INTRODUCTION

It is a challenging task to determine gender in many avian species based on external morphology. Griffits and colleagues reported that more than 50% of bird species are monomorphic (Griffits et al. 1998). Moreover, gender determination is difficult in the juvenile stage, even for dimorphic species (Kahn et al. 1998). The difficulty in gender determination of bird is a hurdle for research, wildlife conservation, and breeding program. Gender determination is necessary for various avian conservation programs that target the protection of numerous species through intensive breeding (Ito et al. 2003). In zoo and breeding centers, the accuracy of gender determination is essential since a massive number of birds are bred, produced, and traded (Vucicevic et al. 2013). Also, gender determination plays a vital role in veterinary science, enhancing the protection and conservation of birds (Lee et al. 2010).

Due to the high demand in the market, parrots (order Psittaciformes) are one of the most traded among all avian orders (Bush et al. 2014). The human demand results in legal and illegal trade of parrots. Understanding of parrot gender will be helpful to breed and protect parrot from natural and human threats such as uncontrolled trade. However, like many other birds, parrots do not show a high degree of sexual dimorphism, even in the mature stage. In this case, applying molecular techniques is necessary for fast and accurate identification of gender in parrots.

In avian species, the male has homogametic sex chromosomes (ZZ), whereas the female has heterogametic sex chromosomes (ZW). The presence of a chromo-helicase- DNA binding protein 1 (CHD1) gene on avian Z and W chromosomes with different size has increased the ability to determine gender for avian species (Griffiths and Tiwari 1995). The CHD1 gene is reported to encode the protein that regulates transcriptional activation on the chromatin level (Ellegren 1996). In chicken (Gallus gallus), CHD1-Z (gene ID: 395783) and CHD1-W (gene ID: 374195) were 48,421 bp and 115,681 bp in size, respectively and each gene includes 37 exons. Even though CHD1 genes encoded on Z and W chromosomes are different in length, the comparison of nucleotide sequences indicated that they are highly conserved (Valadan et al. 2017). For gender determination, based on CHD1 gene sequences of chicken and other animals, different universal primers were designed to flank CHD1 gene fragment with the intron (Griffits et al. 1998;Kahn et al. 1998). The amplified products contain the intron that is different in size between Z and W chromosomes. In chicken, P8/P2 primers bind to exons 23 and 24 and cover the intron between them. PCR amplification for partial CHD1-Z and CHD1-W fragments with P8/P2 primers is a quick and straightforward approach to determine avian gender (Griffits et al. 1998;Jensen et al. 2003). Avian gender is determined according to the observation of gel band where the sizes of the W and Z amplicons are differentiated.

In this study, the blind test to determine the gender of parrots from three Korean zoos was performed based on the amplification of the CHD1 gene. The primer P8/P2 (Griffiths et al. 1998) was used to amplify CHD1 gene fragments from different genders. The PCR products were visualized under UV light to determine the gender of collected parrot samples.

MATERIALS AND METHODS

A set of unknown gender samples of feather and blood examined in this study were obtained from parrots grown in Seoul Zoo (Seoul), Cheongju Zoo (Cheongju) and Uchi Zoo (Gwangju), Korea (Table 1). The ethical clearance number 2019-001 for the study using blood was issued by Seoul Zoo IACUC. Before the study started, all samples were confirmed species names based on cytochrome b gene sequences (data not shown). DNA was isolated from samples by Qiagen DNeasy^® Blood & Tissue Kit (Qiagen Inc., Valencia, CA), following the manufacturer’s instruction. The extracted DNA purity and concentration were measured by MaestroNano spectrophotometer (Maestrogen, Hsinchu, Taiwan).

Partial CHD1 gene was amplified with forward primer P8 (5ʹ-CTCCCAAGGATGAGRAAYTG-3ʹ) and reverse primer P2 (5ʹ-TCTGCATCGCTAAATCCTTT-3ʹ) described by Griffiths et al. (1998). PCR reaction mixture contained 30 μL: 15 μL of 2X DyeMix (Enzynomics, Daejeon, Korea) with 1.0 U of Taq polymerase, 1 μL of each primer (5 pmole μL^-1), 3 μL of DNA and distilled water up to 30 μL. The PCR condition: initial denaturation at 95°C for 2 min; 40 cycles of denaturation at 95°C for 45 s, annealing at 52°C for 30 s, extension at 72°C for 30 s; followed by a final extension at 72°C for 5 min. After staining 5% agarose gel (w/v) with Midori Green Advance (NIPPON Genetics Europe, Dueren, Germany), PCR products were run in 90 minutes at 120 V and visualized under UV light to check there were one or two bands according to parrot gender. For estimation of CHD1 gene fragment size, 100 bp step DNA ladder (Bionics, Korea) was run together with the PCR products. For confirmation of CHD1 gene amplification, PCR products of monk parakeet (Myiopsitta monachus) were cloned by TOPcloner^TM TA-Blunt kit (Enzynomics, Daejeon, Korea) and sequenced by Sanger sequencing. To check whether PCR products were CHD1 gene, the PCR products of monk parakeet (Myiopsitta monachus) were sequenced and submitted to Genbank (accession numbers: MT316044 for CHD1-Z and MT316045 for CHD1-W).

RESULTS AND DISCUSSION

CHD1 gene fragments from 27 parrot samples were amplified with P8/P2 primes. All samples were run on agarose gel and visualized under UV light. The representatives of male and female samples for each species are presented in Figure 1. The sizes of the CHD1-Z and CHD1-W PCR products were estimated in the ranges of 300-400 bp (Fig. 1). For male samples, there was a single band observed on the agarose gel. For female samples, there were double bands with different lengths on the agarose gel (Fig. 1). Of which, shorter CHD1 fragment was located on Z chromosome and longer CHD1 fragment was located on W chromosome. In most species, there were apparent differences in sizes between double gel bands of female samples. However, in Ara ararauna, differences in sizes of CHD1 genes on Z and W chromosomes were comparatively hard to examine (Fig. 1).

Based on the visualization of PCR products of collected samples on an agarose gel, the gender of parrots was identified (Table 1). There were 27 samples of 13 parrot species determined through this study (Table 1). Cacatua alba, C. ducorpsii, C. galerita, C. goffiniana, and Ara macao had only male samples, while Eclectus roratus had only female samples. The remaining species included both male and female samples.

The sizes of CHD1 gene fragments of M. monachus were 323 bp in Z chromosome for both male and female and 361 bp in W chromosome. Blast results indicated that CHD1-Z and CHD1-W fragment were 99.38% and 99.17% similar to CHD1-Z and CHD1-W sequence of M. monachus on Genbank, respective. The finding demonstrated that CHD1 gene was successfully amplified and its size was different between Z and W chromosomes.

Molecular based sexing has gained the popularity for gender identification in birds (Çakmak et al. 2017). As attractive and common birds in the pet market, parrots were targeted for gender determination in different studies. Together with other birds, the gender of various parrots were determined based on the molecular sexing technique (Griffits et al. 1998;Miyaki et al. 1998;Jensen et al. 2003;Vucicevic et al. 2013). To determine the gender of parrot from Korean zoos, this study applied P8/P2 primer set to amplify partial CHD1 gene, and PCR products were screened by gel electrophoresis. With this method, all collected parrots were determined gender. Our finding is congruent with previous studies on bird sexing based on P8/ P2 primers (Griffits et al. 1998;Jensen et al. 2003). P8/P2 primers were designed to bind two exons and flank an intron (Griffits et al. 1998). The size of PCR products amplified with P8/P2 varied from 300-400 bp and showed the difference between Z and W chromosomes (Griffits et al. 1998). The differences in the sizes of CHD1-Z and CHD1-W fragments resulted in a single (CHD1-Z) band in male parrots and double bands (CHD1-Z and CHD1-W) in female parrots. Two bands in female parrots indicated the presence of a shorter CHD1 gene product of Z chromosome and a longer CHD1 gene of W chromosome (Griffits et al. 1998;Miyaki et al. 1998;Jensen et al. 2003). Therefore, amplification of CHD1 gene with P8/P2 primers is able to discriminate parrot gender.

In A. ararauna (Fig. 1b), the separation between CHD1-Z band and CHD1-W band was not clear. In the previous study, Vucicevic et al. (2013) were unable to identify gender for this species with P8/P2 primers, stating that there was little distance between CHD1-Z and CHD1-W in PCR products. In this study, even though CHD1-Z band and CHD1-W band were close, female individuals of A. ararauna can still be predicted, especially when their gel bands were compared to a thin band of male individual of the same species.

In conclusion, this study determined gender for parrots collected from Korean zoos. The finding confirmed that the analysis of CHD1 sequence based on PCR technique is valuable for gender determination in parrots. These results are helpful for management and caring of parrots in Korean zoos. Due to limitation of samples in the zoos, we could not analyze both male and female for all species. Future studies are recommended to apply molecular sexing to both genders for each species, which is important for research and conservation programs of rare and endangered birds.