Complex Genomic Architecture Research Articles

Complex Genomic Landscape of Inversion Polymorphism in Europe's Most Destructive Forest Pest.

In many species, polymorphic genomic inversions underlie complex phenotypic polymorphisms and facilitate local adaptation in the face of gene flow. Multiple polymorphic inversions can co-occur in a genome, but the prevalence, evolutionary significance, and limits to complexity of genomic inversion landscapes remain poorly understood. Here, we examine genome-wide genetic variation in one of Europe's most destructive forest pests, the spruce bark beetle Ips typographus, scan for polymorphic inversions, and test whether inversions are associated with key traits in this species. We analyzed 240 individuals from 18 populations across the species' European range and, using a whole-genome resequencing approach, identified 27 polymorphic inversions covering ∼28% of the genome. The inversions vary in size and in levels of intra-inversion recombination, are highly polymorphic across the species range, and often overlap, forming a complex genomic architecture. We found no support for mechanisms such as directional selection, overdominance, and associative overdominance that are often invoked to explain the presence of large inversion polymorphisms in the genome. This suggests that inversions are either neutral or maintained by the combined action of multiple evolutionary forces. We also found that inversions are enriched in odorant receptor genes encoding elements of recognition pathways for host plants, mates, and symbiotic fungi. Our results indicate that the genome of this major forest pest of growing social, political, and economic importance harbors one of the most complex inversion landscapes described to date and raise questions about the limits of intraspecific genomic architecture complexity.

Novel toxin biosynthetic gene cluster in harmful algal bloom-causing Heteroscytonema crispum: Insights into the origins of paralytic shellfish toxins.

Caused by both eukaryotic dinoflagellates and prokaryotic cyanobacteria, harmful algal blooms (HABs) are events of severe ecological, economic, and public health consequence, and their incidence has become more common of late. Despite coordinated research efforts to identify and characterize the genomes of HAB-causing organisms, the genomic basis and evolutionary origins of paralytic shellfish toxins (PSTs) produced by HABs remain at best incomplete. The PST saxitoxin has an especially complex genomic architecture and enigmatic phylogenetic distribution, spanning dinoflagellates and multiple cyanobacterial genera. Using filtration and extraction techniques to target the desired cyanobacteria from non-axenic culture, coupled with a combination of short and long read sequencing, we generated a reference-quality hybrid genome assembly for Heteroscytonema crispum UTEX LB 1556, a freshwater, PST-producing cyanobacterium thought to have the largest known genome in its phylum. We report a complete, novel biosynthetic gene cluster for the PST saxitoxin. Leveraging this biosynthetic gene cluster, we find support for the hypothesis that PST production has appeared in divergent Cyanobacteria lineages through widespread and repeated horizontal gene transfer. This work demonstrates the utility of long-read sequencing and metagenomic assembly toward advancing our understanding of PST biosynthetic gene cluster diversity and suggests a mechanism for the origin of PST biosynthetic genes.

Transcriptional Analysis of Different Cultivars and Genome-Wide Identification of FAD Gene Family in Tree Peony

The tree peony (Paeonia ostii), a newly recognized woody oil plant endemic to China, is noteworthy for its high content of unsaturated fatty acids (UFA), particularly alpha-linolenic acid (ALA). Fatty acid desaturases (FADs) are integral to plant development and defense mechanisms. Nonetheless, there is limited understanding of (i) the molecular mechanism underlying FA biosynthesis in various varieties during seed maturation and (ii) a genome-wide analysis of FAD family genes within the tree peony. We selected three distinct cultivars of tree peony for transcriptome sequencing and performed an extensive analysis of PoFAD genes. In total, 67,542 unigenes were acquired and annotated with six protein databases available to the public. Forty-one differentially expressed genes (DEGs) pertinent to FA biosynthesis and lipid metabolism were identified in this study. Notably, genes such as PoFAD2, PoFAD6, and PoSAD were found to be significantly upregulated, contributing to a differential linolenic acid and linoleic acid content across the three cultivars. Herein, 24 PoFADs from the P. ostii genome were recognized and categorized into four distinct clusters according to their conserved structural features. The distribution of PoFADs was found to be random and uneven across five chromosomes, indicating a complex genomic architecture. Six colinear gene pairs were found between P. ostii and V. vinifera, indicating a potential link due to their close relationship. Together, these findings significantly enhance our knowledge of the molecular processes governing fatty acid synthesis, elucidate the functional roles of the FAD gene family, and lay the groundwork for using genetic manipulation to boost lipid levels.

Optimization of Genotyping‐by‐Sequencing (GBS) for Germplasm Fingerprinting and Trait Mapping in Faba Bean

ABSTRACTFaba bean (Vicia faba L.) is an important pulse crop with a wide range of agroecological adaptations. The development of genomic tools and a comprehensive catalog of extant genetic diversity are crucial for developing improved faba bean cultivars. The lack of a cost‐effective genotyping platform limits the characterization of large germplasm collections, understanding of genetic diversity across populations, and implementing breeder's tools like genomic selection. Genotyping‐by‐sequencing (GBS) offers high‐resolution genotyping for both model and crop plant species, even without a reference genome sequence. The genome fragments targeted by GBS depend substantially on the restriction enzyme (RE) used for the complexity reduction step. Species with complex genomic architecture require optimization of GBS with proper RE to realize the full potential of GBS. Here, we evaluated various REs in the GBS method and identified that the combination of ApeKI/MseI proved to be the most appropriate for faba bean based on the best library quality, a high number of genomic loci spread across chromosomes, and high enrichment loci associated with the gene space. With the new optimized protocol, we constructed a genetic map using a recombinant inbred line (RIL) population and identified a QTL for seed hilum color on Chromosome 1. In addition, we also genotyped a diversity panel and performed a genome‐wide association studies (GWAS) for important agronomic traits, including plant height (PH), flowering time (FT), and number of pods per plant (PPP). We identified six SNP markers significantly associated with these traits and listed potential candidate genes. The optimized faba bean‐specific GBS procedure will facilitate access to the untapped genetic diversity for genetic research and breeding and may facilitate functional genomics.

Genomic determinants, architecture, and constraints in drought-related traits in Corymbia calophylla

BackgroundDrought adaptation is critical to many tree species persisting under climate change, however our knowledge of the genetic basis for trees to adapt to drought is limited. This knowledge gap impedes our fundamental understanding of drought response and application to forest production and conservation. To improve our understanding of the genomic determinants, architecture, and trait constraints, we assembled a reference genome and detected ~ 6.5 M variants in 432 phenotyped individuals for the foundational tree Corymbia calophylla.ResultsWe found 273 genomic variants determining traits with moderate heritability (h2SNP = 0.26–0.64). Significant variants were predominantly in gene regulatory elements distributed among several haplotype blocks across all chromosomes. Furthermore, traits were constrained by frequent epistatic and pleiotropic interactions.ConclusionsOur results on the genetic basis for drought traits in Corymbia calophylla have several implications for the ability to adapt to climate change: (1) drought related traits are controlled by complex genomic architectures with large haplotypes, epistatic, and pleiotropic interactions; (2) the most significant variants determining drought related traits occurred in regulatory regions; and (3) models incorporating epistatic interactions increase trait predictions. Our findings indicate that despite moderate heritability drought traits are likely constrained by complex genomic architecture potentially limiting trees response to climate change.

Genomic and transcriptomic analyses of Phytophthora cinnamomi reveal complex genome architecture, expansion of pathogenicity factors, and host-dependent gene expression profiles.

Phytophthora cinnamomi is a hemibiotrophic oomycete causing Phytophthora root rot in over 5,000 plant species, threatening natural ecosystems, forestry, and agriculture. Genomic studies of P. cinnamomi are limited compared to other Phytophthora spp. despite the importance of this destructive and highly invasive pathogen. The genome of two genetically and phenotypically distinct P. cinnamomi isolates collected from avocado orchards in California were sequenced using PacBio and Illumina sequencing. Genome sizes were estimated by flow cytometry and assembled de novo to 140-141 Mb genomes with 21,111-21,402 gene models. Genome analyses revealed that both isolates exhibited complex heterozygous genomes fitting the two-speed genome model. The more virulent isolate encodes a larger secretome and more RXLR effectors when compared to the less virulent isolate. Transcriptome analysis after P. cinnamomi infection in Arabidopsis thaliana, Nicotiana benthamiana, and Persea americana de Mill (avocado) showed that this pathogen deploys common gene repertoires in all hosts and host-specific subsets, especially among effectors. Overall, our results suggested that clonal P. cinnamomi isolates employ similar strategies as other Phytophthora spp. to increase phenotypic diversity (e.g., polyploidization, gene duplications, and a bipartite genome architecture) to cope with environmental changes. Our study also provides insights into common and host-specific P. cinnamomi infection strategies and may serve as a method for narrowing and selecting key candidate effectors for functional studies to determine their contributions to plant resistance or susceptibility.

P17.11.A DEVELOPMENT AND CHARACTERISATION OF THE QCELL-R RECURRENT GLIOBLASTOMA PRIMARY CELL LINE RESOURCE: MODELS TO DRIVE BREAKTHROUGH DISCOVERIES IN BRAIN CANCER

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive brain tumour in adults, with a median survival of only 15 months. Despite aggressive treatment with chemotherapy using temozolomide and radiotherapy, GBM has a high prevalence of recurrence with almost all cases recurring within the first year. This highlights a clear need to develop a deeper understanding of GBM recurrence and to develop effective models to test novel therapies. The vast inter- and intra-tumoural heterogeneity and tumour cell plasticity are complicating the development of effective therapies for brain cancer patients. Large-scale genomic analyses of primary and recurrent human GBM tumour samples have revealed a complex genomic architecture. However, the majority of GBM discovery science has been conducted in treatment naïve tumour models, which fail to capture therapy resistance and tumour evolution. Hence, these changes have been difficult to link to GBM growth and functional cell properties upon recurrence. MATERIAL AND METHODS To address this, we have developed the QCell-R resource, a collection of functional models of recurrent GBM. In collaboration with the Royal Brisbane and Women’s Hospital (RBWH), pair-matched patient tumour samples from the initial resection and at recurrence post-therapy have been collected. From these tissue specimens, in vitro cell lines in both 2D and 3D cultures and in vivo orthotopic xenograft models have been developed. Response to standard of care therapy (temozolomide and radiotherapy) were assessed using IncuCyte proliferation assays. Bulk mRNA, scRNA and exome sequencing were also performed. RESULTS QCell-R consists of 11 patient-derived recurrent GBM models, with the time between resections varying from 5-13 months. There are four pair-matched QCell-R models, as well as three standalone recurrent models. Response to standard of care therapy varied between models, with one model exhibiting a four fold higher growth rate-50 (GR50) after temozolomide treatment and a 2.7 fold increase in radiation resistance compared to the matched treatment naïve counterpart. The mRNA and scRNA sequencing has been compared to publicly available datasets to identify genes of interest that may be targetable in recurrence to test in these models. CONCLUSION QCell-R represents a valuable resource to define novel therapies for recurrent GBM. Elucidation of tumour heterogeneity and tumour evolution will identify targetable genes contributing to therapy resistance in these models. Identifying these targetable genes from QCell-R allows the development of novel treatments, creating strategies that may reduce the rate of recurrence by limiting therapy resistance and inhibiting recurrence-initiating cells.

The mutational landscape of a US Midwestern breast cancer cohort reveals subtype-specific cancer drivers and prognostic markers

BackgroundFemale breast cancer remains the second leading cause of cancer-related death in the USA. The heterogeneity in the tumor morphology across the cohort and within patients can lead to unpredictable therapy resistance, metastasis, and clinical outcome. Hence, supplementing classic pathological markers with intrinsic tumor molecular markers can help identify novel molecular subtypes and the discovery of actionable biomarkers.MethodsWe conducted a large multi-institutional genomic analysis of paired normal and tumor samples from breast cancer patients to profile the complex genomic architecture of breast tumors. Long-term patient follow-up, therapeutic regimens, and treatment response for this cohort are documented using the Breast Cancer Collaborative Registry. The majority of the patients in this study were at tumor stage 1 (51.4%) and stage 2 (36.3%) at the time of diagnosis. Whole-exome sequencing data from 554 patients were used for mutational profiling and identifying cancer drivers.ResultsWe identified 54 tumors having at least 1000 mutations and 185 tumors with less than 100 mutations. Tumor mutational burden varied across the classified subtypes, and the top ten mutated genes include MUC4, MUC16, PIK3CA, TTN, TP53, NBPF10, NBPF1, CDC27, AHNAK2, and MUC2. Patients were classified based on seven biological and tumor-specific parameters, including grade, stage, hormone receptor status, histological subtype, Ki67 expression, lymph node status, race, and mutational profiles compared across different subtypes. Mutual exclusion of mutations in PIK3CA and TP53 was pronounced across different tumor grades. Cancer drivers specific to each subtype include TP53, PIK3CA, CDC27, CDH1, STK39, CBFB, MAP3K1, and GATA3, and mutations associated with patient survival were identified in our cohort.ConclusionsThis extensive study has revealed tumor burden, driver genes, co-occurrence, mutual exclusivity, and survival effects of mutations on a US Midwestern breast cancer cohort, paving the way for developing personalized therapeutic strategies.

Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix.

The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum, are increasingly used as model systems in various contexts. In particular, M. lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here we present such resources for M. cliftonense and M. hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq datasets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227Mb and ∼220Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84-85%), low BUSCO duplication rates (8.3-6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, that can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer excellent foundations for comparative genomic research in this group of organisms.

A complex genomic architecture underlies reproductive isolation in a North American oriole hybrid zone

Natural hybrid zones provide powerful opportunities for identifying the mechanisms that facilitate and inhibit speciation. Documenting the extent of genomic admixture allows us to discern the architecture of reproductive isolation through the identification of isolating barriers. This approach is particularly powerful for characterizing the accumulation of isolating barriers in systems exhibiting varying levels of genomic divergence. Here, we use a hybrid zone between two species—the Baltimore (Icterus galbula) and Bullock’s (I. bullockii) orioles—to investigate this architecture of reproductive isolation. We combine whole genome re-sequencing with data from an additional 313 individuals amplityped at ancestry-informative markers to characterize fine-scale patterns of admixture, and to quantify links between genes and the plumage traits. On a genome-wide scale, we document several putative barriers to reproduction, including elevated peaks of divergence above a generally high genomic baseline, a large putative inversion on the Z chromosome, and complex interactions between melanogenesis-pathway candidate genes. Concordant and coincident clines for these different genomic regions further suggest the coupling of pre- and post-mating barriers. Our findings of complex and coupled interactions between pre- and post-mating barriers suggest a relatively rapid accumulation of barriers between these species, and they demonstrate the complexities of the speciation process.

Different Chromosome Segregation Patterns Coexist in the Tetraploid Adriatic Sturgeon Acipenser naccarii

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a critically endangered tetraploid endemism of the Adriatic region; it has been targeted, over the last 20 years, by different conservation programs based on controlled reproduction of captive breeders followed by the release of their juvenile offspring; its preservation would greatly benefit from the correct and coordinated management of the residual genetic variability available in the different captive stocks. In this sense, the setup of an efficient parental allocation procedure would allow identifying familiar groups and establishing informed breeding plans, effectively preserving genetic variation. However, being the species tetraploid, the analyses often deal with complex genome architecture and a preliminary evaluation of allele segregation patterns at different chromosomes is necessary to assess whether the species can be considered a pure tetraploid, as previously observed at some loci, or if a more complex situation is present. Here we study the segregation at 14 microsatellites loci in 12 familiar groups. Results support in different families the tetrasomic segregation pattern at 11 markers and the disomic segregation at three markers. The Adriatic sturgeon thus shows a mixed inheritance modality. In this species, and likely in other sturgeons, accurate knowledge of the loci used for paternity analysis is therefore required.

Evolution of complex genome architecture in gymnosperms.

Gymnosperms represent an ancient lineage that diverged from early spermatophytes during the Devonian. The long fossil records and low diversity in living species prove their complex evolutionary history, which included ancient radiations and massive extinctions. Due to their ultra-large genome size, the whole-genome assembly of gymnosperms has only generated in the past 10 years and is now being further expanded into more taxonomic representations. Here, we provide an overview of the publicly available gymnosperm genome resources and discuss their assembly quality and recent findings in large genome architectures. In particular, we describe the genomic features most related to changes affecting the whole genome. We also highlight new realizations relative to repetitive sequence dynamics, paleopolyploidy, and long introns. Based on the results of relevant genomic studies of gymnosperms, we suggest additional efforts should be made toward exploring the genomes of medium-sized (5–15 gigabases) species. Lastly, more comparative analyses among high-quality assemblies are needed to understand the genomic shifts and the early species diversification of seed plants.

FISH Mapping of Telomeric and Non-Telomeric (AG3T3)3 Reveal the Chromosome Numbers and Chromosome Rearrangements of 41 Woody Plants.

Data for the chromosomal FISH mapping localization of (AG3T3)3 are compiled for 37 species belonging 27 families; for 24 species and 14 families, this is the first such report. The chromosome number and length ranged from 14–136 and 0.56–14.48 μm, respectively. A total of 23 woody plants presented chromosome length less than 3 μm, thus belonging to the small chromosome group. Telomeric signals were observed at each chromosome terminus in 38 plants (90.5%) and were absent at several chromosome termini in only four woody plants (9.5%). Non-telomeric signals were observed in the chromosomes of 23 plants (54.8%); in particular, abundant non-telomeric (AG3T3)3 was obviously observed in Chimonanthus campanulatus. Telomeric signals outside of the chromosome were observed in 11 woody plants (26.2%). Overall, ten (AG3T3)3 signal pattern types were determined, indicating the complex genome architecture of the 37 considered species. The variation in signal pattern was likely due to chromosome deletion, duplication, inversion, and translocation. In addition, large primary constriction was observed in some species, probably due to or leading to chromosome breakage and the formation of new chromosomes. The presented results will guide further research focused on determining the chromosome number and disclosing chromosome rearrangements of woody plants.

Abstract 680: Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy in prostate cancer

Abstract Prostate cancer (PCa) is the most common malignancy and second leading cause of cancer death in American men. Androgen Receptor (AR) mediated transcriptional program is central to normal prostate homeostasis and drives PCa growth and survival. Chromoplexy, a highly complex genomic architecture with several intra- and inter-chromosomal segments joined in a chain, is among the most prominent genetic alterations that drive both prostate cancer initiation and progression, and often involves sites of AR transcription. Previous studies have shown that AR induced, topoisomerase 2 beta (TOP2B) mediated double strand breaks were recombinogenic and led to de novo formation of TMPRSS2-ERG fusion gene, shedding light on the potential role of TOP2B in chromoplexy formation. However, the precise role of TOP2B in AR transcription was not well understood. Here, we hypothesize that TOP2B is recruited to resolve topological constraints arising during induction of AR transcriptional programs, and its catalytic activity is required to facilitate or maintain chromosomal interactions optimal for transcriptional induction. We performed Chromosomal Conformation Capture related techniques (3C and HiC) on LNCaP cells before and after androgen stimulation and observed an increase in chromatin interactions within 15kb from promoters of AR target genes upon androgen induction. These interactions depended on TOP2B, as TOP2B catalytic inhibition or knockdown reduced them significantly. Furthermore, TOP2B Hi-CHIP revealed that TOP2B is involved in key enhancer-promoter looping and in several interactions among gene body, enhancers, promoters of AR target genes, and nearby topological associated domain borders. We went on to isolate which steps during AR transcription induction required TOP2B by examining chromatin localization of the key factors, including AR, cohesin (SMC1A), CTCF, histone 3 lysine 27 acetylation (H3K27ac), and total and phosphorylated RNA Polymerase II (RNAPII) using ChIP-seq. These experiments revealed that TOP2B was not required for AR binding nor for localization of H3K27ac marks. However, it was required for recruitment of cohesin to AR binding sites as well as to AR target gene promoters and gene bodies, for displacement of CTCF near AR target genes, and for localization and phosphorylation of RNAPII at AR target genes. These data nominate TOP2B as a key AR coactivator, assisting in the proper assembly of cohesin during transcription induction, and maintaining chromosomal interactions optimal for binding and activation of RNAPII. Intriguingly, sites of binding of TOP2B, as well as of cohesin, were highly associated with sites of chromoplexy complex rearrangements in human prostate cancers. Taken together, this work elucidates the role of TOP2B in AR-induced transcription, and implicates its involvement in chromoplexy formation in PCa. Citation Format: Minh-Tam N. Pham, Michael C. Haffner, Heather C. Wick, Jonathan B. Coulter, Anuj Gupta, Roshan V. Chikarmane, Harshath Gupta, Sarah Wheelan, William G. Nelson, Srinivasan Yegnasubramanian. Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 680.

Disparate genomic characteristics of patients with early-stage lung adenocarcinoma manifesting as radiological subsolid or solid lesions

IntroductionEarly-stage lung adenocarcinoma (LUAD) manifesting as subsolid nodules (SSNs) exhibit more favorable prognosis than solid nodules (SNs). However, the genomic underpinnings behind their indolent tumor behavior remain largely unexplained. MethodsWe identified patients with stage I invasive LUAD who underwent complete surgical resection and broad-panel next-generation sequencing (NGS). Comparative genomic profiling was then performed by radiological subtype (SSNs vs. SNs) regarding the general genomic features, driver genes, oncogenic pathways, therapeutic actionability, and evolutionary trajectory. ResultsIn total, 177 SSN-LUADs and 133 SN-LUADs were included. Compared with SNs, SSN-LUADs possessed lower somatic mutation count (P < 0.001), genomic alteration count (P = 0.002), and intra-tumor heterogeneity (P = 0.006). In terms of driver genes, SSNs harbored more EGFR mutation (77% vs. 62%), but had lower frequencies of genes such as TP53, ARID1A, PIK3CA, CDKN2A, and BRAF (FDR q < 0.1). Besides, RBM10 mutation was independently associated with SSN-LUADs in multivariate analysis (P = 0.033). Three oncogenic pathways (p53, cell cycle, PI3K) were altered with statistical significance in SNs, while only RNA splicing/processing pathway was significantly altered in SSNs (FDR q < 0.1). Also, SSNs had significantly lower number of pathway alterations (P < 0.001). Finally, SSNs and SNs showed distinct evolutionary trajectories regarding somatic mutations during early-stage LUAD progression. ConclusionsThis study performed the first direct comparative genomic profiling in pathologic stage I invasive LUAD by radiological subtype, highlighting a less complex genomic architecture of SSNs, which might be the molecular interpretation of their indolent tumor behavior.

Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius.

The widely distributed ray-finned fish genus Carassius is very well known due to its unique biological characteristics such as polyploidy, clonality, and/or interspecies hybridization. These biological characteristics have enabled Carassius species to be successfully widespread over relatively short period of evolutionary time. Therefore, this fish model deserves to be the center of attention in the research field. Some studies have already described the Carassius karyotype, but results are inconsistent in the number of morphological categories for individual chromosomes. We investigated three focal species: Carassius auratus, C. carassius and C. gibelio with the aim to describe their standardized diploid karyotypes, and to study their evolutionary relationships using cytogenetic tools. We measured length ( ) of each chromosome and calculated centromeric index (i value). We found: (i) The relationship between and i value showed higher similarity of C. auratus and C. carassius. (ii) The variability of i value within each chromosome expressed by means of the first quartile () up to the third quartile () showed higher similarity of C. carassius and C. gibelio. (iii) The fluorescent in situ hybridization (FISH) analysis revealed higher similarity of C. auratus and C. gibelio. (iv) Standardized karyotype formula described using value () showed differentiation among all investigated species: C. auratus had 24 metacentric (m), 40 submetacentric (), 2 subtelocentric (), 2 acrocentric (a) and 32 telocentric (T) chromosomes (); C. carassius: ; and C. gibelio: . (v) We developed R scripts applicable for the description of standardized karyotype for any other species. The diverse results indicated unprecedented complex genomic and chromosomal architecture in the genus Carassius probably influenced by its unique biological characteristics which make the study of evolutionary relationships more difficult than it has been originally postulated.

Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities

Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp–50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.

Genome Divergence and Dynamics in the Thin-Tailed Desert Sheep From Sudan

With climate change bound to affect food and feed production, emphasis will shift to resilient and adapted indigenous livestock to sustain animal production. However, indigenous livestock comprise several varieties, strains and ecotypes whose genomes are poorly characterized. Here, we investigated genomic variation in an African thin-tailed Desert Sheep sampled in Sudan, using 600K genotype data generated from 92 individuals representing five ecotypes. We included data from 18 fat-tailed and 45 thin-tailed sheep from China, to investigate shared ancestry and perform comparative genomic analysis. We observed a clear genomic differentiation between the African thin-tailed Desert Sheep and the Chinese thin-tailed and fat-tailed sheep, suggesting a broad genetic structure between the fat-tailed and thin-tailed sheep in general, and that at least two autosomal gene pools comprise the genome profile of the thin-tailed sheep. Further analysis detected two distinct genetic clusters in both the African thin-tailed Desert Sheep and the Chinese thin-tailed sheep, suggesting a fine-scale and complex genome architecture in thin-tailed sheep. Selection signature analysis suggested differences in adaptation, production, reproduction and morphology likely underly the fine-scale genetic structure in the African thin-tailed Desert Sheep. This may need to be considered in designing breeding programs and genome-wide association studies.

Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion–deletion variant located in a region of complex genomic architecture

Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of “third-generation” sequencing for accurate characterisation of pathogenic variants.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

SummaryAmong polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene‐level and transcript‐level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.