Male-specific DNA Fragment Research Articles

Identification of Sex-Specific Markers Reveals Male Heterogametic Sex Determination in Pseudobagrus ussuriensis.

Comprehending sex determination mechanism is a first step for developing sex control breeding biotechnologies in fish. Pseudobagrus ussuriensis, one of bagrid catfishes in Bagridae, had been observed to have about threefold size dimorphism between males and females, but its sex determination mechanism had been unknown. In this study, we firstly used the amplified fragment length polymorphism (AFLP)-based screening approach to isolate a male-specific DNA fragment and thereby identified a 10,569 bp of male-specific sequence and a 10,365 bp of female-related sequence by genome walking in the bagrid catfish, in which a substantial genetic differentiation with 96.35 % nucleotide identity was revealed between them. Subsequently, a high differentiating region of 650 bp with only 70.26 % nucleotide identity was found from the corresponding two sequences, and three primer pairs of male-specific marker, male and female-shared marker with different length products in male and female genomes, and female-related marker were designed. Significantly, when these markers were used to identify genetic sex of the bagrid catfish, only male individuals was detected to amplify the male-specific marker fragment, and female-related marker was discovered to produce dosage association in females and in males. Our current data provide significant genetic evidence that P. ussuriensis has heterogametic XY sex chromosomes in males and homogametic XX sex chromosomes in females. Therefore, sex determination mechanism of P. ussuriensis is male heterogametic XX/XY system.

Conversion of a male-specific RAPD marker into an STS marker in Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is an economically important plant. This species is dioecious, and male plants are considered to be more desirable than females due to their higher yields. To reduce the time required for asparagus breeding, molecular marker techniques have been employed to identify sex-linked DNA markers. In the present study, we converted the male-specific random amplified polymorphic DNA marker T35R54-1600 into a sequence tagged site marker. We cloned a male-specific DNA fragment amplified with the T35R54 primer and determined the sequence of the fragment. The size of T35R54-1600 is 1,586 bp, and this fragment is not homologous to known sex-linked BAC sequences, indicating that this fragment is a new sex-linked region. Within this fragment, we designed the primer pair ‘MSSTS710’ to amplify a 710 bp region. This marker could be used to identify the sex of eight cultivars of A. officinalis: ‘Mary Washington 500W’, ‘UC157’, ‘Harumachi Green’, ‘Super Welcome’, ‘F4’, ‘Pacific 2000’, ‘F2’ and ‘Backlim’. We also analyzed the applicability of this marker to two dioecious Asparagus species, A. schoberioides and A. kiusianus, which are cross-compatible with A. officinalis. Although male-specific DNA fragments of two dioecious Asparagus species, A. schoberioides and A. kiusianus, were generated using the existing male-specific marker Asp1T7sp, no amplicon was obtained using the MSSTS710 marker. Since MSSTS710 can be employed for sex identification only in A. officinalis and not in closely related Asparagus species, the DNA region around the MSSTS710 marker must be variable among Asparagus species.

Discovery of a male-biased mutant family and identification of a male-specific SCAR marker in gynogenetic gibel carp Carassius auratus gibelio

Gibel carp ( Carassius auratus gibelio) is a uniquely gynogenetic species with a minor ratio of males in natural habitats, but its male origin and sex determination mechanisms have been unknown. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific SCAR marker was identified from the mutant family. Normal spermatogenesis was observed in the male testes by immunofluorescence histochemistry. Nearly identical AFLP profiles were observed between males and females, but a male-specific 86 bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the male-specific AFLP fragment, a total of 579 bp sequences were cloned by genome walking. Subsequently, a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and consistently detected only in males.

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17 degrees C and 23 degrees C. However, sex ratios become female-biased at <15 degrees C and male-biased at 25 degrees C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.

Fluorescence body of bovine spermatozoa represents Y-chromosome.

The fluorescence body test would be an inexpensive method of quantifying the Y-chromosomes of mammalian spermatozoa, and could be used to monitor the enrichment of X- and Y-spermatozoa. However, a definitive proof that the fluorescence bodies are markers of the Y-chromosome, is available only for humans and gorillas. In the study described here it is demonstrated for the first time in cattle that the fluorescence bodies of spermatozoa represent the Y-chromosomes. The evidence is furnished by a combination of the fluorescence staining (quinacrine mustard) with the in-situ-hybridization using bovine male-specific DNA fragments. The incidence of f-bodies corresponded with autoradiographic grains caused by the tritiated Y-specific DNA in the examined 144 single cells (75, 5, and 88% agreement).

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5′-TTGCTCACGG TAGATAACAA GAGAG-3′ and 5′-TTGCTCACGG ACCAGGTAGG GAATG-3′) were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32 P -dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.

Isolation of X and Y chromosome-specific DNA markers from a liverwort, Marchantia polymorpha, by representational difference analysis.

The liverwort Marchantia polymorpha has X and Y chromosomes in the respective female and male haploids. Here we report the successful exploitation of representational difference analyses to isolate DNA markers for the sex chromosomes. Two female-specific and six male-specific DNA fragments were genetically confirmed to originate from the X and Y chromosomes, respectively.

Male‐specific DNA in the dioecious species Atriplex garrettii (Chenopodiaceae)

The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment (~1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.

Isolation and Cloning of a Male-specific DNA Fragment from Genomic DNA in Pigs (Sus scrofa)

Isolation and Cloning of a Male-specific DNA Fragment from Genomic DNA in Pigs (Sus scrofa)

Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization

An easy assay for quantification of Y chromosome-bearing sperm (Y-sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male-specific DNA fragment was tested for identification of Y-sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y-sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.

Newly Found Bovine Male-Specific DNA Fragments Amplified by Polymerase Chain Reaction.

Primers, whose base sequence was homologous to the 1-20 and 541-560 by of bovine Y chromosome-specific DNA probe ES8 (630 bp) (Ellis et al., 1988), were prepared. Using these primers, male and female bovine genomic DNA was amplified by PCR. Four unknown DNA fragments were detected in amplified male DNA alone. These fragments were also detected by PCR using 10-6 μg of male DNA.When the base sequences of these DNA fragments were determined by the dideoxy method, none of them matched the base sequence of ES8. When their base sequences were examined for partial matching with the base sequence of ES8, the percentage of agreement was 65% in the case of the 92 by fragment (I0-1), 51% for the 142 bp fragment (I0-2), 57% for the 170 bp fragment (I0-3) and 84% for the 224 bp fragment (I0-4). Hybridization of these sequence-determined DNA fragments to male and female bovine DNA demonstrated the male specificity of these fragments. These results suggest that the 4 DNA fragments obtained in the present study can be utilized as bovine male-specific DNA fragments.

Isolation and characterization of two male‐specific DNA fragments from the bovine gene

Two independent male-specific DNA fragments were isolated from the bovine genome by the technique of cloning by deletion enrichment. Each is represented in the order of one to five copies per haploid genome. Neither fragment was found to be polymorphic between two individuals of different breeds with 12 different restriction enzymes. A 'zoo blot' demonstrated that one fragment had a closely related sequence in the sheep genome, although no other hybridizing sequences were detected in a variety of animal DNAs tested with both probes. The DNA sequence of each fragment is presented; one fragment has a continuous open reading frame and a potential Z-DNA forming region.

Localisation of a male-specific DNA fragment to a sub-region of the human Y chromosome.

A ‘male-specific’ DNA sequence has been localised to human satellite III DNA of the Y chromosome.