Potential of Inter- and Intra-Species Variability of CHD1 Gene in Birds as a Forensic Tool


 Nowadays, illegal trafficking and smuggling of animals are among the greatest threats to many avian species. Most commonly smuggled birds belong to parrots, song birds, raptor species and owls. All of these species are protected by national and international legislations. In order to prevent and reduce wildlife trafficking, DNA methods have become an important forensic tool in species and sex identification. In this study, CHD1R/CHD1F primer pair was used to amplify a part of the CHD1 gene from 65 birds that belong to 43 species. For 36 species this is the first time that the length of CHD1 amplicons was measured for the purpose of species determination. The results were visualized using capillary electrophoresis and enabled simultaneous determination of sex and species. Based on the number of amplicons (two in females, one in males) sex was successfully determined in all species, even in cases where gel electrophoresis failed to give results. Moreover, the species was successfully determined in most bird species based on the species-specific sizes of CHD1 amplicon. The method used in this study is of great importance for veterinary forensic medicine and the prevention of wildlife smuggling. Still, further work is necessary to confirm the effectiveness of the method in all bird species.


INTRODUCTION
Wildlife traffi cking, including illegal poaching, trade, and transport of wildlife are considered global problems, spreading from low-income to wealthy countries [1][2][3]. It is estimated that the annual profi t from this illicit activity reaches 2.3 million euros only in the EU, and up to 23 billion in the USA [3,4]. The main subjects of trade, seized within the EU are live birds, followed by live plants, bird and mammal bodies, parts and derivatives. Even though small songbirds, raptor species, parrots, and owls are protected by CITES (https://www.cites.org), the Bonn convention (https://www. cms.int/) as well as by national legislation [5], they are still subject of reported seizures in 79% of all cases in the EU [3]. This activity puts at risk thousands of species and encompasses a risk of zoonotic diseases host by wild animals [3].
Members of the ordo Psittaciformes are usually caught because of their vocal abilities, the beauty of the feathers and use as companion animals [6]. However, members of other bird families are also at risk of illegal traffi cking. The consequence is that today almost all Psittaciformes species are listed in CITES Appendix I (only three of them are listed in Appendix II). The most commonly smuggled Psittaciformes species are Amazona aestiva, A. ochrocephala, Ara chloroptera, A. ararauna, Pionites melanocephala and Aratinga solstitialis. In 2018 349 live birds were seized in the EU, and 316 of them were parrots such as Agapornis fi scheri, A. personatus (App. II/Annex B) and Psittacus erithacus [3].
To reduce wildlife traffi cking and illegal hunting, international organizations and national agencies highly depend on forensic investigations which include species and sex identifi cation of animals [7][8][9]. In the last decades, molecular techniques have become very valuable forensic tools that enable, among other things, identifi cation of species from a very low quantity of samples [2,10,11] of various types (e.g. tissues, scales, hairs, feathers, eggshells). In forensics, identifi cation of avian species is necessary to determine the origin of fragments of feathers that can be found in smugglers' bags.
The central role in modern forensic approaches is reserved for DNA analysis as an unavoidable step in law enforcement and endangered bird species protection [10]. Different molecular techniques are used for sex and species determination, such as DNA barcoding [12,13], polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [14], metabarcoding avian environmental DNA [15], and DNA sequencing [16,17]. There are plenty of papers related to mammalian species identifi cation [11,18,19], while only few related to bird species identifi cation are available [10].
Along with the application in forensics, species identifi cation and sex determination are very important in the taxonomic classifi cation and genetic diversity studies, conservation programs as well as in bird breeding and farming [20,21]. When it comes to birds' sex determination, there are a lot of different molecular techniques applicable for that task [10,22,23]. The most successful genetic marker for sex determination in avian species is Chromodomain Helicase DNA Binding Protein 1 (CHD1) gene due to both high degree of conservation even among distant species and insertion-deletion polymorphism difference between CHD1 genes on Z and W chromosomes [7]. Two primer sets are commonly used for sex determination, 2550F/2718R and P2/ P8, but to the best of our knowledge, only two studies tackled the extension of CHD1F/CHD1R primer set in species identifi cation through precise fragment length determination [10,24].
Regarding the lack of information on the molecular technique that can be used for both sex and species determination in birds, but also the absence of studies that examined the variability of amplicon lengths across endangered bird species, we tested the performance of CHD1F/CHD1R primers by capillary electrophoresis. We aimed to provide more information on partial CHD1 variability from 43 species that are often the subject of wildlife traffi cking and assess capillary electrophoresis as a method of choice for sex and species determination in birds and the potential of this methodology as a forensics tool in wildlife traffi cking.

Sampling and DNA extraction
A total of 65 samples were collected from 43 bird species protected under the Law of the Republic of Serbia or EU regulations. Ethics committee approval was obtained by the Ministry of Agriculture, Forestry and Water Management (number 323-07-0036412017-0515).
Samples were provided by either Zoological Garden of Belgrade or were taken during post-mortem examination at the Department of Veterinary Forensic medicine, Faculty of Veterinary medicine, University of Belgrade. From each bird, samples were taken by plucking at least three to fi ve thoracic feathers and stored in a separate zip bag. After sampling, all live birds were released unharmed. DNA was extracted from the feathers. Quills were cut into 2-to 5-mm-long pieces in sealed 1.5 ml tubes. Afterwards, DNA was extracted using the DNeasy® Blood & Tissue Kit, Cat. No 69504 (Qiagen, Valencia, CA) following a manufacturer protocol.

Agarose gel electrophoresis
Total of 10 μL of PCR product was used for electrophoresis. After the separation in 2% agarose gel and staining in ethidium bromide, PCR products were visualized with UV light. A commercial O'RangeRulerTM 50bp DNA Ladder (Thermo Scientifi c, Massachusetts, United States) was used as the size marker to determine whether Zand W-bands were obtained.

Capillary electrophoresis
After checking the dye-labelled PCR amplicons on the agarose gel, from the remaining PCR product, 1 μL was used for capillary electrophoresis fragment separation. Capillary electrophoresis was performed using polymer type POP-6, 50 cm long capillaries, and internal size standard LIZ500 (Applied Biosystems, Foster City, USA) on ABI3130 DNA analyzer (Applied Biosystems, Foster City, USA) according to the protocol described by Lee et al. (2010). For data analyzing authors used GeneMapper 3.1 (Applied Biosystems).

RESULTS
The amplifi cation products have been obtained for all analyzed samples by both agarose gel electrophoresis and capillary electrophoresis (Table 1). On capillary electrophoresis, one amplifi cation product was observed in 26 collected samples indicating male gender, while two amplifi cation products were detected in 39 samples indicating the female gender. In all cases, the size of CHD amplicons of Z chromosomes (CHD-Z amplicon) was larger compared to amplicons of W chromosome (CHD-W amplicon). In the case of 41 bird species (out of 43 analyzed in this study), the length of CHD-Z amplicons was identical in all samples originating from the same species. In remained two species involved in this study, Buteo buteo and Cygnus cygnus, CHD-Z amplicons from two samples of the same species differed slightly, 3bp in the case of the former, and 1 bp in the case of the latter (Samples No. 24 and 25, 10 and 11 in Table 1). In case of 42 species, the length of CHD-W amplicons was identical in samples originating from the same species; the only exception was observed in Tyto alba where samples of two females generated CHD-W amplicon that differed in four base pairs (Samples No. 17 and 18 in Table 1). The smallest difference in fragment length (42 and 59 bp) between CHD-Z and CHD-W fragments of the same species was noticed in samples of birds belonging to Strigiformes ( Table 1). The same size of CHD-Z amplicon was detected in some samples originating from different species, but mostly in cases of those that belong to the same order or even family. However, in those samples (from different species but with the same length of CHD-Z sequence) CHD-W amplicons were of different sizes (Samples No. 11/12 and 56; 29 and 36; 23 and 39/40).
In 4 out of 65 birds we found disagreement in sex determination results between two methods. All four birds where disagreements were present originated from ordo Strigiformes. In other bird species, such as birds from ordo Psittaciformes, gender has been successfully determined using gel electrophoresis (Figure 1).

DISCUSSION
This study involved bird species that are protected by national or international regulations and are most often the subject of illegal trade on the international or national level (5, https://www.cites.org). CHD1F/CHD1R primer set was used because it produces smaller fragments more appropriate for capillary electrophoresis [10]. Moreover, this primer set is better for less abundant and degraded DNA samples compared to 2550F/2718R primer set, also commonly used for sex determination.
According to the results of Lee et al. (2010), the size of CHD-Z and CHD-W amplicon can be very similar in species of the same order or family. The same applies to our results (Table 1). For 36 species in this study the lenght of CHD-Z and CHD-W amplicon was analised for the fi rst time.
The results were consistent in terms of the equality in size of CHD-Z amplicon within samples originating from the same bird species, excepting Buteo buteo and Cygnus cygnus. Within certain species analyzed in this study (Buteo buteo, Falco tinnunculus, Cygnus cygnus) there was a difference in CHD-Z amplicons size. As suggested by Lee et al. (2010) intraspecies variations may be the cause of differences in CHD-Z gene segment size. Therefore, it is necessary to examine a larger number of individuals of a certain species. Along with a larger number of samples, both male and female individuals should be examined, because sometimes CHD-W fragment is an even better marker for species determination. This is supported by our results of analyzed Amazona species that revealed CHD-Z amplicons of the same size in more than one species, but in all cases length of CHD-W amplicon size was different (Amazona aestiva and Amazona ochrocephala; Amazona fi nschi, Amazona albiguens and Amazona albifrons in Table 1).
In species Ara chloroptera and A. macao (Psittaciformes) the size of CHD-Z fragment had the same length (521 bp) and therefore cannot be used to distinguish species when only males are analyzed. The absence of species-specifi c length of the CHD-sequences may be due to the existence of more than 300 species in ordo Psittaciformes, meaning that it is almost impossible that each species has unique size of CHD-Z fragment.
Such a problem does not apply to females due to the huge variability in the CHD-W fragment, so the species can be easily determined (sample No 38 and 39 in Table 1).
We believe that for forensic application, a probabilistic approach is the only possible solution for strengthening species identifi cation using capillary electrophoresis. The probability of fragment drop-outs as well as the inclusion of fragment length frequencies, in species where more than one fragment length is observed have to be included. On the other hand, discrimination between species whose fragments differentiate by only one base pair is also questionable since the same PCR product can vary that much even between two consecutive runs [25].
There is a difference in size between intronic sequences within CHD-Z and CHD-W fragments and this size difference allows determination of sex in many bird species by molecular techniques capable to separate fragments that differ in a very small number of base pairs (even only one in some cases). When it comes to sex determination in sampled birds using a dye-labelled CHD1F/CHD1R primer set, capillary electrophoresis proved to be an accurate, fast, and reliable method. Our results confi rmed that the specifi ed method enabled accurate sex determination in all tested species, although end point PCR approach was also highly successful, as shown here, but also in previous studies [7,9,10,26,27].
The study by Griffi ts et al. (1998) [28] has shown that the two PCR primers anneal to conserved exonic regions but then amplify across an intron in both CHD-W and CHD-Z. As these introns are noncoding and consequently less conserved, their lengths usually differ between the genes of different species which is followed by the variation of of PCR product lengths between species.
In all cases processed in this study, the sex obtained on an agarose gel was confi rmed by capillary electrophoresis. On agarose gel, samples from heterozygous female individuals showed two bands while one band was obtained from homozygous males. The exceptions were Strigiformes samples where only one band was observed, regardless of sex. Difference in CHD-Z and CHD-W amplicon size in phylogenetically close species is expected to be 30-50 bp, but may be in range from 10 to 80 bp [7]. In those species, it is diffi cult to distinguish female from male birds on agarose gel because females can often be misinterpreted as male birds [7,21]. Our results confi rm that capillary electrophoresis overcomes those shortages in sex differentiation with simultaneous determination of bird species.

CONCLUSION
Capillary electrophoresis is of great importance for the identifi cation of both sex and species of birds and can be used in ecology, genetics, biodiversity and veterinary forensics cases. Determination of sex and species of birds could be an important forensic tool in preventing wildlife smuggling.