The genomes of all lungfish inform on genome expansion and tetrapod evolution.

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

Intra-Phenotypic and -Genotypic Variations of Beauveria bassiana (Bals.) Vuill. Strains Infecting Aedes aegypti L. Adults.

Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification.

Reassessing the exon-foldon correspondence using frustration analysis.

Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon-intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon-foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them.

Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Identification, Classification, and Transcriptional Analysis of Rab GTPase Genes from Tomato (Solanum lycopersicum) Reveals Salt Stress Response Genes.

Salinity in plants generates an osmotic and ionic imbalance inside cells that compromises the viability of the plant. Rab GTPases, the largest family within the small GTPase superfamily, play pivotal roles as regulators of vesicular trafficking in plants, including the economically important and globally cultivated tomato (Solanum lycopersicum). Despite their significance, the specific involvement of these small GTPases in tomato vesicular trafficking and their role under saline stress remains poorly understood. In this work, we identified and classified 54 genes encoding Rab GTPases in cultivated tomato, elucidating their genomic distribution and structural characteristics. We conducted an analysis of duplication events within the S. lycopersicum genome, as well as an examination of gene structure and conserved motifs. In addition, we investigated the transcriptional profiles for these Rab GTPases in various tissues of cultivated and wild tomato species using microarray-based analysis. The results showed predominantly low expression in most of the genes in both leaves and vegetative meristem, contrasting with notably high expression levels observed in seedling roots. Also, a greater increase in gene expression in shoots from salt-tolerant wild tomato species was observed under normal conditions when comparing Solanum habrochaites, Solanum pennellii, and Solanum pimpinellifolium with S. lycopersicum. Furthermore, an expression analysis of Rab GTPases from Solanum chilense in leaves and roots under salt stress treatment were also carried out for their characterization. These findings revealed that specific Rab GTPases from the endocytic pathway and the trans-Golgi network (TGN) showed higher induction in plants exposed to saline stress conditions. Likewise, disparities in gene expression were observed both among members of the same Rab GTPase subfamily and between different subfamilies. Overall, this work emphasizes the high degree of conservation of Rab GTPases, their high functional diversification in higher plants, and the essential role in mediating salt stress tolerance and suggests their potential for further exploration of vesicular trafficking mechanisms in response to abiotic stress conditions.

Conserved and divergent signals in 5' splice site sequences across fungi, metazoa and plants.

In eukaryotic organisms the ensemble of 5' splice site sequences reflects the balance between natural nucleotide variability and minimal molecular constraints necessary to ensure splicing fidelity. This compromise shapes the underlying statistical patterns in the composition of donor splice site sequences. The scope of this study was to mine conserved and divergent signals in the composition of 5' splice site sequences. Because 5' donor sequences are a major cue for proper recognition of splice sites, we reasoned that statistical regularities in their composition could reflect the biological functionality and evolutionary history associated with splicing mechanisms. Results: We considered a regularized maximum entropy modeling framework to mine for non-trivial two-site correlations in donor sequence datasets corresponding to 30 different eukaryotes. For each analyzed species, we identified minimal sets of two-site coupling patterns that were able to replicate, at a given regularization level, the observed one-site and two-site frequencies in donor sequences. By performing a systematic and comparative analysis of 5'splice sites we showed that lineage information could be traced from joint di-nucleotide probabilities. We were able to identify characteristic two-site coupling patterns for plants and animals, and propose that they may echo differences in splicing regulation previously reported between these groups.

Identification of a second genetic alteration in patients with SHOX deficiency individuals: a potential explanation for phenotype variability.

OBJECTIVE: Our study aimed to assess the impact of genetic modifiers on the significant variation in phenotype that is observed in individuals with SHOX deficiency, which is the most prevalent monogenic cause of short stature. DESIGN AND METHODS: We performed a genetic analysis in 98 individuals from 48 families with SHOX deficiency with a target panel designed to capture the entire SHOX genomic region and 114 other genes that modulate growth and/or SHOX action. We prioritized rare potentially deleterious variants. RESULTS: We did not identify potential deleterious variants in the promoter or intronic regions of the SHOX genomic locus. In contrast, we found eight heterozygous variants in 11 individuals from nine families in genes with a potential role as genetic modifiers. In addition to a previously described likely pathogenic (LP) variant in CYP26C1 observed in two families, we identified LP variants in PTHLH and ACAN, and variants of uncertain significance in NPR2, RUNX2, and TP53 in more affected individuals from families with SHOX deficiency. Families with a SHOX alteration restricted to the regulatory region had a higher prevalence of a second likely pathogenic variant (27%) than families with an alteration compromising the SHOX coding region (2.9%, P = .04). CONCLUSION: In conclusion, variants in genes related to the growth plate have a potential role as genetic modifiers of the phenotype in individuals with SHOX deficiency. In individuals with SHOX alterations restricted to the regulatory region, a second alteration could be critical to determine the penetrance and expression of the phenotype.

Genome-wide analysis and characterization of the LRR-RLK gene family provides insights into anthracnose resistance in common bean.

Anthracnose, caused by the hemibiotrophic fungus Colletotrichum lindemuthianum, is a damaging disease of common beans that can drastically reduce crop yield. The most effective strategy to manage anthracnose is the use of resistant cultivars. There are many resistance loci that have been identified, mapped and associated with markers in common bean chromosomes. The Leucine-rich repeat kinase receptor protein (LRR-RLK) family is a diverse group of transmembrane receptors, which potentially recognizes pathogen-associated molecular patterns and activates an immune response. In this study, we performed in silico analyses to identify, classify, and characterize common bean LRR-RLKs, also evaluating their expression profile in response to the infection by C. lindemuthianum. By analyzing the entire genome of Phaseolus vulgaris, we could identify and classify 230 LRR-RLKs into 15 different subfamilies. The analyses of gene structures, conserved domains and motifs suggest that LRR-RLKs from the same subfamily are consistent in their exon/intron organization and composition. LRR-RLK genes were found along the 11 chromosomes of the species, including regions of proximity with anthracnose resistance markers. By investigating the duplication events within the LRR-RLK family, we associated the importance of such a family with an expansion resulting from a strong stabilizing selection. Promoter analysis was also performed, highlighting cis-elements associated with the plant response to biotic stress. With regard to the expression pattern of LRR-RLKs in response to the infection by C. lindemuthianum, we could point out several differentially expressed genes in this subfamily, which were associated to specific molecular patterns of LRR-RLKs. Our work provides a broad analysis of the LRR-RLK family in P. vulgaris, allowing an in-depth structural and functional characterization of genes and proteins of this family. From specific expression patterns related to anthracnose response, we could infer a direct participation of RLK-LRR genes in the mechanisms of resistance to anthracnose, highlighting important subfamilies for further investigations.

Do not panic: An intron-centric guide to alternative splicing.

This review is an attempt to establish concepts of splicing and alternative splicing giving proper relevance to introns, the key actors in this mechanism. It might also work as a guide for those who found their favorite gene undergoes alternative splicing and could benefit from gaining a theoretical framework to understand the possible impacts of this process. This is not a thorough review of all the work in the field, but rather a critical review of some of the most relevant work done to understand the underlying mechanisms of splicing and the key questions that remain unanswered such as: What is the physiological relevance of alternative splicing? What are the functions of the different outcomes? To what extent do different alternative splicing types contribute to the proteome? Intron retention is the most frequent alternative splicing event in plants and, although scientifically neglected, it is also common in animals. This is a heterogeneous type of alternative splicing that includes different sub-types with features that have distinctive consequences in the resulting transcripts. Remarkably, intron retention can be a dead end for a transcript, but it could also be a stable intermediate whose processing is resumed upon a particular signal or change in the cell status. New sequencing technologies combined with the study of intron lariats in different conditions might help to answer key questions and could help us to understand the actual relevance of introns in gene expression regulation.

Deletion in KARLN intron 5 and predictive relationship with bovine tuberculosis and brucellosis infection phenotype.

Kalirin (gene: KALRN) is a Rho-GEF kinase linked to neurodegenerative diseases in humans. Unexpectedly, various polymorphisms in KALRN gene were previously associated with resistance to bacterial infections in ruminants. In this study, we evaluated the effect of the rs384223075 (RS-075) deletion in KALRN intron 5 on the occurrence of Mycobacterium bovis and Brucella abortus infections in cattle. We performed two separate case-control association analyses: one for bovine tuberculosis (bTB) using 308 Holstein and Jersey cows from three herds with prevalence between 5 and 15% for this infection; and another for brucellosis using 140 Holstein and beef crossbred cows from two herds with high prevalence for brucellosis (> 30%). In the bTB analysis, the RS-075 deletion frequency was higher among cases than controls (p = 0.0001), and the absence of the RS-075 deletion allele was associated with negative PPD-skin test results (p = 0.0009) at genotype level. On the contrary, RS-075 was not associated with Brucella spp. serological status (p = 0.72) but, unexpectedly, the deletion allele was more frequent among controls than cases in the beef crossbred herd (0.31 vs. 0.14, p = 0.02). In concordance with this observation, in vitro assays showed that the RS-075 deletion could be linked to an enhanced cellular response to bacterial antigens and unspecific stimulation in mononuclear cells derived from beef crossbred cows, specifically the reactive nitrogen species production (p = 0.008) and proliferation capacity (p = 0.018). This study is consistent with other reports that support an important role of the KALRN gene and its polymorphisms in the host response to intracellular pathogens.

Comprehensive Analysis of the Catalase (CAT) Gene Family and Expression Patterns in Rubber Tree (Hevea brasiliensis) under Various Abiotic Stresses and Multiple Hormone Treatments.

Catalase (CAT) is one of the key enzymes involved in antioxidant defense systems and mainly scavenges H2O2 and plays a vital role in plant growth, development, and various adverse stresses. To date, a systematic study of the CAT gene family in rubber tree has not been reported. In this study, five HbCAT gene family members were identified from the rubber tree genome, and these were mainly clustered into two subfamilies. Gene structure and motif analysis showed that exon-intron and motif patterns were conserved across different plant species. Sequence analysis revealed that HbCAT proteins contain one active catalytic site, one heme-ligand signature sequence, three conserved amino acid residues (His, Tyr, and Asn), and one peroxisome-targeting signal 1 (PTS1) sequence. Fragment duplication is a selection pressure for the evolution of the HbCAT family based on Ka/Ks values. Analysis of cis-acting elements in the promoters indicated that HbCAT gene expression might be regulated by abscisic acid (ABA), salicylic acid (SA), and MYB transcription factors; furthermore, these genes might be involved in plant growth, development, and abiotic stress responses. A tissue-specific expression analysis showed that HbCATs gradually increased with leaf development and were highly expressed in mature leaves. Gene expression profiling exhibited the differential expression of the HbCATs under cold, heat, drought, and NaCl stresses. Our results provide comprehensive information about the HbCAT gene family, laying the foundation for further research on its function in rubber tree.

Evolutionary analysis of endogenous intronic retroviruses in primates reveals an enrichment in transcription binding sites associated with key regulatory processes.

Background: Endogenous retroviruses (ERVs) are the result of the integration of retroviruses into host DNA following germline infection. Endogenous retroviruses are made up of three main genes: gag, pol, and env, each of which encodes viral proteins that can be conserved or not. ERVs have been observed in a wide range of vertebrate genomes and their functions are associated with viral silencing and gene regulation. Results: In this work, we studied the evolutionary history of endogenous retroviruses associated with five human genes (INPP5B, DET1, PSMA1, USH2A, and MACROD2), which are located within intron sections. To verify the retroviral origin of the candidates, several approaches were used to detect and locate ERV elements. Both orthologous and paralogous genes were identified by Ensembl and then analyzed for ERV presence using RetroTector. A phylogenetic tree was reconstructed to identify the minimum time point of ERV acquisition. From that search, we detected ERVs throughout the primate lineage and in some other groups. Also, we identified the minimum origin of the ERVs from the parvorder Catarrhini to the Homininae subfamily. Conclusions: With the data collected, and by observing the transcription factors annotated inside ERVs, we propose that these elements play a relevant role in gene expression regulation and they probably possess important features for tumorigenesis control.

Acanthamoeba castellanii exhibits intron retention during encystment.

Intron retention (IR) refers to the mechanism of alternative splicing in which an intron is not excised from the mature transcript. IR in the cosmopolitan free-living amoeba Acanthamoeba castellanii has not been studied. We performed an analysis of RNA sequencing data during encystment to identify genes that presented differentially retained introns during this process. We show that IR increases during cyst formation, indicating a potential mechanism of gene regulation that could help downregulate metabolism. We identify 69 introns from 67 genes that are differentially retained comparing the trophozoite stage and encystment after 24 and 48 h. These genes include several hypothetical proteins. We show different patterns of IR during encystment taking as examples a lipase, a peroxin-3 protein, an Fbox domain containing protein, a proteasome subunit, a polynucleotide adenylyltransferase, and a tetratricopeptide domain containing protein. A better understanding of IR in Acanthamoeba, and even other protists, could help elucidate changes in life cycle and combat disease such as Acanthamoeba keratitis in which the cyst is key for its persistence.

VNTR Polymorphism in Intron 4 of the eNOS Gene and the Risk of Gastrointestinal Bleeding: A Case-control Study.

BACKGROUND AND AIMS: Considering the lack of knowledge regarding the influence of the variable number of repeats of 27 pb in intron 4 (4b/4a VNTR - rs61722009) of the endothelial nitric oxide synthase (eNOS) on the drug response, we assessed the influence of this polymorphism for the risk of upper gastrointestinal bleeding (UGIB). METHODS: A case-control study, including 200 cases and 706 controls, was conducted in a Brazilian hospital complex. Cases were participants with UGIB diagnosis. Controls were participants admitted to surgical procedures not related to gastrointestinal problems. The 4b/4a VNTR was determined through polymerase chain reaction followed by fragment analysis. Conditional logistic regression models were designed. The additive interaction between the presence of the 4b/4a VNTR variant and the use of low-dose aspirin (LDA) and nonsteroidal anti-inflammatory drugs (NSAIDs) was calculated by fitting the Cox regression model through the parameters of Synergism index (S) and Relative Excess Risk Due To Interaction (RERI). RESULTS: The presence of the 4b/4a VNTR variant did not increase the risk of UGIB: carriers of the 4a/4a genotype (OR=0.37, 95%CI: 0.09-1.45) and of the variant allele "4a" (OR=0.91, 95%CI: 0.55-1.51). The risk of UGIB in LDA users carriers of the wild genotype (OR=4.96, 95%CI: 2.04- 2.06) and the variant allele "4a" (OR=3.49, 95%CI: 1.18-10.38) is similar, as well as for NSAID users carriers of the wild genotype (OR=5.73, 95%CI: 2.61-12.60) and variant allele "4a" (OR=5.51, 95%CI: 1.42-15.82). No additive interaction was identified between the presence of the genetic variant and the use of LDA [RERI: -1.44 (95%CI: -6.02-3.14; S: 0.63 (95%CI: -1.97-1.15)] and NSAIDs [RERI: -0.13 (95%CI: -6.79-6.53; S: 0.97 (95%CI: -0.23-4.19)] on the UGIB risk. CONCLUSION: Our data suggests that there is no increase in the magnitude of UGIB risk in LDA and NSAIDs users' carrying the variant allele "4a".

NOS3 gene intron 4 a/b polymorphism is associated with ESRD in autosomal dominant polycystic kidney disease patients.

INTRODUCTION: Endothelial nitric oxide synthase (eNOS) genes have been implicated in renal hemodynamics as potent regulators of vascular tone and blood pressure. It has been linked to a reduction in plasma nitric oxide levels. Several studies have recently been conducted to investigate the role of NOS3 gene polymorphisms and end-stage renal disease (ESRD). However, the results are still unclear and the mechanisms are not fully defined. As a result, we conducted a meta-analysis to examine the relationship between NOS3 gene polymorphism and ESRD in autosomal polycystic kidney disease (ADPKD) patients. METHODS: To assess the relationship between NOS3 gene polymorphism and ESRD, relevant studies published between September 2002 and December 2020 were retrieved from the PubMed (Medline), EMBASE, Google Scholar, and Web of Science databases. The pooled odds ratio (OR) and 95 % confidence interval (CI) were calculated using a fixed-effect model. To assess the heterogeneity of studies, we used Cochrane's Q test and the Higgins and Thompson I2 statistics. RESULTS: Our meta-analysis of 13 studies showed that the presence of the two NOS3 gene polymorphisms significantly increased ESRD risk in ADPKD patients with 4a/b gene polymorphism (aa+ab vs. bb: OR=1.95, 95% CI=1.24-3.09, p=0.004). In addition, no significant association was found between the NOS3 894G>T (Glu298Asp) polymorphism and the risk of ESRD in ADPKD patients (GT+TT vs. GG: OR=1.21, 95% CI=0.93-1.58, p=0.157). There was no evidence of publication bias. CONCLUSIONS: The findings of the current meta-analysis suggest that NOS3 intron 4a/b polymorphism plays a vital role in the increasing risk of ESRD in ADPKD patients.

HLA-G genetic diversity and evolutive aspects in worldwide populations.

HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies.

Intronic variants of MITF (rs7623610) and CREB1 (rs10932201) genes may enhance splicing efficiency in human melanoma cell line.

We previously reported that intronic single nucleotide variations (SNVs) in MITF (c.938-325G>A, rs7623610) and CREB1 (c.303+373G>A, rs10932201) genes were associated with risk, aggressiveness, and prognosis of cutaneous melanoma (CM). In this study, we investigated the influence of the above SNVs in splicing patterns and efficiency. We constructed minigenes with wild type and variant alleles from MITF and CREB1 to assess the effect of the SNVs on splicing. The minigenes were transfected in the human melanoma cell line (SK-MEL-28). RT-PCR and DNA sequencing investigated the constructs' splicing patterns. Minigenes constructs' splicing efficiency and HNRNPA1 and SF1 splicing genes' expression were investigated by qPCR. We found that MITF and CREB1 SNVs did not alter the splicing pattern, but they influenced the splicing efficiency. MITF-A (p= 0.03) and CREB1-A (p= 0.005) variant minigenes yielded an increase of mRNA generated from the constructions. Additionally, lower mRNA levels of HNRNPA1 and SF1 were seen in the variant minigenes MITF-A (p= 0.04) and CREB1-A (p= 0.005). We described for the first time the potential importance of MITF rs7623610 and CREB1 rs10932201 SNVs in splicing efficiency and its relationship with CM.

Calophyllaceae plastomes, their structure and insights in relationships within the clusioids.

A complete chloroplast genome is not yet available for numerous species of plants. Among the groups that lack plastome information is the clusioid clade (Malpighiales), which includes five families: Bonnetiaceae, Calophyllaceae, Clusiaceae, Hypericaceae, and Podostemaceae. With around 2200 species, it has few published plastomes and most of them are from Podostemaceae. Here we assembled and compared six plastomes from members of the clusioids: five from Calophyllaceae (newly sequenced) and one from Clusiaceae. Putative regions for evolutionary studies were identified and the newly assembled chloroplasts were analyzed with other available chloroplasts for the group, focusing on Calophyllaceae. Our results mostly agree with recent studies which found a general conserved structure, except for the two Podostemaceae species that have a large inversion (trnK-UUU-rbcL) and lack one intron from ycf3. Within Calophyllaceae we observed a longer LSC and reduced IRs in Mahurea exstipulata, resulting in some genic rearrangement, and a short inversion (psbJ-psbE) in Kielmeyera coriacea. Phylogenetic analyses recovered the clusioids and the five families as monophyletic and revealed that conflicts in relationships reported in the literature for the group agree with nodes concentrating uninformative or conflicting gene trees. Our study brings new insights about clusioid plastome architecture and its evolution.

A novel DMD intronic alteration: a potentially disease-causing variant of an intermediate muscular dystrophy phenotype.

Pathogenic germline variants in DMD gene, which encodes the well-known cytoskeletal protein named dystrophin, are associated with a wide range of dystrophinopathies disorders, such as Duchenne muscular dystrophy (DMD, severe form), Becker muscular dystrophy (BMD, mild form) and intermediate muscular dystrophy (IMD). Muscle biopsy, immunohistochemistry, molecular (multiplex ligation-dependent probe amplification (MLPA)/next-generation sequencing (NGS) and Sanger methods) and in silico analyses were performed in order to identify alterations in DMD gene and protein in a patient with a clinical manifestation and with high creatine kinase levels. Herein, we described a previously unreported intronic variant in DMD and reduced dystrophin staining in the muscle biopsy. This novel DMD variant allele, c.9649+4A>T that was located in a splice donor site within intron 66. Sanger sequencing analysis from maternal DNA showed the presence of both variant c.9649+4A>T and wild-type (WT) DMD alleles. Different computational tools suggested that this nucleotide change might affect splicing through a WT donor site disruption, occurring in an evolutionarily conserved region. Indeed, we observed that this novel variant, could explain the reduced dystrophin protein levels and discontinuous sarcolemmal staining in muscle biopsy, which suggests that c.9649+4A>T allele may be re-classified as pathogenic in the future. Our data show that the c.9649+4A>T intronic sequence variant in the DMD gene may be associated with an IMD phenotype and our findings reinforce the importance of a more precise diagnosis combining muscle biopsy, molecular techniques and comprehensive in silico approaches in the clinical cases with negative results for conventional genetic analysis.

Phylogeography, colouration, and cryptic speciation across the Indo-Pacific in the sea urchin genus Echinothrix.

The sea urchins Echinothrix calamaris and Echinothrix diadema have sympatric distributions throughout the Indo-Pacific. Diverse colour variation is reported in both species. To reconstruct the phylogeny of the genus and assess gene flow across the Indo-Pacific we sequenced mitochondrial 16S rDNA, ATPase-6, and ATPase-8, and nuclear 28S rDNA and the Calpain-7 intron. Our analyses revealed that E. diadema formed a single trans-Indo-Pacific clade, but E. calamaris contained three discrete clades. One clade was endemic to the Red Sea and the Gulf of Oman. A second clade occurred from Malaysia in the West to Moorea in the East. A third clade of E. calamaris was distributed across the entire Indo-Pacific biogeographic region. A fossil calibrated phylogeny revealed that the ancestor of E. diadema diverged from the ancestor of E. calamaris ~ 16.8 million years ago (Ma), and that the ancestor of the trans-Indo-Pacific clade and Red Sea and Gulf of Oman clade split from the western and central Pacific clade ~ 9.8 Ma. Time since divergence and genetic distances suggested species level differentiation among clades of E. calamaris. Colour variation was extensive in E. calamaris, but not clade or locality specific. There was little colour polymorphism in E. diadema.