Zinc‐finger intron 7: a new locus for sex identification of giant panda (Ailuropoda melanoleuca)

We developed a single‐reaction test for identifying the sex of giant panda (Ailuropoda melanoleuca) targeted to co‐amplify homologous fragments with size polymorphism that located at zinc‐finger (ZF) intron 7 by using one pair of primers. This assay produced one sex‐specific fragment in females (XX genotypes) whereas two fragments were produced in males (XY genotypes). Indels (insertion/deletion) in intron 7 of Y‐linked allele provide a significant discrimination between ZFX and ZFY, thus the amplification products can be simply distinguished by agarose gel electrophoresis, exhibiting sex‐specific banding patterns (female, 354 bp; male, 354 bp, 135 bp). The new primer set was successfully tested on known‐sex giant pandas by using template DNA extracted from both blood and fecal samples. Cross‐species test was also performed, revealing that this assay could be applied to other Ursidae species. Zoo Biol 29:526–531, 2010. © 2009 Wiley‐Liss, Inc.     

Y-chromosomal markers for the European brown hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>, Pallas 1778)

Studies on demographic population history and gene flow among populations often rely exclusively on matrilinearly inherited mitochondrial DNA markers. However, by excluding patrilines, such approach introduces an analytical bias into the study. To overcome this bias, we established a set of ten Y-chromosomal markers for the European brown hare (Lepus europaeus), which comprises of three overlapping fragments spanning over the sex-determining region Y, five microsatellite loci (LeMS-Y), and two introns of the Y-linked zinc finger protein (LeZFY). Besides the generation of male specific fragments, both the ZFY and the LeMS-Y01 primer pairs also generated amplification products in females, which are visible in standard agarose gels. These polymerase chain reaction (PCR) products were easily distinguishable from the Y-specific amplicons and thus can function as internal positive PCR control in molecular sexing.     

A PCR-RFLP assay for gender assignment in the three-toed sloths (Bradypus, Pilosa, Bradypodidae)

The three-toed sloths (Bradypus) are slow-moving arboreal neotropical mammals. Understanding demographic variables (such as sex ratio) of populations is a key for conservation purposes. Nevertheless, gender assignment of Bradypus is particularly challenging because of the lack of sexual dimorphism in infants and in adults, particularly B. torquatus, the most endangered of the three-toed sloths, in which sex is attributed by visual observation of the reproductively active males. Here, we standardized a method for sexing Bradypus individuals using PCR-RFLP of sex-linked genes ZFX/ZFY. This assay was validated with known-gender animals and proved accurate to assign gender on three Bradypus species.     

奶牛ZFY、ZFX基因片段的克隆及测序

奶牛的早期胚胎性别鉴定是奶牛胚胎分割及移植技术产生较大经济效益的前提,用PCR技术进行早期胚胎性别鉴定具有准确、快速、灵敏度高的特点.利用人和鼠的性别分化相关的DNA序列ZFY、ZFX基因序列设计的引物对公牛和母牛的染色体DNA、PCR扩增,将扩增产物定向克隆到pUC118上,获得ZFY、ZFX转化子,并测定了ZFY、ZFX基因的序列,发现两者同源性达88.2%,以此为基础可设计引物和探针,以PCR方法进行高灵敏度的奶牛性别鉴定.     

Interfamilial characterization of a region of the ZFX and ZFY genes facilitates sex determination in cetaceans and other mammals

Sequence polymorphism of homologues ZFX and ZFY, in a 604-base pair exon region, was examined in 10 known males and 10 known females across seven cetacean families and used to design a simple, highly sensitive and widely applicable fluorescent 5' exonuclease assay for gender determination in cetaceans. Multiplex amplification, cloning, and sequencing of these previously uncharacterized regions revealed (i) eight fixed differences between ZFX and ZFY, (ii) 29 variable sites between ZFX and ZFY and (iii) very low interspecific nucleotide diversity for both ZFX and ZFY across all families examined. We developed a 5' exonuclease assay that produces a small (105 bp) polymerase chain reaction (PCR) product from both the X and the Y chromosome orthologs, and used double-labelled fluorescent probes to distinguish between the two genes in a real-time PCR assay that is highly reproducible and sensitive. We demonstrated sex specificity for 33 cetacean species in nine families. Given the availability of conserved primers and sequence information for many mammalian species, this approach to designing sexing assays for a wide range of species is both practical and efficient.     

PCR-CTPP: a rapid and reliable genotyping technique based on ZFX/ZFY alleles for sex identification of tiger (<Emphasis Type="Italic">Panthera tigris</Emphasis>) and four other endangered felids

An inexpensive, time-saving and reliable method, polymerase chain reaction with confronting two-pair primers (PCR-CTPP), was developed for sex identification in tiger (Panthera tigris) based on zinc finger alleles (ZFX/ZFY). A site of “C/G” transversion representing fixed differences that discriminated between ZFX and ZFY exons among felids was identified for primers designing. This primer set was successfully tested on samples including blood, shed hairs, dried skin, and stool which contained potential contamination caused by prey DNA. Cross species tests shown that this primer set was also useful for sex identification in four other endangered felids.     

Molecular sexing eutherian mammals

Mammals can be molecular sexed by polymerase chain reaction (PCR) amplification of Y chromosome fragments or coamplification of homologous fragments from both sex chromosomes, which are discriminated by size polymorphism or Y‐specific restriction digestion. Although coamplification of X and Y fragments is more reliable, size polymorphism in homologous fragments is uncommon and Y‐specific restriction site identification requires screening with a battery of enzymes or cloning. Here we describe a simple approach, using ‘double peaks’ in the chromatogram upon direct sequencing of PCR products from males, to identify Y‐specific restriction sites, and demonstrate its utility by application to a range of taxa.     

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.     

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.