A novel molecular genetic marker for gender determination of pigeons

The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. One-hundred and twenty random primers were used for RAPD fingerprinting in order to find any sex-specific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR.     

A novel sex-specific DNA marker in Columbidae birds

That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (～ 3 kb long) revealed the presence of tandem and inverted repeats. The ～ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ～ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.     

The identification of a sex-specific DNA marker in the ostrich using a random amplified polymorphic DNA (RAPD) assay

PCR-based techniques described to date for sex diagnosis in birds are not useful in ratites. We report here the identification of a W-linked marker in the ostrich (Struthio camelus) which allows gender diagnosis in chicks or juvenile birds. DNA from 10 females and 11 males was used to prepare two pools for each sex. Two-hundred different 10-mer primers of arbitrary sequence were used to screen those pools using a random amplified polymorphic DNA (RAPD) assay. One primer (D 10) generated a female-specific band. Sex specificity was confirmed by testing the 21 animals individually. The candidate DNA fragment was cloned and sequenced. Longer primers were designed to optimize a sex-specific PCR which will be useful in diagnosis.     

Identification of a DNA marker linked to sex determination in <Emphasis Type="Italic">Calamus tenuis</Emphasis> Roxb., an economically important rattan species in northeast India

Calamus tenuis (Roxb.), a versatile, dioecious rattan species predominant in northeast India, has emerged as an economical material for light furniture and cottage industries. For the genetic improvement of the species, it is essential to be able to recognize male and female plants at the seedling stage. Screening of genomic DNA with inter-simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. Thirty ISSR primers were screened on female and male C. tenuis plants from five different provinces of Assam, India. A putative female-specific marker was identified. The applicability of ISSR-PCR analysis for development of sex-linked molecular markers in Calamus is discussed.     

Amelogenin cross-amplification in the family Bovidae and its application for sex determination

Sex-specific sequence variability of the amelogenin gene had been observed in a variety of mammalian species. In our study, the suitability of the amelogenin gene for sex determination in different species of the family Bovidae was examined. Based on a sequence insertion/deletion characteristic for X- and Y-specific amelogenin (AMELX and AMELY), PCR amplification on male and female genomic DNA from domestic and wild bovine species, sheep and goat, consistently displayed a sex-specific pattern. Thus, the amelogenin amplification by PCR proved to be a reliable method for sex determination not only in domestic and wild species of the tribe Bovini, but also in the related species sheep and goat. Sex determination using the amelogenin-based assay can be performed with at least 40 pg of genomic DNA. The assay enables the investigation of small amounts of DNA from meat, hair, bones, and embryo biopsies to identify species and sex for a number of applications in animal production, forensics, population research, and monitoring within the family Bovidae. Sequence comparison of the amplified amelogenin gene region specific for male and female animals from domestic and wild bovide species revealed further sequence variations within and between sexes as well as between species. Sequence variations in the AMELX gene can be applied to discriminate Bos and Bison individuals from other bovine species, and also from sheep and goat.     

Reproductive effort and costs of reproduction do not explain female-biased sex ratios in the moss Pseudocalliergon trifarium (Amblystegiaceae)

A fundamental assumption in life-history theory is that reproduction is costly. Higher reproductive investment for fruits than for flowers may result in larger costs of reproduction in females than in males, which is often used to explain male-skewed sex ratios in unisexual seed plants. In contrast, bryophytes have predominantly female-biased sex ratios, suggested to be a product of a higher average cost of sexual reproduction in males. Empirical evidence to support this notion is largely lacking. We investigated sex-specific reproductive effort and costs in the unisexual moss Pseudocalliergon trifarium that has a female-dominated expressed sex ratio and rarely produces sporophytes. Annual vegetative segment mass did not differ among male, female, and non-expressing individuals, indicating that there was no threshold-size for sex expression. Mean and annual mass of sexual branches were higher in females than in males, but branch number per segment did not differ between sexes. Prefertilization reproductive effort for females was significantly greater (11.2%) than for males (8.6%). No cost for sexual branch production in terms of reduced relative vegetative growth or decreased investment in reproductive structures in consecutive years was detected. A higher realized reproductive cost in males cannot explain the unbalanced sex ratio in the study species.     

Bryophyte DNA sequences from faeces of an arctic herbivore, barnacle goose (Branta leucopsis)

We tested DNA extraction methods and PCR conditions for the amplification of bryophyte DNA from barnacle goose (Branta leucopsis) faeces collected from Spitsbergen (Svalbard). Both the Qiagen stool kit and a silica-based extraction method received sufficient DNA from fresh and older droppings, as indicated by successful amplification of the plastid psbA-trnH spacer. Standard Taq polymerase outperformed two hot start polymerases. Sequencing of cloned PCR products revealed at least ten moss and two angiosperm sequences. This first example of identifying bryophyte DNA from faeces will allow analysing moss diets of arctic herbivores with a DNA barcoding approach.     

一个新的泥鳅雄性特异DNA片段（简报）

世界上现存鱼类多达24000余种．是脊椎动物中分布最广,种类最多的类群．具有多种多样的生物学特性和重大的经济价值。与高等脊椎动物相比．其性别决定具有多样性和可塑性。大多数鱼类的性别决定机制很原始。性染色体的分化处于萌芽状态。在已进行细胞遗传学研究的1700多种鱼类中．大约有176种（占10．4％）发现有明显的异型性染色体。     

Sex determination in cattle based on simultaneous amplification of a new male-specific DNA sequence and an autosomal locus using the same primers

A PCR-based method for sex determination of bovine DNA samples and embryo biopsies is presented. Using only one primer pair both the male-specific sequence FBNY (127 bp) and a sex-independent control PCR-fragment, the microsatellite marker FBN17 (136-140 bp) are generated in the same PCR reaction. Synteny mapping assigned the male-specific sequence to bovine chromosome Y (BTA Y), whereas FBN17 was mapped to bovine chromosome 2. Localisation of FBNY on BTA Y was confirmed by fluorescence in hybridisation of two BAC clones containing the male-specific sequence. There was no amplification of the male-specific target sequence FBNY in sheep, pig, goat, mice, man, and several wild species of the tribe Bovini. The bovine male-specific fragment was detected in dilutions containing as little as 10 pg genomic DNA and in blastomeres from embryo biopsies. The PCR assay presented here does require neither restriction endonuclease digestion of the PCR product nor additional nested PCR steps. Owing to the advantage of parallel amplification of the autosomal locus FBN17 no additional control fragment is necessary to detect PCR failure. The results of sex determination in embryo biopsies using FBNY were in agreement with the outcome from a reference assay used in commercial breeding programs.     

Sex determination in biological specimens using the <Emphasis Type="Italic">DYS14</Emphasis> marker

The applicability of real-time PCR amplification of the chromosome Y marker DYS14 for sex determination was studied. With this aim, real-time PCR of DYS14 (located within the TSPY1-encoding gene) was performed in plasma DNA specimens obtained from 30 men and 30 women. The PCR results showed that 30 specimens were of male and the other 30 were of female origin. All the results were confirmed by the tests for the SRY marker conventionally used in forensic examination. The detection limit for the DYS14-containing DNA region was established in dilution experiments and was equal to 6.7 pg of DNA (two copies of the genome), which corresponds to 6.7 ng of DNA (2000 copies of the genome) in 1 ml of blood. This level of sensitivity allows sex determination in specimens with small amounts of genetic material. The method can be used for noninvasive prenatal diagnostics of sex-linked congenital diseases and in forensic medical examination.     

The use of amplified fragment length polymorphism (AFLP) in the isolation of sex-specific markers

Sex identification is a problem in research and conservation. It can often be solved using a DNA test but this is only an option if a sex-specific marker is available. Such markers can be identified using the amplified fragment length polymorphism (AFLP) technique. This is usually a taxonomic method, as it produces a DNA fingerprint of 50-100 PCR bands. However, if male and female AFLP products are compared, sex-specific markers are confined to the heterogametic sex and can rapidly be identified. Once a marker is found, AFLP can be used to sex organisms directly or the marker can be sequenced and a standard PCR test designed.     

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.