Genomic In Situ Hybridization Research Articles

Revealing the abnormal meiosis and the variation of the functional female gametes of aneuploid lily (Lilium) using genomic in situ hybridization (GISH)

Revealing the abnormal meiosis and the variation of the functional female gametes of aneuploid lily (Lilium) using genomic in situ hybridization (GISH)

Chromosome Rearrangement in Elymus dahuricus Revealed by ND-FISH and Oligo-FISH Painting.

As a perennial herb in Triticeae, Elymus dahuricus is widely distributed in Qinghai-Tibetan Plateau and Central Asia. It has been used as high-quality fodders for improving degraded grassland. The genomic constitution of E. dahuricus (2n = 6x = 42) has been revealed as StStHHYY by cytological approaches. However, the universal karyotyping nomenclature system of E. dahuricus is not fully established by traditional fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). In this study, the non-denaturing fluorescent in situ hybridization (ND-FISH) using 14 tandem-repeat oligos could effectively distinguish the entire E. dahuricus chromosomes pairs, while Oligo-FISH painting by bulked oligo pools based on wheat-barley collinear regions combined with GISH analysis, is able to precisely determine the linkage group and sub-genomes of the individual E. dahuricus chromosomes. We subsequently established the 42-chromosome karyotype of E. dahuricus with distinctive chromosomal FISH signals, and characterized a new type of intergenomic rearrangement between 2H and 5Y. Furthermore, the comparative chromosomal localization of the centromeric tandem repeats and immunostaining by anti-CENH3 between cultivated barley (Hordeum vulgare L.) and E. dahuricus suggests that centromere-associated sequences in H subgenomes were continuously changing during the process of polyploidization. The precise karyotyping system based on ND-FISH and Oligo-FISH painting methods will be efficient for describing chromosomal rearrangements and evolutionary networks for polyploid Elymus and their related species.

Autotetraploid Origin of Chinese Cherry Revealed by Chromosomal Karyotype and In Situ Hybridization of Seedling Progenies.

Polyploidy is considered a driving force in plant evolution and diversification. Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don], an economically important fruit crop native to China, has evolved at the tetraploid level, with a few pentaploid and hexaploid populations. However, its auto- or allo-polyploid origin remains unclear. To address this issue, we analyzed the ploidy levels and rDNA chromosomal distribution in self- and open-pollinated seedling progenies of tetraploid and hexaploid Chinese cherry. Genomic in situ hybridization (GISH) analysis was conducted to reveal the genomic relationships between Chinese cherry and diploid relatives from the genus Cerasus. Both self- and open-pollinated progenies of tetraploid Chinese cherry exhibited tetraploids, pentaploids, and hexaploids, with tetraploids being the most predominant. In the seedling progenies of hexaploid Chinese cherry, the majority of hexaploids and a few pentaploids were observed. A small number of aneuploids were also observed in the seedling progenies. Chromosome 1, characterized by distinct length characteristics, could be considered the representative chromosome of Chinese cherry. The basic Chinese cherry genome carried two 5S rDNA signals with similar intensity, and polyploids had the expected multiples of this copy number. The 5S rDNA sites were located at the per-centromeric regions of the short arm on chromosomes 4 and 5. Three 45S rDNA sites were detected on chr. 3, 4 and 7 in the haploid complement of Chinese cherry. Tetraploids exhibited 12 signals, while pentaploids and hexaploids showed fewer numbers than expected multiples. Based on the GISH signals, Chinese cherry demonstrated relatively close relationships with C. campanulata and C. conradinae, while being distantly related to another fruiting cherry, C. avium. In combination with the above results, our findings suggested that Chinese cherry likely originated from autotetraploidy.

Development and Identification of a Wheat-Rye Breeding Line for Harmonious Improvement Between Powdery Mildew Resistance and High Yield Potential.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that seriously threatens wheat yield and quality. To control this disease, host resistance is the preferred measure. However, wheat breeding is a complex process with elusive exchange and recombination of the traits from their parents. Increased resistance often leads to a decline in other key traits, such as yield and quality. Developing breakthrough germplasms with harmonious powdery mildew resistance and other key breeding traits is attractive in wheat breeding. In this study, we developed an ideal wheat breeding line AL46 that pyramided its hexaploid triticale parent-derived desirable yield traits and its wheat parent-derived powdery mildew resistance gene Pm2. Sequential genomic in situ hybridization (GISH), multicolor GISH, multicolor fluorescence in situ hybridization, and molecular marker analyses revealed that AL46 was a wheat-rye T1RS·1BL translocation line. Genetic analysis combined with function marker detection and sequence alignment were used to confirm that AL46 carried the Pm2 gene. Then, we evaluated the powdery mildew resistance and comprehensive traits of AL46, and just as we designed, AL46 showed harmonious powdery mildew resistance with some key breeding traits. This study not only developed an ideal wheat germplasm resource but also provided a successful example for pyramiding breeding, which could be a promising direction for wheat improvement in the future.

Homoeologous Chromosome Pairing and Alien Introgression in Backcrossing Progenies Derived from Hybrids Solanum tuberosum (+) Mexican 2x (1 EBN) B-Genome Potato Species

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production, functional genomics studies and wheat improvement. In this study, a wheat gene related to regeneration, TaWOX5, was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for the presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and the QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analyses indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) regions of the chromosomes in transgenic T. monococcum and hexaploid triticale plants. In the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB and RR genomes. Furthermore, the transgene was almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

The Control of the Crossover Localization in Allium.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

Relocation of Sr48 to Chromosome 2D Using an Alternative Mapping Population and Development of a Closely Linked Marker Using Diverse Molecular Technologies.

The Ug99-effective stem rust resistance gene Sr48 was mapped to chromosome 2A based on its repulsion linkage with Yr1 in an Arina/Forno recombinant inbred line (RIL) population. Attempts to identify markers closely linked to Sr48 using available genomic resources were futile. This study used an Arina/Cezanne F5:7 RIL population to identify markers closely linked with Sr48. Using the Arina/Cezanne DArTseq map, Sr48 was mapped on the short arm of chromosome 2D and it co-segregated with 12 markers. These DArTseq marker sequences were used for BlastN search to identify corresponding wheat chromosome survey sequence (CSS) contigs, and PCR-based markers were developed. Two simple sequence repeat (SSR) markers, sun590 and sun592, and two Kompetitive Allele-Specific PCR (KASP) markers were derived from the contig 2DS_5324961 that mapped distal to Sr48. Molecular cytogenetic analysis using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) identified a terminal translocation of chromosome 2A in chromosome 2DL of Forno. This translocation would have led to the formation of a quadrivalent involving chromosomes 2A and 2D in the Arina/Forno population, which would have exhibited pseudo-linkage between Sr48 and Yr1 in chromosome 2AL. Polymorphism of the closet marker sunKASP_239 among a set of 178 wheat genotypes suggested that this marker can be used for marker-assisted selection of Sr48.

Multispecies polyploidization, chromosome shuffling, and genome extraction in Zea/Tripsacum hybrids.

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenome stability, chromosome pairings and rearrangements, and their impacts on an organism's fitness were investigated by fertility phenotyping and molecular cytogenetic techniques genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n = 35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n = 54, MMMPT) overcame self-incompatibility barriers and produced a self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, the mean chromosome number preferably maintained at the near-tetraploid level (2n = 40) with full integrity of 45S rDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 for maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

Cytogenetic Characterization and Metabolomic Differences of Full-Sib Progenies of Saccharum spp.

Sugarcane smut is a worldwide fungal disease. Disease resistance breeding is the most economical and effective measure to prevent and control sugarcane smut. The cytogenetic characteristics and metabolomic differences of sugarcane F1s are closely related to disease resistance. Zhongzhe 1 and G160 sugarcane from the same parents (ROC25 and Yunzhe89-7) were used; the plants were grown in accordance with the barrel method. When the seedlings had 4-5 leaves, genomic in situ hybridization (GISH) was performed; digoxigenin (DIG)-labeled female parental (ROC25)DNA and biotin-labeled male parental (Yunzhe89-7) DNA were used as probes, and the karyotypes of two hybrids were analyzed. The new sugarcane smut-resistant variety (Zhongzhe 1) and the susceptible variety (G160) derived from the same parent were analyzed via gas chromatography-mass spectrometry technology (GC-MS) to compare the metabolomic differences between them. GISH analysis revealed that the chromosome ploidy number of Zhongzhe 1 sugarcane and G160 sugarcane were 114 and 110, respectively. However, the two contain different numbers of chromosomes from the female (ROC25) and male (Yunzhe89-7) parents. Moreover, 258 significantly changed metabolites were identified in smut-resistant Zhongzhe 1, as compared with the smut-susceptible G160 sugarcane: 56 flavonoids, 52 phenolic acids, 30 lipids, 26 organic acids, 26 amino acids and derivatives, 19 nucleotides and derivatives, 5 alkaloids, 9 terpenoids, and 35 others. Multivariate statistical analysis revealed a distinct difference in metabolic pathways between Zhongzhe 1 sugarcane and G160, and both of these varieties had unique functional metabolites. Differences in chromosome composition may constitute the genetic basis for the difference in resistance to smut disease between Zhongzhe 1 sugarcane and G160 sugarcane, and a high accumulation of flavonoids, lipids, terpenoids and tannins may constitute the basis of resistance to smut disease for the Zhongzhe 1 variety.

Genomic relationships of the polyploid rhizoma peanut (Arachis glabrata Benth.) inferred by genomic in situ hybridization (GISH)

The rhizoma peanut (Arachis glabrata Benth., section Rhizomatosae) is a tetraploid perennial legume. Although several A. glabrata cultivars have been developed as forage and ornamental turf, the origin and genomic constitution of this species are still unknown. In this study, we evaluated the affinity between the genomes of A. glabrata and the probable diploid donors of the sections Rhizomatosae, Arachis, Erectoides and Procumbentes by genomic in situ hybridization (GISH). Single GISH analyses detected that species of the sections Erectoides (E2 subgenome) and Procumbentes (E3 subgenome) were the diploid species with the highest degree of genomic affinity with A. glabrata. Based on single GISH experiments and DNA sequence similarity, three species -A. duranensis, A. paraguariensis subsp. capibarensis, and A. rigonii-, which showed the most uniform and brightest hybridization patterns and lowest genetic distance, were selected as probes for double GISH experiments. Double GISH experiments showed that A. glabrata is constituted by four identical or very similar chromosome complements. In these assays, A. paraguariensis subsp. capibarensis showed the highest brightness onto A. glabrata chromosomes. Thus, our results support the autopolyploid origin of A. glabrata and show that the species with E2 subgenome are the most probable ancestors of this polyploid legume forage.

Formamide-Free Genomic In Situ Hybridization (ff-GISH).

Fluorescence in situ hybridization allows for the mapping of various sequence types in the genomes and is thus widely used in structural, functional, and evolutionary studies. One particular type of in situ hybridization that specifically allows to map whole parental genomes in diploid and polyploid hybrids is genomic in situ hybridization (GISH). The efficiency of GISH, i.e., the specificity of hybridization of genomic DNA probes to the parental subgenomes in hybrids depends, among others, on the age of the polyploids and the similarity of the parental genomes, specifically their repetitive DNA fractions. Typically, high levels of overall repeat similarity between the parental genomes result in lower efficiency of GISH. Here, we present the formamide-free GISH (ff-GISH) protocol that can be applied to diploid and polyploid hybrids of both monocots and dicots. ff-GISH allows higher efficiency of the labeling of the putative parental genomes compared to the standard GISH protocol and allows discrimination of parental chromosome sets that share up to 80-90% repeat similarity. This modified method is nontoxic, is simple, and lends itself to modifications. It can also be used for standard FISH and mapping of individual sequence types in chromosomes/genomes.

Development and chromosomal characterization of interspecific hybrids between common buckwheat (Fagopyrum esculentum) and a related perennial species (F. cymosum).

Common buckwheat (Fagopyrum esculentum) is an annual self-incompatible plant that is widely grown. The genus Fagopyrum comprises more than 20 species, including F. cymosum, a perennial that, unlike common buckwheat, is highly resistant to excess water. In this study, we developed interspecific hybrids between F. esculentum and F. cymosum via embryo rescue, to improve undesirable traits of common buckwheat, such as low tolerance to excess water. The interspecific hybrids were confirmed by genomic in situ hybridization (GISH). We also developed DNA markers to confirm the identity of the hybrids and if genes derived from each genome were inherited by the next generation. Observations of pollen indicated that the interspecific hybrids were essentially sterile. Unpaired chromosomes and abnormal segregation during meiosis were likely responsible for the pollen sterility of the hybrids. These findings could facilitate buckwheat breeding to produce lines that can withstand harsh environments with wild or related species in the genus Fagopyrum.

Visualizing Chromosome Territories and Nuclear Architecture of Large Plant Genomes Using Alien Introgressions.

Visualization of chromosome territories is a challenging task in plant genomes due to the lack of chromosome-specific probes, especially in species with large genomes. On the other hand, combination of flow sorting, genomic in situ hybridization (GISH), confocal microscopy, and employment of software for 3D modeling enables to visualize and characterize chromosome territories (CT) in interspecific hybrids. Here, we describe the protocol for the analysis of CTs in wheat-rye and wheat-barley hybrids, including amphiploids and introgression forms, where a pair of chromosomes or chromosome arms from one species is introgressed into the genome of another species. In this way, the architecture and dynamics of CTs in various tissues and different stages of cell cycle can be analyzed.

Genome elimination from the germline cells in diploid and triploid male water frogs Pelophylax esculentus.

Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog P. esculentus (diploid RL and triploids RLL and RRL), a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic in situ hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%–39%) and SSCs (18%–20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.

Cytogenetics and Consequences of Polyploidization on Different Biotic-Abiotic Stress Tolerance and the Potential Mechanisms Involved.

The application of polyploidy in sustainable agriculture has already brought much appreciation among researchers. Polyploidy may occur naturally or can be induced in the laboratory using chemical or gaseous agents and results in complete chromosome nondisjunction. This comprehensive review described the potential of polyploidization on plants, especially its role in crop improvement for enhanced production and host-plant resistance development against pests and diseases. An in-depth investigation on techniques used in the induction of polyploidy, cytogenetic evaluation methods of different ploidy levels, application, and current research trends is also presented. Ongoing research has mainly aimed to bring the recurrence in polyploidy, which is usually detected by flow cytometry, chromosome counting, and cytogenetic techniques such as fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). Polyploidy can bring about positive consequences in the growth and yield attributes of crops, making them more tolerant to abiotic and biotic stresses. However, the unexpected change in chromosome set and lack of knowledge on the mechanism of stress alleviation is hindering the application of polyploidy on a large scale. Moreover, a lack of cost–benefit analysis and knowledge gaps on the socio-economic implication are predominant. Further research on polyploidy coupling with modern genomic technologies will help to bring real-world market prospects in the era of changing climate. This review on polyploidy provides a solid foundation to do next-generation research on crop improvement.

Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium homoeologous group-6 chromosome disomic substitution line with resistance to leaf rust and stripe rust.

Thinopyrum intermedium (JJJsJsStSt, 2n = 6x = 42), a member of tertiary gene pool of hexaploid wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), provides several beneficial genes for wheat improvement. In this study, line CH51 was developed from the BC1F8 progeny of a partial wheat-Th. intermedium amphiploid TAI8335 (2n = 56) and wheat cultivar (cv.) Jintai 170. Somatic metaphase chromosome counting showed that CH51 had stable 42 chromosomes. Genomic in situ hybridization (GISH) analysis showed that CH51 had 40 wheat chromosomes and two Th. intermedium chromosomes involving translocation between Js- and St-genome chromosomes. Non-denaturing fluorescence in situ hybridization (ND-FISH) analysis revealed that CH51 lacked a pair of wheat chromosome 6B. Wheat 55K SNP array analysis verified that chromosome 6B had the highest percentage of missing SNP loci in both CH51 and Chinese Spring (CS) nullisomic 6B-tetrasomic 6D (CS-N6BT6D) and had the highest percentage of polymorphic SNP loci between CH51 and cv. Jintai 170. We identified that CH51 was a wheat-Th. intermedium T6StS.6JsL (6B) disomic substitution line. Disease resistance assessment showed that CH51 exhibited high levels of resistance to the prevalent Chinese leaf rust and stripe rust races in the field. Therefore, the newly developed line CH51 can be utilized as a potential germplasm in wheat disease resistance breeding.

Molecular cytogenetic analyses of two new wheat-rye 6RL translocation lines with resistance to wheat powdery mildew

Rye (Secale cereale) is a valuable gene donor for wheat improvement, especially for its resistance to diseases. Developing rye-derived resistance sources is important for wheat breeding. In the present study, two wheat-rye derivatives, designated JS016 and JS110, were produced by crossing common wheat cultivar Yangmai 23 with Pakistani rye accession W2A. Using sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (mc-FISH), JS016 and JS110 were identified as a T6BS.6RL translocation line and a T6BS.6BL6RL translocation line, respectively. Ten newly 6RL chromosome arm-specific markers were developed and used to confirm the 6RL translocation. The wheat 55K single-nucleotide polymorphism (SNP) array further verified the molecular cytogenetic identification results above and clarified their breakpoints at 430.9 and 523.0 Mb of chromosome 6B in JS016 and JS110, respectively. Resistance spectrum and allelism test demonstrated that JS016 and JS110 possessed novel powdery mildew resistance gene(s) that was derived from the 6RL translocation but differed from Pm20. Moreover, JS016 and JS110 had better agronomic traits than the previously reported 6RL translocation line carrying Pm20. To efficiently transfer and detect the 6RL translocation from JS016 and JS110, one 6RL-specific Kompetitive allele specific PCR (KASP) marker was developed and validated in high throughput marker-assisted selection (MAS).

Characterization of repetitive sequences in Dendrobium officinale and comparative chromosomal structures in Dendrobium species using FISH

Tandem repeats are one of the most conserved features in the eukaryote genomes. Dendrobium is the third largest genus in family Orchidaceae compromising over 1,200 species. However, the organization of repetitive sequences in Dendrobium species remains unclear. In this study, we performed the identification and characterization of the tandem repeats in D. officinale genome using graph-based clustering and Fluorescence in situ hybridization (FISH). Six major clusters including five satellite DNAs (DofSat1-5) and one 5S rDNA repeat (Dof5S) were identified as tandem repeats. The tandem organization of DofSat5 was verified by PCR amplification and southern blotting. The chromosomal locations of the repetitive DNAs in D. officinale were investigated by FISH using the tandem repeats and oligos probes. The results showed that each of the DofSat5, 5S and 45S rDNA had one pair of strong signals on D. officinale chromosomes. The distribution of repetitive DNAs along chromosomes was also investigated based on genomic in situ hybridization (GISH) among four Dendrobium species. The results suggested complex chromosomal fusion/segmentation and rearrangements during the evolution of Dendrobium species. In conclusion, the present study provides new landmarks for unequival differentiation of the Dendrobium chromosomes and facilitate the understanding the chromosome evolution in Dendrobium speceis.

Cytogenetic Analysis in the Toad Species Bufo spinosus, Bufotes viridis and Epidalea calamita (Anura, Bufonidae) from the Mediterranean Area.

Taxonomy in Bufonidae witnessed notable transformations. Bufotes viridis and Epidalea calamita, previously included in genus Bufo, were relocated in other genera, while the genus Bufo was restricted to members of the earlier Bufo bufo group. On the other hand, Bufo bufo sensu lato now includes four species: Bufo bufo, Bufo spinosus, Bufo verrucosissimus and Bufo eichwaldi. In this study, we examined three species of three Bufonidae genera (B. spinosus, B. viridis and E. calamita) by conventional (C-banding and Ag-NOR staining) and molecular (in situ hybridization with probes for telomeric repeats and rDNA loci, and genomic in situ hybridization (GISH)) cytogenetic methods. C-banding patterns are reported for the first time for B. spinosus and E. calamita populations from Iberian Peninsula and for B. viridis from Greece, and reveal several differences with the reported C-banded karyotypes described for other European populations of these species. Silver staining shows size heteromorphisms of the signals at the Nucleolar Organizing Region (NOR). By contrast, FISH with ribosomal probes only reveal size heteromorphism of rDNA sequences in E. calamita, suggesting that the differences observed after silver staining in B. spinosus and B. viridis should be attributed to differences in chromosomal condensation and/or gene activity rather than to differences in the copy number for ribosomal genes. Regarding telomeric repeats, E. calamita is the only species with interstitial telomeric sequences (ITS) located on centromeric regions, probably originated by accumulation of telomeric sequences in the centromeric heterochromatin. Finally, we analyzed the composition and distribution of repetitive sequences by genome in situ hybridization. These experiments reveal the accumulation of repetitive sequences in centromeric regions of the three species, although these sequences are not conserved when species from different genera are compared.