Molecular cytogenetic mapping of Humulus lupulus sex chromosomes

Dioecy is relatively rare in plants and sex determination systems vary among such species. A good example of a plant with heteromorphic sex chromosomes is hop (Humulus lupulus). The genotypes carrying XX or XY chromosomes correspond to female and male plants, respectively. Until now no clear cytogenetic markers for the sex chromosomes of hop have been established. Here, for the first time the sex chromosomes of hop are clearly identified and characterized. The high copy sequence of hop (HSR1) has been cloned and localized on chromosomes by fluorescence in situ hybridization. The HSR1 repeat has shown subtelomeric location on autosomes with the same intensity of the signal. The signal has been present in the subtelomeric region of the long arm and in the near-centromeric region but absent in the telomeric region of the short arm of the X chromosome. At the same time the signal has been found in the telomeric region only of the long arm of the Y chromosome. This finding indicates that the sex chromosomes of hop have evolved from a pair of autosomes via ancient translocation or inversion. The observation of the meiotic configuration of the sex bivalents shows the location of a pseudoautosomal region on the long arms of X and Y chromosomes.     

Gender in plants: sex chromosomes are emerging from the fog

Although most plants have flowers with both male and female sex organs, there are several thousands of plant species where male or female flowers form on different individuals. Surprisingly, the presence of well-established sex chromosomes in these dioecious plants is rare. The best-described example is white campion, for which large sex chromosomes have been identified and mapped partially. A recent study presented a comprehensive genetic and physical mapping of the genome of dioecious papaya. It revealed a short male specific region on the Y chromosome (MSY) that does not recombine with the X chromosome, providing strong evidence that the sex chromosomes originated from a regular pair of autosomes. The primitive papaya Y chromosome thus represents an early event in sex chromosome evolution. In this article, we review the current status of plant sex-chromosome research and discuss the advantages of different dioecious models.     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.)

In dioecious plants of hemp ( Cannabis sativa L.), males are regarded as heterogametic XY and females as homogametic XX, although it is difficult to discriminate the X cytologically from the Y. The Y chromosome is somewhat larger than the X. Our aim was to analyse AFLP markers on X and Y, and to use them to gain some insight into the structure of the sex chromosomes. Markers located on the sex chromosomes can be grouped into different classes, depending on the presence or absence of a fragment on the X and/or the Y. They are detected by separately analysing male and female progenies of a single cross. Five markers were found to be located on both chromosomes. A few recombinants were observed for marker pairs of this class in the male progenies. Two completely linked markers located on the Y chromosome in the male parent show a recombination rate of r = 0.25 with sex. Recombination must have occurred between the sex chromosomes in the male parent. The recombination analysis led to the conclusion that there is a pseudoautosomal region (PAR) on the sex chromosomes, allowing recombination between the X and the Y chromosome. The other regions of the sex chromosomes show only a few recombination events, for the Y as well as for the X. These results are discussed in comparison to other dioecious plants.     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

Swine in Biomedical Research: Creating the Building Blocks of Animal Models

Abstract

Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)‐specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y‐specific primers, and analyzed by ethidium bromide‐staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos.

Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y‐chromosome‐specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day‐old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, while the remainder of the embryo was prepared for in situ hybridization using the biotinylated probe. In situ hybridization performed on embryos shown to be male by PCR, showed pinpoint fluorescence within the nuclei, similar to that obtained when male porcine lymphocytes were hybridized. No evidence of fluorescence was seen when in situ hybridization was performed in parallel on embryos determined to be female by the PCR.

The PCR was found to be a relatively fast, accurate and reproducible means of sex determination of swine preimplantation embryos. This capability could have significant impact on animal breeding and production programs by using PCR as a screening tool for traits of economic importance.     

Male specific genes from dioecious white campion identified by fluorescent differential display

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.     

Sex Determination by Sex Chromosomes in Dioecious Plants

Abstract: Sex chromosomes have been reported in several dioecious plants. The most general system of sex determination with sex chromosomes is the XY system, in which males are the heterogametic sex and females are homogametic. Genetic systems in sex determination are divided into two classes including an X chromosome counting system and an active Y chromosome system. Dioecious plants have unisexual flowers, which have stamens or pistils. The development of unisexual flowers is caused by the suppression of opposite sex primordia. The expression of floral organ identity genes is different between male and female flower primordia. However, these floral organ identity genes show no evidence of sex chromosome linkage. The Y chromosome of Rumex acetosa contains Y chromosome-specific repetitive sequences, whereas the Y chromosome of Silene latifolia has not accumulated chromosome-specific repetitive sequences. The different degree of Y chromosome degeneration may reflect on evolutionary time since the origination of dioecy. The Y chromosome of S. latifolia functions in suppression of female development and initiation and completion of anther development. Analyses of mutants suggested that female suppressor and stamen promoter genes are localized on the Y chromosome. Recently, some sex chromosome-linked genes were isolated from flower buds of S. latifolia.     

Development of a sex-specific molecular marker for Japanese hop Humulus japonicus Siebold & Zucc

Japanese hop (Humulus japonicus Siebold & Zucc.) is a dioecious plant and a suitable model for studying the XX/XY1Y2 system of sex chromosomes. To develop a sex-specific marker, 12 RAPD and 36 ISSR markers were analyzed on the basis of pools of male and female plants identified after flowering. We were the first to identify ISSR marker K-16, which manifested stable amplification of an approximately 300-bp fragment in male plants and the absence of amplification in female plants in the populations examined. Marker effectiveness was confirmed in several Japanese hop populations of different origin.     

C-Banding/DAPI and in situ hybridization reflect karyotype structure and sex chromosome differentiation in Humulus japonicus Siebold & Zucc

Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.     

利用SRY基因和微卫星标记鉴定反刍动物性别

以反刍动物为研究对象,应用多重PCR技术扩增绵羊基因组中X、Y染色体上的4个微卫星标记和SRY基因, 根据基因型进行性别鉴定,试图通过一次DNA扩增同时提供性别鉴定和基因分型的信息。结果表明所设计SRY基因的引物具有高度特异性,是性别鉴定的主要依据,而Y染色体上的MCM158、MAF45两标记由于特异性不好,因此不适用于性别鉴定,对于X染色体上所选的两标记MILVET09和AE25只能进一步验证所鉴定的雄性个体。得出结论在被检个体中,能同时扩增出SRY基因、MCM158、MAF45,X染色体上MILVET09和AE25,且X染色体上的MILVET09、AE25基因型为纯合子的个体为正常的雄性;被检个体中只有Y染色体上MCM158、MAF45和X染色体上MILVET09、AE25的扩增产物,而没有SRY基因的扩增产物,则被检个体为雌性,且MILVET09、AE25的基因型对雌性个体的性别判断无影响。MCM158、MAF45两标记基因型不影响个体的性别鉴定结果。
     

Widespread intersex differentiation across the stickleback genome – The signature of sexually antagonistic selection?

Females and males within a species commonly have distinct reproductive roles, and the associated traits may be under perpetual divergent natural selection between the sexes if their sex‐specific control has not yet evolved. Here, we explore whether such sexually antagonistic selection can be detected based on the magnitude of differentiation between the sexes across genome‐wide genetic polymorphisms by whole‐genome sequencing of large pools of female and male threespine stickleback fish. We find numerous autosomal genome regions exhibiting intersex allele frequency differences beyond the range plausible under pure sampling stochasticity. Alternative sequence alignment strategies rule out that these high‐differentiation regions represent sex chromosome segments misassembled into the autosomes. Instead, comparing allele frequencies and sequence read depth between the sexes reveals that regions of high intersex differentiation arise because autosomal chromosome segments got copied into the male‐specific sex chromosome (Y), where they acquired new mutations. Because the Y chromosome is missing in the stickleback reference genome, sequence reads derived from DNA copies on the Y chromosome still align to the original homologous regions on the autosomes. We argue that this phenomenon hampers the identification of sexually antagonistic selection within a genome, and can lead to spurious conclusions from population genomic analyses when the underlying samples differ in sex ratios. Because the hemizygous sex chromosome sequence (Y or W) is not represented in most reference genomes, these problems may apply broadly.     

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.     

Plant sex determination and sex chromosomes

Sex determination systems in plants have evolved many times from hermaphroditic ancestors (including monoecious plants with separate male and female flowers on the same individual), and sex chromosome systems have arisen several times in flowering plant evolution. Consistent with theoretical models for the evolutionary transition from hermaphroditism to monoecy, multiple sex determining genes are involved, including male-sterility and female-sterility factors. The requirement that recombination should be rare between these different loci is probably the chief reason for the genetic degeneration of Y chromosomes. Theories for Y chromosome degeneration are reviewed in the light of recent results from genes on plant sex chromosomes.     

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.     

Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes

In the medaka, Oryzias latipes, sex is determined chromosomally. The sex chromosomes differ from those of mammals in that the X and Y chromosomes are highly homologous. Using backcross panels for linkage analysis, we mapped 21 sequence tagged site (STS) markers on the sex chromosomes (linkage group 1). The genetic map of the sex chromosome was established using male and female meioses. The genetic length of the sex chromosome was shorter in male than in female meioses. The region where male recombination is suppressed is the region close to the sex-determining gene y, while female recombination was suppressed in both the telomeric regions. The restriction in recombination does not occur uniformly on the sex chromosome, as the genetic map distances of the markers are not proportional in male and female recombination. Thus, this observation seems to support the hypothesis that the heterogeneous sex chromosomes were derived from suppression of recombination between autosomal chromosomes. In two of the markers, Yc-2 and Casp6, which were expressed sequence-tagged (EST) sites, polymorphisms of both X and Y chromosomes were detected. The alleles of the X and Y chromosomes were also detected in O. curvinotus, a species related to the medaka. These markers could be used for genotyping the sex chromosomes in the medaka and other species, and could be used in other studies on sex chromosomes.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Molecular markers for sex determination in papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

We have developed molecular markers tightly linked to Sex1, the gene that determines plant sex in papaya ( Carica papaya L.). Three RAPD products have been cloned and a portion of their DNA sequenced. Based on these sequences SCAR primers were synthesized. SCAR T12 and SCAR W11 produce products in hermaphrodite and male plants and only rarely in females. SCAR T1 produces a product in all papayas regardless of plant sex. SCAR T12 and SCAR W11 showed no recombination in a population of 182 F2 plants from a 'SunUp' by 'Kapoho' cross. Based on these results a PCR-based technique for rapidly and accurately determining the sex of papaya plants was developed using either W11 or T12 to detect the hermaphrodite or male allele and T1, which amplifies a product regardless of sex type, as a positive control. The sexing technique, using SCAR T12 and SCAR T1 as a positive control, was used to correctly predict hermaphrodite papaya plants in a population of seedlings with an overall accuracy of 99.2%.     

运用PCR对小鼠植入前胚胎进行性别诊断

根据C57BL6小鼠Y染色体重复序列145C5的碱基顺序,设计并合成一对引物,运用PCR扩增昆明白小鼠入前胚胎卵裂球DNA,以确定其性别,共对108枚活检胚胎的相应卵裂球进行了性别诊断,获雄性胚46枚,雌性胚62枚,移植后分别获雄性仔鼠4只,准确率100％（4／4）,雌性仔鼠9只,准确率70％（9／13）,本研究结果表明小鼠Y染色体重复序列145C5的碱基顺序在C57BL6小鼠和昆明白小鼠中基本一致,为农牧业动物进行性别选择和运用PCR进行单基因病植入前遗传学诊断提供了方法学基础。