieve acceptable
small confidence limits on the occurrence of null alleles
(e.g. 100 individuals for a confidence limit of 0Ð4% when
all heterogametic individuals carry the marker; Lessells &
Mateman 1998). Such large samples are obviously difficult to attain, but a more efficient check for null alleles is
probably with individuals taken from a wide geographical range rather than a single population.
The a priori expectation for the occurrence of null
alleles can also be reduced by using a PCR test based on
an AFLP marker rather than using the AFLP directly. If a
multiplex PCR test is used at moderate stringency, only a
mutation of the 3' primer ends (6 bp) is likely to stop
amplification. The additional cost of designing this multiplex PCR test is in sequencing a Y-linked AFLP product
and selecting positive control primers. This cost is worthwhile when many samples are to be processed and
accuracy is required (Fig. 3).
In summary, AFLP may be more technically demanding
than RAPD but the increased power makes it less
demanding to isolate a sex-linked marker. This is true, not
only for the avian examples described here but any organism with genetical sex determination.
Acknowledgements
Many thanks to the critiques Kate Lessells, Bob Dawson (International Foundation for the Conservation and Development of
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R. GRIFFITHS AND K. ORR
Fig. 2 Three AFLP profiles from two male
and two female birds highlighting the Wlinked markers (W). Sections 1 and 2 are
from the ostrich illustrated in Fig. 1 whilst
section 3 was obtained with the EACT/M-CAC primer combination from
the shag.
Fig. 3 Sex identification in the ostrich with two different multiplex
PCRs. Lane 1 is a 1 kb marker (BRL), lanes 2 and 3 are ostriches
sexed with the ScW1 primers (161 bp band) and lanes 4 and 5 are
ostriches sexed with ScW2 (104 bp). The P8/P18 CHD primers
provide the internal positive controls in lanes 2Ð5 (≈ 295 bp).
Wildlife) and the referees; to Michelle and Pete Hollingsworth
(AFLP), D. C. Deeming and the Ayres (ostriches), Francis Daunt
(shags) and to BBSRC for the funding.
Richard and Kate work in the Molecular Evolution Unit where
they refine DNA sex identification and study evolutionary ecology.
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