Comparison of three different primer sets for sexing birds

Abstract

Because many bird species are monomorphic or only sexually dimorphic in adult stages, it is difficult to determine their sexes, which may cause significant problems in population and conservation studies. DNA-based sexing relies on the chromodomain helicase DNA binding ( CHD) gene located on the W chromosome and its homolog on the Z chromosome, giving distinct banding patterns on agarose gel as a result of length differences in intronic regions within this gene. We used 3 specific primer sets, CHD1F/CHD1R, 2550F/2718R, and P2/P8, for sex determination of 230 samples from 77 avian species. We report here the records for 70 of those species analyzed using the CHD1F/CHD1R primer set, and 49 species using 2550F/2718R, and 46 species using P2/P8. CHD1F/CHD1R PCR products on agarose gel generally showed an apparent single band in males and 2 bands in females, but the products of 2550F/2718R (61%) and P2/P8 (42%) showed distinct banding patterns for separate bird orders. However, when PCR products of these last 2 primer pairs labeled with fluorescent dye were run in a capillary gel and detected using a DNA analyzer, P2/P8 gave 2 distinguishable peaks in females, whereas 2550F/2718R results remained the same. DNA sexing with any of those 3 primer sets can be used for all sexually monomorphic avian taxa although the primer sets should be compared before choosing the most efficient one for molecular sexing of the studied species.

A molecular genetic approach for sex determination on helmeted hornbill (Rhinoplax vigil) casque: a forensic casework

“…Similarly, according to agarose gel electrophoresis, the success rates of primer sets CHD1F/CHD1R, 2550F/2718R, and P2/P8 were 100% (n = 22), 100% (n = 22), and 40.90% (n =22) for blood samples; 100% (n = 2), 100% (n = 2), and 0 % (n = 2) for feather samples. This can be directly related to the fact that DNA in feather samples generally submit lower copy numbers than DNA obtained from blood [25] and it occurs for keratinous samples also. Another possibility is perhaps DNA from feather and keratinous samples may get more easily degraded [25], so the quality and quantity of DNA extracted from feather and keratinous material would be correspondingly low.…”

“…This can be directly related to the fact that DNA in feather samples generally submit lower copy numbers than DNA obtained from blood [25] and it occurs for keratinous samples also. Another possibility is perhaps DNA from feather and keratinous samples may get more easily degraded [25], so the quality and quantity of DNA extracted from feather and keratinous material would be correspondingly low. Another study reported that the polymorphic nature of the intron length, along with the power of variability in electrophoresis resolution, does not allow for accurate sex identification in some species of bird [14, 20,26].…”

“…PCR cycling condition was as follows; 94°C for 2 min, then 10 cycles of 94°C for 30 s, 54°C for 15 s and 72°C for 30 s, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s, and finally 72°C for 5 min modified from [24]. The PCR cycling for P2/P8 was 94°C for 4 min for initial incubation, followed by 40 cycles at 94°C for 30 s, 51°C for 45 s, and 72°C for 45 s, and further extension at 72°C for 5 min [25]. For amplification of 2550F/2718R we used a touchdown scheme where annealing temperature was reduce 1°C per cycle, starting from 57°C until it reached 50°C, followed by 30 cycles, and final extension at 74°C for 5 min [25].…”

“…The PCR cycling for P2/P8 was 94°C for 4 min for initial incubation, followed by 40 cycles at 94°C for 30 s, 51°C for 45 s, and 72°C for 45 s, and further extension at 72°C for 5 min [25]. For amplification of 2550F/2718R we used a touchdown scheme where annealing temperature was reduce 1°C per cycle, starting from 57°C until it reached 50°C, followed by 30 cycles, and final extension at 74°C for 5 min [25]. Agarose gel electrophoresis (2%) and fluorosafe staining were subsequently carried out to visualize the products.…”

Molecular sexing in Japanese murrelet (Synthliboramphus wumizusume) and a tandem-repeat polymorphism on the W chromosome

“…Although sex identification is very important for biological and ecological field studies, it is difficult to distinguish males and females of this species by the traditional method of inspecting external features, given that the species exhibits monomorphic characteristics. In other species, molecular sexing using PCR methods targeting the CHD1 gene has made it possible to identify the sex rapidly and accurately using non-invasive samples 6,7,13,14 . Japanese murrelets are morphologically monomorphic birds, meaning that it is hard to distinguish between males and females.…”