MOLECULAR TECHNIQUES FOR SEX IDENTIFICATION IN
BIRDS 3
BIOLOGICAL LETT. 2006, 43(1): 3.12
Available online at http://www.biollett.amu.edu.pl
Molecular techniques for sex identification in birds
ANNA DUBIEC and MAGDALENA ZAGALSKA-NEUBAUER
Department of Ornithology, Polish Academy of Sciences, Nadwi.lañska 108, 80-680 Gdañsk, Poland
(Received on 20th July 2005; Accepted on 10th December 2005)
Abstract: In many bird species, males and females cannot be discriminated on the basis of external
characteristics. The problems associated with reliable sex identification were overcome by introducing
the molecular techniques in the 1990s. These techniques are generally based on DNA hybridization or
polymerase chain reaction (PCR). This paper reviews the sexing techniques, highlighting their advantages
and drawbacks. To date, the amplification of primers related to the CHD gene is the most rapid, cheap
and reliable technique of almost universal application. The description of sexing techniques is accompanied
with a short overview of DNA sampling, storage and extraction methods.
Key words: AFLP, CHD gene, molecular sexing, PCR-based methods, RAPD, W-linked markers, sex
chromosomes
INTRODUCTION
The basic information about each individual includes its sex. However, telling
the difference between the sexes in various taxonomic groups is not always as easy
as in humans. In birds that are sexually dimorphic, such as the house sparrow (Passer
domesticus Linneus, 1758), mallard (Anas platyrhynchos Linneus, 1758) and collared
flycatcher (Ficedula albicollis Temminck, 1815), it is very easy to distinguish
between males and females. However, males and females of many species have very
similar phenotypic traits (sexual monomorphism), so that even experienced ornithologists
may have problems with unambiguous sex identification. For example, at least
60% of all passerine species are sexually monomorphic in colour (PRICE & BIRCH
1996). In nestlings it is even more difficult than in the adults to distinguish between
the two sexes. This applies to almost all bird species, as even in those sexually dimorphic
as adults and juveniles, the males and females do not differ in morphological
traits shortly after hatching. Usually the distinct differences between the sexes
appear at the earliest a few weeks after hatching. In passerines, for example, this
results in the lack of data on sex ratio in broods, as young birds leave the nest before
they can be sexed on the basis of morphological traits.
4 A. Dubiec and M. Zagalska-Neubauer
The development of molecular sexing techniques constituted a breakthrough
in reliability and rapidity of sex identification in birds. By the time of their common
application, sex had been identified on the basis of: (1) behavioural observations,
(2) presence of brooding patch, (3) differences in morphometric traits, (4) examination
of the gonads by laparotomy or laparoscopy, and (5) examination of sex chromosomes.
The first two methods can be generally applied only during the breeding
season, and the analyses of morphometric traits may be ambiguous. The examination
of gonads may be difficult in the adult birds outside the breeding season (when
the gonads regress) and in nestlings because of their small body size (PRUS &
SCHMUTZ 1987). Cytological sex identification is based on differences in morphology
of sex chromosomes. In birds, unlike in mammals, females are the heterogametic sex,
i.e. have 2 different sex chromosomes (ZW), while males are the homogametic sex
(ZZ) (SOLARI 1994). Bird sex chromosomes have evolved from a pair of autosomes
(OHNO 1967, FRIDOLFSSON et al. 1998). During this process, the W chromosome lost
many of its genes, so its size was reduced, while the Z chromosome has not changed
the gene content. The W chromosome is rich in heterochromatic, repetitive DNA of
the satellite type and both chromosomes contain a small pseudoautosomal region.
The examination of the morphology of sex chromosomes requires establishing the
cell cultures and stopping the mitotic division at the metaphase stage, when chromosomes
are well visible and separate (DELHANTY 1989). Chromosome spreads are most
commonly based on the cells from the pulp of growing feathers, which may be collected
during moult in the adult birds and from the nestlings. Despite the large number
of chromosomes (most birds have around 80 chromosomes), the sex chromosomes
are usually easy to distinguish from one another, because they differ in size. The Z
chromosome falls into the group of large chromosomes, called macrochromosomes,
usually being comparable in size with chromosomes 4 and 5, while the W chromosome
represents smaller chromosomes, called microchromosomes (TEGELSTRÖM &
RYTTMAN 1981, FILLON & SEQUELA 1995). However, chromosome spreads of good
quality are very laborious. Nowadays there exist simpler, cheaper and more effective
techniques, so cytological sex identification is only very rarely used (e.g. ARCHAWARANON
2004).
This paper reviews molecular techniques for sex identification in birds to help
the researchers who are unfamiliar with this type of sexing, to select the most suitable
method. The techniques are divided into two groups: (1) based on DNA hybridization,
where sex-specific sequences are detected in genomic DNA by complementary
DNA probes; and (2) based on polymerase chain reaction (PCR), in which
sex-specific DNA is located by primers and then amplified. The description of molecular
sexing techniques is accompanied with a short overview of DNA sampling,
storage and extraction methods.
DNA SAMPLING, STORAGE AND EXTRACTION
In birds, DNA is most commonly obtained from blood, as avian erythrocytes
are a source of nuclear DNA. A small amount of blood is collected into nonheparinized
capillaries by puncture of the wing or leg vein (depending on bird species
and age). Another method of DNA sampling includes the collection of feathers,
preferably growing ones and down. DNA is extracted from the cells from the basal
MOLECULAR TECHNIQUES FOR SEX IDENTIFICATION IN BIRDS
5
tip of the calamus (MORIN et al. 1994) or from the blood clot embedded in the shaft
(HORVÁTH et al. 2005). Dead adults, nestlings and embryos are sampled for a piece
of tissue, preferably from the brain or liver. Samples should be stored under conditions
that prevent DNA degradation. Most commonly they are preserved in 96%
ethanol, EDTA buffer or Queen.s lysis buffer (10 mM Tris, 10 mM NaCl, 10 mM
EDTA, 1% n-lauroylsarcosine pH 7.5; SEUTIN et al. 1991). DNA in ethanol is stable
at room temperature and may be stored in this way for several years. The stability of
DNA may be further prolonged by storing the samples frozen at .20°C or .80°C.
Blood samples in EDTA are stored at 4°C (if they are analysed soon after collection)
or in a freezer, while samples in Queen.s lysis buffer, at room temperature. An alternative
to those methods is FTA® databasing paper, which to date has been mainly
applied to human forensic analysis (SMITH & BURGOYNE 2004). FTA® cards (Whatman
International) consist of filter paper impregnated with a mix of chemicals, which
lyse cells, prevent the growth of bacteria and protect the DNA in the sample. The
DNA entangled in the paper can be used for the analyses either in situ (i.e. on the
paper) or following elution from the paper. FTA® cards ensure a long-term stability
(in forensic analysis, blood samples have been found to be stable for more than a
decade) and safe archiving at room temperature.
DNA can be isolated from the sample by any of several methods. They differ
in the effectiveness, costliness and intensiveness of labour. Those features, as well
as the chosen sexing technique, decide which extraction method is the most suitable.
The great variety of available commercial kits enables the isolation of very highquality
DNA. However, ready kits are costly, which noticeably increases the total
costs of sex typing when large-scale sex identification is needed. The cheaper alternatives
include, e.g. the phenol-chloroform method (SAMBROOK et al. 1989), guanidium
thiocyanate method (HAMMOND et al. 1996) and Chelex extraction (W ALSH
et al. 1991). The first two methods are laborious, time-consuming and employ risky
substances, but they yield DNA of optimum quality and quantity. Chelex extractions
are very rapid, which is of great importance when the sexing results are required at
a short time, and produce good quality DNA.
SEXING TECHNIQUES BASED ON DNA HYBRIDIZATION
The W chromosome contains a large proportion of repetitive sequences, which
may be searched for W-linked markers. Those repetitive sequences include tandemly
repeated DNA: micro- and minisatellites. Microsatellites form clusters of usually
less than 150 bp in length, with repeat units up to 13 bp, while minisatellites are
longer, with clusters up to 20 kb in length and repeat units up to 25 bp (BROWN 2002).
To detect micro- or minisatellite sex-linked markers, the DNA is digested with restriction
enzymes, which cleave the DNA at specific sites defined by the nucleotide
sequence. Obtained DNA fragments are next separated by size with electrophoresis
on an agarose gel and transferred to a nylon membrane (Southern blotting), which is
subsequently incubated in a solution containing probes, labelled DNA fragments.
Probes hybridize with genomic DNA, i.e. .stick. to complementary sequences. Hybridization
is detected depending on the type of probe labelling (usually radioactively
with 32P or with biotin) by the autoradiogram, fluorescence, or enzymatic reaction.
As a result, a specific profile of DNA fragments is obtained. In order to visualize
6 A. Dubiec and M. Zagalska-Neubauer
whether the probe generates female-specific fragment(s), the DNA profiles of reference
males and females should be compared. For example, a trinucleotide repeat
(TCC)n was shown to be sex-linked in pigeons and chickens (EPPLEN et al. 1991).
LONGMIRE et al. (1993) tested 4 probes: 3 dinucleotide repeats (CT)n, (GT)n, (CG)n
and 1 trinucleotide repeat (TCC)n in combination with 1 of the 3 restriction enzymes
(HaeIII, HinfI, PstI) in 9 bird species from 6 orders. The best probe (CT)n detected
female-specific sequences in 6 species, while the dinucleotide repeat (CG)n did not
produce any sex-specific fragments in any of the tested species. Most commonly used
minisatellite markers include probe 33.15 (JEFFREYS et al. 1985). MIYAKI et al. (1997),
by applying this probe to 36 South American parrot species from 13 genera, were
able to identify female-specific fragments in 28 species. Each species showed a set
of female-linked fragments (between 2 and 4) peculiar to a given species.
SEXING TECHNIQUES BASED ON POLYMERASE CHAIN REACTION (PCR)
Random amplification of polymorphic DNA (RAPD)
RAPD employs in a PCR reaction a single and randomly chosen 10-nucleotide
primer that amplifies genomic fragments. The low annealing temperature (usually 35.
40°C), which is typical for this technique, reduces the specificity of reaction, and
hence the repeatability of the results. However, simultaneously, low temperature
enables the amplification of a wide range of genomic fragments (W ILLIAMS et al.
1990, W ILLIAMS et al. 1993). If one of the bands on a gel is specific only for females,
it most probably reflects a W-linked sequence (GRIFFITHS & TIWARI 1993). A
polymerase chain reaction with a single primer amplifies usually 10-20 DNA fragments.
Therefore, RAPD analyses start with testing a few dozen of primers to detect
those that amplify the products specific for females. However, despite the large number
of tested primers, a female-specific product may not always be found. For example,
LESSELLS & MATEMAN (1998) did not identify suitable primers for 3 out of the 10
studied species after screening up to 69 different primers per species. As RAPD was
developed to study genetic polymorphism, the differences between the 2 individuals
may be generated by autosomal or Z chromosomal polymorphism (W ILLIAMS et al.
1990). Therefore, to locate the marker, which is most likely linked to the W chromosome,
pooled DNA, i.e. DNA sample from different individuals of the same sex,
should be used. In this way, most of the common polymorphisms in a population are
amplified within each pool (bulked segregant analysis), enabling identification of the
products that are found only in the female pool (MICHELMORE et al. 1991). In general,
the PCR products from a pooled sample of 3.5 males should be compared with
the PCR products from a pooled sample of 3.5 females. DNA fragment are resolved
either on an agarose or polyacrylamide gel and fragments selected as a sex-specific
marker should be among the 4 most intensive bands on a gel (GRIFFITHS 2000).
Amplification fragment length polymorphism (AFLP)
The AFLP technique combines PCR with digesting DNA with restriction enzymes
(VOS et al. 1995). A single reaction results in about 100 DNA fragments. The
first step of the AFLP technique includes DNA digestion with 2 different restriction
endonucleases: a 4-base cutter and a 6-base cutter. The next step is ligation, during
MOLECULAR TECHNIQUES FOR SEX IDENTIFICATION IN BIRDS
7
which oligonucleotide adaptors (20.30 bp) attach to .cohesive ends. produced by
endonucleases. This step is followed by a preselective amplification with selected
primers complementary to an adaptor and a single specific nucleotide in the original
fragment sequence. This reduces the pool of fragments from the original mixture.
The last step of AFLP includes a selective amplification with the same primers, but
usually with a 3-nucleotide extension. The products are displayed on polyacrylamide
gels. Similarly to RAPD, the identification of sex-specific markers is difficult because
of the high polymorphism of DNA sequences. However, unlike in RAPD, the
use of pooled DNA samples to detect female-specific fragments is not recommended,
because the PCR products may not always reflect each of the individual samples
in the pooled DNA. Therefore, sex-specific markers should be detected by surveying
the primers in 3 males and 3 females individually (GRIFFITHS 2000).
In both RAPD and AFLP it is advisable to select a positive control: a band of
lower intensity on a gel than a W-linked fragment (GRIFFITHS 2000). The positive
control is necessary to exclude the possibility of incorrect sexing. Such a possibility
may arise when non-optimal PCR reaction results in no visible W-linked marker,
consequently leading to identification of females as males (SABO et al. 1994). A malespecific
DNA profile can be identified only if a positive control is present and a
female-specific sequence is absent on a gel. W-linked sequences located by RAPD
and AFLP may be also used to design the primers that are subsequently used in a
standard PCR (sequence characterised amplified region, SCAR). Application of such
primers would clearly simplify the whole procedure of sex identification and its
costliness in comparison with the AFLP technique. However, PCR results in this case
in 1 band in females and no bands in males, so a positive control is required. This is
achieved by selecting the primers amplifying the DNA fragment in both males and
females, which may be successfully applied together with the primer pair targeted at
a W-linked sequence in a multiplex PCR. Control primers should amplify a DNA
fragment larger and of lower intensity than the sex-specific fragment. The primers
designed on the basis of the W-specific sequence detected by AFLP, have been successfully
used to separate males from females in the ostrich. This species as well as
other ratites cannot be sexed by CHD-based PCR due to undifferentiated sex chromosomes
(GRIFFITHS & ORR 1999).
CHD-based sex identification
Molecular techniques for sex identification in birds were revolutionized by
discovering in 1995 the first gene located in the W chromosome (GRIFFITHS & TIWARI
1995). A very closely related copy of this gene was soon after discovered also
in the Z chromosome (GRIFFITHS & KORN 1997). Genes are excellent markers for
sex identification as they are made of functional DNA and therefore they evolve very
slowly and are conserved between different taxa. So far, only a few genes have been
located on the W chromosome. The most universal tag for sex typing is provided by
the CHD gene. It encodes the chromodomain helicase DNA binding protein and is
located probably in both chromosomes in almost all bird species except ratites, which
have undifferentiated sex chromosomes (ELLEGREN 1996, GRIFFITHS et al. 1996). The
lack of recombination between the Z and W copies of this gene indicates that it is
located outside the pseudoautosomal region (FRIDOLFSSON et al. 1998). CHD con8
A. Dubiec and M. Zagalska-Neubauer
tains at least two introns, e.g. non-coding quickly-evolving DNA fragments, located
between slowly evolving coding fragments, which differ in length in the Z and W
chromosomes. The three CHD-related primer pairs used in sex identification were
designed to flank the fragment of the gene with the intron (Table 1). This allows
discrimination between the products from the Z and W chromosomes on a gel. Consequently,
males are identified by 1 band and females by 2 bands on a gel, with some
exceptions. The 2550F/2718R primers may in some species produce only 1 fragment
both in males and females (FRIDOLFSSON & ELLEGREN 1999). This results from a
preferential amplification of the shorter gene copy from the W chromosome, which
in turn results in no detectable product from the Z chromosome. However, in such
cases the birds can be easily sexed on the basis of the difference in size of both
amplified fragments. The single fragment in males and females was found in the
Anatidae, Gruidae, Scolopacidae, Falconidae and Accipiteridae. The P2/P8 primers
may also produce only 1 fragment in both sexes, as DNA sequence from the Z chromosome
is shorter than from the W chromosome and may be preferentially amplified.
However, in this case, females may be misidentified as males. The P2/P8 primers
(GRIFFITHS et al. 1998) and the 1237L/1272H primers (KAHN et al. 1998) flank
the same intron. Therefore, the size difference in the fragments from the Z and W
chromosomes is identical (JENSEN et al. 2003). Because 1237L/1272H may produce
a larger number of non-specific fragments than P2/P8, it seems advisable to use the
latter pair of primers (JENSEN et al. 2003). In general, the difference in size between
Z- and W-specific fragments amplified with the 2550F/2718R primers ranges from
150 to 250 bp, while for the P2/P8 primers, from 10 to 80 bp (FRIDOLFSSON &
ELLEGREN 1999, JENSEN et al. 2003). Therefore, in some species sexed with the P2/
P8 primer pair, polyacrylamide instead of agarose gels are recommended to separate
the Z- and W-bands, because polyacrylamide gels provide better resolution. For
example, the fragments amplified with the P2/P8 primers cannot be distinguished on
agarose gels in the auklets (DAWSON et al. 2001). Moreover, the assignment of sex
Primers Nucleotide sequence Source
P2
P8
5´-TCTGCATCGCTAAATCCTTT-3´
5´-CTCCCAAGGATGAGRAAYTG-3´
GRIFFITHS et al. 1998
2550F
2718R
5´-GTTACTGATTCGTCTACGAGA-3´
5´-ATTGAAATGATCCAGTGCTTG-3´
FRIDOLFSSON & ELLEGREN 1999
1237L
1272H
5´-GAGAAACTGTGCAAAACAG-3´
5´-TCCAGAATATCTTCTGCTCC-3´
KAHN et al. 1998
Table 1. Nucleotide sequences of CHD-linked primers applied to sex identification in birds
MOLECULAR TECHNIQUES FOR SEX IDENTIFICATION IN BIRDS 9
on the basis of the P2/P8 primers may be in some species difficult because of a
polymorphism in the Z chromosome. To date such polymorphism has been documented
in nearly 20 species, e.g. in 4 auklet species, great tit (Parus major Linneus, 1758),
Eurasian treecreeper (Certhia familiaris Linneus, 1758) and western gull (Larus
occidentalis Audubon, 1839) (DAWSON et al. 2001, Sheffield Molecular Genetics
Facility bird sex-typing database: http://www.shef.ac.uk/misc/groups/molecol). To
avoid these problems, the 2550F/2718R primers instead of P2/P8 should be used,
because they flank a different intron, which is most probably responsible for a polymorphism
of fragments from the Z chromosome. In the whiskered auklet (Aethia
pygmaea J.F. Gmelin, 1789), the difference in size between the Z chromosome fragments
(12 bp) is very similar to the difference in size between fragments visualized
in females (14 bp). Consequently, sex in this species should be identified basing on
the analysis of fragment size and not on the number of fragments (1 or 2). The 2550F/
2718R primers produce in this species only 2 alternative fragments, which differ in
length by 230 bp, so females can be easily separated from males on agarose gels.
If the fragments from the Z and W chromosomes do not show any detectable
differences in size, but are heterogeneous with respect to the nucleotide sequence,
the female- and male-specific profiles may be generated by applying selected restriction
enzymes to the PCR products. For example, the P2/P8 primers produce the fragments
of undetectable size differences in the Eurasian woodcock (Scolopax rusticola
Linneus, 1758). The application of the restriction enzyme BshNI, which recognizes
a cleavage site in the W chromosome, enables a reliable sex identification, based on
3 bands in females and 1 band in males (VÄLI & ELTS 2002).
Another W-located conserved sequence, which may be applied to sex typing,
although not universally, is the EE0.6 sequence (ITOH et al. 2001). EE0.6 is likely to
be a part of a pseudogene and it has been shown to be conserved in the Carinatae
and Ratitae. With a suitable combination of primers for EE0.6 and control primers
for a Z/W-common sequence, 36 species from 16 different orders of the Carinatae
were sexed by PCR (ITOH et al. 2001).
SELECTION OF A SEXING TECHNIQUE
The unquestionable first method of choice while setting about molecular sexing
of a bird species, which has not previously been sexed in this way, is a CHDbased
technique. It is accurate, easy, relatively cheap and fast. The processing of a
sample including DNA extraction, PCR and resolution of PCR products on a gel may
take less than 5 hours. The selection of the most suitable CHD-linked primer pairs
should start with checking which primers were successfully applied in closely related
species. If no such data are available, it is recommended to test both P2/P8 and
2550F/2718R primers for their applicability in a species under study. Moreover, this
technique as well as the others should be tested for reliability with the individuals of
the known sex. The other described techniques may be recommended only when
CHD-based sexing does not work, because they are more laborious, time-consuming
and costly. According to the intensiveness of labour and costliness, they should
be applied in the following order: RAPD, the location of micro- and minisatellite
10 A. Dubiec and M. Zagalska-Neubauer
markers by DNA hybridization, AFLP. AFLP is more repeatable than RAPD and
allows to produce a greater number of bands per assay, what gives a higher chance
of detection of sex-specific fragments. However, due to the complexity of the test
and its costliness, it has been used for sex identification extremely rarely. The analyses
based on DNA hybridization are accurate, but relatively slow. In all 3 methods
the isolated W-linked markers are often non-coding, fast-evolving sequences. Therefore,
markers identified in a given species are of a very narrow application, usually
limited to one or a few closely related species. Consequently, the process of sex
identification may be considerably prolonged if known markers do not apply and it
is necessary to screen genomic DNA in search for W-linked sequences.
Acknowledgements. We thank MACIEJ GROMADZKI, W OJCIECH KANIA, GRZEGORZ NEUBAUER and both
anonymous referees for critical comments on the previous versions of the manuscript. The work was
assisted by the bird sex-typing database maintained by Deborah Dawson at Sheffield Molecular Genetics
Facility (SMGF). The SMGF is funded by the Natural Environmental Research Council, UK.
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