Identification Of Individual Chromosomes Research Articles

Chromosome Karyotyping and Aneuploidy Detection using Deep Learning Networks

Chromosome karyotype analysis is a vital role in the diagnosis of congenital disease, chromosomal aneuploidies. Karyotyping is defined as the process of categorizing chromosomes into one of 24 kinds. Chromosomal aneuploidies can be classified as structural and numerical. Numerical anomalies such as Down syndrome, Williams’s syndrome Pattu syndrome, Turner syndrome, klinefelter syndrome and certain kind of cancers detected with the help of karyotyping. It necessitates rigorous attention to detail and well-trained people. With these objectives in mind, in this work, we automated karyotyping and identified common numerical aneuploidies using deep learning on a dataset of 5474 distinct grayscale metaphase chromosomal impressions from the Bioimage chromosome categorization dataset (BioImLab), which is freely available online. Three stages are included in the proposed classification model. The first stage entails segmenting and individual chromosome identification using bounding box detection and modified binary mask methods. At this point, achieved individual chromosome detection accuracy is 99%. The second stage involves classifying chromosomes using a 144 deep and 1000 FC layer GLNet (GoogleNet) classifier, which has an accuracy rate of 96.33%. The last step is to accurately identify aneuploidies with a 98% detection rate. A robust automatic classification system has been developed. Many scholars have proposed various karyotyping approaches. This research compares and contrasts the outcomes of several approaches with greater accuracy.

Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

Accurate Chromosome Identification in the Prunus Subgenus Cerasus (Prunus pseudocerasus) and its Relatives by Oligo-FISH.

A precise, rapid and straightforward approach to chromosome identification is fundamental for cytogenetics studies. However, the identification of individual chromosomes was not previously possible for Chinese cherry or other Prunus species due to the small size and similar morphology of their chromosomes. To address this issue, we designed a pool of oligonucleotides distributed across specific pseudochromosome regions of Chinese cherry. This oligonucleotide pool was amplified through multiplex PCR with specific internal primers to produce probes that could recognize specific chromosomes. External primers modified with red and green fluorescence tags could produce unique signal barcoding patterns to identify each chromosome concomitantly. The same oligonucleotide pool could also discriminate all chromosomes in other Prunus species. Additionally, the 5S/45S rDNA probes and the oligo pool were applied in two sequential rounds of fluorescence in situ hybridization (FISH) localized to chromosomes and showed different distribution patterns among Prunus species. At the same time, comparative karyotype analysis revealed high conservation among P. pseudocerasus, P. avium, and P. persica. Together, these findings establish this oligonucleotide pool as the most effective tool for chromosome identification and the analysis of genome organization and evolution in the genus Prunus.

Precise Identification of Chromosome Constitution and Rearrangements in Wheat-Thinopyrum intermedium Derivatives by ND-FISH and Oligo-FISH Painting.

Thinopyrum intermedium possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and JS genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), was able to identify each linkage group of the Th. intermedium chromosomes. We subsequently established the first karyotype of Th. intermedium with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–Th. intermedium partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among Th. intermedium chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–Th. intermedium derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.

Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry

Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C - DNA content of winter-hardy Hibiscus (H. syriacus ‘Saejamyung’, H. sinosyriacus ‘Seobong’, H. moscheutos ‘Luna Red’ and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range in length of somatic chromosomes in H. syriacus, H. sinosyriacus, H. moscheutos, and H. paramutabilis is 2.66–7.06, 3.18–7.31, 2.91–5.23, and 4.75–7.60, respectively. The 2C - DNA content of H. syriacus, H. sinosyriacus, and H. paramutabilis are very similar, the amount was 4.06, 4.11, and 4.18 pg, respectively whereas, H. moscheutos has nearly half and that amount was 2.06 pg. These findings will contribute to the detailed cytogenetic assessment of Hibiscus and thus benefit plant breeding in this genus.

Oligo-FISH Paints in Triticeae.

We developed seven oligonucleotide (oligo) pools based on single-copy sequences, targeting chromosomes 1 to 7 of barley (Hordeum vulgare L.) and wheat (Triticum aestivum) for chromosomal Oligo-FISH painting methods. The probes were applied to high-throughput karyotyping for the Triticeae tribe of over 350 species including 30 genera such as Triticum, Hordeum, Secale, Aegilops, Thinopyrum, and Dasypyrum, as well as several wheat alien-derived lines. In combination with other nondenaturing FISH (ND-FISH) procedures using tandem-repeat oligos, the newly developed Oligo-FISH painting technique provides an efficient tool for the identification of individual chromosomes with homologous linkage groups to establish standard karyotypes, particularly with any wild Triticeae species having nonsequenced genomes for chromosome evolutionary analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Oligo-pool probe development Basic Protocol 2: Nondenaturing FISH Basic Protocol 3: Oligo-FISH painting.

Localization of ITS and 5S rDNA on the chromosomes of Ulva prolifera using fluorescence in situ hybridization

ABSTRACT Ulva prolifera has been identified as the dominant species of the world’s largest green tides occurring in the Yellow Sea, China, using the molecular markers of internal transcribed spacer (ITS) and 5S ribosomal DNA (5S rDNA). In this study, these two markers were successfully located on the chromosomes of U. prolifera using the fluorescence in situ hybridization (FISH) technique. The ITS rDNA probe was localized on chromosome number 1 of gametophytes and the 5S rDNA probe appeared on chromosome number 6. Dual-colour FISH was also performed, and the locations of the two markers were consistent with single-colour localization, indicating a precise gene location. The localization of these rDNAs using the FISH technique facilitated the identification of individual chromosomes and a karyotype analysis of Ulva, which is beneficial for understanding the blooming mechanism of green tides in the Yellow Sea.

Karyotypic stability of Fragaria (strawberry) species revealed by cross-species chromosome painting.

Chromosome karyotyping analysis is particularly useful in determining species relationships and the origin of polyploid species. Identification of individual chromosomes is the foundation for karyotype development. For Fragaria (strawberry) species, definitive identification of the individual chromosomes is extremely difficult because of their small size and similar shape. Here, we identified all chromosomes for 11 representative Fragaria species with different ploidy using a set of oligonucleotide-based probes developed in Fragaria vesca. Comprehensive molecular cytogenetic karyotypes were established based on the individually identified chromosomes. In addition, we used oligo probes to assign the 5S and 45S rDNA loci to specific chromosomes in 16 Fragaria species. We found that these Fragaria species maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the chromosomes among these species. Despite karyotypic stability and similarity, variations in the signal intensity of oligo probes were observed among the homologous chromosomes in several polyploid species. Moreover, most Fragaria species also showed differences in the distribution patterns of 45S and 5S rDNA. These data provide new insights into the origins of several polyploid Fragaria species.

Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation.

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.

Chromosomal Mapping of Tandem Repeats Revealed Massive Chromosomal Rearrangements and Insights Into Senna tora Dysploidy.

Tandem repeats can occupy a large portion of plant genomes and can either cause or result from chromosomal rearrangements, which are important drivers of dysploidy-mediated karyotype evolution and speciation. To understand the contribution of tandem repeats in shaping the extant Senna tora dysploid karyotype, we analyzed the composition and abundance of tandem repeats in the S. tora genome and compared the chromosomal distribution of these repeats between S. tora and a closely related euploid, Senna occidentalis. Using a read clustering algorithm, we identified the major S. tora tandem repeats and visualized their chromosomal distribution by fluorescence in situ hybridization. We identified eight independent repeats covering ~85 Mb or ~12% of the S. tora genome. The unit lengths and copy numbers had ranges of 7–5,833 bp and 325–2.89 × 106, respectively. Three short duplicated sequences were found in the 45S rDNA intergenic spacer, one of which was also detected at an extra-NOR locus. The canonical plant telomeric repeat (TTTAGGG)n was also detected as very intense signals in numerous pericentromeric and interstitial loci. StoTR05_180, which showed subtelomeric distribution in Senna occidentalis, was predominantly pericentromeric in S. tora. The unusual chromosomal distribution of tandem repeats in S. tora not only enabled easy identification of individual chromosomes but also revealed the massive chromosomal rearrangements that have likely played important roles in shaping its dysploid karyotype.

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome-specific painting.

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide-based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C.maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.

Karyotype variability of the Ukrainian Mountain-Carpathian sheep breed

Abstract. The main purpose of this work was to describe the karyotype variability of the Ukrainian Mountain-Carpathian sheep breed. Cytogenetic studies were carried out on a group of 25 sheep from that breed. The cultivation of lymphocytes, preparing the cytogenetic samples, classification and registration of chromosome aberrations were held using conventional methods. It was established that the number and structure of the chromosomes of sheep of the Ukrainian Mountain-Carpathian breed correspond to the norm typical for this species of animals. Chromosomal variability of the studied population includes 19.22% of numerical and structural aberrations. By the number and structure of the chromosome set the Ukrainian Mountain-Carpathian sheep breed does not differ from other breeds of sheep. Accurate identification of individual chromosomes with routine coloring allows using cytogenetic studies in breeding practice in sheep breeding.

Molecular cytogenetic analysis reveals evolutionary relationships between polyploid Aegilops species

We applied fluorescent in situ hybridization (FISH) using (GAA)10 microsatellite repeat alone or in combination with oligo pSc119.2-1, pTa535-1, pAs1-1, 5S and 45S rDNA probes to identify individual chromosomes and produce banded karyotypes for five allopolyploid Aegilops species that were thought to possess M subgenome. Labelling patterns of GAA repeats allow identification of individual chromosomes in the studied species including diploid Ae. comosa subsp. heldreichii (2n = MhMh) and Ae. umbellulata (2n = UU); and allopolyploid Ae. biuncialis (2n = UbUbMbMb), Ae. geniculata (2n = UgUgMgMg), Ae. columnaris (2n = UcUcXcXc), Ae. neglecta (2n = UnUnXnXn) and Ae. crassa (2n = 4x = D1D1XcrXcr and 2n = 6x = D1D1XcrXcrDcrDcr). Intraspecific polymorphisms of GAA labelling patterns were observed between different genotypes in each species. FISH results showed that the X* subgenomes of Ae. columnaris and Ae. neglecta are highly similar, implying that both species originated from a common ancestor. However, these X* subgenomes were also distinct from and more heterochromatic when compared to the M* subgenomes of Ae. biuncialis and Ae. geniculata. This suggests that the X* subgenomes may not have originated from the Ae. comosa, as previously thought. Although (GAA)n bands are relatively few and weak in Ae. tauschii (DD genome), all 4x Ae. crassa (D1D1XcrXcr genome) chromosomes showed distinct GAA patterns. (GAA)10 probe alone was not sufficient for the discrimination of all the chromosomes of 6x Ae. crassa (D1D1XcrXcrDcrDcr), which needs a combination of (GAA)10 with the pTa535-1 or pAs1-1 probes.

A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin.

Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.

Comprehensively Characterizing the Cytological Features of Saccharum spontaneum by the Development of a Complete Set of Chromosome-Specific Oligo Probes.

Chromosome-specific identification is a powerful technique in the study of genome structure and evolution. However, there is no reliable cytogenetic marker to unambiguously identify each of the chromosomes in sugarcane (Saccharum spp., Poaceae), which has a complex genome with a high level of ploidy and heterozygosity. In this study, we developed a set of oligonucleotide (oligo)-based probes through bioinformatic design and massive synthetization. These probes produced a clear and bright single signal in each of the chromosomes and their eight homologous chromosomes in the ancient species Saccharum spontaneum (2n = 8x = 64). Thus, they can be used as reliable markers to robustly label each of the chromosomes in S. spontaneum. We then obtained the karyotype data and established a nomenclature based on chromosomal sizes for the eight chromosomes of the octoploid S. spontaneum. In addition, we also found that the 45S and 5S rDNAs demonstrated high copy number variations among different homologous chromosomes, indicating a rapid evolution of the highly repeated sequence after polyploidization. Our fluorescence in situ hybridization (FISH) assay also demonstrated that these probes could be used as cross-species markers between or within the genera of Sorghum and Saccharum. By comparing FISH analyses, we discovered that several chromosome rearrangement events occurred in S. spontaneum, which might have contributed to the basic chromosome number reduction from 10 in sorghum to 8 in sugarcane. Consistent identification of individual chromosomes makes molecular cytogenetic study possible in sugarcane and will facilitate fine chromosomal structure and karyotype evolution of the genus Saccharum.

Comparative Oligo-FISH Mapping: An Efficient and Powerful Methodology To Reveal Karyotypic and Chromosomal Evolution.

Developing the karyotype of a eukaryotic species relies on identification of individual chromosomes, which has been a major challenge for most nonmodel plant and animal species. We developed a novel chromosome identification system by selecting and labeling oligonucleotides (oligos) located in specific regions on every chromosome. We selected a set of 54,672 oligos (45 nt) based on single copy DNA sequences in the potato genome. These oligos generated 26 distinct FISH signals that can be used as a "bar code" or "banding pattern" to uniquely label each of the 12 chromosomes from both diploid and polyploid (4× and 6×) potato species. Remarkably, the same bar code can be used to identify the 12 homeologous chromosomes among distantly related Solanum species, including tomato and eggplant. Accurate karyotypes based on individually identified chromosomes were established in six Solanum species that have diverged for >15 MY. These six species have maintained a similar karyotype; however, modifications to the FISH signal bar code led to the discovery of two reciprocal chromosomal translocations in Solanum etuberosum and S. caripense We also validated these translocations by oligo-based chromosome painting. We demonstrate that the oligo-based FISH techniques are powerful new tools for chromosome identification and karyotyping research, especially for nonmodel plant species.

Integrated Karyotyping of Woodland Strawberry (Fragaria vesca) with Oligopaint FISH Probes

Chromosome identification is critical for many aspects of cytogenetic research. However, for Fragaria vesca, definite identification of individual chromosomes is almost impossible because of their small size and high similarity. Here, we demonstrate that bulked oligonucleotide (oligo) probes can be used as chromosome-specific DNA markers for chromosome identification in F. vesca. Oligos specific to entire pseudochromosomes in the draft genome of F. vesca were identified and synthesized as libraries. In all, we synthesized 6 oligo libraries corresponding to 6 pseudochromosomes of F. vesca. These libraries were amplified and labeled as probes for fluorescence in situ hybridization (FISH). Two rounds of multicolor FISH analysis were sequentially conducted on the same metaphase cells with each round including 3 probe libraries, which permitted simultaneous identification of all chromosomes of F. vesca. Moreover, 45S and 5S rDNA were mapped to chromosomes 1, 2, and 7, respectively. A karyotype of metaphase chromosomes was constructed, representing the first FISH-based molecular cytogenetic karyotype of F. vesca. Our study can serve as a basis for future comparative cytogenetic research through cross-species chromosome painting using bulked oligo probes and will facilitate the application of breeding technologies that rely on the identification of chromosomes in the genus Fragaria.

New Insights into Phasmatodea Chromosomes.

Currently, approximately 3000 species of stick insects are known; however, chromosome numbers, which range between 21 and 88, are known for only a few of these insects. Also, centromere banding staining (C-banding) patterns were described for fewer than 10 species, and fluorescence in situ hybridization (FISH) was applied exclusively in two Leptynia species. Interestingly, 10–25% of stick insects (Phasmatodea) are obligatory or facultative parthenogenetic. As clonal and/or bisexual reproduction can affect chromosomal evolution, stick insect karyotypes need to be studied more intensely. Chromosome preparation from embryos of five Phasmatodea species (Medauroidea extradentata, Sungaya inexpectata, Sipyloidea sipylus, Phaenopharos khaoyaiensis, and Peruphasma schultei) from four families were studied here by C-banding and FISH applying ribosomal deoxyribonucleic acid (rDNA) and telomeric repeat probes. For three species, data on chromosome numbers and structure were obtained here for the first time, i.e., S. inexpectata, P. khaoyaiensis, and P. schultei. Large C-positive regions enriched with rDNA were identified in all five studied, distantly related species. Some of these C-positive blocks were enriched for telomeric repeats, as well. Chromosomal evolution of stick insects is characterized by variations in chromosome numbers as well as transposition and amplification of repetitive DNA sequences. Here, the first steps were made towards identification of individual chromosomes in Phasmatodea.

Characterization and physical mapping of nuclear ribosomal RNA (rRNA) genes in the haploid gametophytes of Saccharina japonica (Phaeophyta)

Nuclear tandemly repeated ribosomal RNA genes (18S-5.8S-25S rDNA and 5S rDNA) have been proven to be excellent cytogenetic markers for karyotype analysis of various higher plants by using the fluorescence in situ hybridization (FISH) technique. To illustrate physical mapping of these rDNAs in brown seaweed, Saccharina japonica, chromosomes, an approximately 5400-bp transcription unit of 18S-5.8S-25S rDNA was assembled by cloning the 25S rDNA after paired-end sequencing of two screened clones from a bacterial artificial chromosome library of kelp gametophytes. In contrast to the conserved 18S-5.8S-25S rDNA and ITS1 in S. japonica, the 245-bp ITS2 was variable in sequence. The cloned 5S rDNA revealed that the 120-bp conserved coding region was separated by a diverse intergenic spacer sequence that were 250, 445, 905, or 1335 bp in length. On average, the 18S-5.8S-25S rDNA of kelp female and male gametophytes had 45 and 41 copies per haploid genome, respectively, as detected by quantitative real-time PCR, whereas the 5S rDNA had 2590 and 2648 copies, respectively. Southern hybridization with labeled probes of 18S rDNA or 5S rDNA demonstrated that kelp gametophytes possessed only one locus each of the 18S-5.8S-25S or 5S rDNAs. This was further confirmed by FISH analysis using the same labeled probes, thus illustrating that 18S-5.8S-25S rDNA is located at the intercalary region of chromosome 23, whereas 5S rDNA at the sub-telomeric region of chromosomes 27. The localization of these rDNAs using the FISH technique has facilitated the identification of individual chromosomes and karyotype analysis of this kelp.