Duplicated Region Research Articles

Directed Evolution Detects Supernumerary Centric Chromosomes Conferring Resistance to Azoles in Candida auris.

Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Expression of Concern: FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity.

The Editorial Office has contacted the authors of this Bioscience Reports article with regards to two of the images, Figures 5 and 6. A similar region of the Fig.5C Fasudil (5ug/ml) panel has been noted in the FSD-C10 (15ug/ml) panel (region rotated 180 degrees), along with similar bands being identified in the Fig.6 Western blots (the EAE and Fasudil lanes of panels 6A and 6D appear as the Fasudil and FSD-C10 bands of 6C). The authors have cooperated with the Bioscience Reports Editorial Office and have offered explanations for the duplicated regions, including that one of the lanes was erroneously removed from the western blots whilst compiling the figures. They have stated that the duplicated region (and image reversal) for Figure 5 could also have been due to errors whilst putting together the figures. The authors are currently searching for the original data to correct the images; however, given the age of the article, they have yet to be able to source the data. The authors state that the results of their experiments are unaffected by these issues, and as no further issues have been identified by the Editorial Office, the article will remain published with an Expression of Concern to alert readers to the duplicated regions.

The complete mitochondrial genome study and phylogenetic analysis of Asiatic Water Snake (Fowlea piscator) using ‘Next-Generation Sequencing’ technology

The first complete mitochondrial genome sequencing of Asiatic Water snake or Checkered Keelback or Fowlea piscator (=Xenochrophis piscator) was carried out using Next-Generation Sequencing technology. The complete mitochondrial genome of Asiatic Water snake is 16,999bp long with a base composition of 33% A, 28% T, 12% G and 27% C, with a GC content of 39%. Like the typical snake mitochondrial genome, F. piscator also shows relatively similar mitogenome arrangement comprising 37 genes including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and two non-coding regions or a duplicated control region (D-Loop) along with an origin of replication. Nine genes including eight tRNAs and NAD6 were encoded on the Light or L-strand. Phylogenetic analyses using the complete mitochondrial genome of F. piscator demonstrate a close relationship with the family Colubridae and sub family Natricinae.

Scam Image Detection on Copy-Move by JPEG Features and Classical Block Matching with Improved Variant

Numerous methods have been developed to identify copy-move forgeries, which are among the most often used alteration strategies of digital photographs. The most widely used format of digital photographs is JPEG, which allows for high-rate compression without drastically altering the meaning of the picture. The objective of this work is to develop a system that can automatically identify the forgery type of the suspect image through in a single procedure, without requiring any kind of expert information. A preferable method is to run the same image through multiple algorithms, which saves time and prevents the needless evaluation of multiple detection results, from which it may be difficult to determine the correct output due to the presence of multiple confounding factors. Additionally, it has been shown that the established method is very effective in detecting expert forgeries when the duplicated region is picked in a non-rigid fashion, which is almost hard for the human eye to perform.

RF34 | PMON203 Copy Number Variant Analysis Reveals Novel Candidate Genes Associated with Primary Ovarian Insufficiency

Abstract Primary ovarian insufficiency (POI) is characterized by a heterogeneous genetic background. Moreover, the etiology of most POI cases remains unclear. Herein, our goal was to investigate the presence of rare deletions or duplications in patients presenting with POI, which could lead to identification of novel chromosomal regions and/or genes potentially associated with disease risk. Patients and Methods Sixty-six patients presenting idiopathic POI were evaluated; 44 had primary amenorrhea and 22 secondary amenorrhea. Chromosomal microarray analysis was performed to detect copy number variants (CNVs) using array-CGH or SNP-array. Identified CNVs were validated by population databases of either health individuals (Database of Genomic Variants - DGV) or patients carrying chromosomal structural alterations (DECIPHER). A set of rare CNVs was selected and annotated in regard to gene content and clinical associations using bioinformatic tools (RefSeq, OMIM, PubMed, Ovarian Kaleidoscope Database, and Mouse Genome Informatics). Results Fifteen rare CNVs were found in 13 patients, ten of them with primary amenorrhea. The identified CNVs comprised four deleted and 11 duplicated regions, in which the break points occurred intragenic in 8 of them. Most of the CNVs were mapped in the autosomes. Seven CNVs identified in 5 patients were classified as pathogenic or probably pathogenic; three were novel variants containing candidate genes associated with ovarian development [19q13.43 (NLRP5) and Xq13.2 (XIST)] or function [(2q37.3 (PER2)]. The other pathogenic CNVs were previously implicated in POI etiology; two of them [Xq27.2q28 del (14.5 Mb) and Xq22.1q27.1 dup (41.4 Mb)] were encompassed in a complex chromosome rearrangement, associated with 20p13 dup (2.7 Mb), and the last region was 15q25.2 deletion. Four VUS containing genes associated with ovarian development [2p23.1 (XDH and EHD3), 9p24.1 (KDM4C), 22q12.1 (ZNRF3)] or function [1p31.1 (PTGFR)] were identified. Conclusion CNVs analysis indicated a genetic etiology in 10% of these POI patients. New candidate genes associated with ovarian development and function were revealed. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:54 p.m. - 12:59 p.m.

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

A partial duplication of an X-linked gene exclusive of a primate lineage (Macaca)

Gene duplication plays a significant role in evolution. Paralogous gene copies may be lost due to the successive accumulation of deleterious mutations or remain active in the genome. In this work, a partial duplication of an X-linked region in the Macaca genus is identified and explored. Genomic comparisons reveal that the duplication encompasses the genes encoding ornithine transcarbamylase (OTC) and retinitis pigmentosa GTPase regulator (RPGR), spanning over 0.1 Mb on the chromosome 9 of Macaca. According to our analyses, the duplicated region of chromosome 9 involves partial coding sequences of both OTC and RPGR genes. Analyses of the selective pressures did not reveal significant differences in the ratio between nonsynonymous and synonymous mutations (w<1), suggesting that no selective pressures were acting in the evolutionary process. Reports for a biological role regarding some partial duplications exist in the literature, therefore, although being rare events, partial duplications of functionally important genes are worthy of study so that their impact can be explored.

Deep features for image copy move forgery detection

In this modern era, there has been a increase in the copying and moving of illegalities, distributing them, and then tempered images. These days even the most secured data sometimes go under devastating forgery. Copy forgery is seen to be the easiest copying method without leaving any obvious traces of manipulating an image’s content.Like working on the pixels of an image by geometrical and illumination techniques which are either cut or copied or copied to some other place of the identical image. In this paper, we propose copy paste forgery detection of an image by extracting and grouping SIFT key points for analyzation. The first step is to find suspicious clusters of key points and patches to roughly estimate the dimensionality of SIFT. Secondly, it finds pixel level, scale, and color by comparingsimilar neighborhoods of matching duplicated regions. The results prove to be giving good performance scores, computational timing, and complexity

VP.01 Array comparative genomic hybridisation and droplet digital PCR uncover recurrent copy number variation of the titin segmental duplication region

The sarcomeric giants nebulin and titin both contain intragenic segmental duplication regions. Segmental duplication regions are prevalent throughout the genome and are known potential hotspots for recurrent copy number variation and may harbour pathogenic aberrations. Using our custom comparative genomic hybridization array, we have shown that gains and losses of more than one copy of the repeated block of the nebulin triplicate region are a recessive pathogenic mutation contributing to the spectrum of nemaline myopathy-causing changes. Similar copy number variants have been detected in the segmental duplication region of titin, harboured within its PEVK domain. The exact copy number determination of the segmental duplication region in titin is challenging due to the limitations of the methodology and repetitiveness of the region. We developed three complementary custom droplet digital PCR assays for the titin segmental duplication region to confirm true variation. Two of the assays target the repeated region, and one targets a diploid sequence adjacent to the segmental duplication. By comparing the data acquired by the custom comparative genomic hybridization array and the custom droplet digital PCR assays, we assessed the copy number variation of the region in 55 samples from both healthy individuals and samples from patients with different congenital muscle disorders. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses of the region were detected in both samples from healthy individuals and in samples from patients. While the copy number variation observed in our cohort is mostly unlikely to be pathogenic, it is possible that this region of titin harbours pathogenic copy number variation in a fashion similar to that of nebulin. Putative pathogenic thresholds for gains and losses remain to be studied, but the region should no longer be neglected in genetic analyses.

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

ОКИСЛИТЕЛЬНАЯ МОДИФИКАЦИЯ ДНК ПЛАЗМИД СПОСОБСТВУЕТ ИХ ПРОНИКНОВЕНИЮ В ЦИТОПЛАЗМУ МЕЗЕНХИМНЫХ СТВОЛОВЫХ КЛЕТОК ЧЕЛОВЕКА

It is well known that the action of ionizing radiation causes oxidative stress in the cells of the body and leads to the synthesis of reactive oxygen species, which lead to multiple damage to cellular DNA, including the formation of oxidized bases. In this paper, we consider the effect of radiation-induced oxidative modification of plasmid DNA on their penetration into human mesenchymal stem cells. To study the role of oxidative DNA modification in cell penetration, a genetic construct was created based on the pEGFP-C1 (pC1) vector, GenBankAccession: U55763, containing as an insert an artificially synthesized DNA fragment containing a poly-G region (p12G) serving as target for efficient oxidation of the cloned DNA fragment. As a "marker", the selected vector contains the EGFP fluorescent protein (GFP) gene. Flow cytometry and fluorescence microscopy showed that recombinant constructs based on the pEGFP vector containing duplicated poly-G regions and Gn repeats penetrate cells much more efficiently than the original pEGFP vector. Exposure to radiation at a dose of 50 cGy, which causes an increase in the level of 8-oxodG in plasmids after irradiation, leads to a more intense penetration of oxidized plasmids compared to a similar experiment without irradiation.

Novel variants identified in a three‑generation family with concomitant exotropia

Concomitant exotropia is a condition where there is a misalignment between both eyes, which is more prevalent in Asians than in Caucasians. It is an eye disease related to the neural development of binocular vision and eye movement control. Studies have indicated that genetic factors contribute to the development of concomitant exotropia; however, the underlying mutations have not been thoroughly investigated to date. In the present study, whole-exome sequencing was performed in a three-generation family with concomitant exotropia. In the proband and the proband's father, bioinformatics analyses identified a duplication of the genomic region spanning genes PCDHA1-7 and a heterozygous mutation c.3775G>A (p.A1259T) of the COL3A1 gene, which is located in the conserved COLFI domain and leads to decreased stability of the encoded protein product. Furthermore, a deletion of amino acid S165 in the gene NCOA7 was discovered in the family members, including the proband, the proband's mother and maternal grandfather. S165 was predicted to be a conserved phosphokinase site of CK1/VRK and CK1/CK1. The genes in which these variants reside are all involved in cortical neuronal development. The present study reveals novel variants of concomitant exotropia and suggests that aberrant cortical neuronal development may contribute to the origin of concomitant strabismus.

Microevolution of CG23-I Hypervirulent Klebsiella pneumoniae during Recurrent Infections in a Single Patient.

ABSTRACTCG23-I lineage constitutes the majority of hypervirulent Klebsiella pneumoniae. A diabetic patient suffered six episodes of infections caused by CG23-I K. pneumoniae. A total of nine isolates were collected in 2020. We performed whole-genome sequencing to elucidate the within-patient evolution of CG23-I K. pneumoniae. The maximum pairwise difference among the nine longitudinally collected isolates was five single nucleotide polymorphisms. One of the mutations was at the Asp87 position of GyrA. Four indels were identified, including an initiator tRNAfMet duplication, a tRNAArg deletion, a 7-bp insertion, and a 22-bp deletion. All 9 isolates had the genomic features of CG23-I K. pneumoniae, a chromosome-borne ICEKp10, and a large virulence plasmid. The carriage of a complete set of genes for the biosynthesis of colibactin by ICEKp10 gave the nine isolates an ability to cause DNA damage to RAW264.7 cells. Compared with the initial isolate, the last isolate with an additional copy of initiator tRNAfMet grew faster in a nutrient-limiting condition and exhibited enhanced virulence in BALB/c mice. Collectively, we characterized the within-patient microevolution of CG23-I K. pneumoniae through an in-depth comparison of genome sequences. Using the in vitro experiments and mouse models, we also demonstrated that these genomic alterations endowed the isolates with advantages to pass through in vivo selection.IMPORTANCE CG23-I is a significant lineage of hypervirulent Klebsiella pneumoniae. This study characterizes the within-patient microevolution of CG23-I K. pneumoniae. Selective pressures from continuous use of antibiotics favored point mutations contributing to bacterial resistance to antibiotics. The duplication of an initiator tRNAfMet gene helped CG23-I K. pneumoniae proliferate to reach a maximal population size during infections. For longer persistence inside a human host, the large virulence plasmid evolved with more flexible control of replication through duplication of the iteron-1 region. With the genomic alterations, the last isolate had a growth advantage over the initial isolate and exhibited enhanced virulence in BALB/c mice. This study gives us a deeper understanding of the genome evolution during the within-patient pathoadaptation of CG23-I K. pneumoniae.

Complete Mitogenomes of Polypedates Tree Frogs Unveil Gene Rearrangement and Concerted Evolution within Rhacophoridae.

Simple SummaryDuplicated control regions have been reported several times in the tree frog family Rhacophoridae, and previous studies have mostly relied on sequence analysis to reconstruct their evolution. This is the first study to employ a phylogenetic method to demonstrate the existence of concerted and parallel evolution succinctly and intuitively in the duplicated control regions of the family Rhacophoridae. Phylogenetic relationships were also used to illustrate the parallel evolution of ATP8 loss of function in the genus Polypedates. In general, this study elucidated the evolutionary patterns and pathways of mitochondrial gene rearrangement of the family Rhacophoridae from a phylogenetic perspective, which aids in understanding the evolutionary history of this fascinating tree frog taxon from a molecular evolution standpoint.New developments in sequencing technology and nucleotide analysis have allowed us to make great advances in reconstructing anuran phylogeny. As a clade of representative amphibians that have radiated from aquatic to arboreal habitats, our understanding of the systematic status and molecular biology of rhacophorid tree frogs is still limited. We determined two new mitogenomes for the genus Polypedates (Rhacophoridae): P. impresus and P. mutus. We conducted comparative and phylogenetic analyses using our data and seven other rhacophorid mitogenomes. The mitogenomes of the genera Polypedates, Buergeria, and Zhangixalus were almost identical, except that the ATP8 gene in Polypedates had become a non-coding region; Buergeria maintained the legacy “LTPF” tRNA gene cluster compared to the novel “TLPF” order in the other two genera; and B. buergeri and Z. dennysi had no control region (CR) duplication. The resulting phylogenetic relationship supporting the above gene rearrangement pathway suggested parallel evolution of ATP8 gene loss of function (LoF) in Polypedates and CR duplication with concerted evolution of paralogous CRs in rhacophorids. Finally, conflicting topologies in the phylograms of 185 species reflected the advantages of phylogenetic analyses using multiple loci.

Linkage mapping and genome annotation give novel insights into gene family expansions and regional recombination rate variation in the painted lady (Vanessa cardui) butterfly

Characterization of gene family expansions and crossing over is crucial for understanding how organisms adapt to the environment. Here, we develop a high-density linkage map and detailed genome annotation of the painted lady butterfly (Vanessa cardui) - a non-diapausing, highly polyphagous species famous for its long-distance migratory behavior and almost cosmopolitan distribution. Our results reveal a complex interplay between regional recombination rate variation, gene duplications and transposable element activity shaping the genome structure of the painted lady. We identify several lineage specific gene family expansions. Their functions are mainly associated with protein and fat metabolism, detoxification, and defense against infection - critical processes for the painted lady’s unique life-history. Furthermore, the detailed recombination maps allow us to characterize the regional recombination landscape, data that reveal a strong effect of chromosome size on the recombination rate, a limited impact of GC-biased gene conversion and a positive association between recombination and short interspersed elements.

Low-level complex mosaic with multiple cell lines affecting the 18q21.31q21.32 region in a patient with de novo 18q terminal deletion

We describe a 5-year-old girl who was diagnosed at birth with 18q de novo homogeneous deletion at G-banding karyotype. Her clinical condition, characterized by hypotonia, psychomotor retardation, short stature, deafness secondary to bilateral atresia of the external auditory canals, was in agreement with the 18q deletion syndrome though presence of coloboma of a single eye only suggested a mosaic condition as an unusual sign. By combining multiple technologies including array-CGH, FISH, and WGS, we found that the terminal deletion 18q21.32q23 (21 Mb) was in segmental mosaicism of the proximal region 18q21.31q21.32 (2.7 Mb), which showed a variable number of copies: one, two, or three, in 7, 41 and 55% of the cells respectively. Breakpoint junction analysis demonstrated the presence of an inv-dup del (18q) with a disomic segment of 4.7 kb between the inverted and non-inverted copies of the duplicated region 18q21.31q21.32. From these results, we propose that all three types of abnormal chr18 (the inv-dup del and the two 18q terminal deletions of different sizes) arisen from breaks in a dicentric mirror chromosome 18q, either in more than one embryo cell or from subsequent breaking-fusion-bridge cycles. The duplication region was with identical polymorphisms as in all non-recurrent inv-dup del rearrangements though, in contrast with most of them, the 18q abnormality was of maternal origin. Taking into account that distal 18q deletions are not rarely associated with inv-dup del(18q) cell lines, and that the non-disjunction of chromosome 18 takes place especially at maternal meiosis II rather than meiosis I, multiple rescue events starting from trisomic zygotes could be considered alternative to the postmitotic ones. From the clinical point of view, our case, as well as those of del(18q) in mosaic with the dic(18q), shows that the final phenotype is the sum of the different cell lines that acted on embryonic development with signs typical of both the 18q deletion syndrome and trisomy 18. Asymmetrical malformations, such as coloboma of the iris only in the right eye, confirm the underlying mosaicism regardless of whether it is still detectable in the blood.

Evolution and function of ubiquitin-specific proteases (UBPs): Insight into seed development roles in plants

The tung oil produced by the tung tree (Vernicia fordii) provides resources for the manufacture of biodiesel. Ubiquitin-specific proteases (UBPs) are the largest group of deubiquitinases and play key roles in regulating development and stress responses. Here, 21 UBPs were identified in V. fordii, roughly one-half the number found in Manihot esculenta and Hevea brasiliensis. Most UBP duplications are produced from whole-genome duplication (WGD), and significant differences in gene retention existed among Euphorbiaceae. The great majority of UBP-containing blocks in V. fordii, V. montana, Ricinus communis, and Jatropha curcas exhibited extensive conservation with the duplicated regions of M. esculenta and H. brasiliensis. These blocks formed 14 orthologous groups, indicating they shared WGD with UBPs in M. esculenta and H. brasiliensis, but most of these UBPs copies were lost. The UBP orthologs contained significant functional divergence which explained the susceptibility of V. fordii to Fusarium wilt and the resistance of V. montana to Fusarium wilt. The expression patterns and experiments suggested that Vf03G1417 could affect the seed-related traits and positively regulate the seed oil accumulation. This study provided important insights into the evolution of UBPs in Euphorbiaceae and identified important candidate VfUBPs for marker-assisted breeding in V. fordii.

An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster

BackgroundGenome complexity is largely linked to diversification and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non-duplicated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identification and characterization of complex variability between genomes for plant breeding.ResultsHere we improve and demonstrate the use of CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4.5% and a depth per sample of 11.9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region.ConclusionsCRISPR-Cas9 enrichment is a versatile and powerful tool for long-read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed.

SHFLD3 phenotypes caused by 17p13.3 triplication/ duplication encompassing Fingerin (BHLHA9) invariably

BackgroundSplit-hand/ foot malformation with long bone deficiency 3 (SHFLD3) is an extremely rare condition associated with duplications located on 17p13.3, which invariably encompasses the BHLHA9 gene. The disease inherits with variable expressivity and significant incomplete penetrance as high as 50%.ResultsWe have detected 17p13.3 locus one-allele triplication in a male proband from family 1 (F1.1), and duplication in a male proband from family 2 (F2.1) applying array comparative genomic hybridization (array CGH). The rearrangements mapped to the following chromosomal regions–arr[GRCh38] 17p13.3(960254–1291856)×4 in F1.1 and arr[GRCh38] 17p13.3(1227482–1302716)×3 in F2.1. The targeted quantitative PCR revealed that the 17p13.3 locus was also duplicated in the second affected member from family 2 (F2.2; brother of F2.1). In the next step, we performed segregation studies using quantitative PCR and revealed that F1.1 inherited the triplication from his healthy father—F1.2, whereas the locus was unremarkable in the mother of F2.1 & F2.2 and the healthy son of F2.1. However, the duplication was present in a healthy daughter of F2.2, an asymptomatic carrier. The breakpoint analysis allowed to define the exact size and span of the duplicated region in Family 2, i.e., 78,948 bp chr17:1225063–1304010 (HG38). Interestingly, all symptomatic carriers from both families presented with variable SHFLD3 phenotype. The involvement of secondary modifying locus could not be excluded, however, the Sanger sequencing screening of BHLHA9 entire coding sequence was unremarkable for both families.ConclusionsWe have shed light on the one-allele CNV triplication occurrence that should be considered when a higher probe (over duplication range) signal is noted. Second, all SHFLD3 patients were accurately described regarding infrequent limb phenotypes, which were highly variable even when familial. Of note, all symptomatic individuals were males. SHFLD3 still remains a mysterious ultra-rare disease and our findings do not answer crucial questions regarding the disease low penetrance, variable expression and heterogeneity. However, we have presented some clinical and molecular aspects that may be helpful in daily diagnostic routine, both dysmorphological and molecular assessment, of patients affected with SHFLD3.