Intron Of Gene Research Articles

Genome-wide exploration of the CONSTANS-like (COL) gene family and its potential role in regulating plant flowering time in foxtail millet (Setaria italica)

In photoperiod-sensitive plants, the CO-like gene (CONSTANS-like, COL) has a crucial function in regulating the timing of flowering. The blooming period in foxtail millet is greatly influenced by the duration of daylight; however, there is a scarcity of data regarding the molecular properties of the COL genes in the foxtail millet. In this study, we conducted a comprehensive analysis of the COL gene family in foxtail millet at the genome-wide level. We identified 11 SiCOL genes and performed gene structure analysis, which showed pronounced variation in gene length and intron number among the genes. The examination of COL proteins in foxtail millet and other plant species using phylogenetic analysis revealed that they could be clustered into three distinct groups. Cis-element analysis identified elements related to light-responsiveness, hormones, and abiotic stress in the promoter region of the SiCOL gene. Furthermore, tissue-specific expression analysis showed widespread expression of all 11 SiCOL genes in various foxtail millet tissues and organs, particularly in leaves and panicles. Collinearity analysis identified 14 syntenic gene pairs in both foxtail millet and rice. The results also revealed diurnal oscillations in the transcription levels of SiCOL genes under different light conditions. Moreover, among the 11 genes, SiCO, SiCOL1, and SiCOL6 expression levels were negatively correlated with flowering time variation in two foxtail cultivars. Additionally, upon constructing a network of predicted molecular interactions, FLOWER LOCUS-like (FTL) and Phytochromea A (PHY A) were suggested to potentially interact with SiCO, SiCOL1, and SiCOL6. SiCO, SiCOL1, and SiCOL6 have the potential for flowering and heading in foxtail millet. This research enhances our comprehension of the role and control of the SiCOL gene family constituents in foxtail millet, establishing a basis for future investigations.

Functional characterization of chalcone reductase gene CHR3 through RNAi

In this experiment, the soybean genome "Jilin 30" was used as a template to clone the target gene CHR3 using specific primers. The sense fragment, antisense fragment, and intron of the target gene were ligated into pCAMBIA3300 through double enzyme digestion to form a stem loop structure. We then transferred the constructed RNAi vector pCAMBIA3301-CHR3 RNAi into the recipient soybean genome via Agrobacterium mediated method, specifically reducing the expression of the target gene CHR3. By genetic transformation, positive plants were obtained, and molecular biology methods such as Southern blotting, real-time quantitative PCR, isoliquiritigenin analysis, and identification of resistance to Phytophthora root rot were used to detect the expression level of the CHR3 gene in the positive plants and study its function.The RNAi expression vector pCAMBIA3300-CHR3-RNAi was successfully constructed using the sense and antisense fragments of 428 bp CHR3 gene and an intron of 110 bp was inserted to form the stem-loop structure of the RNAi vector. PCR was used to detect 6 lines of the T1 generation and 15 lines of the T2 generation. The results of Southern analysis confirmed the integration of CHR3 and marker genes into the soybean genome. The expression CHR3 gene in the T1 genotypes decreased by 13.1 to 58.3%; the expression in the root decreased by 0.9 to 47.4%; the expression in the leaf decreased by 20 to 44.2%; and the expression in the stem decreased by 7.6 to 23%. The content of isoliquiritigenin decreased by 26.7% as transgenic shoots had a lower content of isoliquiritigenin and reduced resistance to Phytophthora sojae. Bangladesh J. Bot. 53(3): 627-633, 2024 (September) Special

Comparative Mitogenomics Analysis Revealed Evolutionary Divergence among Purpureocillium Species and Gene Arrangement and Intron Dynamics of Ophiocordycipitaceae.

The species of Purpureocillium are cosmopolitan and multitrophic fungi that can infect a wide range of invertebrate hosts. This study reports the mitogenome of P. atypicola, a specialized spider pathogenic fungus. The 112,465 bp mitogenome encoded genes typically found in fungal mitogenomes, and a total of 52 introns inserted into seven genes. A comparison with three other Purpureocillium species revealed significant differences in length and intron number, primarily due to intron variation; however, there was no dynamic variation in the introns of the cox1 gene within the same species of the Purpureocillium genus. Different mitochondrial protein-coding genes showed variable degrees of genetic differentiation among these species, but they were all under purifying selection. Additionally, frequent intron loss or gain events were detected to have occurred during the evolution of the Ophiocordycipitaceae mitogenomes, yet the gene arrangement remains conserved. A phylogenetic analysis of the combined mitochondrial gene set gave identical and well-supported tree topologies. The estimated age of the crown of Ophiocordycipitaceae and Purpureocillium were around the Early Cretaceous period (127 Mya) and Late Cretaceous period (83 Mya), respectively. The results of this study advance our understanding of the genomics, evolution, and taxonomy of this important fungal group.

316VP What you see is what it is: the tales of two brothers with rare intronic dystrophin gene duplication

316VP What you see is what it is: the tales of two brothers with rare intronic dystrophin gene duplication

Assessment of the genetic polymorphism of genus Pinus L. the oligotrophic peat bog "Boloto Mshana"

Aim. To evaluate the molecular genetic polymorphism of representatives of the genus Pinus L. on the territory of the "Boloto Mshana" nature reserve, as well as to test the hypothesis about their possible local hybridization Methods. DNA extraction using CTAB and DNeasy Plant Pro Kit, polymerase chain reaction (PCR) with primers for the chloroplast DNA and genes encoding β-tubulin (TBP markers), electrophoresis of DNA in a polyacrylamide gel with silver nitrate staining. Results. DNA profiles of P. mugo, P. sylvestris and probably P. uliginosa (21 samples in total) were analyzed using one chloroplast DNA marker and two DNA markers based on introns of β-tubulin genes (ТВР, сТВР). The intraspecific genetic polymorphism of the species was compared and the probability of the existence of hybrid forms was investigated. Conclusions. The marker based on the 2nd intron of the β-tubulin genes turned out to be the most effective and informative DNA marker for studying representatives of the Pinus L. genus. P. sylvestris showed less genetic polymorphism by the investigated genetic markers, compared to P. mugo and P. uliginosa. Possible hybrid forms (P. mugo ✕ P. sylvestris) or P. uliginosa were also discovered.

Cereal barcoding by assessing intron length polymorphism of γ-tubulin genes

Aim. The aim of the study was to obtain typical DNA profiles for common cereal plants using a method of assessing γ-tubulin intron length polymorphism and to evaluate the possibility of using this gene for plant species barcoding. Methods. Cereals, in particular wheat, aegilops, barley, and rice, have been studied. The method of γ-tubulin intron length polymorphism assessment was used. Amplified DNA fragments were separated via polyacrylamide gel electrophoresis under non-denaturating conditions and were visualized via silver nitrate staining. Results. The analyzed cereal species-specific DNA profiles of amplicons of the γ-tubulin gene introns were obtained, which made it possible to differentiate the species. Fingerprinting data was used for cluster analysis and dendrogram construction. Conclusions. The feasibility of using the proposed cereal barcoding method has been established. The studied plant DNA profiles can be effectively used in assessing the quality of food products such as flour.

Development of DNA Markers for Acute Hepatopancreatic Necrosis Disease Tolerance in Litopenaeus vannamei through a Genome-Wide Association Study.

Shrimp aquaculture is facing a serious disease, acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio paraheamolyticus (VpAPHND). For sustainable shrimp aquaculture, massive losses of shrimp infected with VpAPHND must be prevented. Research and selection of shrimp tolerant to VpAPHND infection is a sustainable approach to reducing the risk of AHPND. This study focused on the identification and development of potential DNA markers associated with AHPND using DArT sequencing (DArTSeq) and a genome-wide association study. Three populations of post-larval Litopenaeus vannamei were immersed in VpAPHND to collect susceptible (D) and tolerant (S) samples. The 45 D and 48 S shrimp had their genotypes analyzed using DArTSeq. A total of 108,983 SNPs and 17,212 InDels were obtained from the DArTseq data, while the biallelic 516 SNPs and 2293 InDels were finally filtered with PIC < 0.1, MAF < 0.05, and a call rate ≥ 80%. The filtered variants were analyzed for their association with AHPND tolerance. Although there were no significantly associated SNPs and InDels above the Bonferroni correction threshold, candidate variants, four SNPs and 17 InDels corresponding to p < 0.01, were provided for further validation of the AHPND tolerance trait. The candidate SNPs are located on an exon of the zinc finger protein 239-like gene, an intron of an uncharacterized gene, and in intergenic regions. Most of the candidate InDels are in the intergenic regions, with fewer in the intronic and exonic regions. This study provides information on SNPs and InDels for white shrimp. These markers will support the variant database of shrimp and be useful in shrimp aquaculture for breeding selection.

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

Investigation of Introns in Proteus Mirabilis Bacteria and Extent of their Resistance to Antibiotics

Proteus mirabilis is an opportunistic pathogen meaning that causes infection in people with reduced natural immunity. These bacteria possess many virulence factors or pathogens which is essential for colonizing new areas, including tissues and hosts. The study aimed to investigate phenotypically or genetically the introns (MIs) in P. mirabilis by estimate the extent of its resistance to antibiotics. The results showed that the appearance of genetic packages for all migrated isolates at one level. This is an indication of the binding of the primer to its complementary sequence in the DNA strand. The molecular weights of the resulting genetic packages were estimated based on known standard packages, the apparent molecular weights in the path (M) with a graduated size (100- 200 bp). The results also showed a clear similarity of the molecular weight of the resulting bands (1500 bp) with the molecular weight of the diagnostic gene rRNA 16s. In addition, 20 isolates were examined using PCR technology for the three classes of introns gene. All isolates were carried the chromosome intron gene. The appearance of the intron 1 gene on all isolates was (100%).

Resolving and functional analysis of RNA editing sites in sheep ovaries and associations with litter size

Sheep litter size is a critical trait in mutton production. While litter size regulation in relation to DNA transcription have been rigorously investigated, the function of RNA editing remains less explored. To elucidate the mechanisms controlling sheep fecundity at the RNA editing level and identify pivotal RNA editing sites, this study scrutinised RNA editing sites (RESs) in follicular and luteal phases of ovaries from sheep with high and low fecundity, and the functions of population-specific RESs were subsequently analysed. A total of 2 182 475 RESs, 74.61% of which were A-to-I and C-to-U sites, were identified. These RESs were fairly evenly dispersed over the chromosomes, with 46.8% showing close clustering (inter-site distance < 300 bp). Notably, 93% were primarily situated in intronic and intergenic regions. In the follicular phase, pivotal RESs were found in the introns of genes including LPS responsive beige-like anchor, MCC regulator of Wnt signalling, and RWD domain containing 3, among others, and in the exon region of EvC ciliary complex subunit 2. In the luteal phase, RESs were observed in the introns of genes such as H/ACA ribonucleoprotein assembly factor and SDA1 domain-containing 1, and the exon and 3′UTR regions of polypeptide N-acetylgalactosaminyltransferase 15 and ilvB acetolactate synthase-like, respectively. High-fecundity sheep showed RESs in the follicular phase in genes such as fibrillin 1, cyclin−dependent kinase 6, and roundabout 1, and in genes such as autophagy−related 2B and versican in the luteal phase. Thirteen RESs specific to the follicular phase and eight specific to the luteal phase were identified in high-fecundity sheep ovaries. These RESs offer promising molecular targets and enhance understanding of multiple births in sheep from the perspective of posttranscriptional alterations.

Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing.

Quantitative trait loci analyses have revealed an important role for genetic variants in regulating alternative splicing (AS) and alternative cleavage and polyadenylation (APA) in humans. Yet, these studies are generally performed with mature mRNA, so they report on the outcome rather than the processes of RNA maturation and thus may overlook how variants directly modulate pre-mRNA processing. The order in which the many introns of a human gene are removed can substantially influence AS, while nascent RNA polyadenylation can affect RNA stability and decay. However, how splicing order and poly(A) tail length are regulated by genetic variation has never been explored. Here, we used direct RNA nanopore sequencing to investigate allele-specific pre-mRNA maturation in 12 human lymphoblastoid cell lines. We found frequent splicing order differences between alleles and uncovered significant single nucleotide polymorphism (SNP)-splicing order associations in 17 genes. This included SNPs located in or near splice sites as well as more distal intronic and exonic SNPs. Moreover, several genes showed allele-specific poly(A) tail lengths, many of which also had a skewed allelic abundance ratio. HLA class I transcripts, which encode proteins that play an essential role in antigen presentation, showed the most allele-specific splicing orders, which frequently co-occurred with allele-specific AS, APA or poly(A) tail length differences. Together, our results expose new layers of genetic regulation of pre-mRNA maturation and highlight the power of long-read RNA sequencing for allele-specific analyses.

An Integrated Strategy for Analyzing the Complete Complex Integrated Structure of Maize MON810 and Identification of an SNP in External Insertion Sequences.

Genetically modified maize (Zea mays L.) MON810 was approved for importation into China for feed use in 2004; however, the localization data concerning exogenous insertion sequences, which confer insect resistance, have been questionable. MON810 maize plants discovered in northeastern China were used to analyze the molecular characteristics of the exogenous insertion. Using PacBio-HiFi sequencing and PCR assays, we found the insertion was located in chromosome 8, and there was a CaMV35S promoter, hsp70 intron, and insecticide gene cry1Ab, except for genome sequence insertion in the MON810 event. Importantly, the 5' and 3' flanking sequences were located in the region of 55869747-55879326 and 68416240-68419152 on chromosome 5, respectively. The results of this study correct previous results on the genomic localization of the insertion structure for the MON810 event. We also found a single-nucleotide polymorphism (SNP) in the hsp70 intron, which is most likely the first SNP found in a transgenic insertion sequence. PCR amplification in conjunction with Sanger sequencing, allele-specific PCR (AS-PCR), and blocker displacement amplification (BDA) assays were all effective at detecting the base variance. The integrated strategy used in this study can serve as a model for other cases when facing similar challenges involving partially characterized genetic modification events or SNPs.

Notch-Dependent Expression of the Drosophila Hey Gene Is Supported by a Pair of Enhancers with Overlapping Activities.

Drosophila Hey is a basic helix-loop-helix-orange (bHLH-O) protein with an important role in the establishment of distinct identities of postmitotic cells. We have previously identified Hey as a transcriptional target and effector of Notch signalling during the asymmetric division of neuronal progenitors, generating neurons of two types, and we have shown that Notch-dependent expression of Hey also marks a subpopulation of the newborn enteroendocrine (EE) cells in the midgut primordium of the embryo. Here, we investigate the transcriptional regulation of Hey in neuronal and intestinal tissues. We isolated two genomic regions upstream of the promoter (HeyUP) and in the second intron (HeyIN2) of the Hey gene, based on the presence of binding motifs for Su(H), the transcription factor that mediates Notch activity. We found that both regions can direct the overlapping expression patterns of reporter transgenes recapitulating endogenous Hey expression. Moreover, we showed that while HeyIN2 represents a Notch-dependent enhancer, HeyUP confers both Notch-dependent and independent transcriptional regulation. We induced mutations that removed the Su(H) binding motifs in either region and then studied the enhancer functionality in the respective Hey mutant lines. Our results provide direct evidence that although both enhancers support Notch-dependent regulation of the Hey gene, their role is redundant, as a Hey loss-of-function lethal phenotype is observed only after deletion of all their Su(H) binding motifs by CRISPR/Cas9.

Development of DNA Insertion-specific Markers Based on the Intron Region of Oplegnathus punctatus itih4b for Genetic Sex Identification.

Spotted knifejaw (Oplegnathus punctatus) is a significant marine fish species that exhibits pronounced sexual dimorphism, with males generally exhibiting greater weight and growth rates than females. Therefore, the farming of O. punctatus with a high proportion of males is beneficial for improving the quality and efficiency of the O. punctatus aquaculture industry. Furthermore, the development of a rapid technique in sexing O. punctatus fry will facilitate the selection and breeding of superior male varieties of O. punctatus. In this study, genome-wide scanning, comparative genomics, and structural variation analysis methods were employed to identify and extract the homologous region of the inter-alpha-trypsin inhibitor heavy chain 4 (itih4b) gene on the X and Y chromosomes from the complete genome sequence of O. punctatus. This analysis revealed the presence of a large segment of DNA insertion markers on the Y chromosome in the region. Itih4b plays an important role in the mechanisms that regulate inflammatory and immune responses in multicellular organisms. The method described here involved the design of a pair of primers to amplify two bands of 532bp and 333bp in males (individuals with DNA insertion variants in the intron of the itih4b gene). In females (individuals without DNA insertion), only one band of 333bp could be distinguished by agarose gel electrophoresis. This method shortened the time required to accurately characterize intronic DNA insertion variants and genetic sexes in O. punctatus, thereby improving detection efficiency. This study has significant value for the large-scale breeding of O. punctatus all-male seedlings and provides a reference point for the study of intron variation regulation and RNA shearing in the itih4b gene.

Abstract Tu079: The Calcium Handling Machinery and Electrophysiology is Remodeled in Friedreich’s Ataxia

Background: Friedreich's ataxia (FA) is a progressive neurodegenerative disorder. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. Objective: The objective of our study is to determine whether abnormal calcium handling is a molecular mechanism of cardiac dysfunction in FA. Methods: We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium-handling proteins was assessed using proteomics and westernblot. We used echocardiography and telemetric ECG to assess cardiac function in the mice. We also performed in-vivo programmed electrical stimulation (PES) to assess ventricular arrhythmogenesis in the mouse heart. Results: Proteomics and western blot showed that SERCA2, Ryr2, PLN, CASQ2, and CaMKII were downregulated and that the phosphorylation of Ryr2 and PLN (Ser16 and Thr17) were also significantly decreased in left ventricular tissue from humans with FA and from KO mice. The mitochondrial calcium handling proteins VDAC and MCU were upregulated in left ventricular tissue from humans with FA and from KO mice. On telemetric ECG, the RR tended to prolong in the KO animals compared to wild types (p=0.053), and heart rate variability analysis indicated heightened cardiac parasympathetic input. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. In PES, ventricular arrhythmogenesis was greater in KO mice compared to wild types. Conclusions: The reduced expression of calcium handling proteins may contribute to cardiac contractile and electrophysiological abnormalities in FA. Keywords: Friedreich's ataxia, SERCA2, Ryr2, PLN, CASQ2, and CaMKII.

Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β654-Thalassemia Mouse Model.

β654-thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β654 mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β654-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β654-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β654 mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β654 allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β654 mouse model.

The evaluation of IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms in Egyptian patients with alopecia areata: a case–control study

BackgroundAlopecia areata (AA) is a non-scarring hair loss condition that usually affects the scalp. The exact pathogenesis is poorly understood; however, multiple factors like genetics, environmental, psychological, and immunological factors may have a role. The purpose of this study was to look into possible links between the functional interleukin-4 (IL-4) gene intron 3 variable number of tandem repeats (VNTR) and TNF-(rs1799964) gene polymorphism and AA susceptibility. This case–control study consisted of 79 unrelated patients and 156 age- and sex-matched healthy individuals as a control group. The Severity of Alopecia Tool was used to assess the extent of hair loss from the scalp. Polymerase chain reaction (PCR) with specific primers was used to determine IL-4 gene 70-bp VNTR polymorphism while polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was used to investigate TNF-α (rs1799964) gene polymorphism.ResultsNone of the selected polymorphisms for both genotypes and alleles had statistical significance when patients and controls were compared with each other (p-values for IL-4 VNTR were 0.11, 0.74, 0.052 and 0.27 and for TNF-α polymorphism was 0.71, 0.43, 0.65 and 0.55, respectively, for codominant, dominant, recessive and overdominant models of inheritance, respectively). Furthermore, the same results were retrieved when the genotypes were compared with the patient’s clinical and demographic data (p-value > 0.05).ConclusionThe findings indicate that IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms are not linked to the development of AA in the Egyptian population.

A novel splice-altering TNC variant (c.5247A > T, p.Gly1749Gly) in an Chinese family with autosomal dominant non-syndromic hearing loss

BackgroundThis study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene.MethodsA five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis.ResultsThe family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon–intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19.ConclusionThis novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

Differentiation in detoxification gene complements, including neofunctionalization of duplicated cytochrome P450 genes, between lineages of cotton bollworm, Helicoverpa armigera.

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range.

The evolution of ultraconserved elements in vertebrates.

Ultraconserved elements (UCEs) were discovered two decades ago, arbitrarily defined as sequences that are identical over a length ≥200 bp in the human, mouse and rat genomes. The definition was subsequently extended to sequences ≥100 bp identical in at least three of five mammalian genomes (including dog and cow), and shown to have undergone rapid expansion from ancestors in fish and strong negative selection in birds and mammals. Since then, many more genomes have become available, allowing better definition and more thorough examination of UCE distribution and evolutionary history. We developed a fast and flexible analytical pipeline for identifying UCEs in multiple genomes, dedUCE, which allows manipulation of minimum length, sequence identity, and number of species with a detectable UCE according to specified parameters. We suggest an updated definition of UCEs as sequences ≥100 bp and ≥97% sequence identity in ≥50% of placental mammal orders (12813 UCEs). By mapping UCEs to ∼200 species we find that placental UCEs appeared early in vertebrate evolution, well before land colonisation, suggesting the evolutionary pressures driving UCE selection were present in aquatic environments in the Cambrian-Devonian periods. Most (>90%) UCEs likely appeared after the divergence of gnathostomes from jawless predecessors, were largely established in sequence identity by early Sarcopterygii evolution - before the divergence of lobe-finned fishes from tetrapods - and became near fixed in the amniotes. UCEs are mainly located in the introns of protein-coding and non-coding genes involved in neurological and skeletomuscular development, enriched in regulatory elements, and dynamically expressed throughout embryonic development.