Male-specific Band Research Articles

Identification of male sex-specific markers using genome re-sequencing in the Chinese longsnout catfish Leiocassis longirostris

The Chinese longsnout catfish (Leiocassis longirostris Günther), an indigenous commercially important fish species in China, exhibits markedly sex dimorphism in growth, which the males grow much faster than females. So, we aimed to develop sex-specific DNA markers to accelerate the development of sex control techniques for L. longirostris. In this study, 15 individuals of each sex were sequenced using whole-genome re-sequencing technology. A total of 238.51 Gb clean data was used to detect the sex associated markers and we found that 2472 loci were enriched in the first 18 Mb of Chr 7, the putative sex-determination region for L. longirostris. All candidate markers were male heterozygous, implying that the sex-determination system of the L. longirostris is the XX/XY type. Using the sequence polymorphism of male and female individuals in this region, we designed six pairs of primers to develop sex-specific markers, of which three markers exclusively exhibited male-specific bands. Further validation showed that the accuracy of sex prediction was up to 100% for individuals in the Meishan, Chengdu, Yibin and Shishou regions, suggesting wide applicability among local populations. Importantly, marker 3 was located in the ACAP1 gene, which may be involved in sex determination or differentiation of L. longirostris. This method for developing sex-specific markers is efficient, simple, and provides a useful reference that can be applied to other fish species. The markers developed in this work could be instrumental for sex control breeding to increase the yield in the L. longirostris aquaculture industry.

Identification of Sex-specific Molecular Markers in Barbel and Nile Carp using SCoT and ISSR Markers

The present study aimed to investigate molecular sex differences between males and females of two members of family Cyprinidae in Egypt namely Benni and Lebeis using two different molecular markers i.e., start codon targeted polymorphism (SCoT) and inter simple sequence repeats (ISSR). The bulked DNA sample for each gender of the tested species was screened with eight primers from each marker. However, SCoT marker was more efficient than ISSR marker by showing higher number of possible sex-specific bands in the two tested species. SCoT primers were able to generate 7 and 4 male-specific bands, along with two and 5 female-specific bands for B. bynni and L. niloticus, respectively. Whereas, ISSR primers generated 4 and two male-specific bands, vis-a-vis two and three female-specific bands for B. bynni and L. niloticus, respectively. Interestingly, SCoT-01 primer generated unique common female specific band (690bp) which appeared only in the females of Benni and Lebeis. This band is more possible to be female sex-specific. The results obtained in this study could serve as a keystone for molecular sex differentiation studies in the two tested species and other fish species. However, increasing the number of analyzed individuals is highly recommended.

Confirmation of an interspecific hybrid of Drimia based on mitotic, stomatal and ISSR marker analyses

An interspecific hybrid between Drimia govindappae (♀) (2n = 2x = 20) and D. coromandeliana (♂) (2n = 4x = 40) was generated by artificial hand pollination. Mitotic, stomatal and ISSR analyses confirmed that the F1 plant was intermediate between the parents and exhibited the hybrid nature. The F1 plant had 2n = 3x = 30 chromosomes in root-tip cells. Stomata were anomocyctic and the size 42.59 × 34.38 µm. The ISSR primer analysis revealed transmission of both male and female specific bands to F1 plants thereby confirming their true hybrid nature. Dendrograms based on the discriminative ISSR markers (ISSR834 and ISSR836) also showed the similarity of F1 plant profile to male and female parents.

Assessment of Genetic Relationships between Streptocarpus x hybridus V. Parents and F1 Progenies Using SRAP Markers and FT-IR Spectroscopy.

The genetic relationship among three Streptocarpus parents and twelve F1 hybrids was assessed using sequence-related amplified polymorphism (SRAP) molecular markers and Fourier-transform infrared (FT-IR) spectroscopy. Both methods were able to discriminate F1 hybrids and parents as revealed by cluster analysis. For hybrid identification, the type III SRAP marker was the most effective due to the presence of male-specific bands in the hybrids. Different behaviors in the biochemical variability of DNA samples have been observed by FT-IR spectral analysis, which might be attributed to the inherent nature of the genomic DNA from parents and their F1 progenies. Mantel test was also carried out to compare morphological, SRAP, and FT-IR results based on genetic distances. The highest correlation coefficient was found between morphological and SRAP marker distances (R = 0.607; p ≤ 0.022). A lower correlation was observed between the morphological and FT-IR distance matrix (R = 0.231; p ≤0.008). Moreover, a positive correlation was found between the distances generated with SRAP and FT-IR analyses (R = 0.026) but was not statistically significant. These findings show that both SRAP and FT-IR techniques combined with morphological descriptions can be used effectively for nonconventional breeding programs for Streptocarpus to obtain new and valuable varieties.

Identification of sex-linked markers in the sexually cryptic coco de mer: are males and females produced in equal proportions?

Lodoicea maldivica (coco de mer) is a long-lived dioecious palm in which male and female plants are visually indistinguishable when immature, only becoming sexually dimorphic as adults, which in natural forest can take as much as 50 years. Most adult populations in the Seychelles exhibit biased sex ratios, but it is unknown whether this is due to different proportions of male and female plants being produced or to differential mortality. In this study, we developed sex-linked markers in Lodoicea using ddRAD sequencing, enabling us to reliably determine the gender of immature individuals. We screened 589 immature individuals to explore sex ratios across life stages in Lodoicea. The two sex-specific markers resulted in the amplification of male-specific bands (Lm123977 at 405 bp and Lm435135 at 130 bp). Our study of four sub-populations of Lodoicea on the islands of Praslin and Curieuse revealed that the two sexes were produced in approximately equal numbers, with no significant deviation from a 1:1 ratio before the adult stage. We conclude that sex in Lodoicea is genetically determined, suggesting that Lodoicea has a chromosomal sex determination system in which males are the heterogametic sex (XY) and females are homogametic (XX). We discuss the potential causes for observed biased sex ratios in adult populations, and the implications of our results for the life history, ecology and conservation management of Lodoicea.

Validation of RAPD and ISSR Markers Used for Sex Determination in Date Palm Grown under Sudan Conditions

Abstract Date palm is one of the most important economical crops in the world. Sex determination of date palm in early stage is a prerequisite for breeding and cultivation. The aim of this study is to validate RAPD and ISSR markers for sex identification of date palm genotypes grown under Sudan conditions. DNA was extracted from ten seedlings and five male and female plants using CTAB method. Eight primers, six RAPD and two ISSR primers were examined for their validation in sex determination of date palm genotypes. PCR amplification was performed using these primers. Four RAPD primers OPA02, OPJ-09, RD A02 and RD A21 were amplified male specific band with size of 1000, 1100, 1000 and 1400 pb respectively, while ISSR markers could not. The specific bands were observed clearly among all male genotypes and absent in female samples unknown samples irrespective of genotypes. Our results could be useful for sex determination of date palm sex in seedling stage and would promote date palm cultivation and production.

DNA Fingerprinting of Sex in Jojoba (Simmondsia chinensis) Grown under the Semi-arid Conditions of Sudan

Abstract Jojoba cultivation and production face the challenge of establishing ways to identify the sex at early stage of plant growth. The present study was carried out to identify sex of jojoba at the seedling stage under Sudan condition. Two DNA markers, ISSR (UBC807) and RAPD (OPG-5), were used for sex identification of jojoba genotypes: two known male and females genotypes and four unknown genotypes. ISSR marker, UBC807 was successfully amplified a unique male-specific band at 1200 bp, while RAPD marker, OPG-5 could not amplify a unique band within jojoba sex. The result clearly indicates that ISSR-UBC807 marker can be used for sex identification of jojoba at seedlings stage, a finding that could make the commercial cultivation and production of jojoba possible in Sudan.

Identification of Sex Related SRAP Molecular Marker in Humulus scandens

Humulus scandens is a model material for studying the molecular mechamisms of sex differentiation in dioecious plants.We used SRAP(sequence related amplified polymorphism) to screen male or female specific fragments in H.scandensby using male and female gene pool as template.Fifty pairs of primers showed a good amplification pattern after screening 256 pairs of primers.Polyacrylamide gel electrophoresis amplification patterns of SRAP showed that 671 bands were amplified using 50 primer combinations,of which 247 bands were polymorphic and the polymorphic ratio is 36.81%.With five single male plants and five single female plants as materials,we validated the amplification patterns of primer combinations me15 +em11,me14 +em16,me13 +em12 and me7 + em10 again by agarose gel electrophoresis.Although the amplification patterns were more or less different between polyacrylamide gel electrophoresis and agarose gel electrophoresis,male or female specific bands were relatively stable.If cloning,sequencing and chromosomal locating the obtained sex-related DNA sequences on chromosome of H.scandens by using fluorescence in situ hybridization technique,it will be contributed to studying the molecular mechanisms of sex chromosome evolution in H.scandens.

Generation and validation of unique male sex-specific sequence tagged sites (STS) marker from diverse genotypes of dioecious Jojoba-Simmondsia chinensis (Link) Schneider

DNA fingerprinting studies have been carried out with the physiologically mature male and female plants of Jojoba using 80 ISSR primers with a view to generate sex-linked markers. After bulk segregant analysis, two unique ISSR markers, viz. ISSR8481500 and VIS111317 have been developed which can be used for determining the sex at the seedling stage. Of the eighty primers tested on the pooled male DNA and pooled female DNA samples, six ISSR primers were found to be associated with sex expression. Of the six, only two primers ISSR848 and VIS11 generated unique male sex specific bands of ~1,500 and ~1,300 bp which were consecutively present in all the male genotypes and absent in all the respective female genotypes. The remaining four primers when tried on individuals of different genotypes were confined to their sex specificity in only two female genotypes and absent in their male counterparts. One of the male-sex specific markers, VIS111317 has also been cloned and sequenced which showed homology with a sex linked gene, DD44 from dioecious Silene species. Furthermore, VIS111317 was converted into a male sex-specific sequence tagged sites (STS) marker of 584 bp. The male specific STS marker thus developed has been verified and validated on 100 populations of male and female individuals from ten different genotypes of Jojoba to endorse the diagnostic reliability of the STS marker. This can gainfully be employed for screening of sex at seedling stage which would be quite helpful for uprooting the undesired plants, thereby, saving resources like labor, water, fertilizers and space for highly desirable female plants.

Determination of sex in Zamia fischeri Miq., an endangered gymnosperm

Sex determination of Zamia fischeri Miq., one of the endangered cycads was done using peroxidase profiling, two-dimensional gel electrophoresis of proteins and RAPD-based approaches to find an easy and reliable molecular marker. Sex at pre-flowering stage could not be resolved by peroxidase isozymic profiling. Use of sex organ specific tissues as enzyme source resulted in marked differentiation in peroxidase profiles. Fifteen unique spots of relatively high molecular mass were noted in microspore derived protein (male) sample while twenty five unique spots ranging between 20 to 94 kD were identified in ovule derived protein (female) sample as resolved by two dimensional gel electrophoresis. DNA from leaf samples of contrasting sex resulted in four male and female specific RAPD derived DNA fragments (two each of both sexes) of which one male specific band showed homology with micro satellite sequence of Araucaria angustifolia; this can be used as a convenient molecular marker for sex determination of Zamia fischeri at pre-flowering stage. Key words: Molecular marker, RAPD, Sex determination, Zamia fischeri.

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID-DIPLOID RED ALGA GRACILARIA CHILENSIS(1).

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex-linked molecular markers in the haploid-diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male-specific bands; and one RAPD primer (OPD12), a female-specific band. The sequences of the cloned putative sex-specific PCR fragments were used to design specific primers for the female marker SCAR-D12-386 and the male marker SCAR-G16-486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex-specific association, we were able to amplify SCAR-D12-386 and SCAR-G16-486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%-9% nucleotide divergence for SCAR-D12-386 and SCAR-G16-486, respectively. SCAR-D12-386 and SCAR-G16-486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.

A novel male-specific DNA sequence for river buffaloes

In this paper, we report identification of a novel male-specific DNA marker in Iranian river buffalo presenting the possibility of river buffalo gender determination using Random amplified polymorphic DNA-PCR (RAPD-PCR). Twenty eight random primers used for RAPD fingerprinting in order to isolation and characterization of microsatellite sequences using PIMA (PCR-based isolation of microsatellite array). All sharp bands related to different primers were isolated from the gel and inserted to a cloning vector and transformed to competent cells for nucleotide sequencing. Sequences were used as queries for BLAST tool. BLAST results showed that a sequence had high similarity with a part sequence of cattle Y chromosome sequences deposited in GenBank. A pair of primers (IRBMS-F and -R) was designed based on the sequence for amplifying the male-specific band by polymerase chain reaction (PCR) for Iranian river buffalo gender detection. Gender-specific bands in the gel were observed in all males but not in females. This showed that the gender of Iranian river buffalo could be easily and effectively identified by PCR technique using these primers.

웅성 특이적 SRY 및 ZFY 유전자를 이용한 쇠고기 성(性) 판별

본 연구는 포유류의 Y-염색체상에 존재하는 SRY 및 ZFY의 웅성 특이적 성 결정 유전자를 이용하는 PCR 기법으로 쇠고기 성판별 기술을 개발하고자 수행하였다. 성 결정 유전자 영역의 특정 염기서열을 포함하는 primer를 각각 설계 합성하고 이들 primer를 이용하여 PCR증폭을 실시한 다음, 각각의 증폭산물을 1.5% agarose gel에 전기영동하여 웅성 특이적 DNA band의 증폭여부를 확인하였다. SRY 유전자에서 웅성개체 쇠고기는 1,348 bp 크기의 단편을 가진 DNA band가 검출되었으나, 자성개체의 경우 DNA band가 전혀 검출되지 않은 것을 확인 할 수 있었다. 또한, ZFY 유전자에서 웅성개체의 쇠고기는 979 bp 크기의 단편을 가진 DNA band가 모두 검출되었으나, 자성개체의 쇠고기에서는 역시 DNA band가 전혀 검출되지 않았다. 즉, SRY 및 ZFY 유전자는 모두 숫소에서 유래한 쇠고기에서 웅성 특이적인 DNA band가 정확히 검출된 반면 암소에서 유래한 쇠고기에서는 웅성 특이적 DNA band가 전혀 검출되지 않았다. 또한 쇠고기 성 감별법을 도축 후 가공 유통판매단계에 실제 활용 가능성을 검증하고자 시중에 유통되고 있는 등심 및 갈비 포장육 350시료를 무작위 추출하여 성 판별을 실시한 결과 숫소가 252시료(72%) 그리고 암소가 98두(28%)로 조사되었다. 따라서 본 연구에서 개발한 SRY 또는 ZFY의 웅성 특이적 성 결정 유전자를 이용하는 쇠고기 성 감별기술은 생산단계는 물론 도축 후 가공 유통 판매되고 있는 모든 쇠고기의 암수 성감별을 위한 유용한 DNA 표지인자로 활용할 수 있을 것으로 기대된다. The objective of this study was to develop a rapid and reliable method for the sex determination of beef using the PCR(polymerase chain reaction) technique. We have used two bovine sex determining genes, SRY and ZFY, on the Y-chromosome to identify the sex of Hanwoo and Holstein beet. We attempted to amplify 1,348 bp and 979 bp fragments from male and female genomic DNA corresponding to the SRY and ZFY genes, respectively, using male specific primers. The amplified PCR products were separated by electrophoresis in a 1.5% agarose gel to detect a male specific DNA band. When DNA from male beef was amplified with primers specific for the SRY gene, a DNA band of 1,348 bp was present in all of the male samples, but absent from all of the female samples. Also, when DNA from male beef was amplified with primers specific for the ZFY gene, a DNA band of 979 bp was observed in all of the male samples, but absent from all female samples. In conclusion, the bovine SRY and ZFY genes are typically found only in male beef. For the practical application of this method for the sexing of commercial beef at the processing and marketing stages after slaughter. a total of 350 beef samples collected randomly from local markets were analyzed for sex determination. The proportions of male and female samples were 252 (72%) and 98 (28%), respectively. Therefore. the SRY and ZFY genes. which are specific for the Y-chromosome, may be useful sex-diagnostic DNA markers to distinguish male meat from female meat.

Breaking the intergeneric crossing barrier in papaya using sucrose treatment

A breeding programme was undertaken using Carica papaya var. Surya and Vasconcellea cauliflora with a view to raise progenies resistant to ‘papaya ringspot virus’ (PRSV). Earlier studies have clearly demonstrated the cross incompatibility between these two genera. Hence, an attempt was made to break this barrier using sucrose. The pollen of V. cauliflora was collected and pollination was carried out by smearing sucrose solution in the concentrations of 1, 2, 3, 4 and 5% on to the stigmatic surface of the flower. At 5% sucrose concentration, maximum viable seed set (13.73) was obtained. Sucrose at 5% was observed to break the intergeneric barrier by enhancing the pollen germination. There was drastic reduction in the effect of sucrose with the decrease in the concentration levels. The pollen germination studies carried out with and without sucrose clearly demonstrated the efficacy of sucrose in enhancing pollen germination and pollen tube growth. The intervarietal hybridization carried out between the varieties Surya and Pusa Dwarf showed 91.7% set of the fruits with 300 viable seeds per fruit. The hybridity of the progenies was confirmed using ISSR primers by the amplification of DNA from progenies and parents. Four primers UBC 807, 810, 814 and 861 clearly amplified male specific bands, which were present in progenies, but absent in female parent.

Molecular characterization and evolution of X and Y-borne ATRX homologues in American marsupials

In eutherians, the sex-reversing ATRX gene on the X has no homologue on the Y chromosome. However, testis-specific and ubiquitously expressed X-borne genes have been identified in Australian marsupials. We studied nucleotide sequence and chromosomal location of ATRX homologues in two American marsupials, the opossums Didelphis virginiana and Monodelphis domestica. A PCR fragment of M. domestica ATRX was used to probe Southern blots and to screen male genomic libraries. Southern analysis demonstrated ATRX homologues on both X and Y in D. virginiana, and two clones were isolated which hybridized to a single position on the Y chromosome in male-derived cells but to multiple sites of the X in female cells. In M. domestica, there was a single clone that mapped to the X but not to the Y, suggesting that it represents the M. domestica ATRX. However a male-specific band was detected in Southern blots probed with the D. virginiana ATRY and with a mouse ATRX clone, which implies that the Y copy in M. domestica has diverged further from other ATRX homologues. Thus there appears to be a Y-borne copy of ATRY in American, as well as Australian marsupials, although it has diverged in sequence, as have other Y genes that are testis-specific in both eutherian and marsupial lineages.

Analysis of the gonadal sex of five intersex pigs using Y chromosomal markers.

DNA isolated from gonadal tissue of five intersex pigs was analyzed for the presence of the Y chromosomal markers ZFY and DYZ1. Four cases showed no detectable male-specific DNA-sequences. The fifth case exhibited a weak male-specific band at Southern blot analysis with the human ZFY probe and showed a male-specific PCR-product with DYZ1 primers, indicating an XX/XY gonadal chimera.

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.

Effect of pollination timing on the rate of apomictic reproduction revealed by RAPD markers in paspalum notatum.

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1-3 d pre-anthesis; at anthesis; and 2, 4 and 6 d post-anthesis. Cytoembryological analyses indicated that approx. 17% of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety-five oligonucleotides were assayed on the progenitors in order to search for male-specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3.4% before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off-type plants (16.5%), of which only one was a B(III) hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed.

Rapid DNA extraction and PCR-sexing of mouse embryos.

We have devised a PCR-based sexing method that is quick, simple, and highly reproducible. DNA is first extracted from embryonic mouse yolk sac via a 15 min, two-step incubation procedure utilizing PCR-compatible proteinase K buffer. Without any further manipulation the lysate is subjected to 30 cycles of PCR, optimized to run in less than 1 hr. The reaction includes multiplexed primer pairs for Sry and Myog (myogenin) that generate a male specific band of 441 bp and an internal control band of 245 bp, respectively. This robust method is used routinely in our laboratory and gives rapid genotyping results with 98% reliability and 100% accuracy.

Application of AFLP for the detection of sex‐specific markers in hemp

AbstractTwo dioecious hemp accessions (Can18 and Can17) were tested by bulked segregant analysis for polymorphisms between male and female bulks with amplified fragment length polymorphisms. Thirty‐nine primer combinations were tested and 20 of these yielded one to three male‐specific bands. In contrast, no female‐specific band was detected. Eight of these primer combinations were used for testing 80 progeny plants from a cross between two plants from Can18 and 30 plants from Can17. A total of 16 and 17 male‐specific fragments were obtained for Can 18 and Can 17, respectively. Eleven fragments exhibited the same fragment size in both accessions. All male plants, but not one female plant, showed the respective polymorphic band with each of the eight primer combinations. Problems regarding sex determination under field conditions were successfully overcome by testing plants that had been grown in small pots in a greenhouse. The abundant number of potential markers for the male sex, their complete cosegregation with male plants and the absence of markers for the female sex support the presence of a male sex chromosome in hemp.