Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved. Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished. In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.

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High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis.

Visually identifying the sex of a bird can be difficult. It cannot be done in half the world's species when they are adults, and virtually none can be sexed as chicks. Despite this, the sex of a bird is vital for captive breeding. An increased number of birds are being sexed using DNA amplification techniques. In this approach, the CHD-W and CHD-Z are distinguished by the amplification of an intron present in both genes. PCR products on the gel electrophoresis vary in size revealing one band in males at the CHD-Z, and two bands in females corresponding to both the CHD-W and CHD-Z. Two independent sets of primer (P8/P2 and 2550F/2718R) were used to amplify the CHD gene region from both the Z and W chromosome. One hundred and ten (110) birds were sexed using first pair of primers: (P8/P2). Sexing results indicated that 81.8% were successfully determined, 12.7% failed to be amplified and 5.5% were not perfectly determined because the PCR products showed thick band. The thick band caused misidentified female to male birds. An alternative primer (2550F/2718R) was applied to solve the problem. Two hundreds and twenty-nine birds were sexed and the results showed 100% successfully determined. From this study, it is suggested to use a pair of 2550F and 2718R primers for distinguishing a male from a female bird.

https://journal.ipb.ac.id/index.php/hayati/article/download/6535/6535

Application of Two Molecular Sexing Methods for Indonesian Bird Species: Implication for Captive Breeding Programs in Indonesia

High-throughput gender identification of three Columbidae species using melting curve analysis.

Amplified fragment length polymorphism (AFLP) with multicolored fluorescent molecular markers was used to analyze duck (Anas platyrhynchos) genomic DNA and to construct the first AFLP genetic linkage map. These markers were developed and genotyped in 766 F2 individuals from six families from a cross between two different selected duck lines, brown Tsaiya and Pekin. Two hundred and ninety-six polymorphic bands (64% of all bands) were detected using 18 pairs of fluorescent Taq I/Eco RI primer combinations. Each primer set produced a range of 7 to 29 fragments in the reactions, and generated on average 16.4 polymorphic bands. The AFLP linkage map included 260 co-dominant markers distributed in 32 linkage groups. Twenty-one co-dominant markers were not linked with any other marker. Each linkage group contained three to 63 molecular markers and their size ranged between 19.0 cM and 171.9 cM. This AFLP linkage map provides important information for establishing a duck chromosome map, for mapping quantitative trait loci (QTL mapping) and for breeding applications.

/pdf/duck-anas-platyrhynchos-linkage-mapping-by-aflp-24ofxvrmsm.pdf

Duck ( Anas platyrhynchos ) linkage mapping by AFLP fingerprinting

High-throughput sex identification by melting curve analysis in blue-breasted quail and chicken.

1. The accuracy and reproducibility of AFLP fingerprinting was investigated in the duck (Anas Platyrhynchos), using a multicolour fluorescent labeling technique. The fluorescent labelling fragments were separated on a capillary electrophoresis-base ABI PRISM 3100 Genetic Analyzer. 2. A total of 337 AFLP peaks with 103 of them being polymorphic markers were generated by 16 sets consisting of EcoRI/TaqI primer pair combinations. The number and size range of AFLP polymorphisms detected per primer pair varied from 3 to 11 and 58 to 290 bp, respectively. About 30.6% (103/337) of AFLP peaks were detected polymorphisms, with an average of 6.4 polymorphic markers per primer pair. 3. The clear polymorphic peaks were amplified with EcoR+AC/Taq+AC primer combinations. The AFLP peaks showed high reproducibility. From the family testing, we found that the fingerprints of all the offspring were derived from one or other parent. Therefore, we conclude that AFLP fingerprinting might be a suitable method for duck paternity testing.

Y.-S. Cheng

Papers

AFLP fingerprinting for paternity testing in ducks