Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

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.     

Sex identification and genetic variation of Saccharina (Phaeophyta) gametophytes as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) markers were utilized to investigate the genetic variation between male and female gametophyte populations of strains Rongfu and 901 of Saccharina. In total, 11 ISSR primers were able to generate 135 satisfactory and reproducible loci, of which 134 were polymorphic with 99.26 % polymorphism. The percentages of polymorphism of female gametophyte populations (60 and 62 % for their respective strains) were higher than those of the males (53 %), and the Nei’s genetic diversity and Shannon’s information index showed a similar tendency. The clustering of gametophytes of the same sex from each strain was well resolved by both an unweighted paired group method using the arithmetic average and a principal component analysis, suggesting that any male/female gametophyte pair could represent each strain. However, a single pair was not adequate for germplasm maintenance because the genetic variance among individuals within a population accounted for 57.45 % of the total (P?<?0.0001), as shown by the analysis of molecular variance. The gametophyte sex could be identified by amplification with primer UBC809 because of a differential band present in the females. According to the sequence of this band, a pair of ISSR-derived sequence-characterized amplified region (SCAR) primers was designed. With the primers, one female-specific fragment was detected using PCR and Southern blot hybridization. This converted SCAR marker was localized on one unique chromosome of the female gametophytes of these two strains by use of fluorescence in situ hybridization, confirming that it was a female chromosome-specific marker.     

Identification of a male-specific AFLP marker in a functionally dioecious fig,<Emphasis Type="Italic"> Ficus fulva</Emphasis> Reinw. ex Bl. (Moraceae)

A male-specific amplified fragment length polymorphism (AFLP) marker was identified in the functionally dioecious fig species, Ficus fulva. A total of 89 polymorphic fragments from three primer combinations were produced, of which one (246 bp) was present in all males (n=23) and absent in all females (n=24) of two populations. This strong association suggests a tight chromosomal linkage between the AFLP marker and the sex-controlling locus. Further analysis indicated that the marker segregated in open-pollinated progenies from natural populations in a 1:1 ratio (n=156), implying that males are the heterogametic sex. Chromosome preparations showed no evidence for morphologically distinct sex chromosomes. The low frequencies of associated markers argue against a morphologically cryptic non-recombining sex chromosome. The sex-locus is therefore likely to be autosomal. The male-specific AFLP marker was sequenced and converted into a sequence characterised amplified region (SCAR) marker. This SCAR marker produced a fragment of equal size in males and females, suggesting that sequence divergence between male- and female-specific chromosomal regions is low.Publication 3311 NIOO-KNAW Netherlands Institute of Ecology     

A novel sex-specific DNA marker in Columbidae birds

That most Columbidae birds have no conspicuous sexual dimorphism often makes it difficult to identify their sex on the basis of external morphology. In the present study, we report a novel sex-specific DNA marker in Columbidae birds. DNA was extracted from one member of this bird group, Streptopelia orientalis (S. orientalis, oriental turtle dove), and used to identify a female-specific DNA marker using a random amplified polymorphic DNA (RAPD) fingerprinting. One hundred and sixty random primers were used for the RAPD-PCR reactions. When using the OPAV17 primer, a novel 902 bp sex-specific PCR product was amplified from known female birds. This fragment of DNA was cloned and sequenced. Two primers, TurSexOPAV17-F and TurSexOPAV17-R, were designed from the cloned sex-specific sequence, and were successfully used to amplify a 777 bp female-specific fragment using PCR from S. orientalis DNA. This sex-specific marker was also amplified from genomic DNA samples of two other female Columbidae, S. chinensis and Columba livia. Sequence analysis showed that this novel sex-specific marker was highly conserved amongst these three bird species. In contrast, the PCR product was not amplified from male DNA of these species, nor from either sex of the S. chinensis formosa birds. Therefore, we concluded that our novel marker can be used to rapidly and accurately identify the sex of birds from three species of Columbidae.     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

A male and hermaphrodite specific RAPD marker for papaya ( Carica papayaL.)

The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papaya L., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker), exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps, was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and rapid diagnosis of sex in papaya. Received: 1 February 2001 / Accepted: 22 May 2001     

A SCAR MOLECULAR MARKER SPECIFICALLY RELATED TO THE FEMALE GAMETOPHYTES OF SACCHARINA (LAMINARIA) JAPONICA (PHAEOPHYTA)1

PCR amplification was employed to identify female or male gametophyte associated markers in Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (=Laminaria japonica Aresch.). One pair of the primers, P5, was screened from five pairs designed based on a specific sequence (GenBank accession no. AB069714 ) of Marchantia polymorpha Y chromosome, resulting in a differential band ～500 bp in size between female and male gametophytes of Rongfu strain of S. japonica. According to the SCAR (sequence‐characterized amplified regions) strategies, one pair of primers, P51, was designed on the basis of the sequence of this band that was only present in female gametophytes. A SCAR marker, designated FRML‐494 (494‐bp Female‐Related Marker of S. japonica, GenBank accession no. EU931619 ), was developed successfully by PCR amplification using the designed P51 primer pair. The SCAR marker was verified to be present only in female gametophytes of another variety 901 of this kelp that was a hybrid between S. japonica as paternal and S. longissima (Miyabe) C. E. Lane, C. Mayes, Druehl et G. W. Saunders (=Laminaria longissima Miyabe) as maternal, suggesting that the FRML‐494 marker was specifically related to female gametophytes of the genus. This marker is the first molecular tool reported for sex identification in kelps. This study was beneficial for identifying gametophyte gender during vegetative growth and for judging whether the monogenetic sporophytes came from exclusive male or female gametophytes, as well as for further research on sex determination at the molecular level in kelps.     

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

The first sex-specific molecular marker discovered in the moss Pseudocalliergon trifarium

Most dioecious plants do not exhibit discernible sexual dimorphism before sexual maturity. Therefore, it is impossible to address any sex-related questions during the prereproductive phase unless a genetic sex marker is available for gender determination. The aim of the present study was to develop a genetic sex marker for the moss Pseudocalliergon trifarium to allow gender and sex ratio determination at any stage in the life cycle. A high proportion of P. trifarium populations do not express sex. The screening of genomic DNA with inter simple sequence repeat (ISSR) primers was used to discover sex-specific polymerase chain reaction (PCR) amplification products. A presumably female-specific band was found, excised from the gel, cloned, and sequenced. A sequence-walking method was used to characterize the same region in males. A primer pair was designed to allow the amplification of a 159-bp portion of the female-specific DNA region. All tested material, up to 16-year-old herbarium specimens, provided unambiguous amplification products. This study successfully provides, for the first time in a moss, a sex-specific DNA marker. It allows reliable determination of gender and sex ratios. The short length of the amplification product is an advantage as satisfactory PCR products are more likely when the targeted sequence is short. The amount of variation in the DNA region shared by both sexes was relatively high. If the male sequence can be better characterized, the sex-specific regions could possibly be used to evaluate sex-specific phylogeographic patterns.     

The use of amplified fragment length polymorphism (AFLP) in the isolation of sex-specific markers

Sex identification is a problem in research and conservation. It can often be solved using a DNA test but this is only an option if a sex-specific marker is available. Such markers can be identified using the amplified fragment length polymorphism (AFLP) technique. This is usually a taxonomic method, as it produces a DNA fingerprint of 50-100 PCR bands. However, if male and female AFLP products are compared, sex-specific markers are confined to the heterogametic sex and can rapidly be identified. Once a marker is found, AFLP can be used to sex organisms directly or the marker can be sequenced and a standard PCR test designed.     

A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings

Female plants of several dioecious angiosperms are commercially valued for production of fruits or seeds, viz. papaya, nutmeg, pistachio, kiwi fruit and jojoba. To make the cultivation profitable it is necessary to grow more female than male plants. To discriminate between male and female plants, sex-specific molecular markers have been identified in a few dioecious species such as Silene and pistachio. However, accurate and convenient sex diagnostic methods for early sexing of seedlings are not available to date. For the first time, we report here a PCR-based Seedling Sex Diagnostic Assay (SSDA) specially designed for early sexing of papaya seedlings. We have developed a male-specific SCAR marker in papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. The potential of this SCAR marker is further exploited to develop a simplified and highly accurate sex diagnostic assay by (1) including an internal PCR control, (2) following a single-step DNA extraction procedure and (3) optimising the PCR conditions to simultaneously amplify male-specific and control bands from the crude leaf extract. This diagnostic approach would be of great commercial significance to papaya growers as well as to seed companies and plant nurseries for early identification of female seedlings of dioecious species. In principle, this experimental design could be easily applied to molecular analysis of any agriculturally important trait for which specific DNA probes could be identified and hence opens new avenues of research in the field of genetic diagnostics of plants.     

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

A sex‐linked SCAR marker in Bryonia dioica (Cucurbitaceae), a dioecious species with XY sex‐determination and homomorphic sex chromosomes

Genetic crosses between the dioecious Bryonia dioica (Cucurbitaceae) and the monoecious B. alba in 1903 provided the first clear evidence for Mendelian inheritance of dioecy and made B. dioica the first organism for which XY sex‐determination was experimentally proven. Applying molecular tools to this system, we developed a sex‐linked sequence‐characterized amplified region (SCAR) marker for B. dioica and sequenced it for individuals representing the full geographic range of the species from Scotland to North Africa. For comparison, we also sequenced this marker for representatives of the dioecious B. cretica, B. multiflora and B. syriaca, and monoecious B. alba. In no case did any individual, male or female, yield more than two haplotypes. In northern Europe, we found strong linkage between our marker and sex, with all Y‐sequences being identical to each other. In southern Europe, however, the linkage between our marker and sex was weak, with recombination detected within both the X‐ and the Y‐homologues. Population genetic analyses suggest that the SCAR marker experienced different evolutionary pressures in northern and southern Europe. These findings fit with phylogenetic evidence that the XY system in Bryonia is labile and suggest that the genus may be a good system in which to study the early steps of sex chromosome evolution.     

Effects of intrinsic and extrinsic factors on the haemocyte profile of the prawn, Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii was investigated for its total haemocyte count (THC) based on season, sex, size and feeding rate. The THC, when the prawns were subjected to injections of foreign materials was also investigated. The prawns displayed the highest and lowest THC in autumn and winter respectively, with no significant difference between male and female, or among animals with a body weight range of 7-115 g. The prawns displayed the lowest THC at D3 stage, and the highest in C stage. The prawns displayed the lowest THC when they were fed at 0.1% feeding rate among feeding rates of 0.1%, 0.5%, 1.0% and 1.5% body weight x day(-1). Prawns injected with carbon powder and Enterococcus showed increased THC during the first 6 h. Prawns injected with saline and carbon powder had the lowest THC after 30 h, and recovered to the normal value after 54 h. Prawns injected with Enterococcus showed the lowest THC after 42 h, and showed delayed recovery.     

A novel molecular genetic marker for gender determination of pigeons

The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. One-hundred and twenty random primers were used for RAPD fingerprinting in order to find any sex-specific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR.