Chromosomal Hybridization Research Articles

Frequent numerical and structural chromosome changes in early generations of synthetic hexaploid wheat.

Modern hexaploid wheat (Triticum aestivum L.; AABBDD) has evolved from a hybrid of tetraploid wheat (closely related to Triticum turgidum L. ssp. durum (Desf.) Husn., AABB) and goatgrass (Aegilops tauschii Coss., DD). Variations in chromosome structure and ploidy have played important roles in wheat evolution. How these variations occur and their role in expanding the genetic diversity of modern wheat remain largely unknown. Synthetic hexaploid wheat (SHW) can be used to investigate chromosome variations that occur during the early generations of existence. SHW lines derived by crossing durum wheat 'Langdon' with 12 Ae. tauschii accessions were analyzed using oligonucleotide probe multiplex fluorescence insitu hybridization (FISH) of metaphase chromosomes and SNP markers. Cluster analysis based on SNP markers categorizes them into three groups. Among 702 plants from the S8 and S9 generations, 415 (59.12%) carried chromosome variations involving all 21 chromosomes, but with different frequencies for each chromosome and sub-genome. Total chromosome variation frequencies varied between lines, but there was no significant difference among the three groups. The non-random chromosome variations in the SHW lines detected in this study may indicate that similar variations occurred in the early stages of wheat polyploidization and played important roles in wheat evolution.

Cytogenetic evidence supports Avena insularis being closely related to hexaploid oats.

Cytogenetic observations, phylogenetic studies and genome analysis using high-density genetic markers have suggested a tetraploid Avena species carrying the C and D genomes (formerly C and A) to be the donor of all hexaploid oats (AACCDD). However, controversy surrounds which of the three extant CCDD tetraploid species—A. insularis, A. magna and A. murphyi—is most closely related to hexaploid oats. The present work describes a comparative karyotype analysis of these three CCDD tetraploid species and two hexaploid species, A. sativa and A. byzantina. This involved the use of FISH with six simple sequence repeats (SSRs) with the motifs CT, AAC, AAG, ACG, ATC and ACT, two repeated ribosomal sequences, and C genome-specific repetitive DNA. The hybridization pattern of A. insularis with oligonucleotide (AC)10 was also determined and compared with those previously published for A. sativa and A. byzantina. Significant differences in the 5S sites and SSR hybridization patterns of A. murphyi compared to the other CCDD species rule out its being directly involved in the origin of the hexaploids. In contrast, the repetitive and SSR hybridization patterns shown by the D genome chromosomes, and by most of the C genome chromosomes of A. magna and A. insularis, can be equated with the corresponding chromosomes of the hexaploids. Several chromosome hybridization signals seen for A. insularis, but not for A. magna, were shared with the hexaploid oats species, especially with A. byzantina. These diagnostic signals add weight to the idea that the extant A. insularis, or a direct ancestor of it, is the most closely related progenitor of hexaploid oats. The similarity of the chromosome hybridization patterns of the hexaploids and CCDD tetraploids was taken as being indicative of homology. A common chromosome nomenclature for CCDD species based on that of the hexaploid species is proposed.

An integrated job shop scheduling and assembly sequence planning approach for discrete manufacturing

This paper proposes an integrated job shop scheduling and assembly sequence planning (IJSSASP) approach for discrete manufacturing, enabling the part processing sequence and assembly sequence to be optimized simultaneously. The optimization objectives are to minimize the total production completion time and the total inventory time of parts during production. The interaction effects between the job shop schedule and the assembly sequence plan in discrete manufacturing are analyzed, and the mathematical models including the objective functions and the constraints are established for IJSSASP. Based on the above, a non-dominated sorting genetic algorithm-II (NSGA-Ⅱ) with a hybrid chromosome coding mechanism is applied to solve the IJSSASP problem. Through the case studies and comparison tests for different scale problems, the proposed IJSSASP approach is verified to be able to improve the production efficiency and save the manufacturing cost of the discrete manufacturing enterprise more effectively.

Influence of the large-Z effect during contact between butterfly sister species.

Recently diverged butterfly populations in North America have been found to exhibit high levels of divergence on the Z chromosome relative to autosomes, as measured by fixation index, Fst. The pattern of divergence appears to result from accumulation of incompatible alleles, obstructing introgression on the Z chromosome in hybrids (i.e., the large‐Z effect); however, it is unknown whether this mechanism is sufficient to explain the data. Here, we simulate the effects of hybrid incompatibility on interbreeding butterfly populations using a model in which populations accumulate cross‐incompatible alleles in allopatry prior to contact. We compute statistics for introgression and population divergence during contact between model populations and compare our results to those for 15 pairs of butterfly species interbreeding along a suture zone in central Texas. Time scales for allopatry and contact in the model are scaled to glacial and interglacial periods during which real populations evolved in isolation and contact. We find that the data for butterflies are explained well by an otherwise neutral model under slow fusion conditions. In particular, levels of divergence on the Z chromosome increase when interacting clusters of genes are closely linked, consistent with clusters of functionally related genes in butterfly genomes.

Sex chromosomes in meiotic, hemiclonal, clonal and polyploid hybrid vertebrates: along the 'extended speciation continuum'.

We review knowledge about the roles of sex chromosomes in vertebrate hybridization and speciation, exploring a gradient of divergences with increasing reproductive isolation (speciation continuum). Under early divergence, well-differentiated sex chromosomes in meiotic hybrids may cause Haldane-effects and introgress less easily than autosomes. Undifferentiated sex chromosomes are more susceptible to introgression and form multiple (or new) sex chromosome systems with hardly predictable dominance hierarchies. Under increased divergence, most vertebrates reach complete intrinsic reproductive isolation. Slightly earlier, some hybrids (linked in ‘the extended speciation continuum') exhibit aberrant gametogenesis, leading towards female clonality. This facilitates the evolution of various allodiploid and allopolyploid clonal (‘asexual’) hybrid vertebrates, where ‘asexuality' might be a form of intrinsic reproductive isolation. A comprehensive list of ‘asexual' hybrid vertebrates shows that they all evolved from parents with divergences that were greater than at the intraspecific level (K2P-distances of greater than 5–22% based on mtDNA). These ‘asexual' taxa inherited genetic sex determination by mostly undifferentiated sex chromosomes. Among the few known sex-determining systems in hybrid ‘asexuals', female heterogamety (ZW) occurred about twice as often as male heterogamety (XY). We hypothesize that pre-/meiotic aberrations in all-female ZW-hybrids present Haldane-effects promoting their evolution. Understanding the preconditions to produce various clonal or meiotic allopolyploids appears crucial for insights into the evolution of sex, ‘asexuality' and polyploidy.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Goyal-Naqvi Syndrome (Concurrent Trisomy 10p and Terminal 14q Deletion): A Review of the Literature.

Goyal-Naqvi syndrome (GNS) is a newly documented clinical entity that comprises trisomy 10p and terminal 14q deletion, though trisomy 10p and terminal 14q deletion have been discovered as distinct conditions in 1974 and 1997, respectively. Nevertheless, to date, the total number of reported cases of each of these conditions is estimated to be in double digits. Both manifest as a constellation of features like craniofacial dysmorphism, hypotonia, intellectual impairment and global developmental delay. Characteristic facies include protruded forehead, hypertelorism, epicanthic folds, down slanting palpebral fissures, flat nasal bridge, long philtrum, thin upper lip, carp-shaped mouth, retro-micrognathia and low set ears. Besides, trisomy 10p is strikingly associated with clinodactyly and camptodactyly which aids in clinical diagnosis, apart from other musculoskeletal deformities like hip dysplasia and pes planus. Intersex conditions have been found to commonly co-exist. As other systems also display involvement frequently, trisomy 10p is a discernible multiple congenital anomalies/mental retardation (MCA/MR) syndrome. On the other hand, with terminal 14q deletion, increased risk of certain types of cancer was predicted as specific tumor suppressor genes are lost in the deletion and thus, screening was recommended. Genetic workup using techniques like fluorescence in situ hybridization (FISH), spectral karyotyping (SKY) and chromosomal microarray-based comparative genomic hybridization (CGH) was found to be helpful in diagnosis of trisomy 10p and 14q deletion. Prenatal diagnosis of these conditions has been well documented too. Intrauterine growth retardation has been observed to be related to trisomy 10p. There is a paucity of literature on the management of children diagnosed with trisomy 10p or with terminal 14q deletion. Although management of a child diagnosed with concomitant occurrence of trisomy 10p and terminal 14q deletion by a multidisciplinary approach emphasizing physiotherapeutic intervention has shown remarkable improvement in motor skills, the care of children diagnosed with these genetic aberrations needs further investigation. Documentation of more such cases will help to expand phenotypic spectrum for early identification and to delineate natural history for a life span approach. Early identification and intervention facilitate tapping of the maximum neuroplastic potential for better neurodevelopmental outcomes. We present a review of current literature on this novel syndrome to identify gaps in knowledge to build future research.

Satellitome Analysis of Rhodnius prolixus, One of the Main Chagas Disease Vector Species.

The triatomine Rhodnius prolixus is the main vector of Chagas disease in countries such as Colombia and Venezuela, and the first kissing bug whose genome has been sequenced and assembled. In the repetitive genome fraction (repeatome) of this species, the transposable elements represented 19% of R. prolixus genome, being mostly DNA transposon (Class II elements). However, scarce information has been published regarding another important repeated DNA fraction, the satellite DNA (satDNA), or satellitome. Here, we offer, for the first time, extended data about satellite DNA families in the R. prolixus genome using bioinformatics pipeline based on low-coverage sequencing data. The satellitome of R. prolixus represents 8% of the total genome and it is composed by 39 satDNA families, including four satDNA families that are shared with Triatoma infestans, as well as telomeric (TTAGG)n and (GATA)n repeats, also present in the T. infestans genome. Only three of them exceed 1% of the genome. Chromosomal hybridization with these satDNA probes showed dispersed signals over the euchromatin of all chromosomes, both in autosomes and sex chromosomes. Moreover, clustering analysis revealed that most abundant satDNA families configured several superclusters, indicating that R. prolixus satellitome is complex and that the four most abundant satDNA families are composed by different subfamilies. Additionally, transcription of satDNA families was analyzed in different tissues, showing that 33 out of 39 satDNA families are transcribed in four different patterns of expression across samples.

DOP64 Endoscopically injected allogeneic mesenchymal stromal cells alter the mucosal immune cell compartment in patients with ulcerative proctitis

Abstract Background Local mesenchymal stromal cell (MSC)-therapy is approved for the treatment of Crohn’s disease-associated perianal fistulas. However, little is known about the working mechanism of local MSC-therapy. For the first time we evaluated engraftment and immunoregulatory effects of local MSC-therapy in patients with refractory proctitis. To do so, we analyzed biopsies and serum from patients with ulcerative proctitis before and after treatment with endoscopically injected MSCs in a phase IIa clinical trial (EudraCT number 2017-003524-75). Methods Thirteen therapy-refractory ulcerative proctitis patients were endoscopically injected bone marrow-derived allogeneic MSCs from healthy donors. Clinical efficacy was evaluated by the endoscopic and full Mayo score. Engraftment of the MSCs was investigated using fluorescence in-situ hybridization (FISH) of sex chromosomes on post-treatment biopsies. The presence of anti-HLA-antibodies against the MSC-donor was determined in the serum. Changes in immune cell subsets were evaluated using cytometry-by-time-of-flight (CyTOF) analysis. Results Thirteen patients with an endoscopic Mayo score of 2 (n=3) or 3 (n=10) of the rectum were treated with local MSC-therapy. Although complete remission was not achieved, full Mayo score was improved at week 6 (median 8 [IQR 6–10]) compared to baseline (median 11 [IQR 9.5–12]) (p=0.001). Preliminary data using FISH on the Y-chromosome, indicated the presence of MSCs in the rectum biopsies of female patients treated with male donor derived-MSCs at week 6. At baseline, HLA-antibodies were present in four patients. Six weeks after local injection of the MSCs, two out of thirteen patients developed new class I and II HLA-antibodies against the MSCs. Interestingly, in two patients pre-existing HLA-antibodies showed increased/boosted levels after local MSC-therapy, while one additionally developed new HLA-antibodies. CyTOF analysis of inflamed rectal biopsies 6 weeks after MSC treatment revealed significantly increased frequencies of several myeloid subsets (i.e. CD11b+CD14+CCR7+/-CD127+CD25+HLADR+ and CD14+HLA-DR-CD123-CCR7+) and a subset of CD4+ memory T cells with a more exhausted/regulated phenotype (PD-1+TIGIT+CD69+CD38+CD69). Conclusion Local MSC-therapy in patients with refractory proctitis changed the rectal immune profile characterised by a significant increase in a subset of effector memory CD4+ cells and several myeloid subsets, which might be associated with immune modulation. These results provide the basis for future studies on the mechanism of action of MSCs on rectal mucosa. New anti-HLA class antibodies developed in 2/13 patients after local administration. Whether these latter results have consequences for MSC-donor selection deserves further study.

Molecular characterization of a carbapenem-resistant Enterobacter hormaechei ssp. xiangfangensis co-harbouring blaNDM-1 and a chromosomally encoded phage-linked blaCTX-M-15 genes.

Enterobacter cloacae complex (ECC) members are rapidly emerging as successful nosocomial pathogens, especially, with the emergence of carbapenem-resistant clones. In this study, we performed a comprehensive molecular characterization of a carbapenem-resistant E. hormaechei ssp. xiangfangensis LAU_ENC1. hsp60 and average nucleotide identity (ANI) were used for its identification. The repertoire of resistance genes and phage content were analyzed. Plasmid sequences were extracted and compared to closest references. The isolate LAU_ENC1 was identified as an E. hormaechei ssp. xiangfangensis and belonged to ST-114A sub-cluster. blaNDM-1, blaCTX-M-15, blaOXA-1, and blaACT-16 genes were detected as β-lactam resistance determinants. A chromosomal hybrid intact phage, Enterobacter phage LAU1, with blaCTX-M-15 integrated in its direct vicinity within an ISEcp1 - blaCTX-M-15 - wbuC - ∆Tn2 rare cassette was detected. blaNDM-1 was integrated within a novel IncFII conjugative plasmid, pLAU_ENC1, through an IS3000- ΔISAba125-blaNDM-1-bleMBL-//-Tn5403 cassette. To our knowledge, this is the first report of a multi-drug resistant (MDR) E. hormaechei ssp. xiangfangensis carrying a blaCTX-M-15 integrated within the proximity of a provirus chromosomal region. Treatment options for MDR ECC members are becoming scarce, thus warranting an increased monitoring of the dissemination of these pathogens in clinical settings.

Maternal karyogene and cytoplasmic genotype affect the induction efficiency of doubled haploid inducer in Brassica napus

BackgroundArtificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far.ResultsIn this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration.ConclusionThe induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.

Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer.

ObjectiveHematogenous metastasis is essential for the progression of ovarian cancer (OC), and circulating tumor cells (CTCs) are part of the metastatic cascade. However, the detection rate of CTC is low due to the use of less sensitive detection methods. Therefore, this study aimed to detect CTCs and circulating tumorigenic endothelial cells (CTECs) in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization (SE-iFISH). MethodsWe enrolled a total of 56 subjects, including 20 OC patients and 36 ovarian benign tumor patients. CTCs and CTECs were captured by subtraction enrichment (SE) and counted and classified according to immunofluorescence staining of tumor markers (TMs) carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) combined with fluorescence in situ hybridization (iFISH) of chromosome 8 (Chr8) aneuploidy. The diagnostic value and subtype characteristics of CTCs and CTECs were investigated. ResultsThe detection rate of CTCs by SE-iFISH was high. Compared with CA125 and HE4, Chr8 aneuploidy was the major identification feature of CTC. CTC counts in OC were statistically higher than those in benign groups. CTC and CTEC with ≥pentaploidy were detected in both groups, illustrating the poor diagnostic value of CTC or CTEC. Distributions of triploid and tetraploid CTC subtypes were significantly different, and combined detection of triploid and tetraploid CTCs showed the best diagnostic value. In contrast, the distribution of CTECs in the OC and benign groups had no statistically significant difference. Small CTCs accounted for over 1/3 of the total CTC count. We also found that small CTCs and CTECs primarily comprised triploid cells, while large CTCs and CTECs mainly comprised pentaploidy and beyond.ConclusionsThe application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC. Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Circulating Tumor Cells Correlate With Prognosis in Head and Neck Squamous Cell Carcinoma.

Background:To investigate the relationship of circulating tumor cells (CTCs) and the clinical characteristic parameters and prognosis in patients with head and neck squamous cell carcinoma (HNSCC).Methods:The retrospective clinical study included 95 patients with HNSCC who after surgery in Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine between December 2015 and December 2016. All patients were followed up for survival until the end of June 2019. The CTCs detection was performed by negative enrichment (NE) immunofluorescence-in situ hybridization (im-FISH) of chromosome 8.Results:Patients with higher CTCs counts are associated with a worse prognosis with an area under the receiver operator characteristic (ROC) curve of 0.756 [95% confidence interval (CI) 0.640-0.872, p = 0.001]. The CTCs-positive rate of HNSCC patients was 58.9% (56/95) by using the cut-point of 3. Both the chi-square test and binary logistic regression analysis showed that the N stage and clinical stage were significantly associated with CTCs-positive in patients with HNSCC (p < 0.05). Further Non-parametric test analysis indicated that more CTCs counts were detected in late N and clinical stages patients (p < 0.001). The Kaplan-Meier survival analysis indicated that CTCs-positive were correlated with shorter progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p = 0.001). Further, the CTCs-positive was an independent prognostic factor for PFS and OS according to the Cox multivariate regression analysis (p < 0.05).Conclusion:More CTCs were associated with N stage, clinical stage, poor prognosis in patients with HNSCC, which might be used as a prognostic biomarker.

Morphological markers with a triploid set of chromosomes by breeding of apple tree at the polyploid level

The results many years of research in the conditions of the Altai low mountains are given the assessment of hybrid apple populations from heterploid crosses according to the characteristics: resistance to scab, degree of culture, growth strength, stem thickness, leaf thickness, leaf index, morphometry of the graft and stipules, ploidy. The correlation was established by the studied indications with the triploid set of chromosomes of hybrids from heteroploidy’s crosses. It is shown that the cytological assessment of karyotypes selected for a complex of morphological features is required in a significantly smaller volume, and varieties of siberian selection are capable for partial formation of germ sacs with a diploid set of chromosomes. Morphological markers for apple tree selection at the polyploid level were determined by identifying seedlings in the juvenile stage with a triploid set of chromosomes inside a hybrid population. The application of morphological markers makes it possible to identify from 14 to 100% of seedlings with a triploid set of chromosomes in dependence on the original tetraploid form.

Visualizing Meiotic Chromosome Pairing and Segregation in Interspecific Hybrids of Rice by Genomic in situ Hybridization

Meiotic disturbances in F1 hybrids and their progenies are still major problems in wide hybridization. To investigate the genome affinity reflected in chromosome pairing and segregation, we studied chromosome behaviors during meiosis in two interspecific F1 hybrids [O. minuta×O. australiensis (Om×Oa, BCE genome) and Oa×O. ridleyi (Or, EHJ genome)] by using both traditional staining methods and genomic in situ hybridization (GISH). GISH analysis has been successfully performed on mitotic chromosomes to distinguish different Oryza genomes, but relatively fewer systematic analyses of meiotic chromosomes of interspecific hybrids have been reported. In the hybrids, highly irregular chromosome behaviors through meiosis resulted in producing microspores with unbalanced genome. At diakinesis of these two hybrids, most chromosomes present as univalent, with low frequency as bivalents and occasionally as trivalents. In a pollen mother cell, 2 to 8 bivalents and 0 to 4 trivalents were observed in the hybrid Oa×Or, and 1 to 8 bivalents and 0 to 5 trivalents were observed in the hybrid Om×Oa. GISH results indicated that 51.52% bivalents in Oa×Or and 79.65% bivalents in Om×Oa involved allosyndetic association, which indicates that recombination and introgression should be possible if viable backcrosses can be recovered even from triploid hybrids. In this study, we revealed that the meiotic disturbance due to low affinities between parental genomes is the major reason for the sterility of these two triploid interspecific hybrids. The two hybrids showing vigor in reproductive growth are potential genetic resources in future breeding programs. A better understanding of genomic affinities between these distant Oryza species can facilitate planning an effective breeding program by using wide hybridization, and efficient and routine GISH analysis is helpful to monitor alien introgression in the process.

Interleukin-4 and Interleukin-5 Map to Human Chromosome 5 in a Region Encoding Growth Factors and Receptors and Are Deleted in Myeloid Leukemias With a del(5q)

Interleukin-4 (IL-4) is a potent mediator of growth and differentiation of cells of several hematopoietic lineages. Interleukin-5 (IL-5) is a lineage-specific hematopoietic growth factor that stimulates the production of eosinophils and eosinophil colonies from normal human bone marrow cells. By using somatic cell hybrids and in situ chromosomal hybridization, we localized the IL-4 and IL-5 genes to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, the IL-4 and IL-5 genes were found to be deleted in the 5q- chromosome of four patients with refractory anemia (RA) or therapy-related acute nonlymphocytic leukemia (t-ANLL), who had a del(5q). Thus a small segment of chromosome 5 contains IL-4, IL-5, IL-3, and GM-CSF as well as other genes such as CD14 and EGR1. Our findings that each of these genes was deleted in the 5q- chromosome suggest that loss of function of one or more of these genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).

Xp;Yq Unbalanced Translocation with Pseudoautosomal Region Aberrations in a Natural Two-Generation Transmission.

Translocations involving X and Y chromosomes rarely occur in humans and may affect reproductive function. We investigated an Xp:Yq unbalanced translocation with pseudoautosomal region (PAR) aberrations in a natural two-generation transmission. We report the case of an azoospermic male and his fertile mother without any other abnormal clinical phenotypes, except for short stature. Cytogenetic methods, including karyotyping and fluorescence in situ hybridization (FISH), revealed the translocation. Chromosomal microarray comparative genomic hybridization (array-CGH) was used to investigate the regions of Xp partial deletion and Yq partial duplication. Final chromosome karyotypes in the peripheral blood of the infertile male and his mother were 46,Y,der(X)t(X;Y)(p22.33;q11.22) and 46,X,der(X)t(X;Y)(p22.33;q11.22), respectively. Short-stature-homeobox gene deletion was responsible for the short stature in both subjects. PAR aberrations and AZFc duplication may be a direct genetic risk factor for spermatogenesis. This report further supports the use of routine karyotype analysis, FISH-based technology, and array-CGH analysis to identify derivative chromosomes in a complex rearrangement.

Diagnosis of Arboleda-Tham syndrome by whole genome sequencing in an Asian boy with severe developmental delay

Diagnosis of a 9-month-old boy brought to our genetics clinic with chief complaints of developmental delay (DD), failure to thrive, microcephaly, trunk hypotonia and hypertonia of the extremities. Multiple congenital defects but no significant syndromes or diseases were impressed. The chromosomal analysis and array comparative genomic hybridization (aCGH) revealed no significant pathogenic changes. Whole Genome Sequencing (WGS) identified a p.Glu1139fs de novo mutation of the KAT6A gene. The patient's phenotype was consistent clinically with Arboleda-Tham syndrome (ARTHS). Reviewing the literature showed that this is the first patient in Taiwan detected by WGS and that it involves a novel mutation. Comparing the highly variable clinical presentations of this syndrome with our patient, this boy's features and severe developmental defects seem to be due to a late-truncating mutation at the carboxyl end of the KAT6A protein. Our study demonstrates the power of WGS to confirm a diagnosis within 4 weeks for this rare condition.

Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

Karyotype and leaf epidermis histology traits of Digitaria abyssinica (Hochst. Ex A. Rich.) (Poaceae)

Digitaria abyssinica (African couchgrass) has economic potential as forage. This species is taxonomically complex, with a high number of synonyms, basic chromosome number x = 9, and diploid and tetraploid cytotypes. Anatomical, histological, and cytotaxonomic studies may contribute to the taxonomic description of this species and reveal its polyploidization and hybridization events. To this end, this study aimed to characterize the karyotype and leaf epidermis histology of two D. abyssinica genotypes. Chromosome characterization was performed based on chromosome banding using chromomycin A (CMA) and 4,6-diamidino-2-phenylindole (DAPI) and identification of 35S and 5S rDNA sites by fluorescent in situ hybridization. Nuclear DNA quantification was performed by flow cytometry. Paradermal sections were obtained for the leaf epidermis study by light and electron microscopy. The results confirmed that both genotypes are tetraploid (2n = 36). The number and distribution of 35S and 5S rDNA sites suggest the occurrence of postpolyploidization structural chromosomal changes or hybridization processes. Despite intraspecific variation in the number of 5S rDNA sites and CMA+/DAPI+ bands, no changes were identified in karyotypical symmetry or genome size. Leaf epidermis histology traits and cytogenetic data can support breeding programs and germplasm banks in identifying species or cultivars.

Incomplete Sterility of Chromosomal Hybrids: Implications for Karyotype Evolution and Homoploid Hybrid Speciation.

Heterozygotes for major chromosomal rearrangements such as fusions and fissions are expected to display a high level of sterility due to problems during meiosis. However, some species, especially plants and animals with holocentric chromosomes, are known to tolerate chromosomal heterozygosity even for multiple rearrangements. Here, we studied male meiotic chromosome behavior in four hybrid generations (F1–F4) between two chromosomal races of the Wood White butterfly Leptidea sinapis differentiated by at least 24 chromosomal fusions/fissions. Previous work showed that these hybrids were fertile, although their fertility was reduced as compared to crosses within chromosomal races. We demonstrate that (i) F1 hybrids are highly heterozygous with nearly all chromosomes participating in the formation of trivalents at the first meiotic division, and (ii) that from F1 to F4 the number of trivalents decreases and the number of bivalents increases. We argue that the observed process of chromosome sorting would, if continued, result in a new homozygous chromosomal race, i.e., in a new karyotype with intermediate chromosome number and, possibly, in a new incipient homoploid hybrid species. We also discuss the segregational model of karyotype evolution and the chromosomal model of homoploid hybrid speciation.