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Neurodevelopmental disorder with microcephaly, ataxia, and seizures (NEDMAS) syndrome is a rare neurodevelopmental disorder characterized by moderate intellectual disability (ID), thin body habitus, microcephaly, seizures, ataxia, muscle weakness, and speech impairment. So far, only two families with NEDMAS have been reported. We report the clinical and molecular characteristics of three unrelated Turkish families with four NEDMAS patients. Whole-exome sequencing was used to search for the disease-causing variant. The main manifestations of the probands are severe developmental delay and ID, thin body habitus, and severe hypotonia. Brain imaging revealed bilateral cerebral and cerebellar diffuse atrophy. Sequencing results showed that both patients carried a novel missense variant c.1196C>T (p.Thr399Met) in the seryl-tRNA synthetase gene. Our findings help expand the variant spectrum of NEDMAS and provide additional information for diagnosing cases with atypical features. © 2022 Lippincott Williams and Wilkins. All rights reserved.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight, one of the most devastating diseases of rice. Here, a hypervirulent strain, C9-3, defeating Xa1, Xa10, xa13, and Xa23 resistance genes, was used to extract genomic DNA for single molecule real-time (SMRT) sequencing. After assembly, the genome consists of a single-circular chromosome with the size of 4,924,298 bp with G+C content of 63.7% and contains 4,715 genes. Annotation and analysis of the TALE genes using a suite of applications named AnnoTALE suggested that 17 transcription activator-like effectors, including 15 typical TALEs and 2 iTALEs/truncTALEs, were encoded in the genome. The approach and genome resource will contribute to the discovery of new virulence effectors and understanding on rice-X. oryzae pv. oryzae interactions.

Primary Sjogren’s syndrome (pSS) is a complex systemic autoimmune disease, which is difficult to accurately diagnose due to symptom diversity in patients, especially at earlier stages. We tried to find potential single nucleotide polymorphisms (SNPs), copy number variations (CNVs) and related signaling pathways. Genomic DNA was extracted from peripheral blood of 12 individuals (7 individuals from 3 pSS pedigrees and 5 sporadic cases) for whole-exome sequencing (WES) analysis. SNPs and CNVs were identified, followed by functional annotation of genes with SNPs and CNVs. Gene expression profile (involving 64 normal controls and 166 cases) was downloaded from the Gene Expression Omnibus database (GEO) dataset for differentially expression analysis. Sanger sequencing and in vitro validation was used to validate the identified SNPs and differentially expressed genes, respectively. A total of 5 SNPs were identified in both pedigrees and sporadic cases, such as FES, PPM1J, and TRAPPC9. A total of 3402 and 19 CNVs were identified in pedigrees and sporadic cases, respectively. Fifty-one differentially expressed genes were associated with immunity, such as BATF3, LAP3, BATF2, PARP9, and IL15RA. AMPK signaling pathway and cell adhesion molecules (CAMs) were the most significantly enriched signaling pathways of identified SNPs. Identified CNVs were associated with systemic lupus erythematosus, mineral absorption, and HTLV-I infection. IL2-STAT5 signaling, interferon-gamma response, and interferon-alpha response were significantly enriched immune related signaling pathways of identified differentially expressed genes. In conclusion, our study found some potential SNPs, CNVs, and related signaling pathways, which could be useful in understanding the pathological mechanism of pSS.

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.

PIWI-interacting RNAs (piRNAs), small noncoding RNAs 24–35 nucleotides long, are essential for animal fertility. They play critical roles in a range of functions, including transposable element suppression, gene expression regulation, imprinting, and viral defense. In mammals, piRNAs are the most abundant small RNAs in adult testes and the only small RNAs that direct epigenetic modification of chromatin in the nucleus. The production of piRNAs is a complex process from transcription to post-transcription, requiring unique machinery often distinct from the biogenesis of other RNAs. In mice, piRNA biogenesis occurs in specialized subcellular locations, involves dynamic developmental regulation, and displays sexual dimorphism. Furthermore, the genomic loci and sequences of piRNAs evolve much more rapidly than most of the genomic regions. Understanding piRNA biogenesis should reveal novel RNA regulations recognizing and processing piRNA precursors and the forces driving the gain and loss of piRNAs during animal evolution. Such findings may provide the basis for the development of engineered piRNAs capable of modulating epigenetic regulation, thereby offering possible single-dose RNA therapy without changing the genomic DNA. In this review, we focus on the biogenesis of piRNAs in mammalian adult testes that are derived from long non-coding RNAs. Although piRNA biogenesis is believed to be evolutionarily conserved from fruit flies to humans, recent studies argue for the existence of diverse, mammalian-specific RNA-processing pathways that convert precursor RNAs into piRNAs, perhaps associated with the unique features of mammalian piRNAs or germ cell development. We end with the discussion of major questions in the field, including substrate recognition and the birth of new piRNAs.

Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present research aimed to establish a rapid, sensitive and visual method for genotyping of germline genome-edited mutants with small genomic fragment deletion. The genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limit of detection (LOD) of 0.1 ng. The proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-edited mutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.

Mutations in the SLC12A3 gene have been reported to cause Gitelman syndrome (GS). This study aimed to investigate the genetic mutations and clinical features of patients with GS. Four pedigrees (4 GS patients and 14 family members) were enrolled. The symptoms, laboratory results, management, and genotypes were analyzed. Genomic DNA was screened for gene variations using Sanger sequencing. DNA sequences were compared with reference sequences. The effects of the mutations were predicted using prediction tools (Mutation Taster, PolyPhen-2, SIFT, and PROVEAN). Genetic analysis revealed six genetic variants of SLC12A3, including three novel heterozygous mutations (c.2T > C, c.1609C > T, c.3055G > A) and three previously characterized mutations (c.1456G > A, c.2542G > A, c.1077C > G). These mutations were predicted to exert a damaging effect based on predictive in silico tools. GS patients had low blood pressure and low levels of serum K+, serum Mg2+, and 24-h urinary Ca2+ but high levels of 24-h urinary K+. These clinical manifestations and genotypes were consistent with the diagnostic criteria of GS. The study described the phenotypes and genotypes of 4 pedigrees involving GS patients, demonstrating the importance of SLC12A3 gene screening for GS.

Dilated cardiomyopathy (DCM) is one of the most common cardiac phenotypes caused by mutations of lamin A/C (LMNA) gene in humans. In our study, a cohort of 57 patients who underwent heart transplant for dilated cardiomyopathy was screened for variants in LMNA. We identified a synonymous variant c.936G>A in the last nucleotide of exon 5 of LMNA in a DCM family. Clinically, the LMNA variant carriers presented with severe familial DCM, conduction disease, and high creatine-kinase level. The LMNA c.936G>A variant is novel and has not been reported in current genetic variant databases. Sanger sequencing results showed the presence of LMNA c.936G>A variant in the genomic DNA but not in the cDNA derived from one family member's heart tissue. Real-time quantitative polymerase chain reaction showed significantly lower LMNA mRNA levels in the patient's heart compared to the controls, suggesting that the c.936G>A LMNA variant resulted in reduced mRNA and possibly lower protein expression of LMNA. These findings expand the understanding on the association between synonymous variant of LMNA and the molecular pathogenesis in DCM patients.

Codonopsis lanceolata is an herbaceous perennial plant predominantly cultivated in East Asia and used for medicinal purposes. However, genetic information of C. lanceolata is lacking. Therefore, we sequenced genomic DNA using next-generation sequencing (NGS) and searched for simple sequence repeats (SSRs) to develop molecular markers in C. lanceolata. A total of 250,455 SSRs were identified, and di-nucleotides and tri-nucleotides accounted for the majority of all the SSRs. Among these SSRs, we designed 26,334 primer sets from di- to octa-nucleotide motifs. We used an in silico approach to investigate 2626 SSRs (tri- to penta-nucleotide motifs) and found 573 SSRs showing polymorphism. Of the 573 SSRs showing polymorphism in silico, we randomly selected 39 SSRs and verified polymorphism in 16 C. lanceolata accessions. The number of alleles ranged from 2 to 13, and the mean polymorphic information content value was 0.54. Therefore, we successfully designed 39 SSR markers for use in breeding and genetic studies of C. lanceolata.

DNA metabarcoding is a rapid, high-resolution tool used for biomonitoring complex zooplankton communities. However, diversity estimates derived with this approach can be biased by the co-detection of sequences from environmental DNA (eDNA), nuclear-encoded mitochondrial (NUMT) pseudogene contamination, and taxon-specific PCR primer affinity differences. To avoid these methodological uncertainties, we tested the use of metatranscriptomics as an alternative approach for characterizing zooplankton communities. Specifically, we compared metatranscriptomics with PCR-based methods using genomic (gDNA) and complementary DNA (cDNA) amplicons, and morphology-based data for estimating species diversity and composition for both mock communities and field-collected samples. Mock community analyses showed that the use of gDNA mitochondrial cytochrome c oxidase I (mtCO1) amplicons inflates species richness due to the co-detection of extra-organismal eDNA. Significantly more amplicon sequence variants, nucleotide diversity, and indels were observed with gDNA amplicons than with cDNA, indicating the presence of putative NUMT pseudogenes. Moreover, PCR-based methods failed to detect the most abundant species in mock communities due to priming site mismatch. Overall, metatranscriptomics provided estimates of species richness and composition that closely resembled those derived from morphological data. The use of metatranscriptomics was further tested using field-collected samples, with the results showing consistent species diversity estimates among biological and technical replicates. Additionally, temporal zooplankton species composition changes could be monitored using different mitochondrial markers. These findings demonstrate the advantages of metatranscriptomics as an effective tool for monitoring diversity in zooplankton research.

Purpose: To study the genetic basis and clinical manifestations of Wolfram syndrome in a multi-affected family. Methods: Complete clinical examinations including urological, ophthalmic, neurological, and endocrinologic assessment were performed for three affected family members. Genomic DNA was extracted from peripheral blood leukocytes with salting out method and all WFS1 exons and their flanking regions were sequenced. Candidate variation was screened for segregation in the pedigree by Sanger sequencing. Results: A known pathogenic missense mutation in WFS1 gene (c.1885C>T which leads to p.Arg629Trp in the encoded protein) was identified in all affected individuals. Both clinical and genetic investigations confirmed Wolfram syndrome diagnosis with variable phenotypic features. Conclusion: Identical mutations in the Wolfram syndrome causative gene can lead to variable manifestations of the syndrome even in the same family. Although the medical findings and clinical examination are imperative for the diagnosis of Wolfram syndrome, genetic testing is useful to confirm the diagnosis, especially in cases with possible reduced penetrance of the characteristic signs.

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

Species identification using molecular techniques has recently become common for various taxa. Loop-mediated isothermal amplification (LAMP) is one of the easiest and least expensive molecular identification methods. Although few studies have developed LAMP assays for amphibians, we believe that LAMP is also useful for identifying endangered amphibians. Hynobius tokyoensis and H. lichenatus occur in Honshu, Japan, and have parapatric distributions. They are similar morphologically, especially at early developmental stages, including eggs and larvae. Hynobius tokyoensis has been listed as a national endangered species in Japan since 2020, and unambiguous identification of these species is therefore important for their conservation and management. In this study, we developed a LAMP primer set for the mitochondrial cytochrome b region to detect H. tokyoensis, and we evaluated the LAMP assay using total genomic DNA from four H. tokyoensis and three H. lichenatus individuals from across most of their ranges. Our LAMP primer set could distinguish these two species. This study should help to establish LAMP assays for other endangered species and morphologically similar species., 希少種のサンショウウオの簡易DNA判別が可能に --違法売買防止への大きな一手--. 京都大学プレスリリース. 2021-11-15.

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.

Paternal genome elimination—a type of reproduction in which males inherit but fail to pass on their father’s genome—evolved independently in 6–8 arthropod clades. Thousands of species, including several important for agriculture, reproduce via this mode of reproduction. While paternal genome elimination is well established in some of the clades, the evidence in globular springtails (Symphypleona) remains elusive, even though they represent the oldest and most species-rich clade putatively reproducing via paternal genome elimination. We sequenced genomic DNA from whole bodies of Allacma fusca males with high fractions (>27.5%) of sperm to conclusively confirm that all the sperm carry 1 parental haplotype only. Although it is suggestive that the single haplotype present in sperm is maternally inherited, definitive genetic proof of the parent of origin is still needed. The genomic approach we developed allows for the detection of genotypic differences between germline and soma in all species with sufficiently high fraction of germline in their bodies. This opens new opportunities for scans of reproductive modes in small organisms.

Objective: To determine the proportion of fertility in Pakistani infertile females and discover if there are considerable connection among BMP15 gene polymorphism, follicle maturation and hormonal regulation in Pakistani infertile females. Methods: All selected participants were initially examined through follicle-stimulating hormones (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), Prolactin, and Trans-vaginal scan (TVS). BMP15 gene polymorphism among infertile and fertile females was done by extracted Genomic DNA from whole blood. Sanger sequencing was performed for the identification of mutation in exons-intron boundaries of the BMP15 gene. Bioinformatics tools were used to assess the protein structure. Results: The total five mutations including two novel missense variants of BMP15 in exon 2, whereas three previously reported i.e. two cosmic mutations (c.615delC), (c.584InsG) and one frame shift mutations (c.635delA) were also observed. The first novel mutation was found at (c.1038InsGG) (p.346Gln

Objectives Profiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes. Methods Oral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with HpaII restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis. Results Sixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with HpaII distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective. Conclusion PCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories.

A novel COL3A1 variant was identified in a Japanese case of Ehlers-Danlos syndrome type IV (EDS-IV) with a characteristic "Madonna" face, fragile uterus, and easy bruising in addition to a history of cavernous sinus fistula. We confirmed variable diameters of collagen fibrils in the dermis and decrease in type 3 collagen production from cultured fibroblasts. Genomic DNA sequencing of the COL3A1 region and COL3A1 cDNA sequence expressing in cultured fibroblasts identified that a nucleotide variation at c.951+2T>G on intron 14 leads to skipping of exon 14 in COL3A1 cDNA. The novel variation in the splice site of COL3A1 region g.IVS14+2T>G was not listed in the EDS-IV pathogenic genetic databases including Human Gene Mutation Database, ClinVar, and Leiden Open Variation Database. Using the whole genome sequence database of 8380 Japanese individuals reported by the Tohoku Medical Megabank Organization (ToMMo) cohort study, we also confirmed that COL3A1 g.IVS14+2T>G was not a common single nucleotide variation in the Japanese population, although 13 EDS-related COL3A1 variants were identified in the ToMMo database of 8380 Japanese individuals. These results demonstrated that our case of EDS-IV was a result of the novel variation of COL3A1 g.IVS14+2T>G. These statistical genetics approaches with the combination of the ToMMo database of 8380 Japanese individuals and pathogenic genetic databases are a useful method to confirm the uniqueness of novel variation in Japanese.

To investigate how exogenous DNA concatemerizes to form episomal artificial chromosomes (ACs), acquire equal segregation ability and maintain stable holocentromeres, we injected DNA sequences with different features, including sequences that are repetitive or complex, and sequences with different AT-contents, into the gonad of Caenorhabditis elegans to form ACs in embryos, and monitored AC mitotic segregation. We demonstrated that AT-poor sequences (26% AT-content) delayed the acquisition of segregation competency of newly formed ACs. We also co-injected fragmented Saccharomyces cerevisiae genomic DNA, differentially expressed fluorescent markers and ubiquitously expressed selectable marker to construct a less repetitive, more complex AC. We sequenced the whole genome of a strain which propagates this AC through multiple generations, and de novo assembled the AC sequences. We discovered CENP-AHCP-3 domains/peaks are distributed along the AC, as in endogenous chromosomes, suggesting a holocentric architecture. We found that CENP-AHCP-3 binds to the unexpressed marker genes and many fragmented yeast sequences, but is excluded in the yeast extremely high-AT-content centromeric and mitochondrial DNA (> 83% AT-content) on the AC. We identified A-rich motifs in CENP-AHCP-3 domains/peaks on the AC and on endogenous chromosomes, which have some similarity with each other and similarity to some non-germline transcription factor binding sites.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per μg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.

A Gram-stain-negative, rod-shaped (0.3–0.6 × 0.9–2.2 µm), facultatively aerobic, non-motile and yellow-coloured bacterium, designated strain F6397T, was isolated from a marine sediment in Weihai, PR China. Growth of strain F6397T occurred at 4–37 °C (optimum, 30–33 °C), pH 6.0–9.0 (optimum, 7.5) and in the presence of 1–12% (optimum, 3.0%) (w/v) NaCl. Strain F6397T showed oxidase- and catalase-positive activities. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain was assigned to the genus Winogradskyella. Strain F6397T exhibited 95.5–98.1% sequence similarities to recognized species of the genus Winogradskyella. The average nucleotide identity (ANI) scores with Winogradskyella ludwigii HL116T, Winogradskyella litoriviva KMM 6491 T and Winogradskyella thalassocola KMM 3907 T were 80.1, 78.9 and 82.6%, respectively. The digital DNA–DNA hybridization (dDDH) scores were 23.5, 22.9 and 25.7%, respectively. The genomic DNA G + C content was 33.5 mol%. Strain F6397T contained iso-C15:0, iso-C15:1G, iso-C15:0 3-OH and iso-C17:0 3-OH as the predominant fatty acid and MK-6 as the predominant menaquinone. The polar lipid profiles contained phosphatidylethanolamine, three unidentified aminolipids and four unidentified lipids. On the basis of the evidence presented in this study, strain F6397T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella marina sp. nov. is proposed. The type strain is F6397T (= KCTC 82422 T = MCCC 1H00438T).

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium-mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer-DNA (T-DNA) copies in the host genome. Quantifying the copy number of T-DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)-based method to detect a single copy of a T-DNA insertion in transformants. The competitor line BHK- -1 that contains a single copy of competitor BHK- (BHK, Basta, Hygromycin, Kanamycin-resistant genes) was crossed with test transformants and the genomic DNA of F1 plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T-DNA insertion. We also generated the control lines BHK±1:1 and BHK±2:1 , which contain the target (BHK+ ) and competitor (BHK- ) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern-blot analysis to identify a single copy of T-DNA insertion in transformants.

Background A novel variable surface antigens (VSAs), Surface-associated interspersed proteins (SUFRINs), is a protein that is modified on the surface of infected red blood cell (iRBC). Modified proteins on the iRBC surface cause severe malaria, which can lead to death throughout the life cycle of a malaria parasite. Previous study suggested that SURFIN1.1 is an immunogenic membrane-associated protein which was encoded by using the surf1.1 gene expressed during the trophozoite and schizont stages. This study aimed to identify the regions of SURFIN1.1 and investigate the genetic diversity of the extracellular region of the surf1.1 gene. Methods A total of 32 blood samples from falciparum malaria cases that were diagnosed in Si Sa Ket Province, Thailand were collected. Plasmodium genomic DNA was extracted, and the extracellular region of surf1.1 gene was amplified using the polymerase chain reaction (PCR). A sequence analysis was then performed to obtain the number of haplotypes (H), the haplotype diversity (Hd), and the segregating sites (S), while the average number of nucleotide differences between two sequences (Pi); in addition, neutrality testing, Tajima’s D test, Fu and Li’s D* and F* statistics was also performed. Results From a total of 32 patient-isolated samples, 31 DNA sequences were obtained and analysed for surf1.1 gene extracellular region polymorphism. Researchers observed six distinct haplotypes in the current research area. Haplotype frequencies were 61.3%, 16.2%, and 12.9% for H1, H2, and H3, respectively. The remaining haplotype (H4-H6) frequency was 3.2% for each haplotype. Hd was 0.598 ± 0.089 with the Pi of 0.00381, and S was 15. The most common amino acid polymorphic site was E251Q; other sites included N48D, I49V, E228D, E235S, L265F, K267T, E276Q, and S288F. Fu and Li’s D* test value was − 1.24255, Fu and Li’s F* test value was − 1.10175, indicating a tendency toward negative balancing selection acting on the surf1.1 N-terminal region. The most polymorphic region was variable 2 (Var2) while cysteine-rich domain (CRD) was conserved in both the amino acid and nucleotide extracellular region of surf1.1 gene. Conclusions The Thai surf1.1 N-terminal region was well-conserved with only a few polymorphic sites remaining. In this study, the data regarding current bearing on the polymorphism of extracellular region of surf1.1 gene were reported, which might impact the biological roles of P. falciparum. In addition, may possibly serve as a suitable candidate for future development of SURFIN-based vaccines regarding malaria control. Graphic abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12936-021-03876-y., Highlights The regions of SURFIN1.1 were identified: SURFIN1.1 is comprised of extracellular, transmembrane (TM), and intracellular regions.Nucleotide and amino acid sequences of the extracellular region of P. falciparum SURFIN1.1 from a total of 31 field isolates were obtained and analyzed for genetic polymorphism: six different haplotypes were identified.The extracellular region of the SURFIN1.1 among field isolates was conserved, especially in the cysteine-rich domain (CRD) sub-region.High polymorphism was shown in the variable region 2 (Var2), followed by N-terminal (N-ter) and variable region 1 (Var1), respectively.The findings presented herein may enable the discovery and development of a novel SURFIN-based vaccine for prevention and control of malaria. Supplementary Information The online version contains supplementary material available at 10.1186/s12936-021-03876-y.

Objectives In this study, we analyzed the whole exomes of CTSC gene in a family with history of PLS. Materials and methods Genomic DNA was extracted from peripheral blood and genotype analysis was performed. The mutated protein sequence was used to find the best possible tertiary structure for homology modeling. The homology modeling of the novel mutation was then performed using the online Swiss‐Prot server. The results were also analyzed for to verify its validity. Results The analysis of CTSC gene elucidated a novel insertion GAC. The novel mutation was proved by analyzing 50 healthy control volunteers. Modeling of the novel found mutation in CTSC gene revealed structural defects that may have caused the functional abnormalities. Conclusions The structural analysis of the mutated protein model identifies changes in the stereo‐chemical and the energy level of the mutated protein. Since this protein play a role in the activation of granule serine proteases from cytotoxic T lymphocytes, natural killer cells, mast cells, such structural defects may lead to its malfunction causing dysfunctioning of immune defense mechanisms.

Background Adenylate kinase (AK) deficiency is a rare red cell enzymopathy associated with moderate to severe congenital nonspherocytic hemolytic anemia, along with mental and psychomotor retardation (in exceptional cases). Only ten mutations have been detected in the AK1 gene to date. In this study, we aimed to diagnose the unexplained issue of haemolytic anaemia and offer antenatal screening to the family. Methods Genomic DNA was isolated from whole blood by a standard protocol. Targeted next-generation sequencing (t-NGS) was performed to identify pathogenic variants in the patient and control samples. A chronic villus sample was collected at 11 weeks of gestation from the mother, and molecular testing was performed. Genetic confirmation was concluded by Sanger DNA sequencing. Bioinformatics tools predicted the pathogenicity of the variant. Results t-NGS revealed a homozygous variant (c.301C > A, p. Gln101Lys) in the AK1 gene in the patient and heterozygosity in the fetus and parental samples. The prediction tools SIFT, Polyphen2, Provean, PMUT, Mutation taster, and Mutation Assessor, confirmed the damaging effect of the variant on the AK1 protein structure Conclusion We have presented a novel mutation in the AK1 gene (p. Gln101Lys) associated with adenylate kinase deficiency. It is the first prenatal diagnosis of AK deficiency in India, where heterogeneity is exceptionally high.

Analyses of large-scale cancer sequencing data have revealed that mutagenic processes can create distinctive patterns of base substitutions, called mutational signatures. Interestingly, mutational patterns resembling some of these signatures can also be observed in normal cells. To determine whether similar patterns exist more generally, we analyzed large data sets of genetic variation, including mutations from 7 model species and single nucleotide polymorphisms in 42 species, totaling >1.9 billion variants. We found that base substitution patterns for most species closely match single base substitution (SBS) mutational signature 5 in the Catalog of Somatic Mutations in Cancer (COSMIC) database. SBS5 is ubiquitous in cancers and also present in normal human cells, suggesting that similar patterns of genetic variation across so many species are likely due to conserved biochemistry. We investigated the mechanistic origins of the SBS5-like mutational pattern in Saccharomyces cerevisiae, and show that translesion DNA synthesis and sugar metabolism are directly linked to this form of mutagenesis. We propose that conserved metabolic processes in cells are coupled to continuous generation of genetic variants, which can be acted upon by selection to drive the evolution of biological entities.

Cystic echinococcosis (CE) is a neglected zoonotic tropical disease caused by Echinococcus granulosus sensu lato. The aim of this study was to investigate the genetic variation of hydatid cyst isolates obtained from surgically confirmed paediatric cases originating from two different regions in eastern Turkey. Seventeen paediatric cases aged between 6 and 16 were operated by open surgery, and the germinal layers of their cysts were obtained for further molecular analyses. After genomic DNA isolation, 875 bp mt-CO1 gene fragments were amplified in all samples by PCR. Then, the unidirectional sequence analyses of the PCR products were carried out. According to the BLAST analyses of 17 sequences, 16 of these sequences were matched with E. granulosus sensu stricto, while one sequence was identified as E. canadensis (G6/G7) for the first time in paediatric cases in Turkey. High haplotype diversity and low nucleotide diversity were observed in the E. granulosus s.s. sequences.

A Gram-stain-negative, oval or short rod-shaped, non-motile, aerobic bacterium, designated strain S1109LT, was isolated from a marine sediment in Weihai, PR China. Cells were oxidase positive and catalase positive. Growth of strain S1109LT occurred at 10–40 °C (optimum, 30–33 °C), pH 6.5–10.0 (optimum, 7.0–8.0) and in the presence of 1–21% (optimum, 4–6%) (w/v) NaCl. 16S rRNA gene sequence phylogeny indicated that strain S1109LT was associated with the genus Pontibaca of the family Rhodobacteraceae because it showed the highest sequence similarity to Pontibaca methylaminivorans KCTC 22497T (97.5%). The average nucleotide identity (ANI) and the digital DNA–DNA hybridization (dDDH) scores between strain S1109LT and Pontibaca methylaminivorans KCTC 22497T were 74.6% and 18.7%. The major cellular fatty acids of strain S1109LT were C19:0 cyclo ω8c and C18:1 ω7c. The polar lipids profiles of strain S1109LT were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and two unidentified lipids. Strain S1109LT contained ubiquinone-10 as the major respiratory quinone. The genomic DNA G + C content was 55.9 mol%. On the basis of the evidence presented in this study, strain S1109LT is considered to represent a novel species of the genus Pontibaca, for which the name Pontibaca salina sp. nov. is proposed. The type strain of is S1109LT (= KCTC 82411T = MCCC 1H00441T).

Community-based diversity analyses, such as metabarcoding, are increasingly popular in the field of metazoan zooplankton community ecology. However, some of the methodological uncertainties remain, such as the potential inflation of diversity estimates resulting from contamination by pseudogene sequences. Furthermore, primer affinity to specific taxonomic groups might skew community composition and structure during PCR. In this study, we estimated OTU (operational taxonomic unit) richness, Shannon’s H’, and the phylum-level community composition of samples from a coastal zooplankton community using four approaches: complement DNA (cDNA) and genomic DNA (gDNA) mitochondrial COI (Cytochrome oxidase subunit I) gene amplicon, metatranscriptome sequencing, and morphological identification. Results of mismatch distribution demonstrated that 90% is good threshold percentage to differentiate intra- and inter-species. Moderate level of correlations appeared upon comparing the species/OTU richness estimated from the different methods. Results strongly indicated that diversity inflation occurred in the samples amplified from gDNA because of mitochondrial pseudogene contamination (overall, gDNA produced two times more richness compared with cDNA amplicons). The unique community compositions observed in the PCR-based methods indicated that taxonomic amplification bias had occurred during the PCR. Therefore, it is recommended that PCR-free approaches be used whenever resolving community structure represents an essential aspect of the analysis.

A bacterial strain designated KDG-16T is isolated from a freshwater waterfall in Taiwan and characterized to determine its taxonomic affiliation. Cells of strain KDG-16T are Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and form light yellow colonies. Optimal growth occurs at 20-25 oC, pH 6-7, and with 0% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set reveal that strain KDG-16T is affiliated with species in the genus Flavobacterium. Analysis of 16S rRNA gene sequences shows that strain KDG-16T shares the highest similarity with Flavobacterium terrigena DSM 17934T (97.7%). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain KDG-16T and the closely related Flavobacterium species are below the cut-off values of 95-96, 90 and 70%, respectively, used for species demarcation. Strain KDG-16T contains iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the predominant fatty acids. The polar lipid profile consists of phosphatidylethanolamine, one uncharacterized aminophospholipid, one uncharacterized phospholipid, two uncharacterized aminolipids and two uncharacterized lipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. Genomic DNA G+C content of strain KDG-16T is 31.6%. Based on the polyphasic taxonomic data obtained, strain KDG-16T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium difficile sp. nov. is proposed. The type strain is KDG-16T (=BCRC 81194T =LMG 31332T).

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is an ancient prokaryotic defense system that precisely cuts foreign genomic DNA under the control of a small number of guide RNAs. The CRISPR-Cas9 system facilitates efficient double-stranded DNA cleavage that has been recently adopted for genome editing to create or correct inherited genetic mutations causing disease. Congenital heart disease (CHD) is generally caused by genetic mutations such as base substitutions, deletions, and insertions, which result in diverse developmental defects and remains a leading cause of birth defects. Pediatric CHD patients exhibit a spectrum of cardiac abnormalities such as septal defects, valvular defects, and abnormal chamber development. CHD onset occurs during the prenatal period and often results in early lethality during childhood. Because CRISPR-Cas9-based genome editing technology has gained considerable attention for its potential to prevent and treat diseases, we will review the CRISPR-Cas9 system as a genome editing tool and focus on its therapeutic application for CHD.

Cystic echinococcosis (CE) is a zoonotic disease, caused by parasite known as Echinococcus granulosus sensu lato complex, leading to substantial economic losses in rural areas with public health problems. This study was carried out to understand the haplotypic profiles of the partial mitochondrial cytochrome c oxidase (mt-CO1) gene of cattle lung hydatid cyst samples which were obtained from two provinces in Turkey. In this study, forty (n = 40) hydatid cyst samples from the lungs of slaughtered cattle were obtained. The germinal layers were taken separately for each individual cyst then stored in 70% ethanol. From each individual cyst sample, total genomic DNA was extracted. Amplification of the partial mt-CO1 gene (875 bp) was performed using a specific primer set by PCR, and then, the amplicons were sequenced. All sequences were analyzed individually, followed by alignment, and haplotype and phylogenetic analyses were then performed. By the sequence alignment process, 39 out of the 40 sequences were characterized as E. granulosus sensu stricto. However, one of them was matched with E. canadensis (G6/G7). The haplotype analyses of the E. granulosus s.s. isolates were arranged in a star-like orientation with a main haplotype, which was separated from other haplotypes by 1–10 mutation steps, and 26 haplotypes were identified. In the mt-CO1 sequences, 29 polymorphic sites were determined, and 34.5% (10/29) of them were parsimony informative. The findings of this study provide the first report of E. canadensis (G6/G7 genotype) among cattle in Turkey.

Starch is a natural stored carbohydrate resource for the human diet, synthesized and stored in amyloplasts in higher plants. Starch synthase III is directly linked to the amylopectin chain’s elongation in wheat grains, but global warming is a critical barrier to its activity. The present investigation includes the characterization of the TaSS-IIIa1D gene in two wheat genotypes, viz. PBW-343 (heat susceptible) and IC252874 (heat tolerant). The genomic DNA of the above genotypes was used for PCR amplification of TaSS-IIIa1D gene using overlapping gene-specific primers and cloning the gene using the pJET1.2/blunt cloning vector, followed by sequencing and detection of SNPs. In-silico analysis revealed that the TaSS-III gene contains two homologs, TaSS-IIIa and TaSS-IIIb, which were found on the plus strand of chromosome 1 (1A, 1B and 1D) and minus strand of chromosome 2 (2A, 2B and 2D), respectively. The genomic size of the TaSS-IIIa1D gene was approximately 10,529 bp. All homeologous copies of the gene contained 16 exons. Twenty-nine specific SNPs were identified in PBW343 and twenty in IC252874 genotypes for the gene vis-a-vis with the reference copy of Chinese spring cultivar. There were 14 intronic and 15 exonic SNPs contributing to 18 transitions and 9 transversions in the genotype PWB-343, reflecting the transition bias, while in genotype IC252874, nine transitions, nine transversions, and two deletions contribute to 6 intronic and 14 exonic SNPs showing no such bias. Eighteen SNPs detected between the two genotypes for the gene consisting of 11 transitions and 7 transversions also showed transition bias. Third exon found most variable with the maximum number of SNPs. Seven SNPs were found associated with SBD-1, SBD-2, and SS-CD domains of the TaSS-IIIa1D protein. SNPs identified between the heat-sensitive and heat-tolerant genotypes for the heat-sensitive enzyme coding gene could be utilized for wheat improvement after proper validation.

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

Purpose This study aims to identify genetic causes of female infertility associated with recurrent failure of assisted reproductive technology (ART) characterized by embryonic developmental arrest. Methods We recruited infertile patients from two consanguineous families from the Reproductive Medicine Center of Guizhou Provincial People’s Hospital. Peripheral blood was collected for genomic DNA extraction. Two affected individuals and their family members were performed with whole-exome sequencing and Sanger validation in order to identify possible causative genes. For further analyzing the effect of splicing mutation on mRNA integrity in vivo, TLE6 cDNA from the peripheral blood lymphocyte of the affected individual was sequenced. In addition, the possible impact of the pathogenic mutation on the structure and function of the protein were also assessed. Results Two novel homozygous mutations in the peptidylarginine deiminase type VI (PADI6) and the transducin-like enhancer of split 6 (TLE6) genes were identified in the two families. One patient carried the frameshift deletion mutation c.831_832del:p.S278Pfs*59 of the PADI6 gene and the other patient carried the splicing mutation c.1245-2 A>G of the TLE6 gene. The analysis of the mRNA from the proband’s peripheral blood leukocytes confirmed aberrant splicing. Conclusions Our findings expand the mutational spectrum of PADI6 and TLE6 associated with embryonic developmental arrest and deepen our understanding of the genetic causes of infertility with recurrent ART failure.

Primary congenital glaucoma (PCG) (OMIM#231,300) can be caused by pathogenic sequence variations in CYP1B1, LTBP2, MYOC and PXDN genes. The purpose of this study was to investigate mutations in the CYP1B1 gene in families affected with primary congenital glaucoma (PCG) using linkage analysis and Sanger sequencing. A total number of four families with nine affected PCG patients during six months were included in this study. The mutations were identified by homozygosity mapping to find the linked loci and then direct sequencing of all coding exons, the exon–intron boundaries and the 5' untranslated region of CYP1B1 using genomic DNA obtained from affected family members and their parents. Moreover, bioinformatic tools were applied to study mutation effect on protein structure and function. A total of four mutations were identified, and three of these were novel. Two were missense mutations: One was truncating mutation, and the other was an in-frame deletion. Mutations in CYP1B1 could fully explain the PCG phenotype in all of the patients. Also, the bioinformatic study of the mutations showed the structure of the protein is affected, and it is well conserved among similar species. In this study, we identified 4 CYP1B1 mutations, 3 of which were novel. In silico analysis of identified mutations confirmed their molecular pathogenicity. A similar analysis will help understand the biological role of CYP1B1 and the effect of mutations on the regulatory and enzymatic functions of CYP1B1 that result in PCG. Not relevant

A Gram-stain-negative, rod-shaped, aerobic and non-motile bacterium, designated P2-65T, was isolated from Moonsan stream water in the Republic of Korea. The temperature, NaCl concentration and pH ranges for growth of strain P2-65T were 10–37 °C, 0.0–3.0% (w/v) and 6.5–8.5 with optimum growth at 25–30 °C, 0.0–1.0% and 7.0–7.5, respectively. Comparison of 16S rRNA gene sequence showed that strain P2-65T was closely related to Flavobacterium cauense (95.4%) and Flavobacterium cheniae (95.3%). The major fatty acids were iso-C15:0, iso C17:0 3-OH, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0) and iso-C15:0 3-OH. The predominant respiratory quinone was menaquinone-6 (MK-6). The major polar lipids detected in the strain were phosphatidylethanolamine, one aminophospholipid, one unidentified aminolipid and one unidentified polar lipid. The G + C content of the genomic DNA was 39.7%. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values for strain P2-65T with closely related Flavobacterium species were below 74.8% and 20%, respectively. Based on polyphasic features, strain P2-65T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium inviolabile sp. nov. is proposed. The type strain is P2-65T (= KCTC 62055T = NBRC 112953T).

Understanding how the epigenome deteriorates with age and subsequently impacts on biological function may bring unique insights to ageing-related disease mechanisms. As a central cellular apparatus, tRNAs are fundamental to the information flow from DNA to proteins. Whilst only being transcribed from ~46kb ( < 0.002%) of the human genome, their transcripts are the second most abundant in the cell. Furthermore, it is now increasingly recognised that tRNAs and their fragments also have complex regulatory functions. In both their core translational and additional regulatory roles, tRNAs are intimately involved in the control of metabolic processes known to affect ageing. Experimentally DNA methylation can alter tRNA expression, but little is known about the genomic DNA methylation state of tRNAs. Here, we find that the human genomic tRNA loci (610 tRNA genes termed the tRNAome) are enriched for ageing-related DNA hypermethylation. We initially identified DNA hypermethylation of 44 and 21 specific tRNA genes, at study-wide (p < 4.34 × 10 − 9 ) and genome-wide ( p < 4.34 × 10 − 9 ) significance, respectively, in 4,350 MeDIP-seq peripheral blood DNA methylomes (16 - 82 years). This starkly contrasted with 0 hypomethylated at both these significance levels. Further analysing the 21 genome-wide results, we found 3 of these tRNAs to be independent of major changes in cell-type composition (tRNA-iMet-CAT-1-4, tRNA-Ser-AGA-2-6, tRNA-Ile-AAT-4-1). We also excluded the ageing-related changes being due to the inherent CpG density of the tRNAome by permutation analysis (1,000x, Empirical p-value < 1 × 10 − 3 ). We additionally explored 79 tRNA loci in an independent cohort using Fluidigm deep targeted bisulfite-sequencing of pooled DNA (n=190) across a range of 4 timepoints (aged ~4, ~28, ~63, ~78 years). This revealed these ageing changes to be specific to particular isodecoder copies of these tRNA (tRNAs coding for the same amino acid but with sequence body differences) and included replication of 2 of the 3 genome-wide tRNAs. Additionally, this isodecoder-specificity may indicate the potential for regulatory fragment changes with age. In this study we provide the first comprehensive evaluation at the genomic DNA methylation state of the human tRNAome, revealing a discreet and strongly directional hypermethylation with advancing age.

Body weight is one of the most important economically useful traits, which is characterized by complex inheritance. Therefore, a search for genetic mechanisms affecting its formation is of increased scientific interest. This work presents for the first time the results of the genome-wide association studies in the resource population of sheep (Ovis aries) from backcross (Romanov × Katah-din) × Romanov family, whose body weight was recorded in age dynamics, and SNP profiles were obtained using a high-density DNA chip. We identified 38 SNPs significantly associated with body weight (p < 0.00001) and functional candidate genes which affect skeletal muscle growth, bone scaffold formation, lipid, and carbohydrate metabolism. In addition, age-related changes in the set of significantly significant SNPs are shown. Our aim was to search for genomic variants that affect the body weight of (Romanov × Katahdin) × Romanov backcrosses from the resource population at different age periods. The study was performed on 95 sheep from backcross (Romanov × Katahdin) × Romanov family (Ernst Federal Research Center for Animal Husbandry, 2018-2021). Ear notch samples were taken from backcrosses for extraction of genomic DNA, which isolated using DNA-Extran-2 kits (Syntol LLC, Russia). Animals were genotyped using a high-density DNA chip Ovine Infinium® HD SNP BeadChip (Illumina, Inc., USA) containing ~600 thousand SNP markers. The body weight was recorded at the ages of 6(BW6), 42(BW42), 90(BW90), 180(BW180) and 270 days (BW270). To study genome-wide associations with body weight, we used regression analysis implemented in PLINK 1.90 (--assoc --adjust --qt-means). To confirm the significant effect of SNP and to identify significant regions in the genome of the studied sheep, a test was performed to check null hypotheses according to Bonferroni at a threshold value of p < 1.09×10-7, 0.05/459868. The search for candidate genes located in the region of identified SNPs was performed using the VEP (Ensembl Variant Effect Predictor) tool of Ensembl genome browser 103 (https://www.ensembl.org/index.html). After quality control, 459,868 SNPs were used to perform genome-wide association studies (GWAS). Average body weights in the studied sample were 3.28±0.07, 8.03±0.21, 13.74±0.39, 20.19±0.51, and 22.51±0.50 kg at the age of 6, 42, 90, 180, and 270 days, respectively. We found that in different age periods the set of SNPs associated with the integral indicator of the growth rate, the animal body weight, was different. Thus, out of 38 identified SNPs, 18 SNPs located on OAR2, OAR4, OAR9, and OAR15 were reliably associated with BW6 (p < 0.00001); 3 SNPs located on OAR6 and OAR11 with BW42 (p < 0.00001); 2 SNPs located on OAR10 and OAR19 with BW90 (p < 0.00001); 6 SNPs (p < 0.00001) located on OAR1 and OAR13 with BW180 (p < 0.00001) and 6 SNPs located on OAR1 with BW270 (p

Body weight is one of the most important economically useful traits, which is characterized by complex inheritance. Therefore, a search for genetic mechanisms affecting its formation is of increased scientific interest. This work presents for the first time the results of the genome-wide association studies in the resource population of sheep (Ovis aries) from backcross (Romanov × Katah-din) × Romanov family, whose body weight was recorded in age dynamics, and SNP profiles were obtained using a high-density DNA chip. We identified 38 SNPs significantly associated with body weight (p < 0.00001) and functional candidate genes which affect skeletal muscle growth, bone scaffold formation, lipid, and carbohydrate metabolism. In addition, age-related changes in the set of significantly significant SNPs are shown. Our aim was to search for genomic variants that affect the body weight of (Romanov × Katahdin) × Romanov backcrosses from the resource population at different age periods. The study was performed on 95 sheep from backcross (Romanov × Katahdin) × Romanov family (Ernst Federal Research Center for Animal Husbandry, 2018-2021). Ear notch samples were taken from backcrosses for extraction of genomic DNA, which isolated using DNA-Extran-2 kits (Syntol LLC, Russia). Animals were genotyped using a high-density DNA chip Ovine Infinium® HD SNP BeadChip (Illumina, Inc., USA) containing ~600 thousand SNP markers. The body weight was recorded at the ages of 6(BW6), 42(BW42), 90(BW90), 180(BW180) and 270 days (BW270). To study genome-wide associations with body weight, we used regression analysis implemented in PLINK 1.90 (--assoc --adjust --qt-means). To confirm the significant effect of SNP and to identify significant regions in the genome of the studied sheep, a test was performed to check null hypotheses according to Bonferroni at a threshold value of p < 1.09×10-7, 0.05/459868. The search for candidate genes located in the region of identified SNPs was performed using the VEP (Ensembl Variant Effect Predictor) tool of Ensembl genome browser 103 (https://www.ensembl.org/index.html). After quality control, 459,868 SNPs were used to perform genome-wide association studies (GWAS). Average body weights in the studied sample were 3.28±0.07, 8.03±0.21, 13.74±0.39, 20.19±0.51, and 22.51±0.50 kg at the age of 6, 42, 90, 180, and 270 days, respectively. We found that in different age periods the set of SNPs associated with the integral indicator of the growth rate, the animal body weight, was different. Thus, out of 38 identified SNPs, 18 SNPs located on OAR2, OAR4, OAR9, and OAR15 were reliably associated with BW6 (p < 0.00001); 3 SNPs located on OAR6 and OAR11 with BW42 (p < 0.00001); 2 SNPs located on OAR10 and OAR19 with BW90 (p < 0.00001); 6 SNPs (p < 0.00001) located on OAR1 and OAR13 with BW180 (p < 0.00001) and 6 SNPs located on OAR1 with BW270 (p

Nucleosomes in all eukaryotic cells are organized into higher order structures that facilitate genome compaction. Visualizing these organized structures is an important step in understanding how genomic DNA is efficiently stored yet remains accessible to information-processing machinery. Arrays of linked nucleosomes serve as useful models for understanding how the properties of both DNA and protein partners affect their arrangement. A number of important questions are also associated with understanding how the spacings between nucleosomes are affected by the histone proteins, chromatin remodelers, or other chromatin-associated protein partners. Contrast variation small angle X-ray scattering (CVSAXS) reports the DNA conformation within protein-DNA complexes and here is applied to measure the conformation(s) of trinucleosomes in solution, with specific sensitivity to the distance between and relative orientation of linked nucleosomes. These data are interpreted in conjunction with DNA models that account for its sequence dependent mechanical properties, and Monte-Carlo techniques that generate realistic structures for comparison with measured scattering profiles. In solution, trinucleosomes segregate into two dominant populations, with the flanking nucleosomes stacked or nearly equilaterally separated, e.g. with roughly equal distance between all pairs of nucleosomes. These populations are consistent with previously observed magnesium-dependent structures of trinucleosomes with shorter linkers.