Sex determination in 6 bovid species by duplex PCR

Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae,viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, andOvis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including theSRY gene and theGAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.     

Genetic sex determination of mice by simplex PCR

Background

Investigating fetal development in mice necessitates the determination of fetal sex. However, whilst the sex of adult and juvenile mice can be readily distinguished from anogenital distance, the sex of fetal and neonatal mice cannot be identified visually. Instead, genetic sex must be determined by PCR amplification of X chromosome genes with divergent Y chromosome gametologs. Existing simplex PCR methods are confounded by small size differences between amplicons, amplification of unexpected products, and biased amplification of the shorter amplicon.

Results

Primers were designed flanking an 84 bp deletion of the X-linked Rbm31x gene relative to its Y-linked gametolog Rbm31y. A single product was amplified from XX samples, with two products amplified from XY samples. Amplicons were resolved by gel electrophoresis for 20 min, with unbiased amplification of both products observed in XY samples.

Conclusion

This method achieves rapid and unequivocal genetic sex determination of mice in low volume PCR reactions, reducing reagent usage and simultaneously eliminating shortcomings of previous methods.

     

Sex determination in goat by amplification of the HMG box using duplex PCR

The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.     

PCR hot-start using duplex primers

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.     

人类的性别与性别畸形

从细胞遗传学和分子遗传学的角度阐述了人类性别的形成机理和性别畸形的致病机理。人类性别的形成是以SRY基因为主导的、多基因参与和调控的、有序表达的生理过程。性别畸形的形成是由于性染色体数目或结构异常、与性别形成有关的基因缺失、突变或与其表达调控相关的其他基因突变所致。     

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.     

Plant sex determination and sex chromosomes

Sex determination systems in plants have evolved many times from hermaphroditic ancestors (including monoecious plants with separate male and female flowers on the same individual), and sex chromosome systems have arisen several times in flowering plant evolution. Consistent with theoretical models for the evolutionary transition from hermaphroditism to monoecy, multiple sex determining genes are involved, including male-sterility and female-sterility factors. The requirement that recombination should be rare between these different loci is probably the chief reason for the genetic degeneration of Y chromosomes. Theories for Y chromosome degeneration are reviewed in the light of recent results from genes on plant sex chromosomes.     

Bird sex determination

During the evolution, sex determination occurred early. Sex determining factors were progressively isolated from other genes in sexual chromosomes, or gonosomes. Among vertebrates, evolution took two opposite pathways : in mammals, the system of XX:XY sex determination, with Y chromosome, induces male differentiation. In contrast, in birds, the system ZZ:ZW, with the W chromosome, induces female differentiation. But comparative studies show that the two pathways are not so simple. In the chicken as in the lower vertebrates, estrogens play a central role in gonadal sex differentiation. Several genes, show to be critical for mammalian determination, are also expressed in the chicken but their expression pattern differs, indicating functional plasticity. The W-linked female determinants remains still unknown. But comparative studies of the two pathways, with conserved and divergent elements, are broadening our understanding of sex determination.     

Human Y chromosome, sex determination, and spermatogenesis- a feminist view

In this review I want to argue that, far from being a macho entity with an all-powerful role in male development, the human Y chromosome is a "wimp." It is merely a relic of the X chromosome, and most or all of the genes it bears-including the genes that determine sex and control spermatogenesis-are relics of genes on the X chromosome that have other functions altogether.     

Freemartinism and FecX allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR

A new naturally occurring mutation in the fecundity gene BMP15 in the Rasa Aragonesa sheep breed (Ovis aries) has been found to affect prolificacy. This mutation (FecX^R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX^R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX^R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercial flocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX^R rams. This analysis identified 103 rams hemizygous for the FecX^R allele and 1 heterozygous ram. Because this gene is located on the X chromosome, this heterozygous animal is a freemartin ram that is co-amplifying the DNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplex PCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highly prolific ewes in commercial flocks and to assist breeders in selection of repository lambs.     

Germ cell sex determination

Whether a germ cell embarks on oogenesis, the female gametogenic pathway, or spermatogenesis, the male pathway, may be determined cell-autonomously, by the germ cell's own genes, or by the tissue environment in which it is located. The decision may or may not be associated with the time of entry into meiosis, and this in turn may be controlled wholly by the germ cell's own genes, or in part by the environment. These issues will be explored with reference to Caenorhabditis elegans, Drosophila and the mouse.     

Multilocus autosomal sex determination

The two basic one locus sex determination models, diploid individual sex determination and parental sex determination, are generalized to the multilocus framework. As in the single locus case, it is established that there are two classes of polymorphic equilibria, equilibria with even sex ratio and equilibria with equal allele frequencies in the two sexes. The condition for external stability of this second class equilibria to invasion by a new mutant allele is that a new appropriately averaged sex ratio near the equilibrium be moved closer to the even sex ratio. However, stable polymorphisms with noneven sex ratio are not those that have a sex ratio as close as possible to 1/2, in contrast to the single locus case.Research supported in part by NIH grants GM 28016 and GM 10452 and a grant from the U.S.-Israel Binational Science Foundation     

Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.     

The primary sex ratio under environmental sex determination

The ESS primary sex ratio (male/female) under environmental sex determination (ESD) is shown to be equal to the ratio of the average fertility of a female to the average fertility of a male. Thus, depending upon how male and female fertility change over the environmental variable causing ESD, the primary sex ratio may be either male or female biased, or neither. The primary sex ratio thus contains information as to how male and female fertilities change with the environment.