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Autoinhibition is a prevalent allosteric regulatory mechanism in signaling proteins. Reduced autoinhibition underlies the tumorigenic effect of some known cancer drivers, but whether autoinhibition is altered generally in cancer remains elusive. Here, we demonstrate that cancer-associated missense mutations, in-frame insertions/deletions, and fusion breakpoints are enriched within inhibitory allosteric switches (IASs) across all cancer types. Selection for IASs that are recurrently mutated in cancers identifies established and unknown cancer drivers. Recurrent missense mutations in IASs of these drivers are associated with distinct, cancer-specific changes in molecular signaling. For the specific case of PPP3CA, the catalytic subunit of calcineurin, we provide insights into the molecular mechanisms of altered autoinhibition by cancer mutations using biomolecular simulations, and demonstrate that such mutations are associated with transcriptome changes consistent with increased calcineurin signaling. Our integrative study shows that autoinhibition-modulating genetic alterations are positively selected for by cancer cells.

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Within anatomical willed body programs and skeletal collections, whole bodies and their disassociated limbs and organs are identified and tracked. However, if these tracking mechanisms fail, DNA recovered from the formalin-fixed tissues/organs could provide an additional layer of quality assurance. Embalming fluids preserve biological tissues; however, they also damage, fragment, and cross-link DNA and protein molecules. This project investigated the success of STR-typing from various soft tissue and bone samples that were fixed with embalming solutions with a range of formaldehyde concentrations. Formalin-fixed samples dissected from five cadavers, including skin, muscle, bone, heart, and kidney were used in Phase 1 of this study. In Phase 2, an additional 57 tissue samples from various embalmed organs and body parts were collected to demonstrate long-term fixation and direct applicability within a body donor program. DNA was extracted from the samples using the QIAamp® FFPE Tissue Kit (QIAGEN), quantified with the Investigator® QuantiPlex® Pro RGQ qPCR Kit (QIAGEN), and amplified using the Investigator® 24plex and 26plex QS Kits and the Investigator® DIPplex Kit (QIAGEN). The results show the DNA was severely damaged, degraded, and often in low amounts (after one year post-embalming). Sampling from skin and muscle tissues embalmed with ~2.5%-5% formaldehyde solutions appears to be the best strategy for identification, while also maintaining the preservation of the tissues. The results of this project can provide informative data when determining which genotyping strategy may be best suited for the identification, re-association, and establishment of a database for the provenance of formalin-fixed human remains.

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Abaca (Musa textilis Née) is an economically important fiber crop in the Philippines. Its economic potential, however, is hampered by biotic and abiotic stresses, which are exacerbated by insufficient genomic resources for varietal identification vital for crop improvement. To address these gaps, this study aimed to discover genome-wide polymorphisms among abaca cultivars and other Musa species and analyze their potential as genetic marker resources. This was achieved through whole-genome Illumina resequencing of abaca cultivars and variant calling using BCFtools, followed by genetic diversity and phylogenetic analyses. A total of 20,590,381 high-quality single-nucleotide polymorphisms (SNP) and DNA insertions/deletions (InDels) were mined across 16 abaca cultivars. Filtering based on linkage disequilibrium (LD) yielded 130,768 SNPs and 13,620 InDels, accounting for 0.396 ± 0.106 and 0.431 ± 0.111 of gene diversity across these cultivars. LD-pruned polymorphisms across abaca, M. troglodytarum, M. acuminata and M. balbisiana enabled genetic differentiation within abaca and across the four Musa spp. Phylogenetic analysis revealed the registered varieties Abuab and Inosa to accumulate a significant number of mutations, eliciting further studies linking mutations to their advantageous phenotypes. Overall, this study pioneered in producing marker resources in abaca based on genome-wide polymorphisms vital for varietal authentication and comparative genotyping with the more studied Musa spp.

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Human gut microbiota is associated with human health and disease, and is known to have the second-largest genome in the human body. The microbiota genome is important for their functions and metabolites; however, accurate genomic access to the microbiota of the human gut is hindered due to the difficulty of cultivating and the shortcomings of sequencing technology. Therefore, we applied the stLFR library construction method to assemble the microbiota genomes and demonstrated that assembly property outperformed standard metagenome sequencing. Using the assembled genomes as references, SNP, INDEL, and HGT gene analyses were performed. The results demonstrated significant differences in the number of SNPs and INDELs among different individuals. The individual displayed a unique species variation spectrum, and the similarity of strains within individuals decreased over time. In addition, the coverage depth analysis of the stLFR method shows that a sequencing depth of 60X is sufficient for SNP calling. HGT analysis revealed that the genes involved in replication, recombination and repair, mobilome prophages, and transposons were the most transferred genes among different bacterial species in individuals. A preliminary framework for human gut microbiome studies was established using the stLFR library construction method.

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Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

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Mutations in simple sequence repeat loci underlie many inherited disorders in humans, and are increasingly recognized as important determinants of natural phenotypic variation. In eukaryotes, mutations in these sequences are primarily repaired by the MutSß mismatch repair complex. To better understand the role of this complex in mismatch repair and the determinants of simple sequence repeat mutation predisposition, we performed mutation accumulation in yeast strains with abrogated MutSß function. We demonstrate that mutations in simple sequence repeat loci in the absence of mismatch repair are primarily deletions. We also show that mutations accumulate at drastically different rates in short (<8 bp) and longer repeat loci. These data lend support to a model in which the mismatch repair complex is responsible for repair primarily in longer simple sequence repeats.

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Complex kinship analysis has been widely applied in disaster victim identification and criminal investigations. A larger number of genetic markers is required to improve the discrimination power of the system in complex kinship analysis compared to that in paternity testing, as distant relatives share fewer genetic segments. Genetic markers, including short tandem repeats (STRs), single-nucleotide polymorphisms (SNPs), and insertions-deletions (indels), play complementary roles in kinship analysis. Few studies have systematically analyzed the system discrimination power of a new combination of different types of genetic markers before using these markers in practice. Here, we tested the ability of a set of 56 STRs available in commercial panels on complex kinship analysis. We next introduced a combination marker set of STRs, indels, and SNPs and evaluated the system discrimination power of 72 indels + 52 SNPs to improve the weight of 56 STRs. Statistical analysis of complex kinship within third-degree kinship testing was performed to compare 56 STRs or 72 indels + 52 SNPs alone. True samples were assessed, including 99 full siblings, 112 uncle/aunt-nephew/niece, 43 grandfather/grandmother-grandson/granddaughter, 63 first cousins, and 5931 unrelated pairs. Simulation was also performed using 10,000 pairs of relatives and 10,000 unrelated individuals. The effectiveness of the three marker sets in kinship testing was ranked as follows: 56 STRs + 72 indels + 52 SNPs > 56 STRs > 72 indels + 52 SNPs. All three marker sets were powerful in first-degree kinship testing; 56 STRs and 56 STRs + 72 indels + 52 SNPs could distinguish most second-degree relatives from unrelated pairs. However, only a portion of third-degree relatives was correctly determined from unrelated individuals using 56 STRs + 72 indels + 52 SNPs. In relationship testing, 56 STRs and 56 STRs + 72 indels + 52 SNPs were powerful enough to distinguish first-degree relatives from second-degree or third-degree relatives. Our results provide a strategy and guidance applicable in forensic practice for complex kinship analysis by combining STRs, SNPs, and indels.

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Idiopathic superior oblique muscle palsy is a major type of paralytic, non-comitant strabismus and presents vertical and cyclo-torsional deviation of one eye against the other eye, with a large vertical fusion range and abnormal head posture such as head tilt. Genetic background is considered to play a role in its development, as patients with idiopathic superior oblique muscle palsy have varying degrees of muscle hypoplasia and, rarely, the complete absence of the muscle, that is, aplasia. In this study, whole genome sequencing was performed, and single nucleotide variations and short insertions/deletions (SNVs/InDels) were annotated in two patients each in three small families (six patients in total) with idiopathic superior oblique muscle palsy, in addition to three normal individuals in one family. At first, linkage analysis was carried out in the three families and SNVs/InDels in chromosomal loci with negative LOD scores were excluded. Next, SNVs/InDels shared by the six patients, but not by the three normal individuals, were chosen. SNVs/InDels were further narrowed down by choosing low-frequency (<1%) or non-registered SNVs/InDels in four databases for the Japanese population, and then by choosing SNVs/InDels with functional influence, leading to one candidate gene, SSTR5-AS1 in chromosome 16. The six patients were heterozygous for 13-nucleotide deletion in SSTR5-AS1, except for one homozygous patient, while the three normal individuals were wild type. Targeted polymerase chain reaction (PCR) and direct sequencing of PCR products confirmed the 13-nucleotide deletion in SSTR5-AS1. In the face of newly-registered SSTR5-AS1 13-nucleotide deletion at a higher frequency in a latest released database for the Japanese population, the skipping of low-frequency and non-registration sorting still resulted in only 13 candidate genes including SSTR5-AS1 as common variants. The skipping of linkage analysis also led to the same set of 13 candidate genes. Different testing strategies that consisted of linkage analysis and simple unintentional bioinformatics could reach candidate genes in three small families with idiopathic superior oblique muscle palsy.

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The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

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In our previous study, in which array CGH was used on 19 Lebanese ASD subjects and their parents, we identified rare copy number variants (CNVs) in 14 subjects. The five remaining subjects did not show any CNVs related to autism spectrum disorders (ASD). In the present complementary study, we applied whole-exome sequencing (WES), which allows the identification of rare genetic variations such as single nucleotide variations and small insertions/deletions, to the five negative CNV subjects. After stringent filtering of initial data on the five families, three novel genes potentially related to neurodevelopment were identified, including a de novo mutation in the MIS18BP1 gene. In addition, genes already known to be related to ASD contained sequence variations. Our findings outline the potential involvement of the novel de novo mutation in the MIS18BP1 gene in the genetic etiology and pathophysiology of ASD and highlights the genetic complexity of these disorders. Further studies with larger cohorts of subjects are needed to confirm these observations, and functional analyses need to be performed to understand the precise pathophysiology in these cases.

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PURPOSE: The objective of this study is to validate the existence of dual cores within the typical phosphotyrosine binding (PTB) domain and to identify potentially damaging and pathogenic nonsynonymous coding single nuclear polymorphisms (nsSNPs) in the canonical PTB domain of the CCM2 gene that causes cerebral cavernous malformations (CCMs). METHODS: The nsSNPs within the coding sequence for PTB domain of human CCM2 gene, retrieved from exclusive database searches, were analyzed for their functional and structural impact using a series of bioinformatic tools. The effects of mutations on the tertiary structure of the PTB domain in human CCM2 protein were predicted to examine the effect of nsSNPs on the tertiary structure of PTB Cores. RESULTS: Our mutation analysis, through alignment of protein structures between wildtype CCM2 and mutant, predicted that the structural impacts of pathogenic nsSNPs is biophysically limited to only the spatially adjacent substituted amino acid site with minimal structural influence on the adjacent core of the PTB domain, suggesting both cores are independently functional and essential for proper CCM2 PTB function. CONCLUSION: Utilizing a combination of protein conservation and structure-based analysis, we analyzed the structural effects of inherited pathogenic mutations within the CCM2 PTB domain. Our results predicted that the pathogenic amino acid substitutions lead to only subtle changes locally, confined to the surrounding tertiary structure of the PTB core within which it resides, while no structural disturbance to the neighboring PTB core was observed, reaffirming the presence of independently functional dual cores in the CCM2 typical PTB domain.

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Sperm-associated antigen 17 (SPAG17) gene encodes a central pair protein, which is involved in flagellar motility, male fertility and skeletal growth in ruminants. The insertions/deletions (indels) and copy number variations (CNVs) influence phenotypic traits by altering the sequences and copy numbers of functional genes, respectively. This study identified a novel 8-bp indel of SPAG17 gene in 1520 individuals from eight different cattle breeds, as well as a novel CNV region in 355 animals. The correlation analysis of indel showed that the individuals of ID genotype had superior performance traits such as body height (p = 0.038) and body slanting length (p = 0.041) as compared to other genotypes in Xianan cattle. For the CNV, different copy numbers were closely related to the body height in Qinchuan (p = 0.045) and body weight in Xianan (p = 0.036) breeds. Importantly, significant difference was observed between the 8-bp indel and the copy number loss in Xianan breed (p < 0.01). These findings indicated that the variations within the bovine SPAG17 gene can be considered as an effective DNA molecular marker for beef cattle breeding.

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Introduction: The world is still struggling against the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in 2022. The pandemic has been facilitated by the intermittent emergence of variant strains, which has been explained and classified mainly by the patterns of point mutations of the spike (S) gene. However, the profiles of insertions/deletions (indels) in SARS-CoV-2 genomes during the pandemic remain largely unevaluated yet. Methods: In this study, we first screened for the genome regions of polymorphic indel sites by performing multiple sequence alignment; then, NCBI BLAST search and GISAID database search were performed to comprehensively investigate the indel profiles at the polymorphic indel hotspot and elucidate the emergence and spread of the indels in time and geographical distribution. Results: A polymorphic indel hotspot was identified in the N-terminal domain of the S gene at approximately 22,200 nucleotide position, corresponding to 210-215 amino acid positions of SARS-CoV-2 S protein. This polymorphic hotspot was comprised of adjacent 3-base deletion (5'-ATT-3'; Spike_N211del) and 9-base insertion (5'-AGCCAGAAG-3'; Spike_ins214EPE). By performing NCBI BLAST search and GISAID database search, we identified several types of tandem repeats of the 9-base insertion, creating an 18-base insertion (Spike_ins214EPEEPE, Spike_ins214EPDEPE). The results of the searches suggested that the two-cycle tandem repeats of the 9-base insertion were created in November 2021 in Central Europe, whereas the emergence of the original one-cycle 9-base insertion (Spike_ins214EPE) would date back to the middle of 2020 and was away from the Central Europe. The identified 18-base insertions based on 2-cycle tandem repeat of the 9-base insertion were collected between November 2021 and April 2022, suggesting that these mutations could not survive and have been already eliminated. Discussion: The GISAID database search implied that this polymorphic indel hotspot to be with one of the highest tolerability for incorporating indels in SARS-CoV-2 S gene. In summary, the present study identified a variable number of tandem repeat of 9-base insertion in the N-terminal domain of SARS-CoV-2 S gene, and the repeat could have occurred at different time from the insertion of the original 9-base insertion.

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Structural variation (SV) describes a broad class of genetic variation greater than 50 bp in size. SVs can cause a wide range of genetic diseases and are prevalent in rare developmental disorders (DDs). Individuals presenting with DDs are often referred for diagnostic testing with chromosomal microarrays (CMAs) to identify large copy-number variants (CNVs) and/or with single-gene, gene-panel, or exome sequencing (ES) to identify single-nucleotide variants, small insertions/deletions, and CNVs. However, individuals with pathogenic SVs undetectable by conventional analysis often remain undiagnosed. Consequently, we have developed the tool InDelible, which interrogates short-read sequencing data for split-read clusters characteristic of SV breakpoints. We applied InDelible to 13,438 probands with severe DDs recruited as part of the Deciphering Developmental Disorders (DDD) study and discovered 63 rare, damaging variants in genes previously associated with DDs missed by standard SNV, indel, or CNV discovery approaches. Clinical review of these 63 variants determined that about half (30/63) were plausibly pathogenic. InDelible was particularly effective at ascertaining variants between 21 and 500 bp in size and increased the total number of potentially pathogenic variants identified by DDD in this size range by 42.9%. Of particular interest were seven confirmed de novo variants in MECP2, which represent 35.0% of all de novo protein-truncating variants in MECP2 among DDD study participants. InDelible provides a framework for the discovery of pathogenic SVs that are most likely missed by standard analytical workflows and has the potential to improve the diagnostic yield of ES across a broad range of genetic diseases.

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BACKGROUND: Oat (Avena sativa L.) is a recognized health-food, and the contributions of its different candidate A-genome progenitor species remain inconclusive. Here, we report chloroplast genome sequences of eleven Avena species, to examine the plastome evolutionary dynamics and analyze phylogenetic relationships between oat and its congeneric wild related species. RESULTS: The chloroplast genomes of eleven Avena species (size range of 135,889-135,998 bp) share quadripartite structure, comprising of a large single copy (LSC; 80,014-80,132 bp), a small single copy (SSC; 12,575-12,679 bp) and a pair of inverted repeats (IRs; 21,603-21,614 bp). The plastomes contain 131 genes including 84 protein-coding genes, eight ribosomal RNAs and 39 transfer RNAs. The nucleotide sequence diversities (Pi values) range from 0.0036 (rps19) to 0.0093 (rpl32) for ten most polymorphic genes and from 0.0084 (psbH-petB) to 0.0240 (petG-trnW-CCA) for ten most polymorphic intergenic regions. Gene selective pressure analysis shows that all protein-coding genes have been under purifying selection. The adjacent position relationships between tandem repeats, insertions/deletions and single nucleotide polymorphisms support the evolutionary importance of tandem repeats in causing plastome mutations in Avena. Phylogenomic analyses, based on the complete plastome sequences and the LSC intermolecular recombination sequences, support the monophyly of Avena with two clades in the genus. CONCLUSIONS: Diversification of Avena plastomes is explained by the presence of highly diverse genes and intergenic regions, LSC intermolecular recombination, and the co-occurrence of tandem repeat and indels or single nucleotide polymorphisms. The study demonstrates that the A-genome diploid-polyploid lineage maintains two subclades derived from different maternal ancestors, with A. longiglumis as the first diverging species in clade I. These genome resources will be helpful in elucidating the chloroplast genome structure, understanding the evolutionary dynamics at genus Avena and family Poaceae levels, and are potentially useful to exploit plastome variation in making hybrids for plant breeding.

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High-throughput sequencing technologies have rapidly developed during the past years and have become an essential tool in plant sciences. However, the analysis of genomic data remains challenging and relies mostly on the performance of automatic pipelines. Frequently applied pipelines involve the alignment of sequence reads against a reference sequence and the identification of sequence variants. Since most benchmarking studies of bioinformatics tools for this purpose have been conducted on human datasets, there is a lack of benchmarking studies in plant sciences. In this study, we evaluated the performance of 50 different variant calling pipelines, including five read mappers and ten variant callers, on six real plant datasets of the model organism Arabidopsis thaliana. Sets of variants were evaluated based on various parameters including sensitivity and specificity. We found that all investigated tools are suitable for analysis of NGS data in plant research. When looking at different performance metrics, BWA-MEM and Novoalign were the best mappers and GATK returned the best results in the variant calling step.

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The co-occurrence among single nucleotide polymorphisms (SNPs), insertions-deletions (InDels), and oligonucleotide repeats has been reported in prokaryote, eukaryote, and chloroplast genomes. Correlations among SNPs, InDels, and repeats have been investigated in the plant family Araceae previously using pair-wise sequence alignments of the chloroplast genomes of two morphotypes of one species, Colocasia esculenta belonging to subfamily Aroideae (crown group), and four species from the subfamily Lemnoideae, a basal group. The family Araceae is a large family comprising 3,645 species in 144 genera, grouped into eight subfamilies. In the current study, we performed 34 comparisons using 27 species from 7 subfamilies of Araceae to determine correlation coefficients among the mutational events at the family, subfamily, and genus levels. We express strength of the correlations as: negligible or very weak (0.10-0.19), weak (0.20-0.29), moderate (0.30-0.39), strong (0.40-0.69), very strong (0.70-0.99), and perfect (1.00). We observed strong/very strong correlations in most comparisons, whereas a few comparisons showed moderate correlations. The average correlation coefficient was recorded as 0.66 between "SNPs and InDels," 0.50 between "InDels and repeats," and 0.42 between "SNPs and repeats." In qualitative analyses, 95-100% of the repeats at family and sub-family level, while 36-86% of the repeats at genus level comparisons co-occurred with SNPs in the same bins. Our findings show that such correlations among mutational events exist throughout Araceae and support the hypothesis of distribution of oligonucleotide repeats as a proxy for mutational hotspots.

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Lysine demethylase 6A (KDM6A, also known as UTX), which belongs a type of histone demethylase, is essential for spermatogenesis and embryo development. However, in pig, the coding sequence, mRNA expression and insertion/deletion (indel) variants of KDM6A remain unclear. Herein, the open-reading frame (ORF) of pig KDM6A gene was cloned and characterized. The entire ORF sequence was 4206 bp in length encoding a deduced protein of 1401 amino acid residues. The phylogenetic tree and signatures of selection on the pig KDM6A gene were calculated, and the results revealed that the pig KDM6A gene has been under strong positively selection. Expression analysis results showed that KDM6A gene was expressed in all tissues tested both in male piglets and adult boars. Meanwhile, with testicular development, the KDM6A expression levels in testis were significantly increased. Additionally, three intronic indels of 11-bp insertion, 17-bp deletion and 16-bp insertion were identified. Association analyses revealed that the 11-bp indel was associated with testicular short perimeter (TSP) (P < 0.01), testicular long perimeter (TLP) (P < 0.01) and testicular weight (TW) (P < 0.01) in 15-day-old Yorkshire and TLP (P < 0.01) in 40-day-old Yorkshire. The 16-bp indel was associated with TLP, TSP and TW (P < 0.05) in 15-day-old Yorkshire. All these findings would provide a foundation for the further research of KDM6A gene and the application of marker-assisted selection to pig breeding.

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Owing to its great medicinal and ornamental values, Dendrobium officinale is frequently adulterated with other Dendrobium species on the market. Unfortunately, the utilization of the common DNA markers ITS, ITS2, and matK+rbcL is unable to distinguish D. officinale from 5 closely related species of it (D. tosaense, D. shixingense, D. flexicaule, D. scoriarum and D. aduncum). Here, we compared 63 Dendrobium plastomes comprising 40 newly sequenced plastomes of the 6 species and 23 previously published plastomes. The plastomes of D. officinale and its closely related species were shown to have conserved genome structure and gene content. Comparative analyses revealed that small single copy region contained higher variation than large single copy and inverted repeat regions, which was mainly attributed to the loss/retention of ndh genes. Furthermore, the intraspecific sequence variability among different Dendrobium species was shown to be diversified, which necessitates a cautious evaluation of genetic markers specific for different Dendrobium species. By evaluating the maximum likelihood trees inferred from different datasets, we found that the complete plastome sequence dataset had the highest discriminatory power for D. officinale and its closely related species, indicating that complete plastome sequences can be used to accurately authenticate Dendrobium species.

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INTRODUCTION: Multidrug resistance in Streptococcus pneumoniae has emerged as a serious problem to public health. A further understanding of the genetic diversity in antibiotic-resistant S. pneumoniae isolates is needed. METHODS: We conducted whole-genome resequencing for 25 pneumococcal strains isolated from children with different antimicrobial resistance profiles. Comparative analysis focus on detection of single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) was conducted. Moreover, phylogenetic analysis was applied to investigate the genetic relationship among these strains. RESULTS: The genome size of the isolates was ~2.1 Mbp, covering >90% of the total estimated size of the reference genome. The overall G+C% content was ~39.5%, and there were 2,200-2,400 open reading frames. All isolates with different drug resistance profiles harbored many indels (range 131-171) and SNPs (range 16,103-28,128). Genetic diversity analysis showed that the variation of different genes were associated with specific antibiotic resistance. Known antibiotic resistance genes (pbps, murMN, ciaH, rplD, sulA, and dpr) were identified, and new genes (regR, argH, trkH, and PTS-EII) closely related with antibiotic resistance were found, although these genes were primarily annotated with functions in virulence as well as carbohydrate and amino acid transport and metabolism. Phylogenetic analysis unambiguously indicated that isolates with different antibiotic resistance profiles harbored similar genetic backgrounds. One isolate, 14-LC.ER1025, showed a much weaker phylogenetic relationship with the other isolates, possibly caused by genomic variation. CONCLUSION: In this study, although pneumococcal isolates had similar genetic backgrounds, strains were diverse at the genomic level. These strains exhibited distinct variations in their indel and SNP compositions associated with drug resistance.