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


RESULTS
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.


CONCLUSIONS
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.
Chang
Hsueh-Wei
HW
Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan. changhw@kmu.edu.tw
Cheng
Chun-An
CA
Gu
De-Leung
DL
Chang
Chia-Che
CC
Su
San-Hua
SH
Wen
Cheng-Hao
CH
Chou
Yii-Cheng
YC
Chou
Ta-Ching
TC
Yao
Cheng-Te
CT
Tsai
Chi-Li
CL
Cheng
Chien-Chung
CC
D000818:Animals; D052160:Benzothiazoles; D001717:Birds / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D003959:Diamines; D004586:Electrophoresis / Q000379:methods*; D005260:Female; D008297:Male; D015336:Molecular Probe Techniques; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D012730:Sex Chromosomes / Q000235:genetics*; D012732:Sex Determination Analysis / Q000379:methods*
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA