Fluorescence In Situ Hybridization Mapping Research Articles

Molecular Cytogenetics in Domestic Bovids: A Review.

The discovery of the Robertsonian translocation (rob) involving cattle chromosomes 1 and 29 and the demonstration of its deleterious effects on fertility focused the interest of many scientific groups on using chromosome banding techniques to reveal chromosome abnormalities and verify their effects on fertility in domestic animals. At the same time, comparative banding studies among various species of domestic or wild animals were found useful for delineating chromosome evolution among species. The advent of molecular cytogenetics, particularly the use of fluorescence in situ hybridization (FISH), has allowed a deeper investigation of the chromosomes of domestic animals through: (a) the physical mapping of specific DNA sequences on chromosome regions; (b) the use of specific chromosome markers for the identification of the chromosomes or chromosome regions involved in chromosome abnormalities, especially when poor banding patterns are produced; (c) better anchoring of radiation hybrid and genetic maps to specific chromosome regions; (d) better comparisons of related and unrelated species by comparative FISH mapping and/or Zoo-FISH techniques; (e) the study of meiotic segregation, especially by sperm-FISH, in some chromosome abnormalities; (f) better demonstration of conserved or lost DNA sequences in chromosome abnormalities; (g) the use of informatic and genomic reconstructions, in addition to CGH arrays, to predict conserved or lost chromosome regions in related species; and (h) the study of some chromosome abnormalities and genomic stability using PCR applications. This review summarizes the most important applications of molecular cytogenetics in domestic bovids, with an emphasis on FISH mapping applications.

Accumulation of retrotransposons contributes to W chromosome differentiation in the willow beauty Peribatodesrhomboidaria (Lepidoptera: Geometridae)

The W chromosome of Lepidoptera is typically gene-poor, repeat-rich and composed of heterochromatin. Pioneering studies investigating this chromosome reported an abundance of mobile elements. However, the actual composition of the W chromosome varies greatly between species, as repeatedly demonstrated by comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH). Here we present an analysis of repeats on the W chromosome in the willow beauty, Peribatodesrhomboidaria (Geometridae), a species in which CGH predicted an abundance of W-enriched or W-specific sequences. Indeed, comparative analysis of male and female genomes using RepeatExplorer identified ten putative W chromosome-enriched repeats, most of which are LTR or LINE mobile elements. We analysed the two most abundant: PRW LINE-like and PRW Bel-Pao. The results of FISH mapping and bioinformatic analysis confirmed their enrichment on the W chromosome, supporting the hypothesis that mobile elements are the driving force of W chromosome differentiation in Lepidoptera. As the W chromosome is highly underrepresented in chromosome-level genome assemblies of Lepidoptera, this recently introduced approach, combining bioinformatic comparative genome analysis with molecular cytogenetics, provides an elegant tool for studying this elusive and rapidly evolving part of the genome.

Comparative Fluorescence In Situ Hybridization (FISH) Mapping of Twenty-Three Endogenous Jaagsiekte Sheep Retrovirus (enJSRVs) in Sheep (Ovis aries) and River Buffalo (Bubalus bubalis) Chromosomes.

Simple SummaryThe genome of domestic sheep (Ovis aries) harbors at least twenty-seven copies of enJSRVs, endogenous retroviruses (ERVs) highly related to the exogenous and pathogenic Jaagsiekte sheep betaretrovirus (JSRV). Interestingly, some of these loci are insertionally polymorphic, that is they are present only in some individuals or populations of their host species. This differential distribution of enJSRVs has provided important insights into tracing host and viral evolution. In this study, we report the first comparative fluorescent in situ hybridization (FISH) mapping of genetically characterized enJSRVs in domestic sheep (2n = 54) and river buffalo (Bubalus bubalis, 2n = 50), and reveal a high conservation of enJSRVs chromosome localization between these two species.Endogenous retroviruses (ERVs) are the remnants of ancient infections of host germline cells, thus representing key tools to study host and viral evolution. Homologous ERV sequences often map at the same genomic locus of different species, indicating that retroviral integration occurred in the genomes of the common ancestors of those species. The genome of domestic sheep (Ovis aries) harbors at least twenty-seven copies of ERVs related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRVs), thus referred to as enJSRVs. Some of these loci are unequally distributed between breeds and individuals of the host species due to polymorphic insertions, thereby representing invaluable tools to trace the evolutionary dynamics of virus populations within their hosts. In this study, we extend the cytogenetic physical maps of sheep and river buffalo by performing fluorescent in situ hybridization (FISH) mapping of twenty-three genetically characterized enJSRVs. Additionally, we report the first comparative FISH mapping of enJSRVs in domestic sheep (2n = 54) and river buffalo (Bubalus bubalis, 2n = 50). Finally, we demonstrate that enJSRV loci are conserved in the homologous chromosomes and chromosome bands of both species. Altogether, our results support the hypothesis that enJSRVs were present in the genomes of both species before they differentiated within the Bovidae family.

EP060: Characterizing the complex chromosomal rearrangements in a Philadelphia negative BCR/ABL1 positive case

The Philadelphia chromosome (Ph), produced by the reciprocal translocation t(9;22)(q34;q11.2), is a derivative chromosome 22 with a chimeric BCR/ABL1 fusion gene encoding constitutively active tyrosine kinase protein. The Ph occurs in 95% of chronic myeloid leukemia (CML) cases and the BCR/ABL1 fusion is a diagnostic hallmark for CML. A small subset of patients (around 5-10%), displaying typical features of CML, lack a Ph by chromosome analysis, but are shown to carry BCR/ABL1 fusion either by fluorescence in situ hybridization (FISH) or molecular analysis. Two major mechanisms have been proposed to explain how BCR/ABL1 rearrangements arise in these Ph-negative BCR/ABL1 positive cases: a cryptic insertion between chromosomes 9 and 22; or via two sequential translocations where a standard t(9;22) translocation is followed by an additional reverse translocation resulting in the reconstitution of the normal morphology of the partner chromosomes. The bone marrow specimen of a 66 year old female patient, presented with persistent leukocytosis with myelocytes and metamyelocytes, along with thrombocytosis, was sent to our lab for both BCR/ABL1 RT-PCR analysis and chromosome analysis, due to suspicious myeloproliferative neoplasm. The RT-PCR analysis showed the presence of the BCR/ABL1 major (p210) breakpoint fusion transcript b2a2 (e13a2). Initial chromosome analysis did not identify a typical t(9;22)(q34;q11.2) reciprocal translocation; instead, structural abnormalities were observed on the short arm of chromosome 12 and the long arm of chromosome 22. Overall, the abnormal chromosome 22 resembles the morphology of a “Philadelphia chromosome” while both homologs of chromosome 9 show normal morphology. This leads us to hypothesize that a complex rearrangement involving chromosomes 9, 12, and 22 might be contributing to this case. Metaphase fluorescence in situ hybridization (FISH) analysis was performed to test our hypothesis using BCR/ABL1 fusion probes, where the only cryptic BCR/ABL1 fusion signal was mapped to the long arm of chromosome 9 that has normal morphology at band q34, while the long arm of the derivative chromosome 22 is absent for BCR/ABL1 fusion signals, suggesting that the Ph-like derivative chromosome 22 is not a real Philadelphia chromosome, making this case a true Ph negative case. The final ISCN for the case is designated as: 46,XX,der(9)(9pter->9q34::22q11.2->22q11.2::9q34->9qter),der(12)(22qter->22q11.2::12p13->12qter),der(22)(22pter->22q11.2::12p13->12pter), and this can be explained by an initial three way translocation between chromosomes 9, 12, and 22 followed by an additional sequential reverse translocation between the derivative chromosomes 9 and 22, leading the cryptic BCR/ABL1 fusion being inserted into the long arm of chromosome 9 and bringing chromosome 9 back to a normal morphology. Cryptic BCR/ABL1 fusion has been characterized as a variant rearrangement in CML patients lacking Ph, and up to 50% of these cases with variant BCR/ABL1 fusion exhibit a del(9q) of the derivative chromosome 9, which has been considered to be a marker for poor prognosis in CML patients. Thus, chromosomal microarray analysis (CMA) was also performed for this case, and no deletions in 9q were identified. Here, we presented a case where FISH mapping was utilized to further characterize the complex chromosomal rearrangements between chromosomes 9, 12, and 22 in a Ph negative BCR/ABL1 positive case, which reveals that the cryptic BCR/ABL1 is inserted into the long arm of chromosome 9 with normal morphology at band q34, while the Ph-like derivative chromosome 22 is not a true Ph chromosome. Our study illustrates the challenge of recognizing the complex pattern of cytogenetic aberrations in these Ph negative BCR/ABL1 positive CML cases with variant BCR/ABL1 rearrangements, and highlights the importance of utilizing comprehensive cytogenomic and molecular techniques (including metaphase FISH, RT-PCR and CMA) in characterizing these complex chromosomal abnormalities for proper diagnosis and prognosis prediction.

Comparative chromosome studies in species of subtribe Orchidinae (Orchidaceae).

In our study, FISH mapping using 18S-5.8S-25S rDNA and 5S rDNA sequences was performed for the first time on Ophrystenthredinifera Willdenow, 1805, Serapiasvomeracea (Burman f., 1770) Briquet, 1910 and Himantoglossumhircinum (Linnaeus, 1753) Sprengel, 1826. A detailed study was also performed on O.tenthredinifera using Giemsa-staining, silver-staining, CMA fluorescence banding and fluorescence in situ hybridisation (FISH) with rDNA probes. We analysed two subspecies, i.e. O.tenthrediniferasubsp.neglecta (Parlatore, 1860) E.G. Camus, 1908 and O.tenthrediniferasubsp.grandiflora (Tenore, 1819) Kreutz, 2004 by the traditional Feulgen method and constructed the karyotype. The cytotaxonomic implications for both taxa are also discussed. In Himantoglossumhircinum, FISH and silver staining highlighted differences in the number of two rDNA families (35S and 5S) with respect to Barliarobertiana (Loiseleur-Deslongchamps, 1807) Greuter, 1967. In addition, fluorescence in situ hybridisation was also applied to diploid (2n = 2x = 36) and triploid (2n = 3x = 54) Anacamptismorio (Linnaeus, 1753) R.M. Bateman, Pridgeon et M.W. Chase, 1997. As far as we are aware, this is the first case of autotriploidy observed in A.morio.

Comparative FISH mapping of 45S and 5S rDNA in the genus Gambierdiscus advances understanding of the cytogenetic diversity and mitosis of dinoflagellates

ABSTRACT Dinoflagellates are protists with exceptionally large genomes and chromosomes that lack nucleosomes. To date, information on their genome organization is scarce. Studies investigating the distribution of ribosomal RNA genes (rDNA) in plants and animals by fluorescence in situ hybridization (FISH) have provided important information to elucidate the genome organization and evolution of many species. In the present study, FISH was used to analyse the chromosomal organization of 45S and 5S rDNA and the extent of rDNA variation among five species of Gambierdiscus (G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus). The results, the intensity and location of the FISH signals, are summarized as follows: (1) All Gambierdiscus species have extremely high copy number of 45S rDNA repeat units arranged in a large nucleolus situated in the concave region of the typical interphasic bean-shaped nucleus. In addition, outside the large nucleolus, G. silvae and G. carolinianus have minor clusters of 45S rDNA repeats. (2) Many copies of 5S rDNA organized in one or a few clusters, depending on the species, are physically separated from the 45S rDNA but located close, peripherally, to the nucleolus. This nuclear organization is preserved during cell division. (3) Gambierdiscus australes was the species with the lowest 5S rDNA FISH signals clustered into a single site. In contrast, one major and several minor 5S rDNA clusters of FISH signals were observed in G. excentricus. We find that the species analysed can be differentiated by their species-specific FISH rDNA phenotypes demonstrating the usefulness of FISH in comparative cytogenomic studies of Gambierdiscus. In addition, using G. australes as a model, we demonstrate the usefulness of 45S and 5S rDNA as chromosomal markers to study dinomitosis, the atypical nuclear division that solely occurs in the class Dinophyceae.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

Molecular Genetics of Myeloid Leukemia: Identification of the Commonly Deleted Segment of Chromosome 20

A deletion of the long arm of chromosome 20 [del(20q)] is a recurring abnormality in malignant myeloid disorders. The occurrence of the del(20q) in a broad spectrum of myeloid disorders suggests that the loss of genetic material on 20q could provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor-suppressor gene. We have examined a series of patients with the del(20q) using fluorescence in situ hybridization (FISH) with unique sequence probes that map along the length of 20q, and have delineated a segment that is deleted in 95% of all patients examined (18 of 19). In addition, we have shown that the deletions are interstitial rather than terminal. This region of deletion extends from 20q11.2 to q12, and is flanked by the RPN2 (proximal) and D20S17 loci (distal). The SRC and ADA genes are located within the commonly deleted segment. Our findings emphasize the importance of FISH and other molecular mapping techniques in defining such a region. The delineation of a commonly deleted segment in 20q11.2-q12 will facilitate the identification of candidate tumor-suppressor genes on 20q.

A New Recurrent Translocation, t(5;11)(q35;p15.5), Associated With del(5q) in Childhood Acute Myeloid Leukemia

AbstractPartial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harveyras-1–related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Sequence Analysis and FISH Mapping of Four Satellite DNA Families among Cervidae.

Centromeric and pericentromeric chromosome regions are occupied by satellite DNA. Satellite DNAs play essential roles in chromosome segregation, and, thanks to their extensive sequence variability, to some extent, they can also be used as phylogenetic markers. In this paper, we isolated and sequenced satellite DNA I-IV in 11 species of Cervidae. The obtained satellite DNA sequences and their chromosomal distribution were compared among the analysed representatives of cervid subfamilies Cervinae and Capreolinae. Only satI and satII sequences are probably present in all analysed species with high abundance. On the other hand, fluorescence in situ hybridisation (FISH) with satIII and satIV probes showed signals only in a part of the analysed species, indicating interspecies copy number variations. Several indices, including FISH patterns, the high guanine and cytosine (GC) content, and the presence of centromere protein B (CENP-B) binding motif, suggest that the satII DNA may represent the most important satellite DNA family that might be involved in the centromeric function in Cervidae. The absence or low intensity of satellite DNA FISH signals on biarmed chromosomes probably reflects the evolutionary reduction of heterochromatin following the formation of chromosome fusions. The phylogenetic trees constructed on the basis of the satellite I-IV DNA relationships generally support the present cervid taxonomy.

Fluorescence in situ hybridization of 35S rDNA sites and karyotype of wild Lilium (Liliaceae) species from China: taxonomic and phylogenetic implications

Most recent studies on Lilium species have been descriptive observations based on FISH (fluorescence in situ hybridization) analyses or phylogenetic studies focusing on only one section without taking the evolutionary framework of Lilium into account. To better understand the taxonomic status, evolution, and karyotype diversity of the genus Lilium, we conducted FISH mapping using 35S rDNA sequences in 29 important wild Lilium species covering four sections of the genus in China. Their molecular phylogenetic relationships were analyzed based on the internal transcribed spacer (ITS) region. For 35S rDNA, the hybridization loci always existed in pairs on homologous chromosomes. The number of hybridization signals varied from 4 to 14 in diploid species, and the signals were mainly located on the proximal region of the short arm of chromosomes. Among all the sections, sect. Sinomartagon had the most 35S rDNA signals. Genotype grouping based on 35S signal loci was largely consistent with groups in the phylogenetic tree constructed using ITS sequences, and different species in a sect./subsect. shared some common molecular and cytogenetic features. The results indicated that sect. Martagon is monophyletic in a single clade; Lophophorum should be treated as a different taxon from Sinomartagon; Lilium taliense and Lilium wardii Stapf ex Stern show close molecular and cytological phylogenetic relationships; Lilium duchartrei Franch. is more distantly related to other sect. Sinomartagon species; Lilium lophophorum (Bureau & Franch.) Franch. is distinct from other Lophophorum species; Leucolirion species form two relatively independent groups (6a and 6b, with Lilium henryi Baker and Lilium rosthornii Diels in 6a); and Nomocharis-like Lilium species should be treated as a different taxon, like sect. Lophophorum. Based on previous studies and our findings, we discuss the evolutionary trends of Lilium with references to its geographical distribution. We propose that sect. Lophophorum is a relatively primitive group, and species in subsect. Leucolirion 6b may be more evolved than those in 6a. These findings provide a new reference for the taxonomy, phylogeny, and breeding of Lilium.

Insight into the Chromosome Structure of the Cultivated Tetraploid Alfalfa (Medicago sativa subsp. sativa L.) by a Combined Use of GISH and FISH Techniques.

Cytogenetic research in Medicago sativa subsp. sativa L., the cultivated tetraploid alfalfa (2n = 4x = 32), has lagged behind other crops mostly due to the small size and the uniform morphology of its chromosomes. However, in the last decades, the development of molecular cytogenetic techniques based on in situ hybridization has largely contributed to overcoming these limitations. The purpose of this study was to extend our knowledge about the chromosome structure of alfalfa by using a combination of genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) techniques. The results of self-GISH (sGISH) suggested that a substantial part of the repetitive fraction of the genome of subsp. sativa is constituted by tandem repeats typical of satellite DNA. The coincidence of sGISH and C-banding patterns supported this assumption. The FISH mapping of the Arabidopsis-type TTTAGGG telomeric repeats demonstrated, for the first time, that the alfalfa telomeres consist of this type of sequence and revealed a massive presence of interstitial telomeric repeats (ITRs). In the light of this finding M. sativa appears to be a suitable material for studying the origin and function of such extra telomeric repeats. To further exploit this result, investigation will be extended to the diploid subspp. coerulea and falcata in order to explore possible connections between the distribution of ITRs, the ploidy level, and the evolutionary pathway of the taxa.

FISH mapping of rDNA and telomeric repeats in 10 Senna species

Plants of the genus Senna (Fabaceae) are valued for industrial and medicinal properties that have spurred crop improvement through breeding programs. However, there is very little chromosomal data for this genus, limiting the potential for cytogenetics and genomics research. Here, we used fluorescence in situ hybridization (FISH) to perform a comparative analysis of 10 Senna spp. karyotypes (2n = 28 except S. tora with 2n = 26). We detected differences in the distributions of rDNA gene clusters and Arabidopsis-type telomeric repeats (TTTAGGG)n. Only one 5S rDNA pair was detected in all species, but we observed more interspecies variation for 45S rDNA: four pairs in S. didymobotrya, S. spectabilis, and S. viarum; two pairs in S. candolleana; and one pair in S. bauhinioides, S. multiglandulosa, S. occidentalis, and S. tora. In both S. leandrii and S. sulfurea, 45S rDNA distribution was hemizygous. While all chromosomes possessed telomeric signals in the terminal region, this signal was also present in the interstitial regions of all S. tora chromosomes and, less obviously (fewer chromosomes, weaker signals), in S. occidentalis. These preliminary data demonstrate interspecific karyotype variation that reveals Senna genome dynamics. Our results facilitate cytogenetic mapping of other major repeats, thus contributing to an improved understanding of Senna genome structure and evolutionary history.

A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

The major drawbacks of standard plant fluorescence in situ hybridization (FISH) designed for double-stranded DNA probes include requirement for experimentally determined heat denaturation of chromosomes at high temperatures and at least overnight hybridization. Consequently, processing with chromosomal preparations may easily result in heat-induced deterioration of chromosomal structural details, is time-consuming, and involves the use of toxic formamide and formaldehyde. Here, I have described a simple and appealing non-toxic procedure with ethylene carbonate (EC)—a formamide-substituting solvent and double-stranded repetitive DNA probes. Applying EC as a component of the hybridization solution at 46 °C not only allowed successful overnight hybridization but also gave a possibility to reduce the hybridization time to 3 h, hence converting the technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve well chromosome structural details, e.g., DAPI-positive bands, thus facilitating simultaneous FISH mapping and chromosome banding on the same slide. The procedure requires no formaldehyde and RNA-se treatment of chromosomes, and no heat denaturation of chromosomal DNA. The key condition is to obtain high-quality cytoplasm-free preparations. The method was reproducible in all the plants studied (Allium, Nigella, Tradescantia, Vicia), giving a species-specific signal pattern together with clear DAPI bands on chromosomes. The procedure described here is expected to give a positive stimulus for improving gene-mapping approaches in plants.

Segmental and tandem chromosome duplications led to divergent evolution of the chalcone synthase gene family in Phalaenopsis orchids.

Background and AimsOrchidaceae is a large plant family, and its extraordinary adaptations may have guaranteed its evolutionary success. Flavonoids are a group of secondary metabolites that mediate plant acclimation to challenge environments. Chalcone synthase (CHS) catalyses the initial step in the flavonoid biosynthetic pathway. This is the first chromosome-level investigation of the CHS gene family in Phalaenopsis aphrodite and was conducted to elucidate if divergence of this gene family is associated with chromosome evolution.MethodsComplete CHS genes were identified from our whole-genome sequencing data sets and their gene expression profiles were obtained from our transcriptomic data sets. Fluorescence in situ hybridization (FISH) was conducted to position five CHS genes to high-resolution pachytene chromosomes.Key ResultsThe five Phalaenopsis CHS genes can be classified into three groups, PaCHS1, PaCHS2 and the tandemly arrayed three-gene cluster, which diverged earlier than those of the orchid genera and species. Additionally, pachytene chromosome-based FISH mapping showed that the three groups of CHS genes are localized on three distinct chromosomes. Moreover, an expression analysis of RNA sequencing revealed that the five CHS genes had highly differentiated expression patterns and its expression pattern-based clustering showed high correlations between sequence divergences and chromosomal localizations of the CHS gene family in P. aphrodite.ConclusionsBased on their phylogenetic relationships, expression clustering analysis and chromosomal distributions of the five paralogous PaCHS genes, we proposed that expansion of this gene family in P. aphrodite occurred through segmental duplications, followed by tandem duplications. These findings provide information for further studies of CHS functions and regulations, and shed light on the divergence of an important gene family in orchids.

Improved accuracy assessment for 3D genome reconstructions

BackgroundThree dimensional (3D) genome spatial organization is critical for numerous cellular functions, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Genome conformation had been difficult to elucidate but the advent chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin architecture and yielded numerous biological insights. Although most of these findings have flowed from analysis of proximity data produced by these assays, added value in generating 3D reconstructions has been demonstrated, deriving, in part, from superposing genomic features on the reconstruction. However, advantages of 3D structure-based analyses are clearly conditional on the accuracy of the attendant reconstructions, which is difficult to assess. Proponents of competing reconstruction algorithms have evaluated their accuracy by recourse to simulation of toy structures and/or limited fluorescence in situ hybridization (FISH) imaging that features a handful of low resolution probes. Accordingly, new methods of reconstruction accuracy assessment are needed.ResultsHere we utilize two recently devised assays to develop methodology for assessing 3D reconstruction accuracy. Multiplex FISH increases the number of probes by an order of magnitude and hence the number of inter-probe distances by two orders, providing sufficient information for structure-level evaluation via mean-squared deviations (MSD). Crucially, underscoring multiplex FISH applications are large numbers of coordinate-system aligned replicates that provide the basis for a referent distribution for MSD statistics. Using this system we show that reconstructions based on Hi-C data for IMR90 cells are accurate for some chromosomes but not others. The second new assay, genome architecture mapping, utilizes large numbers of thin cryosections to obtain a measure of proximity. We exploit the planarity of the cryosections – not used in inferring proximity – to obtain measures of reconstruction accuracy, with referents provided via resampling. Application to mouse embryonic stem cells shows reconstruction accuracies that vary by chromosome.ConclusionsWe have developed methods for assessing the accuracy of 3D genome reconstructions that exploit features of recently advanced multiplex FISH and genome architecture mapping assays. These approaches can help overcome the absence of gold standards for making such assessments which are important in view of the considerable uncertainties surrounding 3D genome reconstruction.

FISH mapping of 45S rRNA and IHHB genes on autosomes and B chromosome of cichlid fish Astatotilapia latifasciata

B chromosomes (B) are supernumerary, additional and non-homologous to standard (A) chromosomes. Significant biological questions concerning the origin and structural organization of Bs are under discussion. Previous reports found that B chromosomes are enriched with repetitive elements and can carry functional genes. The present study is aimed to extend our knowledge of the structure and composition of B chromosomes. We have combined cytogenetics and genomics approaches to undertsand the B chromosome problem. Cytogenetics analysis, applied to cichlid fish Astatotilapia latifasciata, revealed the presence of 1 or 2 B chromosomes. Next generation sequencing data analysis, based on differential coverage approach, among B- (individual without B) and B+ (individual with B) samples, detected a higher number of copies for the Indian Hedgehog b (Ihhb) gene and 45S RNA transcriptional gene-cluster. Fluorescence in situ hybridization (FISH) confirmed that these genes are located on the B chromosome. In addition, the excessively abundant and strong signals of IHHB on B shows that this gene has accumulated and amplified its copies due to frequent duplication events. The findings also demonstrate that B chromosome have incorporated the entire 45S RNA cluster (18S ribosomal RNA, internal transcribed spacer 1, 5.8S ribosomal RNA, internal transcribed spacer 2, and 28S ribosomal RNA) from the A complement set. Our study contributes to explain that these genic sequences are vital components in constitution of B chromosomes.

Development and applications of fluorescence in situ hybridization using oligonucleotide-based probes

Fluorescence in situ hybridization (FISH) has been the most important technique in plant cytogenetic research. However, application of FISH techniques in most plant species has been hindered by the lack of cloned probes that generate distinct signals on chromosomes. We have recently developed a procedure to develop FISH probes by selecting a large number of oligonucleotides (oligos) derived from single copy DNA sequences. The bioinformatically selected oligos are then massively synthesized de novo in parallel, amplified, and labeled as FISH probes. The “oligo-FISH probes” designed from conserved DNA sequences generate distinct FISH signals on related plant species that have diverged for more than 10 million years. We have conducted oligo-FISH in several distantly related species in the Solanaceae family. This comparative FISH mapping effort has revealed karyotype evolution and discovered species-specific chromosomal translocations in several Solanum species.

Physical Location of New PCR-Based Markers and Powdery Mildew Resistance Gene(s) on Rye (Secale cereale L.) Chromosome 4 Using 4R Dissection Lines.

Rye (Secale cereale L.) 4R chromosome contains elite genes that are applicable for wheat (Triticum aestivum L.) cultivar improvement. PCR-based 4R-specific markers can benefit the detection of elite genes on 4R in wheat backgrounds. In this study, a new fluorescence in situ hybridization (FISH) map of the 4RKu chromosome of rye Kustro has been constructed. A set of 4RKu dissection lines was obtained and 301 new 4RKu-specific markers were developed using specific length amplified fragment sequencing (SLAF-seq) technology. These markers were combined with the 99 4RKu-specific markers previously developed, and were physically mapped to 4RKu chromosome using the new FISH map and the 4RKu dissection lines. A total of 338 of the 400 markers have been successfully mapped to six regions of 4RKu chromosome. Additionally, the powdery mildew resistance gene(s) on the 4RLKu arm was located to the segment between L.4 and L.8, the same region where 115 4RLKu-specific markers were mapped. The markers developed in this study can be used to identify a specific segment of 4R chromatin in wheat backgrounds, help construct a high-density physical map of 4R chromosome, and facilitate the utilization of elite genes on 4R chromosome in wheat breeding programs.

Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species

BackgroundPreparation of good chromosome spreads without cytoplasmic contamination is the crucial step in cytogenetic mapping. To date, cytogenetic research in the Orchidaceae family has been carried out solely on mitotic metaphase chromosomes. Well-spread meiotic pachytene chromosomes can provide higher resolution and fine detail for analysis of chromosomal structure and are also beneficial for chromosomal FISH (fluorescence in situ hybridization) mapping. However, an adequate method for the preparation of meiotic pachytene chromosomes in orchid species has not yet been reported.ResultsTwo Taiwanese native Phalaenopsis species were selected to test the modified drop method for preparation of meiotic pachytene chromosomes from pollinia. In this modified method, pollinia were ground and treated with an enzyme mixture to completely remove cell walls. Protoplasts were resuspended in ethanol/glacial acetic acid and dropped onto a wet inclined slide of 30° from a height of 0.5 m. The sample was then flowed down the inclined plane to spread the chromosomes. Hundreds of pachytene chromosomes with little to no cytoplasmic contamination were well spread on each slide. We also showed that the resolution of 45S rDNA-containing chromosomes at the pachytene stage was up to 20 times higher than that at metaphase. Slides prepared following this modified drop method were amenable to FISH mapping of both 45S and 5S rDNA on pachytene chromosomes and, after FISH, the chromosomal structure remained intact for further analysis.ConclusionThis modified drop method is suitable for pachytene spreads from pollinia of Phalaenopsis orchids. The large number and high-resolution pachytene spreads, with little or no cytoplasmic contamination, prepared by the modified drop method could be used for FISH mapping of DNA fragments to accelerate the integration of cytogenetic and molecular research in Phalaenopsis orchids.