GISH painting of the Y chromosomes suggests advanced phases of sex chromosome evolution in three dioecious Cannabaceae species (Humulus lupulus, H. japonicus, and Cannabis sativa).

In plants, dioecy is relatively rare, and it involves sex chromosome systems that often developed independently over time. These characteristics make dioecious plants an attractive model to study sex chromosome evolution. To clarify the patterns of plant sex chromosome evolution, studies should be performed on a wide range of dioecious species. It is interesting to study the sex chromosomes in related species that evolved during a long period of independent sex chromosome evolution. The Cannabaceae family includes three dioecious species with heteromorphic sex chromosomes. Cannabis sativa and Humulus lupulus use the XX/XY chromosome system, whereas Humulus japonicus contains multiple sex chromosomes (XX/XY1Y2). To better understand sex chromosome evolution and the level of genomic divergence of these three related species, we undertook self-GISH and comparative GISH analyses. The self-GISH allowed visualization of the Y chromosomes of C. sativa, H. lupulus, and H. japonicus. The self-GISH signal was distributed along the entire Y chromosome, excluding the pseudo-autosomal region (PAR). Our results indicate that the male-specific region of the Y chromosome (MSY) spans the overwhelming majority of the Y chromosomes of all three species studied. The self-GISH results reveal the accumulation of repetitive DNA sequences in the Y chromosomes of all three species studied. This sequences presented in autosomes and/or chromosome X at a lower copy number than in Y. In comparative GISH experiments where the probe DNA of one species was applied to another species, a weak signal was exclusively detected on 45S rDNA sites, indicating a high level of genomic differentiation of the species used in this study. We demonstrate small PAR size and opposing large MSY and its positions on Y chromosomes. We also found that these genomes are highly differentiated. Furthermore, the data obtained in this study indicate a long period of independent and advanced sex chromosome evolution. Our study provides a valuable basis for future genomic studies of sex and suggests that the Cannabaceae family offers a promising model to study sex chromosome evolution.

Study and Physical Mapping of the Species-Specific Tandem Repeat CS-237 Linked with 45S Ribosomal DNA Intergenic Spacer in Cannabis sativa L.

Hemp (Cannabis sativa L.) is a valuable crop and model plant for studying sex chromosomes. The scientific interest in the plant has led to its whole genome sequencing and the determination of its cytogenetic characteristics. A range of cytogenetic markers (subtelomeric repeat CS-1, 5S rDNA, and 45S rDNA) has been mapped onto hemp's chromosomes by fluorescent in situ hybridization (FISH). In this study, another cytogenetic marker (the tandem repeat CS-237, with a 237 bp monomer) was found, studied, and localized on chromosomes by FISH. The signal distribution and karyotyping revealed that the CS-237 probe was localized in chromosome 6 with one hybridization site and in chromosome 8 with two hybridization sites, one of which colocalizes with the 45S rDNA probe (with which a nucleolus organizer region, NOR, was detected). A BLAST analysis of the genomic data and PCR experiments showed that the modified CS-237 monomers (delCS-237, 208 bp in size) were present in the intergenic spacers (IGSs) of hemp 45S rDNA monomers. Such a feature was firstly observed in Cannabaceae species. However, IGS-linked DNA repeats were found in several plant species of other families (Fabaceae, Solanaceae, and Asteraceae). This phenomenon is discussed in this article. The example of CS-237 may be useful for further studying the phenomenon as well as for the physical mapping of hemp chromosomes.

The Development of New Species-Specific Molecular Markers Based on 5S rDNA in Elaeagnus L. Species.

The Elaeagnus L. species are trees and bushes that mainly grow in temperate zones of Western Europe; Minor, Central, and Southeast Asia; the Far East; and North America. Some species are used as fruit or ornamental plants and have economic value. Problems with the identification of species in the Elaeagnus genus by molecular genetical methods arise in the study of populations, systematics, breeding, and other areas of plant science and practice. Recently, the polymorphism of 5S ribosomal DNA non-transcribed spacers (5S rDNA NTSs) in Elaeagnaceae Adans. has been described. The results were used in our study as a basis for development of new species-specific molecular markers for some members of the Elaeagnus genus. The author's method was applied for finding regions that were potentially applicable for species-specific primer design. As a result, some species-specific molecular markers were developed for Elaeagnus angustifolia L., E. commutata Bernh., E. pungens Thunb., and E. multiflora Thunb. These markers were tested in a range of samples and showed the presence of amplified fragments in lanes of the marked species only. Samples of other species showed no amplifications. Thus, the developed markers may be useful for the species identification of the studied Elaeagnus plants in botanical, dendrological, and genetic research (especially in a leafless period of year), as well as in breeding and hybridization experiments.

A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species.

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95-96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

Molecular cytogenetic analysis of monoecious hemp (Cannabis sativa L.) cultivars reveals its karyotype variations and sex chromosomes constitution.

Hemp (Cannabis sativa L., 2n = 20) is a dioecious plant. Sex expression is controlled by an X-to-autosome balance system consisting of the heteromorphic sex chromosomes XY for males and XX for females. Genetically monoecious hemp offers several agronomic advantages compared to the dioecious cultivars that are widely used in hemp cultivation. The male or female origin of monoecious maternal plants is unknown. Additionally, the sex chromosome composition of monoecious hemp forms remains unknown. In this study, we examine the sex chromosome makeup in monoecious hemp using a cytogenetic approach. Eight monoecious and two dioecious cultivars were used. The DNA of 210 monoecious plants was used for PCR analysis with the male-associated markers MADC2 and SCAR323. All monoecious plants showed female amplification patterns. Fluorescence in situ hybridization (FISH) with the subtelomeric CS-1 probe to chromosomes plates and karyotyping revealed a lack of Y chromosome and presence of XX sex chromosomes in monoecious cultivars with the chromosome number 2n = 20. There was a high level of intra- and intercultivar karyotype variation detected. The results of this study can be used for further analysis of the genetic basis of sex expression in plants.

Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system.

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

Sex chromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846 (Cannabaceae) revealed by fluorescence in situ hybridization of subtelomeric repeat.

Humulus japonicus Siebold et Zucc (Japanese hop) is a dioecious species of the family Cannabaceae. The chromosome number is 2n = 16 = 14 + XX for females and 2n = 17 = 14 + XY1Y2 for male. To date, no fluorescence in situ hybridization (FISH) markers have been established for the identification of Humulus japonicus sex chromosomes. In this paper, we report a method for the mitotic and meiotic sex chromosome differentiation in Humulus japonicus by FISH for HJSR, a high copy subtelomeric repeat. The signal is present in the subtelomeric region of one arm of the X chromosome. We demonstrate that males have two Y chromosomes that differ in FISH signal with the HJSR probe. Indeed, the HJSR probe hybridizes to a subtelomeric region on both arms of chromosome Y1 but not of chromosome Y2. The orientation and position of pseudoautosomal regions (PAR1 and PAR2) were also determined.