Male-specific DNA Markers Research Articles

Evidence of sterility of the male sporophytes of the brownalga Saccharina japonica (Phaeophyceae) in cultureirrespective of their ploidy levels.

Monoclonal female gametophytes of Saccharina japonica, when cultured independently, can develop into female sporophytes. Previous research has shown that the chromosomes in female sporophytes of S. japonica may naturally duplicate, forming diploids, and these diploid female sporophytes are capable of forming sori and releasing zoospores. In contrast, male sporophytes derived from culturing monoclonal male gametophytes failed to form sori and produce meiospores; thus, it was hypothesized that these male sporophytes are haploid and unable to perform meiotic division. In order to explore whether the chromosomes in male sporophytes can naturally duplicate and whether such diploid male sporophytes can effectively form sori and produce meiospores, we obtained 62 male sporophytes and cultivated them into adults. Only male-specific DNA markers were detected in all of these male sporophytes, indicating their male nature. Ten microsatellite markers were used to estimate the relatedness of the parental gametophytes and the corresponding sporophytes. Results revealed that the genotypes of the male sporophytes matched exactly with the corresponding male gametophytes. Both diploid and haploid sporophytes were detected in these 62 male sporophytes when analyzed by flow cytometry. After 16 months of cultivation, none of the male sporophytes formed sori regardless of their ploidy. As controls, both female and hybrid sporophytes developed sori and released viable zoospores. These findings suggest that the sterility of male sporophytes in S. japonica is not related to their ploidy; rather, it is attributed to the absence of essential elements for sori formation that may be present only in the female.

Identification of male-specific DNA markers and generation of genetically all-female offspring using natural pseudomales in Amur catfish (Silurus asotus)

Amur catfish (Silurus asotus) is an economically important species. It demonstrates sexual dimorphism in growth, the females grow faster than the males. Therefore, production of all-female fries for Amur catfish has high commercial potential. However, sex-specific molecular markers are not available for Amur catfish, which hinders the attempt to breed and apply all-female seed for this species. In this study, we established 3 male-specific markers for Amur catfish by restriction-site associated DNA sequencing (RAD-seq) and revealed that Amur catfish has a male heterogametic (XX female, XY male) sex-determination system. Spontaneous pseudomales (individuals that are physiological male and genetic female) were identified from the wild populations of Amur catfish. Genetically all-female larvae for Amur catfish were obtained by crossing the spontaneous pseudomales with the normal females. The male-specific markers for Amur catfish are valuable tools for detecting the genetic sex of the brood fish. Furthermore, generating all-female offspring using natural pseudomales can considerably save the breeding time, which offers a new option for commercial Amur catfish seed production.

Validation of a male-specific DNA marker confirms XX/XY-type sex determination in several Hungarian strains of African catfish (Clarias gariepinus)

African catfish (Clarias gariepinus) is a promising food fish species with significant potential and growing mass of production in freshwater aquaculture. Male African catfish possess improved production characteristics over females, therefore the use of monosex populations could be advantageous for aquaculture production. However, our knowledge about the sex determination mechanism of this species is still limited and controversial. A previously isolated male-specific DNA marker (CgaY1) was validated using offspring groups from targeted crosses (n = 630) and it was found to predict the sex of 608 individuals correctly (96.43% accuracy). Using the proportion of recombinants, we estimated the average genetic distance between the potential sex determination locus and the sex-specific marker to be 3.57 cM. As an earlier study suggested that both XX/XY and ZZ/ZW systems coexist in this species, we tested the applicability of their putative ‘moderately sex-linked loci’ and found that no sex-specific amplification could be detected for any of them. In addition, temperature-induced masculinization suggested by others was also tested, but no such effect was detected in our stocks when the published parameters were used for heat treatment. Altogether, our results support an exclusive XX/XY sex determination system in our African catfish stock and indicate a good potential for the future use of this male-specific DNA marker in research and commercial production.

Successful Production of an All-Female Common Carp (Cyprinus carpio L.) Population Using cyp17a1-Deficient Neomale Carp

Due to sexual dimorphism in the growth of certain cultured fish species, the production of monosex fishes is desirable for the aquaculture industry. Nowadays, the most widely practiced technique available for the mass production of monosex fish populations is sex steroid-induced sex reversal. Here, a novel strategy for the successful production of all-female (AF) common carp (Cyprinus carpio L.), to take advantage of the sexual dimorphism in growth documented in this species, has been developed using genetic engineering via single gene-targeting manipulation without any exogenous hormone treatments. Male and female heterozygous cyp17a1-deficient common carp were first obtained using the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) technique. An all-male phenotype for homozygous cyp17a1-deficient carp, regardless of the individuals’ sex-determination genotypes (XY or XX), has been observed. A male-specific DNA marker newly identified in our laboratory was used to screen the neomale carp population with the XX genotype from the cyp17a1-deficient carp. These neomale carp develop a normal testis structure with normal spermatogenesis and sperm capacity. The neomale common carp were then mated with wild-type (WT) females (cyp17a1+/+ XX genotype) using artificial fertilization. All the AF offspring sample fish from the neomale-WT female mating were confirmed as having the cyp17a1+/− XX genotype, and normal development of gonads to ovaries was observed in 100.00% of this group at eight months post-fertilization (mpf). A total of 1000 carp fingerlings, 500 from the WT male and female and 500 from the neomale and WT female mating, were mixed and reared in the same pond. The average body weight of cyp17a1+/− XX females was higher by 6.60% (8 mpf) and 32.66% (12 mpf) than that of the control common carp. Our study demonstrates the first successful production of a monosex teleost population with the advantages of sexual dimorphism in growth using genetic manipulation targeting a single locus.

Two generations of meiotic gynogenesis significantly elevate homogeneity and confirm genetic mode of sex determination in bighead carp (Hypophthalmichthys nobilis)

Gynogenesis is an effective technique for establishing pure line and validating possible chromosomal mode of sex determination in fish. As one of the major aquaculture fish historically cultured in China, bighead carp (Hypophthalmichthys nobilis) is famous for its market value of the head, but slow in its genetic improvement due to a long period of sexual maturation. In this study, females of bighead carp (F0 dams) with fast growth and bigger head were chose to induce first and second generation of meiotic gynogenesis (GF1 and GF2 for short) by using UV-irradiated heterologous sperm of common carp (Cyprinus carpio). On average, diploid gynogens were obtained by cold shock with a survival rate of 7.36% for GF1 and 6.24% for GF2, respectively. Results of morphology comparison and parentage assignment showed that all survival gynogens were genetically derived from the dams. Compared with their F0 parents, heterozygosities of GF1 and GF2 gynogens decreased by 42% and 60% when analyzed using 50 SSRs, and by 35% and 53% when using 1505 SNP markers, respectively. Sexing of the GF1 and GF2 gynogens showed all-female genotypes and phenotypes when examined by male-specific DNA markers, histology section and morphology of gonads. Overall, large number of meiotic gynogens have been successfully induced in two consecutive generations, providing valuable resources for fast domestication and genetic improvement in bighead carp. Our evidence of sex ratios in both GF1 and GF2 gynogens also strongly supports a proposed chromosomal mechanism of sex determination (XX/XY) for this large-size cyprinid fish.

Development of a male specific genetic marker for Garcinia subelliptica Merr. tree

ABSTRACT Garcinia subelliptica Merr. (called “fukugi” in Japanese) is a dioecious tree species, which has traditionally been planted as windbreak forests in the coast of Ryukyu islands. Although effective afforestation and maintenance of G. subelliptica would greatly be assisted by early sex diagnosis and selection at the seedling stage, the genome and the sex-determination system of G. subelliptica are still poorly understood. In this study, we estimated the genome size of G. subelliptica to be approximately ~ 12 Gb for female and male, based on k-mer analysis. Furthermore, we developed a male-specific DNA marker for G. subelliptica, using the restriction site-associated DNA sequencing (RAD-seq) method. The 660 bp marker fragment, named FSDM (“fukugi” sex determination marker), was amplified in all males but not in the female G. subelliptica genome analyzed so far. In addition, the segregation ratio of the marker was 1:1 among the tested population. The DNA sequence of FSDM showed a significant similarity to the antifungal protein ginkbilobin-2 gene. The development of the sex-specific genetic marker of G. subelliptica may allow more effective conservation of traditional landscape and future design of villages around the northern part of the main island of Okinawa, a promising candidate site for World Natural Heritage.

Applicability of two male specific DNA markers for sex identification of various Asparagus species

Applicability of two male specific DNA markers for sex identification of various <i>Asparagus</i> species

Isolation of a Male-Specific Molecular Marker and Development of a Genetic Sex Identification Technique in Spotted Knifejaw (Oplegnathus punctatus).

Sex-specific DNA markers are very helpful for identifying genetic sex and studying sex determination mechanisms in fish. To identify the sex-specific markers of spotted knifejaw (Oplegnathus punctatus), we performed a comparative analysis of the female and male genomes. In this study, an 18bp insertion was identified in the male genome after verification by sequencing depth and PCR. An effective and rapid method based on PCR was then developed to identify the genetic sex. A male-female-shared primer pair and a male-specific primer were designed for PCR amplification to avoid false-negative phenomena. To examine the primers in practice, we utilized hundreds of spotted knifejaw fish from different groups to identify their genetic sex, and the results were consistent with their phenotypic sex. The male-specific DNA marker would be helpful for artificial breeding, Y chromosome assembly and further study of the sex determination mechanism. This study is the first to identify an effective sex-specific marker in spotted knifejaw.

Improvement of the Pacific bluefin tuna (Thunnus orientalis) reference genome and development of male-specific DNA markers

The Pacific bluefin tuna, Thunnus orientalis, is a highly migratory species that is widely distributed in the North Pacific Ocean. Like other marine species, T. orientalis has no external sexual dimorphism; thus, identifying sex-specific variants from whole genome sequence data is a useful approach to develop an effective sex identification method. Here, we report an improved draft genome of T. orientalis and male-specific DNA markers. Combining PacBio long reads and Illumina short reads sufficiently improved genome assembly, with a 38-fold increase in scaffold contiguity (to 444 scaffolds) compared to the first published draft genome. Through analysing re-sequence data of 15 males and 16 females, 250 male-specific SNPs were identified from more than 30 million polymorphisms. All male-specific variants were male-heterozygous, suggesting that T. orientalis has a male heterogametic sex-determination system. The largest linkage disequilibrium block (3,174 bp on scaffold_064) contained 51 male-specific variants. PCR primers and a PCR-based sex identification assay were developed using these male-specific variants. The sex of 115 individuals (56 males and 59 females; sex was diagnosed by visual examination of the gonads) was identified with high accuracy using the assay. This easy, accurate, and practical technique facilitates the control of sex ratios in tuna farms. Furthermore, this method could be used to estimate the sex ratio and/or the sex-specific growth rate of natural populations.

A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.

A method for sex identification in asparagus using DNA from seeds

Asparagus (Asparagus officinalis L.) is a dioecious species, with both male and female individuals. Male plants are more desirable to cultivate than female plants because they have higher yields, and, unlike female plants, they do not have a weed problem resulting from fallen seeds. A male-specific DNA marker is currently available to identify the sex of asparagus individuals using total DNA extracted from cladodes and roots. However, no published method is currently available for DNA extraction and PCR amplification from asparagus seeds. In this study, we tested several heat-resistant DNA polymerases for PCR and several methods for extracting DNA from asparagus seeds and successfully established a method for identifying the sex of asparagus seeds using this male-specific DNA marker. We found that PCR amplification of DNA extracted from asparagus seeds using simple methods such as single-step DNA extraction requires the use of high efficiency DNA polymerase. By contrast, many types of heat-resistant DNA polymerases can be used for PCR amplification of high-quality DNA extracted from asparagus seeds using a commercially available DNA extraction kit. Our method for sex identification of asparagus seeds could facilitate quality checking of all-male asparagus seeds and accelerate the screening of super-male asparagus.

Identification of a male-specific DNA marker in the large yellow croaker (Larimichthys crocea)

Understanding sex determination mechanisms is the first and most paramount step in the development of sex control breeding biotechnologies for fish. The large yellow croaker (Larimichthys crocea) is an economically vital fish in China and East Asia, which exhibits a sex-related growth dimorphism where females grow significantly faster than males. However, the lack of a rapid and accurate sex specific marker for the large yellow croaker has hampered the application of sex control in the breeding process, as well as sex-determination investigations of this species. In this study, a male-specific 15bp deletion in the third intron of the Dmrt1 gene was identified by comparative analysis of genomic re-sequencing data from male and female samples. Two pairs of primers (MS and MFS) were carefully designed; thus, the male-specific marker could be genotyped by PCR amplification and agarose gel electrophoresis. More importantly, this male-specific marker could also be applied to precisely identify the genetic sex of multiple fish. This male specific DNA marker will have potential applications in the elucidation of the sex determination mechanism and will also accelerate the development of sex control breeding in the large yellow croaker. Moreover, our current data further validated an significant genetic evidence that an XY-male determination mechanism is utilized in the large yellow croaker.

Development of a RAPD-Based Male-Specific Molecular Marker in Japanese Hop (Humulus japonicus Siebold &amp; Zucc.)

Abstract The male-specific DNA markers are very useful in molecular sexing of non-flowering plants and seeds of dioecious species. In this paper we identified ten Y chromosome-specific RAPD primers suitable for identification of male plants in three Cannabaceae species with sex chromosomes (Humulus lupulus, XX/XY; H. japonicus, XX/XY1Y2; Cannabis sativa, XX/XY). Basing on the nucleotide sequence of the OPJ-09 RAPD product we developed the HJY09 SCAR marker, which is very efficient in sexing of Japanese hop.

A male-specific SCAR DNA marker and sex ratio of seedlings in salak (Salacca zalacca var. zalacca)

A male-specific SCAR DNA marker was developed using a RAPD DNA marker specific for male plants of Salacca zalacca var. zalacca (salak palm). The marker is 1579 bp long and has a GC content of 38.5 %. Its sequence contains 1 or 2 open reading frames, indicating the marker is probably a coding region. No highly similar sequences were found in a search of the GenBank database. Sexes were identified using the SCAR DNA marker for three kinds of seedlings grouped by the number of seeds per fruit (1, 2 or 3). The sex ratio of female to male did not differ significantly from 1:1 for the three kinds of seedlings, implying that the number of seeds per fruit is not a reliable index to identify the sex of a seedling.

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers' genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.

Another CAPS DNA marker for sex identification in jojoba seedlings

Jojoba [Simmondsia chinensis (Link) Schneider] plant has immense industrial value. Jojoba is grown for its seed oil, which is a unique liquid wax consisting of esters formed from acids and alcohols with chain lengths of 20 or 22 carbon atoms. Higher seed yield is obtained when well balanced male and female plants are established in jojoba orchard. However, the sex of jojoba plants cannot be determined with morphological characters until the plants reach reproductive maturity. DNA markers are commonly used for plant genetic studies [1,2,3,4], Up-to-date several male and female specific DNA markers have been developed for sex identification in jojoba seedlings [5,6]. This study reports another cleavage-amplified polymorphic sequence (CAPS) assay, which easily identify male individuals. Genomic DNAs of 16 male and 16 female jojoba plants growing in an orchard were extracted using a DNA extraction protocol [4]. A touch-down PCR approach was used to amplify genomic DNAs of jojoba using J818F 5'-AGGGGATAAATGAGCCGAAT-3' and J818R 5'-GACCCAGAGGATGAGGAATG-3' primer pair [6]. Amplified products were digested using Hind III restriction enzyme. Electrophoresis separation of the digested products as shown in Figure 1 indicated that male and female jojoba plant can be easily identified. CAPS marker reported in this study could be used in breeding studies and in the sex allocation of seedlings in seed orchard establishment.

Cytomorphological and molecular characterization of interspecific F1 hybrid of Momordica dioica Roxb. Momordica subangulata subsp. renigera (G. Don) de Wilde

Interspecific hybrid (F1) between spine gourd (Momordica dioica) and teasle gourd (Momordicasubangulata Blume subsp. Renigera (G. Don) WJJ de Wilde) produced through bagging and hand pollination showed vigorous growth habit with less fertility. Morphological parameters along with meiotic, mitotic chromosome behaviors and Randomly Amplified Polymorphic DNA (RAPD) analysis, confirmed interchanges of genetic characters in the new hybrids. The interspecific hybrid showed a vigorous foliage with intermediate flower characters than that of parental species. Self pollination and backcrossing of F1plants to either parent confirmed presence of both male and female sterility, that might be due to improper meiotic chromosome pairing. The characters like petal spot and time of anthesis (early morning) were similar to that of pollen parent while the diameter and size of leaves and flowers, pedicel length and plant growth were intermediate as compared to parents. Somatic chromosome analysis revealed 2n = 28 in M.dioica – the female parent, 2n = 56 in M. subangulata ssp. renigera - the male parent and 2n = 42 in F1hybrid of M. dioica × M. subangulata ssp. renigera. RAPD analysis of both the parents and F1 hybrid confirmed more genetic affinity (60.95%) with female parent than the male parent (32.35%). Primer specific DNA markers confirmed the introgression of male genetic elements into the newly developed amphidiploids. Incorporation of male specific DNA markers into the F1 hybrid is discussed. Preliminary evaluation indicates that the F1 plants sprouted early, which is lacking in the mother parent and this character could be exploited by restoring of fertility by chromosome doubling. Key words: Momordica, cytomorphology, F1 hybrid, karyotype, RAPD markers.

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.

A reliable gender diagnostic PCR assay for jojoba (Simmondsia chinensis (Link) Schneider)

Jojoba (Simmondsia chinensis (Link) Schneider) is an obligate cross-pollinated shrub native to the Sonora desert. The most valuable product of the jojoba seeds is the liquid wax or jojoba oil which is marketed extensively in the cosmetic industry. Differing from the most of the cultivated crop species, jojoba has slow-growing habit, difficulties in the determination of sex at the early seedling stages, a male-biased ratio and low seed yield. In jojoba, the development of molecular strategies for the identification of sexes at early stages has been a priority in plantations and breeding programs. Two previous studies reported two candidate male-specific jojoba DNA markers. However, present study indicated that these markers were not useful in jojoba sex identification. A reliable gender diagnostic marker for jojoba is, therefore, needed. In the present study a novel jojoba male-specific touch-down polymerase chain reaction based DNA marker (JMS900) was reported using a total of 120 individual jojoba plants bulked into 16 samples. This sex specific DNA marker may have considerable theoretical and practical applications in the establishment of jojoba plantation and breeding studies.

Knowing your DNA database: Issues with determining ancestral Y haplotypes in a Y-Filer database

Y-chromosome STR profiles are being increasingly used in forensic investigations. Differences between populations in male-specific DNA markers can be large (relative to autosomal ones) and this knowledge has implications for appropriate database construction. Also, in cosmopolitan populations, such as Australia, asymmetrical admixture between groups can have important effects. This paper reports on these issues as they relate to the South Australian Y chromosome Aboriginal database.