Molecular sexing of pine marten (Martes martes): how many replicates?

We used primers developed for the SRY gene in otters (Lutra lutra) to determine sex in pine marten (Martes martes). The otter SRY primers worked accurately for pine marten and assigned sex correctly in most replicates. These primers can be used on tissue and noninvasively collected hair samples for the identification of the animal's sex. We found that, based on five sets of replicates, DNA extracted from leg muscle and hair gave significantly better results than DNA extracted from ear tissue. Finally, results indicate the optimum number of PCR replicates to accurately assign sex using this technique.     

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.     

利用孕妇血浆DNA检测胎儿性别的研究

本文探讨应用孕妇血浆中游离DNA进行无创性产前性别诊断的可行性。用柱分离法提取73例孕妇血浆中DNA,用巢式PCR技术检测其胎儿SRY基因。 结果73位孕妇血浆DNA含量为0.0062~0.3399μg/μL。巢式PCR检测胎儿SRY基因的灵敏度为97.37%（37/38）,假阴性率2.86%(1/35),特异度85.71%（30/35）,假阳性率13.16%(5/38),总符合率91.78%（67/73）。采用孕妇血浆胎儿DNA和巢式PCR技术可以快速简便的进行无创性产前性别诊断,诊断结果的准确率为91.8%,对性连锁遗传病的预防具有重要意义。 Abstract:To investigate the feasibility and possibility of application of fetal DNA from maternal plasma for noninvasive prenatal diagnosis of fetal sex,plasma DNAs in blood samples of 73 pregnant women at the gestational period of 26 to 41 weeks were extracted by column separation and nested polymerase chain reaction were employed to amplify the SRY gene.A comparison was made between the amplification results and the real sex of the fetus after their delivery.The concordance rate of SRY gene amplification results of plasma free DNA with real fetal sex was 91.78% (67/73),the sensitivity rate was 97.37% (37/38),and the specific rate was 85.71% (30/35).The cell-free fetal DNA in maternal blood can be one of the valuable material sources for noninvasive prenatal diagnosis and the method of nested PCR could be useful for fetal sex determination.The specific rate of the test was 91.78%.It is of significance to prevent sex-linked inheritant diseases.     

A simple and improved PCR-based technique for white-tailed deer (<Emphasis Type="Italic">Odocoileus virginianus</Emphasis>) sex identification

We describe a simple single-reaction technique for identifying the sex of white-tailed deer (Odocoileus virginianus) based on the PCR amplification of a zinc-finger intron using one pair of primers. Although Sry-coamplification confirmed sex identities, use of the Sry marker was unnecessary due to dimorphic alleles on the X and Y chromosomes at the zinc-finger locus. Insertions in intron 7 of the Y-linked allele (417 bp) make it nearly twice as long as the X-linked allele (236 bp) and thus the amplification products are easily discernable by simple agarose gel electrophoresis. The relatively short size of these products makes them useful for DNA-based sex identification from potentially low-yield tissue samples (e.g., hair, feces). This technique will provide ecologists, conservation geneticists and wildlife managers with a mechanism to readily and reliably identify the sex of unknown white-tailed deer tissue samples, and likely similar samples from other cervid species.     

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.     

哺乳动物性别决定和性反转

目前已知SRY仅是涉及性别决定过程的基因之一.近年来又发现和克隆了许多可能参与性腺分化与发育的基因,如副中肾抑制基因MIS,也称抗副中肾激素基因AMH;SRY相关基因SOX9;编码甾类因子的基因SFI;X-连锁的DAX基因;Wilm′s肿瘤抑制基因WTI;以及X-连锁的剂量敏感基因DSS等,并新建立了性别决定的Z-基因模型,DSS-基因模型和Jimenez等的模型,较合理地解释了哺乳动物性别决定的分子机理和以前难以解释的各种奇特的性反转现象,使性别决定的研究取得了长足的进展,但仍有一些悬而未决的问题有待于进一步探索.     

Accuracy in molecular sexing of martens (Martes americana and Martes caurina) varies among sample types

We evaluated the accuracy of sex identification using the SRY marker for American marten (Martes americana) and Pacific marten (Martes caurina) using samples collected from commercial trappers and those obtained via noninvasive sampling. We determined that sex identification from Lut-SRY primers is accurate at >90% for muscle and hair samples collected noninvasively. We found much lower accuracy when using hair samples plucked from trapper-killed carcasses, errors presumably incurred from contamination from the DNA of trappers, who were entirely male. We also found that the sequence generated from Lut-SRY primers, originally developed for Eurasian otters (Lutra lutra), differed slightly for martens, and recommend that new primers be developed from the sequences we provide. The SRY marker can be reliably used for sex identification in both species of marten, provided that samples have low probabilities of contamination. Researchers should avoid samples collected from external locations on trapper-killed carcasses for DNA-based analyses.     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

Sex‐linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up‐AC‐2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction‐amplified in three female gametophytes. This putative female‐specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex‐related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.     

Sry基因的研究进展

性别决定基因（Sex region of Y chromosome, 人类以SRY,小鼠以Sry表示）的研究进展是近几年来人类在性别决定,性别分化研究中获得的最大的突破性成果,该文从SRY（Sry)发现前关于性别决定因子的研究,SRY（Sry)的确定,小鼠Sry的结构研究,小鼠Sry的表达研究及Sry下游基因的确定等5个方面对小鼠Sry的研究进展进行综述,对进一步深入研究Sry下游基因存在的瓶颈问题人了一定的分析,并提出核移植技术可能对研究Sry的调节及其下游基因所需的特殊实验材料展现了新的希望。     

A universal and reliable assay for molecular sex identification of three‐spined sticklebacks (Gasterosteus aculeatus)

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex‐related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three‐spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex‐linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single‐marker‐based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost‐efficient tool for universal sex identification of three‐spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.     

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.     

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads

In sharp contrast to birds and mammals, most cold‐blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X‐Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex‐determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex‐linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F₁ offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex‐specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite‐containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex‐determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.     

Demography of Unexploited Beavers in Southern Illinois

ABSTRACT Beavers (Castor canadensis) are important in ecosystems and to humans. Although beavers are increasingly protected from harvest, relatively few studies of unexploited beaver populations have been reported. Furthermore, few radiotelemetry studies exist for beavers because no practical method of attaching a radiotransmitter to beavers was available until recently. We used radiotelemetry, remote videography, and trapping data to quantify survival, dispersal, and natality of unexploited beavers in southern Illinois, USA, during 2004 to 2006. Beaver colony density was one of the highest reported in the wildlife literature at 3.27 colonies/km². We monitored 62 beavers for survival; all mortalities (n = 15) occurred during the fall and winter seasons. The pooled annual survival rate for adult and juvenile females was (x̄±SE) 0.76±0.05. Annual survival rates for adult and juvenile males were 0.87±0.04 and 0.55±0.07, respectively. Seasonal survival only differed among sex classes and age classes during the fall. Dispersal rates for juvenile beavers ranged from 0.38±0.13 to 0.59±0.13 and did not vary by sex or age. To quantify natality and recruitment, we captured and euthanized 79 beavers adjacent to our live-capture area; we found a low pregnancy rate of adult females (36%), and no juveniles were bred. Natality of bred females was 3.6 offspring per adult female, and 0.36 kits were recruited per adult female. Apparent kit survival was 28%. Our research provides information to wildlife managers about beaver demographics for a high-density population, based on relatively large sample sizes and novel research techniques for the species.     

Phylogeny of sex‐determining mechanisms in squamate reptiles: are sex chromosomes an evolutionary trap?

Squamate reptiles possess two general modes of sex determination: (1) genotypic sex determination (GSD), where the sex of an individual is determined by sex chromosomes, i.e. by sex‐specific differences in genotype; and (2) temperature‐dependent sex determination (TSD), where sex chromosomes are absent and sex is determined by nongenetic factors. After gathering information about sex‐determining mechanisms for more than 400 species, we employed comparative phylogenetic analyses to reconstruct the evolution of sex determination in Squamata. Our results suggest relative uniformity in sex‐determining mechanisms in the majority of the squamate lineages. Well‐documented variability is found only in dragon lizards (Agamidae) and geckos (Gekkota). Polarity of the sex‐determining mechanisms in outgroups identified TSD as the ancestral mode for Squamata. After extensive review of the literature, we concluded that to date there is no known well‐documented transition from GSD to TSD in reptiles, although transitions in the opposite direction are plentiful and well corroborated by cytogenetic evidence. We postulate that the evolution of sex‐determining mechanisms in Squamata was probably restricted to the transitions from ancestral TSD to GSD. In other words, transitions were from the absence of sex chromosomes to the emergence of sex chromosomes, which have never disappeared and constitute an evolutionary trap. This evolutionary trap hypothesis could change the understanding of phylogenetic conservatism of sex‐determining systems in many large clades such as butterflies, snakes, birds, and mammals. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 168–183.     

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.     

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal

Summary XY females (n=17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.     

High error rates for avian molecular sex identification primer sets applied to molted feathers

ABSTRACT Molted feathers are becoming increasingly important as a source of DNA for identifying the sex of individuals, and accurate methods for molecular sex identification are needed. Three molecular sex identification primer sets have been developed for use in nearly all nonratite birds, but performance of these primer sets has not been evaluated for molted feathers. For two species of birds, the Ring‐necked Pheasant (Phasianus colchicus) and the Scarlet Macaw (Ara macao), we evaluated success and error rates among primer sets using DNA from molted feathers and assessed the percentage of times an incorrect sex would be assigned when analyses are completed in duplicate. Amplification success rates differed among the primer sets for both species, ranging from 67.5% to 89.2% (P= 0.0002 and 0.009), and error rates were high, ranging from 1.9% to 24.2%. Success rates and error rates were not consistent between species and among primer sets. To improve the accuracy of molecular sex identification tests when using molted feathers, we suggest determining acceptable confidence levels in the accuracy of sex assignment, conducting pilot tests to evaluate the performance of different primer sets, and using high‐resolution electrophoresis systems to increase detection of errors.