The genomes of seven economic Caesalpinioideae trees provide insights into polyploidization history and secondary metabolite biosynthesis.

The Caesalpinioideae subfamily contains many well-known trees that are important for economic sustainability and human health, but a lack of genomic resources has hindered their breeding and utilization. Here, we present chromosome-level reference genomes for the two food and industrial trees Gleditsia sinensis (921 Mb) and Biancaea sappan (872 Mb), the three shade and ornamental trees Albizia julibrissin (705 Mb), Delonix regia (580 Mb), and Acacia confusa (566 Mb), and the two pioneer and hedgerow trees Leucaena leucocephala (1338 Mb) and Mimosa bimucronata (641 Mb). Phylogenetic inference shows that the mimosoid clade has a much higher evolutionary rate than the other clades of Caesalpinioideae. Macrosynteny comparison suggests that the fusion and breakage of an unstable chromosome are responsible for the difference in basic chromosome number (13 or 14) for Caesalpinioideae. After an ancient whole-genome duplication (WGD) shared by all Caesalpinioideae species (CWGD, ∼72.0 million years ago [MYA]), there were two recent successive WGD events, LWGD-1 (16.2-19.5 MYA) and LWGD-2 (7.1-9.5 MYA), in L. leucocephala. Thereafter, ∼40% gene loss and genome-size contraction have occurred during the diploidization process in L. leucocephala. To investigate secondary metabolites, we identified all gene copies involved in mimosine metabolism in these species and found that the abundance of mimosine biosynthesis genes in L. leucocephala largely explains its high mimosine production. We also identified the set of all potential genes involved in triterpenoid saponin biosynthesis in G. sinensis, which is more complete than that based on previous transcriptome-derived unigenes. Our results and genomic resources will facilitate biological studies of Caesalpinioideae and promote the utilization of valuable secondary metabolites.

X chromosome rearrangement associated with premature ovarian insufficiency as diagnosed by molecular cytogenetic methods: a case report and review of the literature.

BACKGROUND: Premature ovarian insufficiency (POI) is a clinical condition characterized by ovarian dysfunction in women under 40. The etiology of most POI cases remains unidentified and is believed to be multifactorial, including factors such as autoimmunity, metabolism, infection, and genetics. POI exhibits significant genetic heterogeneity, and it can result from chromosomal abnormalities and monogenic defects. CASE PRESENTATION: The study participant, a 33-year-old woman, presented with a history of irregular menstruation that commenced two years ago, progressing to prolonged menstrual episodes and eventual cessation. The participant exhibits a rearrangement of the X chromosome, characterized by heterozygosity duplication on the long arm and heterozygosity deletion on the short arm by whole exome sequencing(WES) combined with cell chromosome detection. CONCLUSIONS: This study expands the spectrum of mutations associated with POI resulting from X chromosomal abnormalities. WES-Copy number variation analysis, in conjunction with chromosome karyotype analysis and other detection techniques, can provide a more comprehensive understanding of the genetic landscape underlying complex single or multi-system diseases.

A word of caution: T-DNA-associated mutagenesis in plant reproduction research.

T-DNA transformation is prevalent in Arabidopsis research and has expanded to a broad range of crops and model plants. While major progress has been made in optimizing the Agrobacterium-mediated transformation process for various species, a variety of pitfalls associated with the T-DNA insertion may lead to the misinterpretation of T-DNA mutant analysis. Indeed, secondary mutagenesis either on the integration site or elsewhere in the genome, together with epigenetic interactions between T-DNA inserts or frequent genomic rearrangements, can be tricky to differentiate from the effect of the knockout of the gene of interest. These are mainly the case for genomic rearrangements that become balanced in filial generations without consequential phenotypical defects, which may be confusing particularly for studies that aim to investigate fertility and gametogenesis. As a cautionary note to the plant research community studying gametogenesis, we here report an overview of the consequences of T-DNA-induced secondary mutagenesis with emphasis on the genomic imbalance on gametogenesis. Additionally, we present a simple guideline to evaluate the T-DNA-mutagenized transgenic lines to decrease the risk of faulty analysis with minimal experimental effort.

Preimplantation genetic testing in couples with balanced chromosome rearrangement: a four-year period real world retrospective cohort study.

BACKGROUND: Couples with balanced chromosome rearrangement (BCR) are at high risk of recurrent miscarriages or birth defects due to chromosomally abnormal embryos. This study aimed to provide real-world evidence of the euploidy rate of blastocysts from couples with BCR using preimplantation genetic testing (PGT) and to guide pretesting genetic counselling. METHODS: A continuous four-year PGT data from couples with BCR were retrospectively analyzed. Biopsied trophectoderm cells were amplified using whole genome amplification, and next-generation sequencing was performed to detect the chromosomal numerical and segmental aberrations. Clinical data and molecular genetic testing results were analyzed and compared among the subgroups. RESULTS: A total of 1571 PGT cycles with 5942 blastocysts were performed chromosomal numerical and segmental aberrations detection during the four years. Of them, 1034 PGT cycles with 4129 blastocysts for BCR couples were included; 68.96% (713/1034) PGT cycles had transferable euploid embryos. The total euploidy rate of blastocysts in couples carrying the BCR was 35.29% (1457/4129). Couples with complex BCR had euploid blastocyst rates similar to those of couples with non-complex BCR (46.15% vs. 35.18%, P > 0.05). Chromosome inversion had the highest chance of obtaining a euploid blastocyst (57.27%), followed by Robertsonian translocation (RobT) (46.06%), and the lowest in reciprocal translocation (RecT) (30.11%) (P < 0.05). Couples with males carrying RobT had higher rates of euploid embryo both in each PGT cycles and total blastocysts than female RobT carriers did, despite the female age in male RobT is significant older than those with female RobT (P < 0.05). The proportions of non-carrier embryos were 52.78% (95/180) and 47.06% (40/85) in euploid blastocysts from couples with RecT and RobT, respectively (P > 0.05). RecT had the highest proportion of blastocysts with translocated chromosome-associated abnormalities (74.23%, 1527/2057), followed by RobT (54.60%, 273/500) and inversion (30.85%, 29/94) (P < 0.05). CONCLUSIONS: In couples carrying BCR, the total euploidy rate of blastocysts was 35.29%, with the highest in inversion, followed by RobT and RecT. Even in couples carrying complex BCR, the probability of having a transferable blastocyst was 46.15%. Among the euploid blastocysts, the non-carrier ratios in RecT and RobT were 52.78% and 47.06%, respectively. RecT had the highest proportion of blastocysts with translocated chromosome-associated abnormalities.

Karyotype Variability in Wild Narcissus poeticus L. Populations from Different Environmental Conditions in the Dinaric Alps.

Narcissus poeticus L. (Amaryllidaceae), a facultative serpentinophyte, is a highly variable species and particularly important ancestor of cultivated daffodils, but is rarely studied in field populations. This study, based on natural populations in the Balkans, focused on karyotype variability, genome size, ploidy and the presence of B chromosomes. Thirteen native populations from different environmental and soil conditions were collected and analyzed using flow cytometry to estimate nuclear genome size, fluorescence in situ hybridization (FISH) for physical mapping of rDNA, fluorochrome labeling (chromomycin and Hoechst) for heterochromatin organization and silver nitrate staining of nucleoli for determining rRNA gene activity. The organization of rDNA and natural triploids is reported here for the first time. The presence of individuals with B chromosomes (in 9/13 populations) and chromosomal rearrangements was also detected. The observed B chromosome showed three different morphotypes. The most frequent submetacentric type showed four different patterns, mainly with active ribosomal genes. The results obtained show that N. poeticus has a dynamic genome with variable genome size due to the presence of polyploidy, B chromosomes and chromosomal rearrangements. It is hypothesized that the observed changes reflect the response of the genome to different environmental conditions, where individuals with B chromosomes appear to have certain adaptive advantages.

Evaluation of genetic risk of apparently balanced chromosomal rearrangement carriers by breakpoint characterization.

PURPOSE: To report genetic characteristics and associated risk of chromosomal breaks due to chromosomal rearrangements in large samples. METHODS: MicroSeq, a technique that combines chromosome microdissection and next-generation sequencing, was used to identify chromosomal breakpoints. Long-range PCR and Sanger sequencing were used to precisely characterize 100 breakpoints in 50 ABCR carriers. RESULTS: In addition to the recurrent regions of balanced rearrangement breaks in 8q24.13, 11q11.23, and 22q11.21 that had been documented, we have discovered a 10-Mb region of 12q24.13-q24.3 that could potentially be a sparse region of balanced rearrangement breaks. We found that 898 breakpoints caused gene disruption and a total of 188 breakpoints interrupted genes recorded in OMIM. The percentage of breakpoints that disrupted autosomal dominant genes recorded in OMIM was 25.53% (48/188). Fifty-four of the precisely characterized breakpoints had 1-8-bp microhomologous sequences. CONCLUSION: Our findings provide a reference for the evaluation of the pathogenicity of mutations in related genes that cause protein truncation in clinical practice. According to the characteristics of breakpoints, non-homologous end joining and microhomology-mediated break-induced replication may be the main mechanism for ABCRs formation.

Haplotype-resolved chromosome-level genome of hexaploid Jerusalem artichoke provides insights into its origin, evolution, and inulin metabolism.

Jerusalem artichoke (Helianthus tuberosus) is a global multifunctional crop. It has wide applications in the food, health, feed, and biofuel industries and in ecological protection; it also serves as a germplasm pool for breeding of the global oil crop common sunflower (Helianthus annuus). However, biological studies of Jerusalem artichoke have been hindered by a lack of genome sequences, and its high polyploidy and large genome size have posed challenges to genome assembly. Here, we report a 21-Gb chromosome-level assembly of the hexaploid Jerusalem artichoke genome, which comprises 17 homologous groups, each with 6 pseudochromosomes. We found multiple large-scale chromosome rearrangements between Jerusalem artichoke and common sunflower, and our results show that the hexaploid genome of Jerusalem artichoke was formed by a hybridization event between a tetraploid and a diploid Helianthus species, followed by chromosome doubling of the hybrid, which occurred approximately 2 million years ago. Moreover, we identified more copies of actively expressed genes involved in inulin metabolism and showed that these genes may still be undergoing loss of function or sub- or neofunctionalization. These genomic resources will promote further biological studies, breeding improvement, and industrial utilization of Helianthus crops.

Development of a Set of Wheat-Rye Derivative Lines from Hexaploid Triticale with Complex Chromosomal Rearrangements to Improve Disease Resistance, Agronomic and Quality Traits of Wheat.

An elite hexaploid triticale Yukuri from Australia was used as a bridge for transferring valuable genes from Secale cereale L. into common wheat for enriching the genetic variability of cultivated wheat. Non-denaturing-fluorescence in situ hybridization (ND-FISH) identified that Yukuri was a secondary triticale with a complete set of rye chromosomes and a 6D(6A) substitution. Seed protein electrophoresis showed that Yukuri had a unique composition of glutenin subunits. A set of Yukuri-derived wheat-rye introgression lines were created from a Yukuri x wheat population, and all lines were identified by ND-FISH with multiple probes and validated by diagnostic molecular marker analysis. A total of 59 wheat-rye introgression lines including modified chromosome structural variations of wheat, and new complex recombinant chromosomes of rye were detected through ND-FISH and Oligo-FISH painting based on oligonucleotide pools derived from wheat-barley genome collinear regions. Wheat lines carrying the 1R chromosome from Yukuri displayed resistance to both stripe rust and powdery mildew, while the lines carrying the 3RL and 7RL chromosome arms showed stripe rust resistance. The chromosome 1R-derived lines were found to exhibit a significant effect on most of the dough-related parameters, and chromosome 5R was clearly associated with increased grain weight. The development of the wheat-rye cytogenetic stocks carrying disease resistances and superior agronomic traits, as well as the molecular markers and FISH probes will promote the introgression of abundant variation from rye into wheat improvement programs.

Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Case Report: Decrypting an interchromosomal insertion associated with Marfan's syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants.

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient's neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.

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.

Lepidopteran Synteny Units reveal deep chromosomal conservation in butterflies and moths.

DNA is compacted into individual particles or chromosomes that form the basic units of inheritance. However, different animals and plants have widely different numbers of chromosomes. This means that we cannot readily tell which chromosomes are related to which. Here, we describe a simple technique that looks at the similarity of genes on each chromosome and thus gives us a true picture of their homology or similarity through evolutionary time. We use this new system to look at the chromosomes of butterflies and moths or Lepidoptera. We term the associated synteny units, Lepidopteran Synteny Units (LSUs). Using a sample of butterfly and moth genomes from across evolutionary time, we show that LSUs form a simple and reliable method of tracing chromosomal homology back through time. Surprisingly, this technique reveals that butterfly and moth chromosomes show conserved blocks dating back to their sister group the Trichoptera. As Lepidoptera have holocentric chromosomes, it will be interesting to see if similar levels of synteny are shown in groups of animals with monocentric chromosomes. The ability to define homology via LSU analysis makes it considerably easier to approach many questions in chromosomal evolution.

Chromosome translocation affects multiple phenotypes, causes genome-wide dysregulation of gene expression, and remodels metabolome in hexaploid wheat.

Chromosomal rearrangements (CRs) may occur in newly formed polyploids due to compromised meiotic fidelity. Moreover, CRs can be more readily tolerated in polyploids allowing their longer-term retention and hence potential spreading/fixation within a lineage. The direct functional consequences of CRs in plant polyploids remain unexplored. Here, we identified a heterozygous individual from a synthetic allohexaploid wheat in which the terminal parts of the long-arms of chromosomes 2D (approximately 193 Mb) and 4A (approximately 167 Mb) were reciprocally translocated. Five homogeneous translocation lines including both unbalanced and balanced types were developed by selfing fertilization of the founder mutant (RT [2DL; 4AL]-ter/1, reciprocal translocation). We investigated impacts of these translocations on phenotype, genome-wide gene expression and metabolome. We find that, compared with sibling wild-type, CRs in the form of both unbalanced and balanced translocations induced substantial changes of gene expression primarily via trans-regulation in the nascent allopolyploid wheat. The CRs also manifested clear phenotypic and metabolic consequences. In particular, the genetically balanced, stable reciprocal translocations lines showed immediate enhanced reproductive fitness relative to wild type. Our results underscore the profound impact of CRs on gene expression in nascent allopolyploids with wide-ranging phenotypic and metabolic consequences, suggesting CRs are an important source of genetic variation that can be exploited for crop breeding.

Genomic rearrangements and evolutionary changes in 3D chromatin topologies in the cotton tribe (Gossypieae).

BACKGROUND: Analysis of the relationship between chromosomal structural variation (synteny breaks) and 3D-chromatin architectural changes among closely related species has the potential to reveal causes and correlates between chromosomal change and chromatin remodeling. Of note, contrary to extensive studies in animal species, the pace and pattern of chromatin architectural changes following the speciation of plants remain unexplored; moreover, there is little exploration of the occurrence of synteny breaks in the context of multiple genome topological hierarchies within the same model species. RESULTS: Here we used Hi-C and epigenomic analyses to characterize and compare the profiles of hierarchical chromatin architectural features in representative species of the cotton tribe (Gossypieae), including Gossypium arboreum, Gossypium raimondii, and Gossypioides kirkii, which differ with respect to chromosome rearrangements. We found that (i) overall chromatin architectural territories were preserved in Gossypioides and Gossypium, which was reflected in their similar intra-chromosomal contact patterns and spatial chromosomal distributions; (ii) the non-random preferential occurrence of synteny breaks in A compartment significantly associate with the B-to-A compartment switch in syntenic blocks flanking synteny breaks; (iii) synteny changes co-localize with open-chromatin boundaries of topologically associating domains, while TAD stabilization has a greater influence on regulating orthologous expression divergence than do rearrangements; and (iv) rearranged chromosome segments largely maintain ancestral in-cis interactions. CONCLUSIONS: Our findings provide insights into the non-random occurrence of epigenomic remodeling relative to the genomic landscape and its evolutionary and functional connections to alterations of hierarchical chromatin architecture, on a known evolutionary timescale.

Higher chromosomal abnormality rate in blastocysts from a subset of patients with pericentric inversion (Inv) 1 variant.

The purpose of this study was to evaluate the incidence of unbalanced chromosome rearrangement in blastocyst-stage embryos from carriers of pericentric inversion of chromosome 1 (PEI-1). A total of 98 embryos from 22 PEI-1 carriers were tested for unbalanced rearrangements, originating from inversion carriers, and overall aneuploidy. Logistic regression analysis indicated that the ratio of inverted segment size to chromosome length was a statistically significant risk factor for unbalanced chromosome rearrangement from PEI-1 carriers (p = 0.003). The optimal cut-off values to predict the risk of unbalanced chromosome rearrangement was 36%, with the incidence being 2.0% in the <36% group and 32.7% in the ≥36% group. The unbalanced embryo rate was 24.4% in male carriers compared to 12.3% in female carriers. Inter-chromosomal effect analysis was performed using 98 blastocysts from PEI-1 carriers and 116 blastocysts from age-matched controls. PEI-1 carriers had similar sporadic aneuploidy rates compared to those of age-matched controls at 32.7 vs. 31.9%, respectively. In conclusion, the risk of unbalanced chromosome rearrangement is affected by inverted segment size in PEI-1 carriers.

Single-gene FISH maps and major chromosomal rearrangements in Elymus sibiricus and E. nutans.

BACKGROUND: Chromosomal variations have been revealed in both E. sibiricus and E. nutans, but chromosomal structural variations, such as intra-genome translocations and inversions, are still not recognized due to the cytological limitations of previous studies. Furthermore, the syntenic relationship between both species and wheat chromosomes remains unknown. RESULTS: Fifty-nine single-gene fluorescence in situ hybridization (FISH) probes, including 22 single-gene probes previously mapped on wheat chromosomes and other newly developed probes from the cDNA of Elymus species, were used to characterize the chromosome homoeologous relationship and collinearity of both E. sibiricus and E. nutans with those of wheat. Eight species-specific chromosomal rearrangements (CRs) were exclusively identified in E. sibiricus, including five pericentric inversions in 1H, 2H, 3H, 6H and 2St; one possible pericentric inversion in 5St; one paracentric inversion in 4St; and one reciprocal 4H/6H translocation. Five species-specific CRs were identified in E. nutans, including one possible pericentric inversion in 2Y, three possible pericentric multiple-inversions in 1H, 2H and 4Y, and one reciprocal 4Y/5Y translocation. Polymorphic CRs were detected in three of the six materials in E. sibiricus, which were mainly represented by inter-genomic translocations. More polymorphic CRs were identified in E. nutans, including duplication and insertion, deletion, pericentric inversion, paracentric inversion, and intra- or inter-genomic translocation in different chromosomes. CONCLUSIONS: The study first identified the cross-species homoeology and the syntenic relationship between E. sibiricus, E. nutans and wheat chromosomes. There are distinct different species-specific CRs between E. sibiricus and E. nutans, which may be due to their different polyploidy processes. The frequencies of intra-species polymorphic CRs in E. nutans were higher than that in E. sibiricus. To conclude, the results provide new insights into genome structure and evolution and will facilitate the utilization of germplasm diversity in both E. sibiricus and E. nutans.

Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line.

BACKGROUND: Karyotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line. RESULTS: We obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling "chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH. CONCLUSIONS: In the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure-function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

A Cre-LoxP-based approach for combinatorial chromosome rearrangements in human HAP1 cells.

Alterations of human karyotype caused by chromosomal rearrangements are often associated with considerable phenotypic effects. Studying molecular mechanisms underlying these effects requires an efficient and scalable experimental model. Here, we propose a Cre-LoxP-based approach for the generation of combinatorial diversity of chromosomal rearrangements. We demonstrate that using the developed system, both intra- and inter-chromosomal rearrangements can be induced in the human haploid HAP1 cells, although the latter is significantly less effective. The obtained genetically modified HAP1 cell line can be used to dissect genomic effects associated with intra-chromosomal structural variations.

Chromosomal segregation analysis and HOST-based sperm selection in a complex reciprocal translocation carrier.

INTRODUCTION: Complex chromosomal rearrangements (CCRs) involve two or more chromosomes and at least three breakpoints. Due to their complexity, they are associated with a high number of unbalanced gametes, whose fertilization is often incompatible with viable fetal development. Preimplantation genetic diagnosis (PGD) is usually offered to those patients and typically shows modest results considering the high number of unbalanced embryos. We previously showed that a sperm selection process using the hypo-osmotic swelling test (HOST) allows for an 83% reduction in the proportion of unbalanced spermatozoa (US) in male rearrangements carriers. This is the first report of the use of this procedure in a CCR carrier. CASE DESCRIPTION: We report on the case of a 36-year-old male t(4;7;14)(q12;p21;q11.2) carrier who presented to our center for infertility. Sperm fluorescent in situ hybridization showed an 88% proportion of unbalanced spermatozoa. After hypo-osmotic incubation and selection of spermatozoa with a specific flagellar conformation, the proportion of unbalanced spermatozoa dropped to 15%. DISCUSSION: In the present case, we show that it is possible to select chromosomally balanced prior to in vitro fertilization in male CCR carriers. This technique has the potential of increasing the proportion of euploid embryos and therefore the chances of healthy pregnancy and birth.

Hi-C Analysis to Identify Genome-Wide Chromatin Structural Aberration in Cancer.

Hi-C is a method that analyzes genome-wide chromatin structure using next-generation sequencer. Chromatin structure is crucial for regulating transcription or replication, and Hi-C has revealed the hierarchical chromatin structures, such as loop, domain , and compartment structures. Aberrant alteration of these structures causes disease, and a number of structural aberrations in cancer cells have been reported recently. Besides, Hi-C can identify chromosome rearrangements that frequently occurred in cancer. Therefore, Hi-C is a powerful technique to analyze epigenomic and genomic aberrations in tumorigenesis. Here we will introduce the basic protocol of Hi-C in experimental and analytical aspects.