Noninvasive and nondestructive sampling for avian microsatellite genotyping: a case study on the vulnerable Chinese Egret (Egretta eulophotes)

Study area and sample collection

Samples of the Chinese Egret (Egretta eulophotes) were collected at Xiaocaiyu Islet (23.47°N, 117.43°E) in Fujian Province, China. The breeding colonies were visited approximately every 5 days during the breeding season from early May to late July, 2011. Fieldwork in the breeding colony was conducted in the morning and restricted to a maximum of 2 h per day. The nests were marked with tags, eggs were numbered with indelible ink and nestlings were correspondingly ringed with a colored leg band. To sample egg swabs the external surface of each egg, when first found, was gently handled and rubbed with a single sterile swab moistened with sterile phosphate-buffered saline (PBS; 0.2 M; pH = 7.2); the cotton end of the swab was then cut off and kept in a 1.5 mL centrifuge tube with 600 μL of a sterile PBS buffer (0.2 M; pH = 7.2) for no longer than 6 h. Buccal swabs were individually collected from about 5 day old nestlings using sterile cotton swabs, mantle feathers were plucked from the back of old nestlings, about 10 days old and kept individually in 95 % ethanol. Additionally, 80 μL of blood was collected from each randomly sampled 15 day old nestling by puncturing its wing vein and immediately stored in 50 mM EDTA. Furthermore, during field surveys, naturally shed feathers with big calami (>2 cm) were collected near the nests and each was placed in a paper envelope. Single fecal pellets were collected from under the nests and stored individually in 95 % ethanol. All of the samples were stored at −70 °C until DNA extraction.

DNA extraction

Total genomic DNA was isolated from the nondestructive and noninvasive samples by the following method. First, samples were incubated overnight at 55 °C in 600 μL of a lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 0.1 mg Protease K and 1 % sodium dodecyl sulphate). Phenol–chloroform-isoamylol (25:24:1) was added for protein precipitation. The mixture was vortexed at low speed until thoroughly homogenized, after centrifugation at 10,000×g for 10 min and the supernatant was transferred to a new 2-mL tube. Phenol-chloroform-isoamylol was then used for the second extraction. This extraction was purified with an Axyprep™ DNA Gel Extraction Kit (Axygen) following the protocol from the manufacturer. Finally, a 60 μL elution buffer (2.5 mM Tris-HCl, pH 7.0–8.5, Axygen) was used to preserve the DNA, of which the concentration was quantified for all samples using a UV–visible spectrophotometer (Thermo Fisher Scientific, USA).

PCR amplifications

In order to test for PCR success rates, DNA samples were amplified for species identification using E. eulophotes-specific ND6 and tRNA^Glu gene primers (Huang et al. 2012a), which respectively amplified 101 and 278 bp fragments of the mitochondrial DNA (mtDNA) control region. PCR amplifications were performed in 20 μL reactions containing 10 μL Premix Taq (Takara), 0.4 μM of each primer and 50 ng of genomic DNA. The PCR procedure included an initial denaturation at 94 °C for 3 min, followed by 32 cycles of 30 s at 94 °C, 40 s at 55 °C, 40 s at 72 °C and a final extension of 5 min at 72 °C. The PCR products were visualized on 1.5 % agarose gels and only DNA samples successfully amplified by mtDNA were used in the amplification of nuclear DNA (nDNA).

The samples which succeeded in species identification were then amplified for sex identification using Ardeidae-specific chromo-helicase-DNA-binding protein (CHD) gene primers (Wang et al. 2011), producing two bands (140 and 250 bp) for females and only one 250-bp band for males. Amplification was carried out in a 20-μL mix containing 10 μL Premix Taq (Takara), 0.4 μM of each primer and 50 ng of genomic DNA. The PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 48 °C for 45 s, 72 °C for 45 s and a final extension at 72 °C for 5 min. The PCR products were visualized on 2 % agarose gels and stained with ethidium bromide. To increase the accuracy of the measurements, sex determination was performed for each sample at least twice and the sex was confirmed only when the results were identical. Eggshell swab samples representing maternal DNA should identify as female, while different types of samples from a single individual should be of the same sex.

To evaluate microsatellite genotyping performances, nine polymorphic microsatellite markers (Huang et al. 2010) were allocated to three multiplex PCR panels using the software MPprimer (Shen et al. 2010), based on size limitations and primer annealing temperatures (Fig. 1). To reduce the occurrence of stutter bands and ambiguity in scoring allele size, only tetranucleotide and pentanucleotide microsatellite loci were used. The 5′ end of the forward primer for each locus was labeled with a HEX, TAMRA or FAM fluorescent dye. The multiplex PCR panels were performed using Multiplex PCR Assay Kits (Takara) in a 20-μL reaction volume containing 10 μL of Multiplex PCR Mix 2, a standard diluted primer mix, 0.1 μL Multiplex PCR Mix 1 and 50 ng of template DNA. The PCR conditions were as follows: denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, annealing temperature for 90 s, extension at 72 °C for 60 s and a final extension for 30 min at 60 °C. The PCR products were run on an ABI PRISM 3730 automated DNA sequencer with a LIZ500 internal size standard (Applied Biosystems) where the alleles were scored with the Genemapper v4.1 (Applied Biosystems). Negative controls containing no DNA template were included in each PCR.

Genetic analysis

To obtain reliable polymorphic information on each microsatellite locus, only DNA extracted from blood samples (n = 16) was used. The software Cervus v3.0 (Kalinowski et al. 2007) was used to estimate expected and observed heterozygosity (H ₀ and H _e), the number of alleles per locus (N _A) and the amount of information on polymorphism (PIC). Genepop v4.0.7 (Rousset 2008) was used to determine departures from the Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) among all pairs of loci. The possible presence of stuttering, null alleles and large allele dropout was evaluated in Micro-checker v2.2.3 (Van Oosterhout et al. 2004). The genotyping performances obtained using different types of samples were evaluated by a multiple-tube approach (Taberlet et al. 1996) with the following modifications. First, DNA samples successfully amplified by mtDNA were typed for nine microsatellite loci: if less than five amplifiable loci were present, the samples were discarded from the subsequent analysis. Second, at least two additional independent PCR replicates were performed using the samples that passed the genotype screening on the first round. Finally, genotypes for each sample and locus were verified by identical alleles in three successful PCRs. Genotypes were checked for estimates of error rates (ADO and FA) by comparing the observed allele sizes with those expected sizes based on an original article by Huang et al. (2010) and by comparing the genotypes obtained in three PCRs. For the calculation of ADO and FA, equations described in Broquet and Petit (2004) and Pompanon et al. (2005) were employed, in which ADO should only be estimated considering heterozygous genotypes. A conservative criterion was adopted, in which a heterozygote was confirmed by the results of two double alleles and a homozygote was confirmed by the results of three single alleles, for allelic dropouts are much more common than false alleles in noninvasive analysis (Gagneux et al. 1997; Baumgardt et al. 2013). Furthermore, the genotyping results could also be mutually confirmed by different DNA sources. For example, genotypes of plucked feathers or buccal swabs could be verified by blood samples from the same individuals, while genotypes of eggshell swab samples could be verified by comparing them with those of other sample types (blood, plucked feathers and buccal swabs), obtained from hatched nestlings in the same nest. Moreover, sample quality indices (SQI) and loci quality indices (LQI), ranging from 0 to 1 (described in Miquel et al. 2006), were calculated for each sample and locus to reflect clearly the reliability of microsatellite genotypes using the multiple-tube approach.

DNA concentration

Amplification success rate and microsatellite genotyping performances

One DNA template obtained from shed feathers and six DNA samples extracted from feces were excluded from further analysis (Table 1), because they were not amplified by the partial ND6 and tRNA^Glu genes, although their DNA concentrations were not statistically lower than the successfully amplified fecal samples (Mann–Whitney U-test, Z = −1.852, p = 0.072). All of the DNA samples extracted from blood, eggshell swabs, plucked feathers and buccal swabs were successfully amplified (Table 2). For the amplification of the CHD gene, two samples of both eggshell swabs and feces were not successfully amplified. All of the other noninvasive, nondestructive and invasive samples were amplified successfully (Table 2). As expected, eggshell swab samples, as verified by sex determination, were identified as female.

The consensus genotypes for each locus and each sample in the repetition experiment were confirmed. Blood samples and buccal swabs produced particularly reliable genotypes with no appearance of ADO or FA (Table 4). In plucked feathers, one ADO (rate 2.67 %) and one FA (1.08 %) were observed at one locus. In eggshell swabs, six loci were affected by ADO (rates 1.45–2.84 %) and one FA (3.03 %) was detected at one locus. In shed feathers, seven loci were affected by ADO with rates ranging from 4.76 % to 19.05 %, while FA was observed in five loci (2.22–4.44 %). The error rate was highest in the fecal samples, in which ADO (6.67–38.10 %) and FA (6.06–15.15 %) occurred in all nine loci (Table 4).

Quality index for each sample and locus

The results from measuring quality indices (QIs) were as expected from theoretical considerations. The buccal swabs (n = 13) sample quality index (SQI) was up to 1.0 (Fig. 2), as good as that of the blood samples. The SQI of the plucked feathers (n = 31) was 0.996, that of eggshell swabs (n = 55) 0.986 and of the shed feathers (n = 15) 0.918. However, the SQI of feces (n = 11) was more variable, ranging from 0.55 to 1.0 (Fig. 2). The quality indices for locus (LQIs) of shed feathers and fecal samples were more variable than those of other sample types (Fig. 3). The average quality indices of nine loci for all samples were high (LQI > 0.9), except for feces which ranged from 0.70 to 0.82.