A DNA Test to Sex Ratite Birds

Molecular Ecology (2002) 11, 851–856

BlackwellA Science LtdDNA test to sex ratite birds

LEON HUYNEN, CRAIG D. MILLAR* and DAVID M. LAMBERT Institute of Molecular BioSciences and Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Private Bag 11–222, Palmerston North, New Zealand

Abstract DNA-based sex tests now exist for many avian species. However, none of these tests are widely applicable to ratites. We present DNA sequence data for a locus that is W chromosome-linked in the kiwi, ostrich, cassowary, rhea, and emu. At the amino acid level, this sequence has significant homology to X-linked genes in platyfish and Caenorhabditis elegans. Polymerase chain reaction (PCR) primers designed to this locus allow the assignment of sex in all species of living ratites. Keywords: birds, RAPD, ratites, sex-assignment, sex-markers, W chromosome Received 4 June 2001; revision received 28 August 2001; accepted 3 January 2002

Random amplification of polymorphic DNA (RAPD) Introduction analysis has been used previously to successfully isolate Many DNA-based sexing methods now exist which are sex-specific markers for chicken (Levin et al. 1993), great tit, capable of sexing a wide range of birds. These methods zebrafinch, jackdaw (Griffiths & Tiwari 1993), and ostrich rely on the detection, by PCR or Southern analysis, of (Bello & Sanchez 1999). We have also used a RAPD sex-specific genetic markers such as CHD1W/CHD1Z approach in an attempt to assign sex in ratites and report (Ellegren 1996; Griffiths et al. 1998; Fridolfsson & Ellegren the detection of a sex-specific sequence in kiwi and the 1999), EE0.6 (Itoh et al. 2001), ATP5A1W/ATP5A1Z subsequent development of a PCR-based test that allows (Dvorák et al. 1992), and Wpkci (Hori et al. 2000; O’Neill the determination of sex in all living ratites. et al. 2000). Collectively, these methods are capable of genetically sexing nearly all species of birds, with the Materials and methods notable exception of the ratites. Sex determination in birds, including ratites, involves DNA samples a ZZ (male), ZW (female) chromosomal mechanism (Ansari et al. 1988), although for a long time, ratites were Samples from the following ratites; rhea (Rhea americana), thought not to have such a system, as their sex chromo- ostrich (Struthio camelus), emu (Dromaius novaehollandiae), somes were shown to be largely homomorphic (Takagi cassowary (Casuarius casuarius manteui), and North Island et al. 1972; De Boer 1980). However, Fluorescence In Situ brown kiwi (Apteryx australis manteui) were obtained as DNA Hybridization (FISH) analysis has shown that distinct precipitates or blood. In all cases the samples were derived genetic differences between the two putative sex chromo- from birds of known sex (determined by morphology/ somes do exist for ostrich (Ogawa et al. 1998) and cas- behaviour, or, for the ratite DNA samples provided by sowary (Nishida-Umehara et al. 1999). Furthermore, PCR- J Halverson, by PCR analysis using species-specific primers). based DNA sex tests have now been developed for Avian DNA was isolated from blood by SDS/proteinase emus, cassowaries (J Halverson pers. comm.; De Kloet K digestion and phenol-chloroform extraction (Sambrook 2001) and ostriches (Bello & Sanchez 1999; Griffiths & Orr et al. 1989). 1999), but these tests are unable to be applied to ratites in general. RAPD screening for sex-specific markers DNA mixtures of known-sex North Island brown kiwi Correspondence: Prof. David Lambert. Fax: + 64 6350 5866; E-mail: [email protected] were subjected to RAPD analysis using 30 10-mer primers *Present address: School of Biological Sciences, University of of arbitrary sequence (Kit A, Operon Technologies Inc.). Auckland, Private Bag 92019, Auckland, New Zealand PCR was carried out in 10 µL volumes consisting of 2–10 ng

of DNA, 2.5 mm MgCl2, 10 mm Tris-HCl pH 8.3, 50 mm KCl, 200 µm of each dNTP, 1.5 µm of each primer, and 0.2 U of AmpliTaq®. (Perkin Elmer). Samples were initially heated to 94 °C for 120 s and then subjected to 10 cycles of PCR amplification at 94 °C for 15 s, 42 °C for 30 s, and 72 °C for 30 s. This was followed by 25 additional cycles of 94 °C for 15 s, 38 °C for 30 s, and 72 °C for 30 s. PCR products were separated by electrophoresis in 2% MS, 1% LE agarose (Boehringer Mannheim) in TBE, stained with ethidium Fig. 1 RAPD screening of rhea, emu, and kiwi DNA for female- specific fragments. Mixes of male (M) and female (F) DNA from bromide and then visualized over UV light. rhea and emu, or individual samples from North Island brown Sex-specific PCR products were recovered from agarose kiwi of known sex were subjected to PCR using the 10-mer by centrifugation through glass wool and purified by OPA18. An amplification product of approximately 700 bp, phenol-chloroform extraction and ethanol precipitation present in female kiwi only, is indicated by the white arrows. (Sambrook et al. 1989). PCR products were sequenced by Molecular weight standards are provided by the 1 Kb™ ladder the dideoxy method and then analysed on an ABI 377 (Gibco BRL®). Cont. and bp refer to the negative control and base automated sequencer. pairs, respectively.

Southern analysis (n = 11). The same primer failed to detect sex-specific dif- Southern analysis was carried out as described in Millar ferences in mixtures of DNA from male and female rheas et al. (1992). and emus (Fig. 1). The kiwi W-specific DNA fragment (kW1) was sequenced and shown to be 676 bp in length (patent pending; Sequence-specific PCR primers GenBank accession number: AF308932). At the nucleotide The primers designed and used in this work are detailed in level, kW1 has no significant homology to sequences in the Figs 2 and 5. Amplification reactions were carried out in GenBank database. However, when the kW1 sequence was 10 µL volumes as described above, but using the following translated and compared to the database at the amino acid conditions; 94 °C for 60 s, then 10 cycles of 94 °C for 15 s, level, significant homology (42 and 52% identity, respec- 55 °C for 20 s, and 72 °C for 20 s, followed by 10 cycles of tively) was found over a region of 80 amino acids with two 94 °C for 15 s, 50 °C for 20 s, and 72 °C for 20 s, and finally X-linked proteins: Xmrk (GenBank accession number: 25 cycles of 94 °C for 15 s, 47 °C for 20 s, and 72 °C for 20 s. AAA93301), an oncogene product found in the platyfish In some instances a second (nested) PCR was carried out Xiphophorus maculatus and thought to play a role in melano- by diluting the first PCR mix 1:10 with water. One µL of genesis (Gutbrod & Schartl 1999); and a putative protein this dilution was then added to the second PCR mix and (GenBank accession number: CAB76746) from Caenorhabditis cycled as described. elegans that contains a CHRomatin Organization MOdifier (CHROMO) domain. The homology kW1 translation products share with these proteins covers an interrupted Results reading frame of approximately 120 amino acids that For RAPD analysis, DNA was extracted from the blood of harbours a conserved integrase motif (Fig. 2). known-sex North Island brown kiwi and quantified by gel The kW1 fragment was used to probe DNA from electrophoresis against standards of known concentration. known-sex emus and was shown to detect several W- Separate mixtures were then made of male and female specific DNA sequences, as well as DNA common to both DNA, such that the DNA from each individual was repres- sexes (Fig. 3). ented equally in the mixture. By using such an approach, PCR primers (k1, k2, k3, and k4), designed from the kW1 it was hoped that non-sex linked DNA polymorphisms sequence, were able to amplify female-specific DNA frag- present in any one individual, which may have led to false ments from a number of ratite species (at least five known positive results, would be effectively diluted. males and five known females were tested for North Island RAPD analysis was initially carried out on DNA mix- brown kiwi, emu, ostrich, rhea, and cassowary). The primers tures from 11 male and 10 female North Island brown kiwi. k1 and k2 consistently amplified at least one female-specific Primers that amplified a putative sex-specific fragment fragment but sometimes failed to amplify from male DNA. were then used to amplify DNA from individual male and For this reason the CHDW1/CHDZ1 gene primers P2 female kiwi. Thirty primers were tested of which only one, and P8 were added to the PCR mix as a reaction control OPA18 (5′-AGGTGACCGT-3′), consistently amplified a (Fig. 4A; Griffiths et al. 1998). A nested PCR, using the k1/ sex-specific fragment in females (n = 10), but not in males k2 primers, followed by the k3/k4 primers, produced a

Fig. 2 Sequence, translation, and peptide line-up of kW1. The nucleotide sequence of kW1 was translated in frame 2 (pep2) or frame 3 (pep3) and compared with the amino acid sequence of Xmrk and a putative CHROMO domain protein from C. elegans. The amino acids in bold type represent a conserved integrase motif. The position of sequence-specific primers k1–4 are indicated. A stop signal is shown with an asterisk.

Fig. 4 Sex-specific amplification of DNA from known-sex rhea, emu, kiwi, ostrich (ostr.), and cassowary (cass.). (A) DNA from male and female ratites amplified using the sequence-specific primers k1/k2. As k1 and k2 sometimes failed to amplify sequences from some male ratites, the CHD1W/CHD1Z primers P2 and P8 (Griffiths et al. 1998) were added to the PCR mix at 1.5 µm to serve as a reaction control. (B) Results of a nested PCR carried out on the same ratite DNA as in (A), using primers k1/k2 followed by k3/k4.

Fig. 3 Southern analysis of DNA isolated from male and female single DNA fragment in male rheas, emus, kiwi, and emus. Genomic DNA was isolated from known-sex emus, ostriches, and two DNA fragments in male cassowaries. In digested with the restriction enzyme HinfI and hybridized to kW1 female ratites the same PCR conditions produced a single labelled with α 32[P]dCTP. Molecular weight standards (DNA std.) are shown in kilobase pairs (kb). Examples of fragments that female-specific fragment in rheas, two female-specific are W chromosome-specific (W) and Z or autosomal chromosome- fragments in emus, kiwi, and ostriches, and three female- specific (Z/A) are indicated. specific fragments in cassowaries (Fig. 4B).

Fig. 5 Line-up (according to sequence homology) of amplified DNA from male and female ratites. The sequence of kW1 and PCR products obtained by nested PCR using k1/k2 followed by k3/k4 were aligned by eye and used to design the primers w1 and k7. The consensus sequence (Con) is shown above the sequence alignment with nucleotide identities depicted as dots (.) and sequence deletions as dashes (–). The percentage sequence homology each sequence has with kW1 is also shown. Respectively, Os, Em, Rh, and Ca refer to ostrich, emu, rhea, and cassowary, while M and F refer to male and female. The numbers refer to the position of nucleotides within the 676 bp kW1 fragment.

Several PCR products (obtained by nested PCR using 200 bp. Each sequence is at least 86% homologous to that k1/k2 followed by k3/k4) were sequenced and aligned by of kW1 (Fig. 5). eye (Fig. 5). Only the ~600 bp products amplified in male The primers w1 and k7 were designed (to the sequences ostrich, emu, and rhea, and the ~380 bp products amplified shown in Fig. 5) to maximize the difference obtained in female ostrich, emu, and cassowary produced clear DNA in amplification products between male and female sequence. Analysis of these sequences indicated that the ratites. Using these primers, we were able to genetically smaller ~380 bp female-specific DNA products differ from sex all extant species of ratites (Fig. 6). All species sexed the larger ~600 bp fragments by a deletion of approximately produced a DNA fragment of about 350 bp, while females

Interestingly, Southern analysis of emu DNA, using kW1 as a probe, shows that the strength of the hybridization signal of all DNA bands in females is as strong (or stronger) than the equivalent bands in males (Fig. 3). Given that males have two Z chromosomes while females have only one, this result is surprising and suggests that if any of these sequences are present on the Z chromosome, there are at least as many copies on the W chromosome. Alternatively the non-W bands may be autosomal in origin. In support of this, Klein & Ellendorf (2000) have Fig. 6 Genetic sexing of ratite species. DNA was supplied or extracted from the blood of known-sex birds and subjected to PCR recently shown that in chicken, several copies of the W- using the primers w1 and k7. A 350 bp PCR product, common to specific Xho1 family of repetitive sequences are also all Struthioniformes, is indicated by the black arrow. White present on various autosomes. arrowheads indicate PCR products that are consistently amplified Sequence analysis indicates that kW1 (when translated) from female DNA only. shares homology with the peptide sequence of two X-linked genes. However, the absence of a single uninterrupted open reading frame in kW1 (or any of the fragments produced an additional PCR product of about 150 bp as sequenced) as well as the lack of third base variance in well as shadow bands slightly smaller or larger than 350 bp. coding triplets (when compared to the coding triplets of Xmrk and the putative Caenorhabditis elegans protein) suggests that these sequences are unlikely to be expressed. Discussion In this report a DNA sequence has been isolated from The sex-specific sequence (kW1) we have isolated from female kiwi that appears to be present on the W chromo- kiwi is conserved on the W chromosome of all species of some of all species of extant ratites. Using this sequence, extant ratites. Primers k1 and k2, designed from the kW1 PCR primers have been designed to this allow the rapid sequence are capable of amplifying DNA from the W and efficient sexing of all living ratite birds. chromosome as well as from other, possibly autosomal chromosomes. A nested PCR, using the primers k1/k2, Acknowledgements followed by primers k3/k4 results in the amplification of 1–2 fragments in males and 2–3 fragments in females This research was made possible by a grant from the Marsden (Fig. 4B). Surprisingly, the fragment amplified in males is Fund ‘Sexing the Lost Giants of New Zealand’ and supported by not amplified in the corresponding females for emus, kiwi, Massey University. North Island brown kiwi blood samples were kindly supplied by Murray Potter (Institute of Natural Resources, ostriches, and cassowaries. The failure to amplify these Massey University, New Zealand), Helen McCormack (Rainbow sequences in females may be due to the preferential & Fairy Springs, Rotorua, New Zealand), and Eric Fox (Otorohanga amplification of the smaller W chromosome-specific Kiwi House, New Zealand). Emu, ostrich, rhea, and cassowary fragments (Toouli et al. 2000). DNA was a kind gift from Joy Halverson (Celera AgGen, CA, Amplification of female ratite DNA using the primers USA). Emu blood samples were provided by Murray Goss (Emu w1 and k7 results in the production of a ~150 bp female- Farm, Feilding, New Zealand). Further thanks to T King, P Ritchie specific fragment as well as 2–3 female-specific shadow and A Roeder for comments on the manuscript. Publication number two from the Allan Wilson Centre for Molecular Ecology bands of approximately 350 bp (Fig. 6). 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