Complete DNA Sequence Research Articles

The complete mitochondrial genome of the true limpet Scurria scurra (Lottiidae)

The complete mitochondrial DNA sequence of the scurrinid limpet Scurria scurra (Lesson 1830) is reported here. This mitogenome is 18,482 bp long and includes two rRNA genes, 13 protein-coding genes, and 24 tRNAs. The genus Scurria is a nascent model group for marine ecological studies. This mitogenome will help to gain a better understanding of the phylogenetic relationships within Patellogastropoda, the most primitive group of extant gastropods.

A new genus name for pygmy lorises, Xanthonycticebus gen. nov. (Mammalia, primates)

Lorisiformes are nocturnal primates from Africa and Asia with four genera, with two (Arctocebus and Loris), three (Perodicticus) and nine (Nycticebus) recognised species. Their cryptic lifestyle and lack of study have resulted in an underappreciation of the variation at the species and genus level. There are marked differences between the pygmy slow loris Nycticebus pygmaeus and the other Nycticebus species and, in the past, several authors have suggested that these may warrant recognition at the generic level. We here combine morphological, behavioural, karyotypical and genetic data to show that these contrasts are, indeed, significantly large and consistent. We propose Xanthonycticebus gen. nov. as a new genus name for the pygmy slow lorises and suggest a common name of pygmy lorises. Based on analysis of complete mitochondrial DNA sequences, we calculate the divergence of pygmy from slow lorises at 9.9–10.0%. The median date, calculated for the divergence between Xanthonycticebus and Nycticebus, is 10.5 Mya (range 4.9–21.0 Mya). Xanthonycticebus differs from Nycticebus by showing sympatry with other slow loris species, by habitually giving birth to twins, by showing seasonal body mass and whole body coat colour changes (absent in other species living at similar latitudes) and a multi-male, multi-female social system. Pygmy lorises are easily recognisable by the absence of hair on their ears and more protruding premaxilla. Xanthonycticebus is threatened by habitat loss and illegal trade despite legal protection across their range and all slow lorises are listed on appendix 1 of CITES. The suggested nomenclatural changes should not affect their legal status.

The mitochondrial genome of Grapsus albolineatus (Decapoda: Brachyura: Grapsidae) and phylogenetic associations in Brachyura

Complete mitochondrial genomes (mitogenomes) can provide useful information for phylogenetic relationships, gene rearrangement, and evolutionary traits. In this study, we determined the complete mitochondrial DNA sequence of the herbivorous crab Grapsus albolineatus. It is a typical metazoan mitochondrial genome. The total size is 15,583 bp, contains the entire set of 37 genes, and has an AT-rich region. Then, 23 of the 37 genes were encoded by the heavy (+) strand while 14 are encoded by the light (−) strand. Compared with the pan-crustacean ground pattern, two tRNA genes (tRNA-His and tRNA-Gln) were rearranged and the tandem duplication/random loss model was used to explain the observed gene rearrangements. The phylogenetic results showed that all Grapsidae crabs clustered together as a group. Furthermore, the monophyly of each family was well supported, with the exception of Menippidae. In general, the results obtained in this study will contribute to the better understanding of gene rearrangements in Grapsidae crab mitogenomes and provide new insights into the phylogeny of Brachyura.

Mitogenome sequence comparison in the endangered congeneric Pinna nobilis and Pinna rudis bivalves.

The pen shells Pinna nobilis and Pinna rudis are large wedge-shaped bivalve molluscs. Both species are threatened by different anthropogenic pressures. In the last few years, P. nobilis populations have significantly reduced due to massive mortality events. The complete mitochondrial DNA sequences of these congeneric species have been determined and compared for the first time. The mitogenome sequences of P. nobilis and P. rudis were 18,919bp and 18,264bp in length, respectively. Each mitogenome is composed of 12 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA (tRNAs) genes and non-coding regions. A putative Adenosine Triphosphate synthase subunit 8 gene could only be proposed for P. nobilis. Both newly sequenced mitogenomes present a conserved gene order between them, comparable to the closely related Atrina pectinata, but global arrangement greatly differs from other available bivalve mitochondrial sequences. Multiple copies of tRNA-Cys were identified, located in different positions probably due to mechanisms of mitochondrial genome rearrangements, and detected 2 and 3 times in P. rudis and in P. nobilis, respectively. A close relationship was shown between Pinna species and Atrina pectinata and a consistent clustering showing a monophyletic origin of Pinnidae family sequences was evidenced. The mitochondrial genomes will provide a valuable genetic resource for further studies on population genetics and species identification.

Genetic profiling of Mitochondrial DNA revealed two variants of U2 haplogroup associated with T2DM in a South Indian population

The present case-control study was conducted to examine the association between mitochondrial DNA and type 2 diabetes mellitus (T2DM) in a South Indian population. The complete mitochondrial DNA sequencing from 438 individuals including 217 T2DM patients and 221 controls was carried out. An association of haplogroup U2 (odds ratio= 3.876; 95% CI: 1.240-12.116; P = 0.011) with an increased risk of T2DM in South Indian population was found by using haplogroup classification and association test. Two marker SNPs of haplogroup U2, m.1811A>G (MT-RNR2) (OR = 1.806, CI = 1.025-3.184, P value = 0.041) and m.16051A>G (D-loop) (OR = 2.511, CI = 1.166-5.409, P value = 0.019) were found to have significant association with occurrence of T2DM even after adjusting for gender and age. Though the results provide evidence that mitochondrial variants are major contributors to T2DM risk, they need to be confirmed with replication study in larger sample size.

Genetic diversity in mitochondrial DNA D-loop region of indigenous pig breeds of India

India is home for at least 18 indigenous pig breeds; however, the genetic diversity of Indian pig, Sus scrofa domesticus, population is poorly known. Here, the hypervariable region (HVR) of mitochondrial DNA D-loop (~487 bp) of 214 pigs representing five indigenous and three exotic breeds was sequenced and analysed with reference sequences from other countries. A total of 54 segregating sites among the sequences revealed 56 different haplotypes. Two, 11, eight, seven and six haplotypes were identified with some haplotype sharing in indigenous breeds: Doom, Ghungroo, Mali, Niang-Megha and Tenyi-Vo, respectively. Population pairwise differences (PhiST) (0.409) were found significant (P<0.001), and variance within breeds (59.1%) was more than that of among breeds (40.9%). Similar topology was noted in phylogeny and median-joining network. Indian domestic pigs from this study were found to possess unique and highly differentiated haplotypes on network analysis. The diverse haplotypes and phylogenetic lineages identified here is the first report on Indian pig breeds that need to be further explored by complete mitochondrial DNA sequencing and analysis. These findings provide indicative insights for conservation and optimum utilization of the porcine genetic resources.

Characterization of the complete mitogenome of Gymnocypris dobula (Günther, 1868) (Cypriniformes: Cyprinidae)

Gymnocypris dobula, classified into the highly specialized Schizothoracine fish, is endemic to Tibet, China. The complete mitochondrial DNA sequence of G. dobula was 16,728 base pairs in length and comprised 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes as well as one control region as in a typical vertebrate mitochondrial DNA gene. The ML and BI trees showed that G. dobula was most closely related to Gymnocypris scleracanthus within the highly specialized group. This mitogenome provides new molecular data for further taxonomic and phylogenetic studies of Schizothoracine fish.

Site-Specific Fluorescent Labeling, Single-Step Immunocytochemistry, and Delivery of Nanobodies into Living Cells.

The smallest natural antibody fragments currently available are single-domain antibodies obtained from camelid species and sharks (variable new antigen receptors). These molecules consist of a single amino acid chain of ~120 amino acids that adopts a typical immunoglobulin fold. Single-domain antibodies (nanobodies) are monovalent and can be isolated from immunized animals, from naïve libraries, or from synthetic libraries. Importantly, their complete DNA sequences are readily obtained by default, which greatly facilitates their rapid manipulation for various applications. Here, a PCR-based protocol for inserting a sortase A recognition sequence at the carboxy-terminus of a nanobody is described. Subsequently, a sortase A-catalyzed biochemical reaction results in tagging of the nanobody with a short carboxy-terminal amino acid sequence that carries a non-canonical residue (propargyl glycine). This allows click chemistry to be performed with an azido-derivatized fluorophore, with the ensuing fluorescent nanobody being covalently and site-specifically labeled. The labeled nanobody can be used directly for immunocytochemistry, omitting the classical secondary antibody step. Also described are methods for delivery of fluorescent nanobodies into the cytoplasm of mammalian cells by photoporation, a very low-toxicity approach involving laser light and graphene quantum dots. The combined protocol embodies a novel route for studying protein function in living cells at high resolution.

First Description of the Mitogenome and Phylogeny of Culicinae Species from the Amazon Region.

The Culicidae family is distributed worldwide and comprises about 3587 species subdivided into the subfamilies Anophelinae and Culicinae. This is the first description of complete mitochondrial DNA sequences from Aedes fluviatilis, Aedeomyia squamipennis, Coquillettidia nigricans, Psorophora albipes, and Psorophora ferox. The mitogenomes showed an average length of 15,046 pb and 78.02% AT content, comprising 37 functional subunits (13 protein coding genes, 22 tRNAs, and two rRNAs). The most common start codons were ATT/ATG, and TAA was the stop codon for all PCGs. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogeny was inferred by analyzing the 13 PCGs concatenated nucleotide sequences of 48 mitogenomes. Maximum likelihood and Bayesian inference analysis placed Ps. albipes and Ps. ferox in the Janthinosoma group, like the accepted classification of Psorophora genus. Ae. fluviatilis was placed in the Aedini tribe, but was revealed to be more related to the Haemagogus genus, a result that may have been hampered by the poor sampling of Aedes sequences. Cq. nigricans clustered with Cq. chrysonotum, both related to Mansonia. Ae. squamipennis was placed as the most external lineage of the Culicinae subfamily. The yielded topology supports the concept of monophyly of all groups and ratifies the current taxonomic classification.

Ancient DNA reveals the maternal genetic history of East Asian domestic pigs

Zoo-archaeological and genetic evidence suggest that pigs were domesticated independently in Central China and Eastern Anatolia along with the development of agricultural communities and civilizations. However, the genetic history of domestic pigs, especially in China, has not been fully explored. In this study, we generate 42 complete mitochondrial DNA sequences from ∼7500- to 2750-year-old individuals from the Yellow River basin. Our results show that the maternal genetic continuity of East Asian domestic pigs dates back to at least the Early to Middle Neolithic. In contrast, the Near Eastern ancestry in European domestic pigs saw a near-complete genomic replacement by the European wild boar. The majority of East Asian domestic pigs share close haplotypes, and the most recent common ancestor of most branches dates back to less than 20,000 years before present, inferred using new substitution rates of whole mitogenomes or combined protein-coding regions. Two major population expansion events of East Asian domestic pigs coincided with changes in climate, widespread adoption of introduced crops, and the development of agrarian societies. These findings add to our understanding of the maternal genetic composition and help to complete the picture of domestic pig evolutionary history in East Asia.

Genetic Analysis of Hexaploid Wheat (Triticum aestivum L.) Using the Complete Sequencing of Chloroplast DNA and Haplotype Analysis of the Wknox1 Gene.

The aim of the presented study is a genetic characterization of the hexaploid wheat Triticum aestivum L. Two approaches were used for the genealogical study of hexaploid wheats—the complete sequencing of chloroplast DNA and PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth-exon region of Wknox1b. The complete chloroplast DNA sequences of 13 hexaploid wheat samples were determined: Free-threshing—T. aestivum subsp. aestivum, one sample; T. aestivum subsp. compactum, two samples; T. aestivum subsp. sphaerococcum, one sample; T. aestivum subsp. carthlicoides, four samples. Hulled—T. aestivum subsp. spelta, three samples; T. aestivum subsp. vavilovii jakubz., two samples. The comparative analysis of complete cpDNA sequences of 20 hexaploid wheat samples (13 samples in this article plus 7 samples sequenced in this laboratory in 2018) was carried out. PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth exon region of Wknox1b of all 20 hexaploid wheat samples was carried out. The 20 hexaploid wheat samples (13 samples in this article plus 7 samples in 2018) can be divided into two groups—T. aestivum subsp. spelta, three samples and T. aestivum subsp. vavilovii collected in Armenia, and the remaining 16 samples, including T. aestivum subsp. vavilovii collected in Europe (Sweden). If we take the cpDNA of Chinese Spring as a reference, 25 SNPs can be identified. Furthermore, 13–14 SNPs can be identified in T. aestivum subsp. spelta and subsp. vavilovii (Vav1). In the other samples up to 11 SNPs were detected. 22 SNPs are found in the intergenic regions, 2 found in introns, and 10 SNPs were found in the genes, of which seven are synonymous. PCR-based haplotype analysis of the fourth intron of Wknox1d and the fifth-to-sixth-exon region of Wknox1b provides an opportunity to make an assumption that hexaploid wheats T. aestivum subsp. macha var. palaeocolchicum and var. letshckumicum differ from other macha samples by the absence of a 42 bp insertion in the fourth intron of Wknox1d. One possible explanation for this observation would be that two Aegilops tauschii Coss. (A) and (B) participated in the formation of hexaploids through the D genome: Ae. tauschii (A)—macha (1–5, 7, 8, 10–12), and Ae. tauschii (B)—macha M6, M9, T. aestivum subsp. aestivum cv. ‘Chinese Spring’ and cv. ‘Red Doly’.

Father-to-offspring transmission of extremely long NOTCH2NLC repeat expansions with contractions: genetic and epigenetic profiling with long-read sequencing

BackgroundGGC repeat expansions in NOTCH2NLC are associated with neuronal intranuclear inclusion disease. Very recently, asymptomatic carriers with NOTCH2NLC repeat expansions were reported. In these asymptomatic individuals, the CpG island in NOTCH2NLC is hypermethylated, suggesting that two factors repeat length and DNA methylation status should be considered to evaluate pathogenicity. Long-read sequencing can be used to simultaneously profile genomic and epigenomic alterations. We analyzed four sporadic cases with NOTCH2NLC repeat expansion and their phenotypically normal parents. The native genomic DNA that retains base modification was sequenced on a per-trio basis using both PacBio and Oxford Nanopore long-read sequencing technologies. A custom workflow was developed to evaluate DNA modifications. With these two technologies combined, long-range DNA methylation information was integrated with complete repeat DNA sequences to investigate the genetic origins of expanded GGC repeats in these sporadic cases.ResultsIn all four families, asymptomatic fathers had longer expansions (median: 522, 390, 528 and 650 repeats) compared with their affected offspring (median: 93, 117, 162 and 140 repeats, respectively). These expansions are much longer than the disease-causing range previously reported (in general, 41–300 repeats). Repeat lengths were extremely variable in the father, suggesting somatic mosaicism. Instability is more frequent in alleles with uninterrupted pure GGCs. Single molecule epigenetic analysis revealed complex DNA methylation patterns and epigenetic heterogeneity. We identified an aberrant gain-of-methylation region (2.2 kb in size beyond the CpG island and GGC repeats) in asymptomatic fathers. This methylated region was unmethylated in the normal allele with bilateral transitional zones with both methylated and unmethylated CpG dinucleotides, which may be protected from methylation to ensure NOTCH2NLC expression.ConclusionsWe clearly demonstrate that the four sporadic NOTCH2NLC-related cases are derived from the paternal GGC repeat contraction associated with demethylation. The entire genetic and epigenetic landscape of the NOTCH2NLC region was uncovered using the custom workflow of long-read sequence data, demonstrating the utility of this method for revealing epigenetic/mutational changes in repetitive elements, which are difficult to characterize by conventional short-read/bisulfite sequencing methods. Our approach should be useful for biomedical research, aiding the discovery of DNA methylation abnormalities through the entire genome.

Avian paramyxovirus 4 isolated from the mallard (&lt;i&gt;Anas platyrhynchos&lt;/i&gt;, Linnaeus, 1758): the first case detected in the Western Caspian region

Aim . The work is aimed at exploring the molecular and genetic properties of avian paramyxovirus 4 (APMV‐4) isolated from the mallard duck. Material and Methods . In 2018, as a part of an influenza A virus surveillance programme, biological samples were collected from wild birds on Lake Adzhi (Papas) in the Republic of Dagestan. The isolation of virus strains from biological samples was performed using 10‐day‐old special pathogen‐free chicken eggs. The primary detection of hemagglutinating agents in the fluid of the allantoic cavity was carried out via hemagglutination assay. The presence of RNA of the influenza A virus was determined using Real Time PCR. Then, for samples negative in PCR for the influenza A virus, the complete genome DNA sequences were established using NGS sequencing. It was determined that the APMV‐4 strain obtained belonged to intraspecific genetic lines was determined using phylogenetic analysis. Results . APMV‐4 strain was isolated for the first time in the Western Caspian region. Phylogenetic analysis of the complete genome and gene F showed that the APMV‐4/mallard/Dagestan/92d/2018 strain belongs to the Eurasian clade of subgenotypes. Analysis of amino acid substitutions showed that the cleavage site of the fusion protein is monobasic which is characteristic of lentogenic strains. Conclusion . During the surveillance program for influenza A viruses in autumn 2018, the APMV‐4 strain was isolated from the mallard duck. Analysis revealed that the APMV‐4 strain was closely phylogenetically related to strains isolated from two wild and one domestic duck from China. This suggests an interconnection between the bird populations in China and the Western Caspian region within the West Asian‐East African migration route.

The complete mitochondrial genome of two-belt cardinal and striped cardinalfish Apogonichthyoides taeniatus (Cuvier, 1828)

The complete mitochondrial DNA sequence of Apogonichthyoides taeniatus (Cuvier, 1828) is determined. The mitochondrial genome is 17,050 in length and has the same composition and gene order like most other vertebrates. The phylogenetic analysis based on 13 concatenated PCGs nucleotide sequences among 20 species showed that this species has high support with the sister branch Jaydia lineata. Our findings provide useful information for phylogenetic and evolutionary research of Kurtiformes species.

The complete mitochondrial genome and phylogenetic analysis of Neorhodomela munita

Neorhodomela munita (Perestenko) Masuda 1982 is distributed in the coastal areas of Shandong and Liaoning in China, and also in Japan. In this study, the complete nucleotide sequence of the circular mitochondrial DNA of the red alga Neorhodomela munita has been determined. The complete mitochondrial DNA sequence of Neorhodomela munita was 25,318 bp in length with an overall GC content of 25.1% and encoded 23 protein-coding genes, two ribosomal RNAs and 24 transfer RNAs. Phylogenetic tree showed that Neorhodomela munita clustered together with Choreocolax polysiphoniae. The phylogenetic analysis may provide a better understanding of the evolution of the Rhodophyta species.

Complete mitochondrial genome of the Japanese field vole microtus montebelli (Milne-Edwards, 1872) (Rodentia: Arvicolinae)

The complete mitochondrial DNA sequence of the Japanese field vole Microtus montebelli was determined using Illumina MiSeq platform. The assembled genome was 16,307 bp in length and contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes. According to phylogenetic analysis of 13 protein-coding genes, M. montebelli and other Microtus species consist of paraphyletic clades and M. montebelli is most closely related to M. kikuchii, a species endemic to Taiwan.

Mitochondrial Genetic Heterogeneity in Leber's Hereditary Optic Neuropathy: Original Study with Meta-Analysis.

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disorder that causes loss of central vision. Three primary variants (m.3460G>A, m.11778G>A, and m.14484T>C) and about 16 secondary variants are responsible for LHON in the majority of the cases. We investigated the complete mitochondrial DNA (mtDNA) sequences of 189 LHON patients and found a total of 54 disease-linked pathogenic variants. The primary variants m.11778G>A and m.14484T>C were accountable for only 14.81% and 2.64% cases, respectively. Patients with these two variants also possessed additional disease-associated variants. Among 156 patients who lacked the three primary variants, 16.02% harboured other LHON-associated variants either alone or in combination with other disease-associated variants. Furthermore, we observed that none of the haplogroups were explicitly associated with LHON. We performed a meta-analysis of m.4216T>C and m.13708G>A and found a significant association of these two variants with the LHON phenotype. Based on this study, we recommend the use of complete mtDNA sequencing to diagnose LHON, as we found disease-associated variants throughout the mitochondrial genome.

The sequence structure and phylogenetic analysis by complete mitochondrial genome of kohlrabi (Brassica oleracea var. gongylodes L.)

Kohlrabi (Brassica oleracea var. gongylodes L.) is an important dietary rhizome vegetable in the Brassicaceae family. However, to date, few mitochondrial genomic resources have been reported for kohlrabi. In this study, we obtained the complete mitochondrial DNA sequence of 219,964 bp from an individual green kohlrabi. A total of 61 genes were annotated, including 33 protein-coding genes, 23 transfer RNA genes, three ribosomal RNA genes, and two pseudo genes. In addition, 1,001 open reading frames and five RNA editing sites were annotated. Relative synonymous codon usage analysis revealed significant difference in usage frequency of synonymous codon. Phylogenetic inference showed that kohlrabi is closely related to B. oleracea var. botrytis. This study provides a good foundation for further understanding the relationship and evolutionary origins among Brassicaceae crops.

Mitochondrial DNA Diversity of the Nangan Islanders Living in the Mazu Archipelago of the Taiwan Strait

This study investigates the genetic relationship of the Mazu peoples on the east coast of China in the Taiwan Strait. Using partial and complete mitochondrial DNA (mtDNA) sequences, we compare Mazu with surrounding East Asia populations. Mazu shows no exclusive affinities with either Southeast or Northeast Asia. High genetic diversity and a very high number of exclusive haplogroups of various Asian origins suggest that Mazu resulted from a process of continuous resettlement that started when it first became an archipelago at the end of the last glacial maximum and that continued till the last century. As a result, genetic drift did not contribute to an exclusive Mazu profile. The structure of haplogroups that show signatures of the Neolithic era (N9a10a), or influx from Island Southeast Asia (F1a4a) suggest recent gene flows and Mazu relationship with it's pre-Neolithic era (presence of pre-E1a or R9/pre-F from Liangdao man) was not seen.

DNA sequence and community structure diversity of multi-year soil fungi in Grape of Xinjiang

This study is designed to understand the community structure and diversity of fungi in the rhizosphere soil of grape. As the sample for this study, the rhizosphere soil of Crimson seedless grape with different planting years was collected from Shihezi in Xinjiang to carry out high-throughput sequencing, by which the complete sequence of soil fungi DNA was identified, and accordingly, the richness and diversity index of fungi were determined. The results showed that the dominant phyla of fungi in the grape rhizosphere soil with different planting years were Ascomycota and Basidiomycota, and the dominant classes of fungi were Sordariomycetes and Dothideomycetes. Soil organic matter, total potassium, total nitrogen and available phosphorus were the main soil fertility factors affecting the abundance and diversity of soil fungal communities, among which soil organic matter had the most significant influence. In addition, the fungal diversity and richness were highest in the middle layer (20–35 cm) of the grape rhizosphere soil with 12 planting years and lowest in the lower layer (35–50 cm) of the grape rhizosphere soil with 5 planting years. Linear discriminant analysis suggested that there were more biomarkers in the vineyard rhizosphere soil with 10 planting years, which meant there were more fungal communities with significant difference in the soil, especially in the middle layer (20–35). The results of this study can provide data reference and theoretical basis for improving vineyard soil quality, evaluating soil microecological effects and improving ecological environment of vineyard soil.