An evaluation of CHD‐Specific primer sets for sex typing of birds from feathers

The amplification of the highly conserved chromo‐helicase‐DNA binding region found in both the Z and W chromosome was evaluated with three sets of primers (P8/P2, 1237L/1272H and 2550F/2718R). DNA extracted from feathers through a simple boiling method was used to address its reliability in generating the sex‐linked bands. All the bird samples, including the seven bird families that have not been reported previously, were successfully amplified with the primer set 2550F/2718R. The resulting polymerase chain reaction products showed clearly resolved fragments on a conventional agarose gel electrophoresis with size differences ranging from 80 to 540 bp between the two respective ZW gene copies. Although the P8/P2 primer was not as effective under the same conditions, it was able to produce well‐resolved Z and W bands from bird species under the Antidea family, whereas the 2250F/2718R primer set only produced a single amplified fragment of a different size between the male and the female. Zoo Biol 27:62–69, 2008. © 2007 Wiley‐Liss, Inc.     

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.     

Morphometric sex determination of Milky and Painted Storks in captivity

Logistic regression was applied to develop a morphometric sexing method of two closely related stork species that were previously sexed through amplification of the CHD gene. Tarsus length (TL) and bill length (BL) measurements were recorded from captive populations of adult Milky Stork (Mycteria cinerea) (n = 60) and Painted Stork (Mycteria leucocephala) (n = 58) at Zoo Negara Malaysia. Despite having monomorphic plumages, both stork species exhibited normal sexual size dimorphism in which males were significantly larger than females in the tested variables. Based on logistic regression analysis, BL correctly classified the sex of sampled individuals from Painted and Milky stork with an overall predicted accuracy of 94.8 and 90.0%, respectively. However, TL measurements generated a lower predicted accuracy level of 86.2% and a same accuracy level of 90% on the sex classification of individuals from Painted and Milky stork, respectively. By comparing the measurements of both species, only the average BL measurements of the Milky storks were significantly lower than that of Painted storks (t-test, P80.001). The logistic regression equation in this study may serve as a simple and more practical option for sexing Milky and Painted storks for their breeding and conservation programmes.     

A comparison of plucked feathers versus blood samples as DNA sources for molecular sexing

ABSTRACT.   Feathers are increasingly collected as a nondestructive source of DNA for avian genetic research. Although feather samples are not optimal in some important ways than more robust blood or tissue samples, feather sampling requires less training for field workers, results in shorter handling times for the organism, generates no hazardous wastes, and requires simpler storage procedures. Along with these largely positive attributes comes a set of challenges, particularly the relatively low copy number of DNA present in feather samples. We compared the utility and reliability of feathers to the more traditional blood samples as sources of DNA for polymerase chain reaction (PCR)-based molecular sexing of Black-capped Chickadees ( Poecile atricapilla ). DNA from 102 individuals was extracted separately from both single rectrices and from blood samples, and the sex of each bird was then determined using standard PCR-based methods. We found complete agreement between sex determinations based on feather versus blood DNA extractions. Slight variations in lab protocols were necessary to obtain consistent results from these two DNA sources; and we briefly discuss other sources of error that could occur in feather-based molecular sexing studies. This controlled comparison of feather versus blood samples demonstrates that plucked rectrices provide a highly reliable source of DNA for molecular sexing of wild birds.     

Sex Determination in 58 Bird Species and Evaluation of CHD Gene as a Universal Molecular Marker in Bird Sexing

The aim of this research was to test the CHD gene (Chromo Helicase DNA‐binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim. Zoo Biol 32:269–276, 2013. © 2012 Wiley Periodicals, Inc.     

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

Many bird species are sexually monomorphic and cannot be sexed based on phenotypic traits. Rapid sex determination is often a necessary component of avian studies focusing on behavior, ecology, evolution, and conservation. While PCR‐based methods are the most common technique for molecularly sexing birds in the laboratory, a simpler, faster, and cheaper method has emerged, which can be used in the laboratory, but importantly also in the field. Herein, we used loop‐mediated isothermal amplification (LAMP) for rapid sex determination of blood samples from juvenile European blackcaps, Sylvia atricapilla, sampled in the wild. We designed LAMP primers unique to S. atricapilla based on the sex chromosome‐specific gene, chromo‐helicase‐DNA‐binding protein (CHD), optimized the primers for laboratory and field application, and then used them to test a subset of wild‐caught juvenile blackcaps of unknown gender at the time of capture. Sex determination results were fast and accurate. The advantages of this technique are that it allows researchers to identify the sex of individual birds within hours of sampling and eliminates the need for direct access to a laboratory if implemented at a remote field site. This work adds to the increasing list of available LAMP primers for different bird species and is a new addition within the Passeriformes order.     

A rapid qPCR method for genetic sex identification of Salmo salar and Salmo trutta including simultaneous elucidation of interspecies hybrid paternity by high‐resolution melt analysis

This study presents an improved duplex quantitative polymerase chain reaction (qPCR) method using the master sex‐determining gene sdY as a marker for simultaneous genetic sex identification of salmonids of the Salmo genus and paternity elucidation for Salmo salar × Salmo trutta hybrids. This method will provide a new, simple and economical molecular tool for ecological studies of these species as well as for aquaculture purposes.     

A cheap,reliable and rapid method of extracting high‐quality DNA from plants

Here we describe a rapid method for extracting DNA from plant material using a microtitre plate system. This extraction procedure has been tested using three species, Brassica napus, Brassica napus/rapa hybrids and Arum maculatum, stored frozen, in silica gel or used fresh. The length of storage was between 7 days and 2 years, and in all cases high molecular weight DNA was reliably purified. Polymerase chain reaction (PCR) testing, using multiplex and single product amplification, inter simple sequence repeats (ISSRs) and microsatellites, was always reliable. This method combines the speed of commercial 96‐well plate methods with the economies associated with readily available laboratory chemicals.     

Sex‐specific prey partitioning in breeding piscivorous birds examined via a novel,noninvasive approach

Piscivorous birds frequently display sex‐specific differences in their hunting and feeding behavior, which lead to diverging impacts on prey populations. Cormorants (Phalacrocoracidae), for example, were previously studied to examine dietary differences between the sexes and males were found to consume larger fish in coastal areas during autumn and winter. However, information on prey partitioning during breeding and generally on sex‐specific foraging in inland waters is missing. Here, we assess sex‐specific prey choice of Great Cormorants (Phalacrocorax carbo) during two subsequent breeding seasons in the Central European Alpine foreland, an area characterized by numerous stagnant and flowing waters in close proximity to each other. We developed a unique, noninvasive approach and applied it to regurgitated pellets: molecular cormorant sexing combined with molecular fish identification and fish‐length regression analysis performed on prey hard parts. Altogether, 364 pellets delivered information on both, bird sex, and consumed prey. The sexes differed significantly in their overall prey composition, even though Perca fluviatilis, Rutilus rutilus, and Coregonus spp. represented the main food source for both. Albeit prey composition did not indicate the use of different water bodies by the sexes, male diet was characterized by higher prey diversity within a pellet and the consumption of larger fish. The current findings show that female and male cormorants to some extent target the available prey spectrum at different levels. Finally, the comprehensive and noninvasive approach has great potential for application in studies of other piscivorous bird species.     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

A modified mini-prep method for economical and rapid extraction of genomic DNA in plants

Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of 700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based markers.     

A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue

We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.     

A novel PCR‐based genetic marker shows that sex of offspring does not account for hatch‐order effects on growth,survival and dominance in Pūkeko Porphyrio melanotus melanotus

Intra‐brood competition can influence a variety of fitness‐related traits in birds. Previous research on the joint‐nesting Pūkeko Porphyrio melanotus melanotus, a New Zealand subspecies of Australasian Swamphen, showed that chicks that hatched earlier in a brood tended to grow faster, were more likely to survive and had higher dominance status as adults than later hatched nest‐mates. However, this finding could be due to changes in offspring sex ratio across hatch order (e.g. if males tend to hatch earlier), which was not previously examined because of methodological challenges associated with sexing nestling Pūkeko. Here, we report a useful PCR‐based genetic marker to determine the sex of Pūkeko. We then used new sex‐specific data to re‐examine patterns of offspring growth, survival and dominance. We found that the sex of offspring does not account for the hatching‐order patterns related to social dominance, growth or survival. Furthermore, changes in offspring sex ratio across hatching‐order were negligible and offspring sex ratios did not differ significantly between the primary female and secondary female broods (in joint‐clutch nests), or when comparing primary female and single female broods. We found no clear evidence for sex ratio bias according to hatching‐order and conclude that hatching‐order and not offspring sex explain patterns of growth, survivorship and adult dominance in Pūkeko.     

Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

A cost-effective,simple and high-throughput method for DNA extraction from insects

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present.     

Isolation and PCR amplification of genomic DNA from dried capsules of cardamon (Elettaria cardamomum M.)

Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage tissue such as cardamom capsules. The yield of DNA was 6–7 μg g⁻¹ tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence Characterised Amplified Regions (SCAR) markers for profiling cardamoms.     

A critique of avian CHD‐based molecular sexing protocols illustrated by a Z‐chromosome polymorphism detected in auklets

The sexes of non‐ratite birds can be determined routinely by PCR amplification of the CHD‐Z and CHD‐W genes. CHD‐based molecular sexing of four species of auklets revealed the presence of a polymorphism in the Z chromosome. No deviation from a 1:1 sex ratio was observed among the chicks, though the analyses were of limited power. Polymorphism in the CHD‐Z gene has not been reported previously in any bird, but if undetected it could lead to the incorrect assignment of sex. We discuss the potential difficulties caused by a polymorphism such as that identified in auklets and the merits of alternative CHD‐based sexing protocols and primers.     

从少量转染细胞中同时快速提取总RNA和基因组DNA

采用4mol / L LiCl将DNA和RNA分相,建立了同时从少量转染细胞中快速提取细胞总RNA和大分子基因组DNA的方法.与以前的方法相比,本法快速、简便、经济,尤其适合应用在哺乳动物细胞基因表达与调控的研究中．