Huiwen Liu,Dan Wang,Chenglin Zhang,Tianchun Pu,Lijuan Xiong,Fuwen Wei,Yibo Hu
Development of short‐target primers for species identification in biological studies of Carnivora
Carnivora,COI,noninvasive sample,species identification
Abstract Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling‐based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high‐performance short‐target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short‐target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short‐target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.
Development of short‐target primers for species identification in biological studies of Carnivora

Noninvasive genetic sampling greatly facilitates studies on the genetics, ecology, and conservation of threatened species. Species identification is often a prerequisite for noninvasive sampling‐based biological studies. Due to the low quantity and quality of genomic DNA from noninvasive samples, high‐performance short‐target PCR primers are necessary for DNA barcoding applications. The order Carnivora is characterized by an elusive habit and threatened status. In this study, we developed three pairs of short‐target primers for identifying Carnivora species. The COI279 primer pair was suitable for samples with better DNA quality. The COI157a and COI157b primer pairs performed well for noninvasive samples and reduced the interference of nuclear mitochondrial pseudogenes (numts). COI157a could effectively identify samples from Felidae, Canidae, Viverridae, and Hyaenidae, while COI157b could be applied to samples from Ursidae, Ailuridae, Mustelidae, Procyonidae, and Herpestidae. These short‐target primers will facilitate noninvasive biological studies and efforts to conserve Carnivora species.

Noninvasive sampling approaches are rapidly becoming the main method of collecting samples to study and conserve threatened species (Kelly et al., 2012), and for some large, threatened animals, this method is the most feasible solution (Laguardia et al., 2015; Schmidt et al., 2017). In this method, noninvasive samples (e.g., scats, hair, feathers, historical specimens) are collected without catching or killing the animals, which causes minimal disturbance to them (Taberlet et al., 1999). Researchers can extract DNA from these noninvasive samples to gain a variety of information, such as species identification, individual identification, sex, diet composition, and gut microbiome (Chaves et al., 2012; DeCandia et al., 2016; Rodgers & Janečka, 2013). In addition, species identification is also a prerequisite for other studies of wild animals, including behavioral (e.g., sexual selection), physiological (e.g., scat hormone measure), and animal medicine (e.g., parasite infection rate) studies (Huang et al., 2014; Schwartz & Monfort, 2008; Wei et al., 2012). Carnivora, as the fourth largest order of Mammalia, harbors many top predators and threatened species (Yu & Zhang, 2006), which receive wide public attention and conservation efforts worldwide. Due to the low population size, high vigilance and elusiveness, and often nocturnal habit of these species, it is difficult to directly observe wild carnivores, which greatly hinders field surveys and conservation biology research. Under these circumstances, noninvasive sampling has become an appropriate and widely used solution for the study and conservation of wild carnivores (Zemanova, 2021), especially Felidae, Canidae, Ursidae, and Musteloidea (e.g., Bischof et al., 2016; Modi et al., 2018; Mumma et al., 2015; Wang et al., 2016). DNA barcoding is a widely used method of molecular species identification in species diversity and conservation genetics studies. Hebert et al. (2003) first proposed the use of the mitochondrial cytochrome C oxidase subunit I (COI) gene fragment as a marker for DNA barcoding. For species identification with Carnivora animals, widely used universal COI primers (e.g., primers developed for metazoans, vertebrates, or mammals) were used in most studies, such as LCO1490/HCO2198 (710‐bp product length) (Folmer et al., 1994), VF1/VR1, VF1d/VR1d, VF1i/VR1i (658‐bp product length) (Ivanova et al., 2006; Ward et al., 2005), GF_1/GR_1 (approximately 650‐bp product length) (Li et al., 2017), and COI_minimm_F/COI_minimm_R (102‐bp product length) (Kocher et al., 2017). Only one study designed specific COI primers, BC‐F2/BC‐F3/BC‐R2 (239‐bp or 188‐bp product length), for Carnivora species (Chaves et al., 2012). The application of universal primers developed for vertebrates or mammals in Carnivora could pose some potential problems. First, these primers are often tested in a limited number of species, which may limit their utility in some particular groups. Second, interference from nuclear mitochondrial pseudogenes (numts) exists in some groups of Carnivora. For example, numts of COI have been reported in Panthera (Kim et al., 2006), Felis (Lopez et al., 1994), and Otocolobus (Pietsch, 2012). These universal primers might amplify numts, which would introduce errors in species identification and phylogenetic analysis (Zhang & Hewitt, 1996). Third, because DNA that is extracted from noninvasive samples is often degraded, some universal primers with relatively long product lengths might fail to amplify efficiently (Taberlet et al., 1999). Therefore, it is necessary and practical to design universal primers not only for identifying Carnivora species but also for noninvasive samples. In this study, we designed three pairs of universal Carnivora primers using the consensus‐degenerate hybrid oligonucleotide primer (CODEHOP) method (Boyce et al., 2009; Rose et al., 1998, 2003) and manual alignment methods and evaluated the performance of these primers using Carnivora samples from 38 species and three subspecies, which consisted of 33 tissue samples from 31 species and 20 scat samples from 19 species.

Tissue samples of dead individuals from 31 Carnivora species in nine families (Canidae, Ursidae, Ailuridae, Mephitidae, Mustelidae, Procyonidae Felidae, Viverridae, and Herpestidae) and scat samples from 19 Carnivora species in six families (Canidae, Ursidae, Ailuridae, Procyonidae, Felidae, and Hyaenidae) were collected with specific sources shown in Table A1. These samples were stored in −80°C freezers. The collections of tissue samples of dead animals and scat samples were approved by the agencies that the samples were affiliated with (Table A1) and conform to the guidelines of animal care and experiments. Tissue DNA was extracted with a DNeasy Blood & Tissue Kit (QIAGEN), and scat DNA was extracted with a QIAamp Fast DNA Stool Mini Kit (QIAGEN) following the manufacturer's instructions. After extraction, DNA was visualized in 1% agarose gels to assess its quality by observing whether the electrophoresis band was clear. Because of the limited scat volume and multiple PCR experiments, some scat samples were not used for all three pairs of primers.

The CODEHOP strategy can be used to design universal primers to perform PCR amplification with distantly related gene sequences. Designed from conserved amino acids within aligned protein sequences, a CODEHOP primer consists of a 3′ degenerate core region and a 5′ nondegenerate ‘clamp’ region. The 3′ core region, with a length of approximately three to four amino acids, is in a highly conserved area. To amplify more potential sequences, the 3′ core region uses a degenerate base in the third position of each codon. The 5′ clamp region, which has a length of approximately five to seven amino acids, uses the most common nucleotide of reference sequences. It does not contain degenerate bases, which ensures amplification stability and efficiency during the annealing process of the later cycles (Boyce et al., 2009; Rose et al., 1998, 2003). Reference COI sequences of 126 Carnivora species were obtained from NCBI GenBank (Table A2). Conserved regions of the COI gene were preliminarily screened in ClustalX (version 2.1; Larkin et al., 2007) by sequence alignment, and the COI codon usage bias of carnivores was calculated by CodonW (version 1.4.2; http://codonw.sourceforge.net/). Reference COI sequences and codon usage bias were imported to Base‐By‐Base (version 3; Tu et al., 2018), a software package including the j‐CODEHOP plug‐in. The max degeneracy parameter was set to 32 to find as many potential universal Carnivora primers as possible, while clamp length was set to 15 bp and core length to four amino acids to gain primers of suitable length. Then, selected primers were evaluated by the online calculator OligoCalc (2021‐3‐14; Kibbe, 2007). In this step, primers were matched according to melting temperature. The poly‐G/C sequence, potential hairpin formation, self‐annealing, and cross‐dimer were checked to filter out improper primers. A universal Carnivora primer pair COI279 (F: 5′‐ATAATGATAGGAGGAttyggnaaytg; R: 5′‐CATTGCAGGAGGTTTcatrttdatdatdat) was selected, in which the uppercase section is for the clamp region and the lowercase section is for the core region. The product length was 279 bp (Figure 1), a length that could be amplified in noninvasive samples (e.g., Huber et al., 2002). Because numts are present in Felidae as reported previously (Kim et al., 2006; Lopez et al., 1994; Pietsch, 2012), we manually designed two shorter universal Carnivora primer pairs to avoid the impacts of numts. Based on the sequence alignment results, we selected the sequences with high conservativeness among felids but multiple variations when comparing with numts, as the potential primers. Then, we used Primer Premier 5 (version 5.00; Lalitha, 2000) to assess the quality of the primer pairs based on the occurrence of hairpins, self‐annealing, and cross dimers. COI157a (F: 5′‐TCAGYCTCYTAATYCGRGCCGA; R: 5′‐AAYGGRACYARYCAGTTTCC) was designed for identifying the Canidae, Felidae, and Viverridae samples, while COI157b (F: 5′‐TCAGYCTWYTAATYCGYGCTGA; R: 5′‐AARGGYACYARYCAGTTMCC) was designed for identifying the samples from the other Carnivora families. The two primer pairs produced 157‐bp PCR products (Figure 1), which was a suitable length for amplifying seriously degraded noninvasive samples (Michalski et al., 2011; Santini et al., 2007).

For the amplification of COI279, COI157a, and COI157b primers, a 30‐μL PCR system was used, including 15 μL of HotStar‐Taq Master Mix (QIAGEN), 0.1 μg/μL BSA (TaKaRa), 0.25–0.5 μM of each COI279 primer or 0.25–0.33 μM of each COI157a/157b primer, and 3–5 μL of DNA template (depending on the PCR amplification performance). For COI279, the amplification procedure consisted of an initial denaturation (15 min at 95°C) followed by touchdown PCR (10 cycles of 30 s at a denaturation temperature of 94°C; 40 s at an annealing temperature of 62–54°C, which was decreased by 2°C every 2 cycles; and 50 s at an extension temperature of 72°C), followed by 30–35 cycles (depending on the DNA quality) of 30 s at 94°C, 40 s at 52°C, and 50 s at 72°C, and a final extension for 10 min at 72°C. For COI157a and COI157b, the amplification procedure consisted of an initial denaturation (15 min at 95°C) followed by touchdown PCR (a total of eight cycles of 30 s at 94°C; 40 s at a temperature from 54 to 52°C, which was decreased by 2°C every four cycles; and 50 s at 72°C), followed by 30–35 cycles of 30 s at 94°C, 40 s at 50°C, and 50 s at 72°C, and a final extension for 10 min at 72°C. All PCR products were visualized in 2% agarose gels. Then, the positive products were recovered separately by Gel DNA Recovery Cleaning Kit (GeneOn Biotech) and cleaned by BigDye Sequencing Clean Up Kit (MCLAB). Finally, the products were sequenced bidirectionally on an ABI 3730XL Analyzer (Thermo Fisher, at SinoGenoMax company) to obtain reliable results.

Bidirectional DNA sequences were assembled by the SeqMan module of DNAStar software (version 7.1.0; Swindell & Plasterer, 1997). Only clear sequence peaks were considered credible. Next, sequence searches were executed by megaBLAST of NCBI to identify the species source of the samples, and the results were then compared with the known species source of the samples.

All three pairs of universal Carnivora primers worked effectively for the Carnivora species samples. The COI279 primer pair performed well for the samples of 26 species from nine families and 23 genera of Carnivora (Table 1). However, this primer pair also had limitations. The first problem was interference from numts. Although the samples of Felis and Otocolobus could be identified successfully, the samples of Panthera, such as leopard and snow leopard samples, could not be identified because of interference from numts, and the chromatograms were disordered as a result. Furthermore, BLAST sequences for the Vulpes species often revealed Nyctereutes procyonoides with a similarity of 86%; thus, COI279 cannot be used to recognize foxes. For noninvasive samples, COI279 identified four of seven scat samples tested (Table 1, Table A5). The COI157a primer pair successfully identified most samples (18 of 22 species tested) from Felidae, Canidae, Viverridae, and Hyaenidae (Table 1). It can successfully overcome the interference from numts for Panthera samples. This primer pair had difficulties distinguishing related Vulpes species, such as Vulpes corsac and V. ferrilata as well as V. lagopus and V. velox. It cannot discriminate Caracal caracal from Prionailurus rubiginosus. For noninvasive samples, the COI157a primer pair successfully identified scat samples from 13 of 15 species tested from three families (Table A3). The COI157b primer pair successfully identified most samples (15 of 16 species tested) from Ailuridae, Ursidae, Herpestidae, Mustelidae, and Procyonidae (Table 1). However, it failed to identify Mephitis mephitis because the PCR was unsuccessful. For noninvasive samples, the COI157b primer pair successfully identified scat samples from four of five species tested from three families (Table A4).

In this study, the universal Carnivora primers COI157a and COI157b performed well for most of the tested species, which represented multiple different families, and for the amplification of noninvasive samples. For most Carnivora species, species identification can be performed by combining two pairs of universal Carnivora primers. Compared to the other primers, COI279 showed higher universality for many Carnivora species, although we lacked enough samples to test whether COI279 is suitable for pinnipeds. Nevertheless, COI279 had some limitations. First, because the product length was 279 bp, COI279 was a better option for good‐quality DNA samples. The second limitation is interference from numts. In addition to being unable to identify Panthera species, COI279 also amplified unknown sequences from Vulpes species. Because the unknown sequences BLASTed to the Vulpes vulpes genome, we speculate that it may be the newly discovered numt of the Vulpes. This is possible because a nearly complete mitochondrial genome was discovered in the nuclear genome of dogs (Canis lupus familiaris) (Verscheure et al., 2015), implying the possible existence of unknown numts in other Canidae species. There is no doubt that numt interference on universal primers is a large obstacle for the popularization and application of the DNA barcoding method. Coamplification could occur easily. However, for ancient and conserved numts relative to homologous mtDNA, universal primers may tend to amplify numts rather than mtDNA (Bensasson et al., 2001). For taxonomical groups with known numts, numts could be avoided by manually designing primers. For taxonomical groups in which the existence of numts is unknown, caution is needed when analyzing the mtDNA data and especially when assembling the mitochondrial genome as a reference for DNA barcoding. Alternative methods of reducing numt interference include extracting mitochondrial DNA to reduce the impacts of numts (Ibarguchi et al., 2006) and recognizing numts by checking for the occurrence of indels, in‐frame stop codons, and changes in nucleotide composition in the sequences (Song et al., 2008). Recent studies have suggested that bioinformatics filtering of high‐throughput sequencing data may be a good choice for discovering unknown numts (Smart et al., 2019; Woerner et al., 2020). For noninvasive samples, we designed COI157a and COI157b universal Carnivora primers with short PCR products, which were also named DNA minibarcoding (Meusnier et al., 2008). In our study, the universal Carnivora primers COI157a and COI157b performed well for noninvasive samples. With the rapid development of next‐generation sequencing technology, the two primer pairs could be used as potential molecular markers for high‐throughput species identification. However, in our study, short products had difficulties in differentiating subspecies or most closely related species, that is, Canis lupus and C. l. familiaris, Felis catus and F. silvestris, Panthera tigris tigris and P. t. altaica, because short products have less variation for subspecies or most closely related species. There were some troubles in PCR experiments of noninvasive samples under some circumstances. In this study, we used touchdown PCR (Korbie & Mattick, 2008) to increase the specificity and sensitivity of PCR. Considering the presence of PCR inhibitors in noninvasive DNA extracts, especially scat samples, we added BSA to the PCR solution. Diluting DNA extracts or reducing the amount of DNA extracts used are also optional solutions (Schrader et al., 2012). In addition, it has been reported that scat samples may coamplify both predator DNA and prey DNA (Symondson, 2002). We did not find the signature of prey DNA sequences in our BLAST identification; however, this remains a problem that should be treated with caution in scat DNA studies. Our universal Carnivora primers for species identification have some advantages over existing similar primers. For COI primers, LCO1490/HCO2198 (Folmer et al., 1994) is a widely used primer pair in Carnivora species identification. However, its 710‐bp product is not suitable for noninvasive samples. Compared with our primers, the BC‐F2/BC‐F3/BC‐R2 primer (Chaves et al., 2012), which is for Carnivora species identification, has a longer product and was tested on fewer families and species of Carnivora. Additionally, compared with other non‐COI Carnivora noninvasive primers (De Barba et al., 2014; Michalski et al., 2011; Rodriguez‐Castro et al., 2020), our primers also have some advantages: for example, using only one pair of primers, obtaining DNA sequences rather than the fragment length, or having wider universality to Carnivora species. In summary, our developed short‐fragment primers could have important applications for species identification in noninvasive studies of Carnivora species, especially in conservation genetics, individual‐based ecology, and metagenomics studies.

Huiwen Liu: Formal analysis (lead); writing – original draft (lead). Dan Wang: Formal analysis (lead). Chenglin Zhang: Resources (equal). Tianchun Pu: Resources (equal). Lijuan Xiong: Formal analysis (supporting). Fuwen Wei: Funding acquisition (equal); resources (equal); writing – review and editing (supporting). Yibo Hu: Conceptualization (lead); funding acquisition (equal); project administration (lead); supervision (lead); writing – review and editing (lead).

The authors declare no conflicts of interest.