Gender identification in Chicken (Gallus gallus) by PCR using whole blood and dried blood spot on filter paper as template: without prior DNA isolation

Abstract

Abstract:Accurate sex identification of pure line chickens in their early age has significant economic impact in breeding industry. In the recent years, range of Polymerase Chain Reaction (PCR) based sex determination techniques are routinely used to identify the sex of parent lines in breeding industries, however purified DNA is a prerequisite. Hence this study was aimed to develop a rapid and inexpensive PCR based gender identification method for chicken using whole blood samples and dried blood spots as template for PCR without DNA extraction. In addition, practicability of two W-chromosome specific gene targets in chicken for sex determination also characterised. Successful amplification of sex specific fragments and an internal control was achieved with the range of 0.125μl and 0.250μl volume of whole blood on filter paper (~1 mm) prepared from chicken and dried blood spot. This technique does not require DNA extraction, freeze/thawing of blood samples, pre-treatment with any reagents, dilution of whole blood or dried blood spots on filter paper. It can be carried out with commercially available Taq polymerase enzymes with increased concentration of MgCl 2 (3 mM) and 0.5% of DMSO without optimisation of PCR buffers. In conclusion, as compared to the existing PCR based sex identification techniques, the present approach is relatively economic, time saving, requires minimal steps and eliminates the need for DNA extraction. Introduction:

No evidence for a link between forest herbicides and offspring sex ratio in a migratory songbird using high-throughput molecular sexing

“…This further reduced processing time, but it also demonstrated our analysis technique is compatible with a sample storage medium that is popular with researchers because it eliminates DNA degradation in the field, and allows for samples to be stored at room temperature for extended periods ( Longmire et al , 1988 ). Despite its clear potential to streamline molecular sexing, it is surprising that direct PCR has seen limited application in avian field studies ( Morinha et al , 2012 ), as this technique has been used to sex birds via analysis of whole blood ( Dhanasekaran et al , 2016 ), dried blood spots ( Quintana et al , 2009 ; Suriyaphol et al , 2014 ; Dhanasekaran et al , 2016 ), and blood stored in ethanol ( Tomasulo et al , 2002 ). Thus, our study provides an important step forward with a streamlined method for sexing nestlings and adults in sexually monomorphic species, and it is broadly applicable for a wide range of research topics given the pervasiveness of sex-related differences in physiology ( Arnold and Itoh, 2011 ), behavior ( Balthazart and Ball, 1995 ), and life history ( Clutton-Brock and Isvaran, 2007 ; Székely et al , 2014 ).…”

“…Although such PCR-based sexing techniques are effective, most require purified DNA for analysis that is obtained through the expensive and time-consuming process of DNA extraction. More recently, direct PCR approaches have been developed that allow for the amplification of DNA fragments directly from blood and other tissues without the need for the DNA extraction step ( Swaran, 2014 ), although this technique has experienced limited application in avian studies ( Morinha et al , 2012 ; but see Tomasulo et al , 2002 ; Quintana et al , 2009 ; Suriyaphol et al , 2014 ; Dhanasekaran et al , 2016 ). Further development of rapid, accurate and cost-effective direct PCR approaches for sexing birds can increase the tractability of large-scale studies of offspring sex ratio and, more broadly, encourage wider application of molecular sexing in avian studies to identify and account for sex differences in physiology, behavior and life history.…”

Short Communication: A new primer set in CHD1 gene for bird sex identification

“…The report on PCR-based gender determination approaches and sex-linked markers in avian species are steadily increasing. A continuous effort was made in PCRbased sex determination techniques to increase accuracy, speed and high-throughput applicability through previous reports (Dhanasekaran et al 2016). DNA-based methods for molecular sexing need sex-linked-markers, mostly using Chromodomain-helicase DNA binding protein-1 (CHD1) gene which is one of the conserved areas in sexlinked Z and W chromosomes (Griffiths et al 1998).…”

“…Some laboratories have used specific primers to get the female-specific bands through PCR and the segments of PCR products have been proved to be different from each other (Ramos et al 2009;Liu et al 2011) i.e sex determination of chicken by PCR using W chromosomespecific primers was already done in two W chromosome linked EST genes targets form cDNA microarray 2d-1H5 (Acc. No: AB188532) and 2d-2D9 (Acc.No: AB188526) by Dhanasekaran et al (2016). One of the most commonly used bird determination sex is P2/P8 in the CHD1 gene, which has been able to sex approximately 80% of nonratite species.…”

Comparison of DNA Isolation and Dominant and Co-dominant Molecular Markers to Reveal the Genetic Sex of Gallus domesticus (Domestic Chickens)

“…Application of the polymerase chain reaction (PCR) to identifi cation of genetic sexes of birds is ideal because it requires only a small sample, such as a drop of blood or a single plucked feather, for DNA extraction, minimising trauma to individual birds (Itoh, 2001). The PCR based sex identifi cation is therefore, considered to be the most accurate and inexpensive method over most of the available sex determining methods (Dhanasekaran et al, 2016).…”