Analysis Of Repetitive Sequences Research Articles

Repetitive Sequences, Codon Usage Bias and Phylogenetic Analysis of the Plastome of Miliusa glochidioides.

Annonaceae is the largest family in Magnoliales, exhibiting the greatest diversity among and within genera. In this study, we conducted an analysis of repetitive sequences and codon usage bias in the previously acquired plastome of Miliusa glochidioides. Using a concatenated dataset of shared genes, we constructed the phylogenetic relationships among 27 Annonaceae species. The results showed that the size of the plastomes in the Annonaceae ranged from 159 to 202kb, with the size of the inverted repeat region ranging from 40 to 65kb. Within the plastome of M. glochidioides, we identified 42 SSRs, 36 tandem repeats, and 9 dispersed repeats. These SSRs consist of three nucleotide types and eight motif types, with a preference for A/T bases, primarily located in the large single-copy regions and intergenic spacers. Tandem and dispersed repeat sequences were predominantly detected in the IR region. Through codon usage bias analysis, we identified 30 high-frequency codons and 11 optimal codons. The plastome of M. glochidioides demonstrated relatively weak codon usage bias, favoring codons with A/T endings, primarily influenced by natural selection. Phylogenetic analysis revealed that all four subfamilies formed monophyletic groups, with Cananga odorata (Ambavioideae) and Anaxagorea javanica (Anaxagoreoideae) successively nested outside Annonoideae + Malmeoideae. These findings improve our understanding of the plastome of M. glochidioides and provide additional insights for studying plastome evolution in Annonaceae.

Phylogenetic relationships, selective pressure and molecular markers development of six species in subfamily Polygonoideae based on complete chloroplast genomes

The subfamily Polygonoideae encompasses a diverse array of medicinal and horticultural plants that hold significant economic value. However, due to the lack of a robust taxonomy based on phylogenetic relationships, the classification within this family is perplexing, and there is also a scarcity of reports on the chloroplast genomes of many plants falling under this classification. In this study, we conducted a comprehensive analysis by sequencing and characterizing the complete chloroplast genomes of six Polygonoideae plants, namely Pteroxygonum denticulatum, Pleuropterus multiflorus, Pleuropterus ciliinervis, Fallopia aubertii, Fallopia dentatoalata, and Fallopia convolvulus. Our findings revealed that these six plants possess chloroplast genomes with a typical quadripartite structure, averaging 162,931 bp in length. Comparative chloroplast analysis, codon usage analysis, and repetitive sequence analysis demonstrated a high level of conservation within the chloroplast genomes of these plants. Furthermore, phylogenetic analysis unveiled a distinct clade occupied by P. denticulatum, while P. ciliinrvis displayed a closer relationship to the three plants belonging to the Fallopia genus. Selective pressure analysis based on maximum likelihood trees showed that a total of 14 protein-coding genes exhibited positive selection, with psbB and ycf1 having the highest number of positive amino acid sites. Additionally, we identified four molecular markers, namely petN-psbM, psal-ycf4, ycf3-trnS-GGA, and trnL-UAG-ccsA, which exhibit high variability and can be utilized for the identification of these six plants.

Orthoptera-TElib: a library of Orthoptera transposable elements for TE annotation

Transposable elements (TEs) are a major component of eukaryotic genomes and are present in almost all eukaryotic organisms. TEs are highly dynamic between and within species, which significantly affects the general applicability of the TE databases. Orthoptera is the only known group in the class Insecta with a significantly enlarged genome (0.93-21.48 Gb). When analyzing the large genome using the existing TE public database, the efficiency of TE annotation is not satisfactory. To address this limitation, it becomes imperative to continually update the available TE resource library and the need for an Orthoptera-specific library as more insect genomes are publicly available. Here, we used the complete genome data of 12 Orthoptera species to de novo annotate TEs, then manually re-annotate the unclassified TEs to construct a non-redundant Orthoptera-specific TE library: Orthoptera-TElib. Orthoptera-TElib contains 24,021 TE entries including the re-annotated results of 13,964 unknown TEs. The naming of TE entries in Orthoptera-TElib adopts the same naming as RepeatMasker and Dfam and is encoded as the three-level form of “level1/level2-level3”. Orthoptera-TElib can be directly used as an input reference database and is compatible with mainstream repetitive sequence analysis software such as RepeatMasker and dnaPipeTE. When analyzing TEs of Orthoptera species, Orthoptera-TElib performs better TE annotation as compared to Dfam and Repbase regardless of using low-coverage sequencing or genome assembly data. The most improved TE annotation result is Angaracris rhodopa, which has increased from 7.89% of the genome to 53.28%. Finally, Orthoptera-TElib is stored in Sqlite3 for the convenience of data updates and user access.

Genome-Wide and Expression Pattern Analysis of the HIT4 Gene Family Uncovers the Involvement of GHHIT4_4 in Response to Verticillium Wilt in Gossypium hirsutum.

Chromatin remodelers are essential for regulating plant growth, development, and responses to environmental stresses. HIT4 (HEAT-INTOLERANT 4) is a novel stress-induced chromatin remodeling factor that has been less studied in abiotic stress and stress resistance, particularly in cotton. In this study, we conducted a comprehensive analysis of the members of the HIT4 gene family in Gossypium hirsutum using bioinformatics methods, including phylogenetic relationships, gene organization, transcription profiles, phylogenetic connections, selection pressure, and stress response. A total of 18 HIT4 genes were identified in four cotton species, with six HIT4 gene members in upland cotton. Based on the evolutionary relationships shown in the phylogenetic tree, the 18 HIT4 protein sequences were classified into four distinct subgroups. Furthermore, we conducted chromosome mapping to determine the genomic locations of these genes and visually represented the structural characteristics of HIT4 in G. hirsutum. In addition, we predicted the regulatory elements in HIT4 in G. hirsutum and conducted an analysis of repetitive sequences and gene collinearity among HIT4 in four cotton species. Moreover, we calculated the Ka/Ks ratio for homologous genes to assess the selection pressure acting on HIT4. Using RNA-seq, we explored the expression patterns of HIT4 genes in G. hirsutum and Gossypium barbadense. Through weighted gene co-expression network analysis (WGCNA), we found that GHHIT4_4 belonged to the MEblue module, which was mainly enriched in pathways such as DNA replication, phagosome, pentose and glucuronate interconversions, steroid biosynthesis, and starch and sucrose metabolism. This module may regulate the mechanism of upland cotton resistance to Verticillium wilt through DNA replication, phagosome, and various metabolic pathways. In addition, we performed heterologous overexpression of GH_D11G0591 (GHHIT4_4) in tobacco, and the results showed a significant reduction in disease index compared to the wild type, with higher expression levels of disease resistance genes in the transgenic tobacco. After conducting a VIGS (virus-induced gene silencing) experiment in cotton, the results indicated that silencing GHHIT4_4 had a significant impact, the resistance to Verticillium wilt weakened, and the internode length of the plants significantly decreased by 30.7% while the number of true leaves increased by 41.5%. qRT-PCR analysis indicated that GHHIT4_4 mainly enhanced cotton resistance to Verticillium wilt by indirectly regulating the PAL, 4CL, and CHI genes. The subcellular localization results revealed that GHHIT4_4 was predominantly distributed in the mitochondria and nucleus. This study offers preliminary evidence for the involvement of the GHHIT4_4 in cotton resistance to Verticillium wilt and lays the foundation for further research on the disease resistance mechanism of this gene in cotton.

Mechanisms of Karyotypic Diversification in Ancistrus (Siluriformes, Loricariidae): Inferences from Repetitive Sequence Analysis.

Ancistrus is a highly diverse neotropical fish genus that exhibits extensive chromosomal variability, encompassing karyotypic morphology, diploid chromosome number (2n = 34-54), and the evolution of various types of sex chromosome systems. Robertsonian rearrangements related to unstable chromosomal sites are here described. Here, the karyotypes of two Ancistrus species were comparatively analyzed using classical cytogenetic techniques, in addition to isolation, cloning, sequencing, molecular characterization, and fluorescence in situ hybridization of repetitive sequences (i.e., 18S and 5S rDNA; U1, U2, and U5 snDNA; and telomere sequences). The species analyzed here have different karyotypes: Ancistrus sp. 1 (2n = 38, XX/XY) and Ancistrus cirrhosus (2n = 34, no heteromorphic sex chromosomes). Comparative mapping showed different organizations for the analyzed repetitive sequences: 18S and U1 sequences occurred in a single site in all populations of the analyzed species, while 5S and U2 sequences could occur in single or multiple sites. A sequencing analysis confirmed the identities of the U1, U2, and U5 snDNA sequences. Additionally, a syntenic condition for U2-U5 snDNA was found in Ancistrus. In a comparative analysis, the sequences of rDNA and U snDNA showed inter- and intraspecific chromosomal diversification. The occurrence of Robertsonian rearrangements and other dispersal mechanisms of repetitive sequences are discussed.

Genomic Characterization of hox Genes in Senegalese Sole (Solea senegalensis, Kaup 1858): Clues to Evolutionary Path in Pleuronectiformes.

The Senegalese sole (Solea senegalensis, Kaup 1858), a marine flatfish, belongs to the Pleuronectiformes order. It is a commercially important species for fisheries and aquaculture. However, in aquaculture, several production bottlenecks have still to be resolved, including skeletal deformities and high mortality during the larval and juvenile phase. The study aims to characterize the hox gene clusters in S. senegalensis to understand better the developmental and metamorphosis process in this species. Using a BAC library, the clones that contain hox genes were isolated, sequenced by NGS and used as BAC-FISH probes. Subsequently the hox clusters were studied by sequence analysis, comparative genomics, and cytogenetic and phylogenetic analysis. Cytogenetic analysis demonstrated the localization of four BAC clones on chromosome pairs 4, 12, 13, and 16 of the Senegalese sole cytogenomic map. Comparative and phylogenetic analysis showed a highly conserved organization in each cluster and different phylogenetic clustering in each hox cluster. Analysis of structural and repetitive sequences revealed accumulations of polymorphisms mediated by repetitive elements in the hoxba cluster, mainly retroelements. Therefore, a possible loss of the hoxb7a gene can be established in the Pleuronectiformes lineage. This work allows the organization and regulation of hox clusters to be understood, and is a good base for further studies of expression patterns.

Chromosomal Diversification in Pseudacanthicus Species (Loricariidae, Hypostominae) Revealed by Comparative Mapping of Repetitive Sequences.

Simple SummaryThe fishes of the Loricariidae family have a huge genetic diversity, mainly involving variations in the number and shape of chromosomes. The recognition of the species genus Pseudacanthicus is complex due to the large diversity of forms and limited knowledge of their genetic diversity. In this study, the karyotypes of three Pseudacanthicus species were comparatively analyzed using classical and molecular methods. They presented the same diploid number, but with different compositions of repetitive DNA sequences. Such information can be useful for the recognition of distinct species, in addition to providing important insights into the real biodiversity of this important group of Neotropical fish.Pseudacanthicus is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three Pseudacanthicus species (P. spinosus, P. leopardus and Pseudacanthicus sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, P. leopardus presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in P. leopardus, and in multiple sites in Pseudacanthicus sp. and P. spinosus. The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although Pseudacanthicus species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.

Performance of HaiYang-2 Altimetric Data in Marine Gravity Research and a New Global Marine Gravity Model NSOAS22

Haiyang-2 (HY-2) missions have accumulated sea surface height (SSH) observations on a global scale for more than 10 years. Four satellites, HY-2A, HY-2B, HY-2C and HY-2D, provide even but differently distributed data, which play a complementary role in marine gravity studies with other missions. Therefore, this paper evaluates the performances of HY-2 altimetric data in marine gravity modeling from the following four perspectives: SSH accuracy, geoid signal resolution ability, vertical deflections and gravity anomaly. First, the centimeter-magnitude accuracy level of HY-2 data is proved by analyzing SSH discrepancies at crossover points within a certain time limit. Second, the spectral analysis of repetitive along-track data sequences in a time domain shows a geoid resolution range from 18 to 24 km. Taking HY-2 exact repeat missions (ERM), for example, the resolution could be remarkably enhanced by stacking repetitive cycles. Third, validation with an XGM2019 model showed that vertical deflections were reliably computed for all HY-2 missions, but HY-2A performed slightly worse than the other HY-2 missions. Meanwhile, HY-2C and HY-2D with a ~66° orbital inclination obviously had an improved ability to capture east–west signals compared to HY-2A and HY-2B. Finally, we constructed global marine gravity results based on three input datasets, HY-2 dataset only, multi-satellite dataset without HY-2 and multi-satellite dataset with HY-2. Validations were performed using published models and shipborne gravimetric data. The results showed that the HY-2 dataset is capable of improving marine gravity anomaly recoveries and that the accuracy of NSOAS22 with incorporated HY-2 data is comparable to DTU21 and SS V31.1. Furthermore, HY-2 observations should not be the only input dataset to construct a 1’ × 1’ resolution marine gravity model.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Detection of β-Lactamase–Producing Proteus mirabilis Strains of Animal Origin in Andhra Pradesh, India and Their Genetic Diversity

Proteus mirabilis is abundant in soil and water. Although this bacterium is part of the normal human intestinal flora, it can cause serious infections in humans, including complicated urinary tract infections. This pathogen is also commonly associated with multidrug resistance. In the present study, analysis of 1,093 samples from foods of animal origin and animal intestinal samples recovered 232 P. mirabilis isolates identified by PCR assay. Of these 232 isolates, 72 produced β-lactamase (determined by both phenotypic and genotypic methods), with the highest prevalence in poultry cloacal swabs (11.82%) followed by mutton (9.18%), khoa (6.32%), pork (5.63%), pig rectal swabs (5.52%), beef (5.45%), and chicken (5.13%) but none from sheep rectal swabs and bovine rectal swabs. Among β-lactamase genes, blaTEM was the predominant gene detected (59 isolates) followed by blaOXA (11 isolates), blaSHV (5 isolates), blaFOX (5 isolates), blaCIT (4 isolates), blaCTX-M1 and blaCTX-M9 (2 isolates each) and blaCTX-M2, blaDHA, and blaEBC (1 isolate each). None of the isolates carried blaACC, blaMOX, or carbapenemase genes (blaVIM, blaIMP, blaKPC, and blaNDM-1). Dendrogram analysis of enterobacterial repetitive intergenic consensus sequences and repetitive extragenic palindromic sequences obtained with PCR analysis of β-lactamase-producing isolates revealed 63 isolates, but 9 isolates did not yield bands. The analysis revealed that 6.58% of the samples had β-lactamase-producing P. mirabilis isolates that may affect food safety and contaminate the environment. Further genotyping revealed the genetic relationships between isolates of different origin. These findings emphasize the need for careful use of antibiotics to control the spread of β-lactamase-producing bacteria.

Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae).

Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

Identification and characterization of centromeric sequences in Xenopus laevis.

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant Centromere Protein A (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences, but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

Genome-wide analyses of tandem repeats and transposable elements in patchouli.

Patchouli, Pogostemon cablin (Blanco) Benth., is a traditional Chinese medicinal plant from the order Lamiales. It is considered a valuable herb due to its essential oil content and range of therapeutic effects. This study aimed to explore the evolutionary history of repetitive sequences in the patchouli genome by analyzing tandem repeats and transposable elements (TEs). We first retrieved genomic data for patchouli and four other Lamiales species from the GenBank database. Next, the content of tandem repeats with different period sizes was identified. Long terminal repeats (LTRs) were then identified with LTR_STRUC. Finally, the evolutionary landscape of TEs was explored using an in-house PERL program. The analysis of repetitive sequences revealed that tandem repeats constitute a higher proportion of the patchouli genome compared to the four other species. Analyses of TE families showed that most of the repetitive sequences in the patchouli genome are TEs, and that recently inserted TEs make up a comparatively larger proportion than older ones. Our analyses of LTR retrotransposons in their host genome indicated the existence of ancient LTR retrotransposon expansion, and the escape of these elements from natural selection revealed their ages. Our identification and analyses of repetitive sequences should provide new insights for further investigation of patchouli evolution.

Genome-wide characterization of the SPL gene family involved in the age development of Jatropha curcas

BackgroundSPL (SQUAMOSA-promoter binding protein-like) proteins form a large family of plant-specific transcription factors that play essential roles in various aspects of plant growth and development. They are potentially important candidates for genetic improvement of agronomic traits. However, there were limited information about the SPL genes in Jatropha curcas, an important biofuel plant.ResultsIn Jatropha, 15 JcSPL genes were identified. Phylogenetic analysis revealed that most of the JcSPLs were closely related to SPLs from woody plant rather than herbaceous plant and distantly related to monocotyledon SPLs. Gene structure, conserved motif and repetitive sequence analysis indicated diverse and specific functions of some JcSPL genes. By combination of target prediction and degradome sequencing analysis, 10 of the 15 JcSPLs were shown to be targets of JcmiR156. Quantitative PCR analysis showed diversified spatial-temporal expression patterns of JcSPLs. It is interesting that the expression levels of JcSPL3 were the highest in all tissues examined in 7- or 10-year-old plants and exhibited increasing trend with plant age, suggesting its important role in the regulation of age development in Jatropha. Overexpression of JcSPL3 in Arabidopsis resulted in earlier flowering time, shorter silique length and reduced biomass of roots.ConclusionsThrough comprehensive and systematic analysis of phylogenetic relationships, conserved motifs, gene structures, chromosomal locations, repetitive sequence and expression patterns, 15 JcSPL genes were identified in Jatropha and characterized in great detail. These results provide deep insight into the evolutionary origin and biological significance of plant SPLs and lay the foundation for further functional characterization of JcSPLs with the purpose of genetic improvement in Jatropha.

Genome size and identification of repetitive DNA sequences using low coverage sequencing in Hancornia speciosa Gomes (Apocynaceae: Gentianales).

Repetitive DNA is an important component of eukaryotic genomes, accounting for more than 90% of the genome size of some species, including mobile elements and satellite DNA sequences. The aim of study was to characterize the genome of Hancornia speciosa Gomes using C-value genome size estimate and repetitive DNA sequences analysis. The genome size estimate was obtained by flow cytometry and the repetitive DNA sequences were accessed using graph-based clustering. Evolutionary relationships among species of Apocynaceae was obtained using reads of Catharanthus roseus L., Rhayza stricta Decne, and Asclepias syriaca L. from the NCBI and analyzed by graph-based clustering. The genome size estimates in two botanical varieties showed 2C-values ranging from 0.88 to 1.08 pg, indicating small genome size. Clusters representing repeats making up at least 0.01% of the genome revealed the proportion of repetitive DNA ranging from 19.87% (H. speciosa) to 51.674% (A. syriaca), of which the mobile elements were more abundant. Satellite DNA sequences were not found in H. speciosa and R. stricta, while at least one satellite was detected in C. roseus and A. syriaca, suggesting that the LTR retrotransposon Ty3/Gypsy/Chromovirus may have replaced the satellite DNA in H. speciosa and R. stricta.

Evolution of the Proto Sex-Chromosome in Solea senegalensis.

Solea senegalensis is a flatfish belonging to the Soleidae family within the Pleuronectiformes order. It has a karyotype of 2n = 42 (FN = 60; 6M + 4 SM + 8 St + 24 T) and a XX/XY system. The first pair of metacentric chromosomes has been proposed as a proto sex-chromosome originated by a Robertsonian fusion between acrocentric chromosomes. In order to elucidate a possible evolutionary origin of this chromosome 1, studies of genomic synteny were carried out with eight fish species. A total of 88 genes annotated within of 14 BACs located in the chromosome 1 of S. senegalensis were used to elaborate syntenic maps. Six BACs (BAC5K5, BAC52C17, BAC53B20, BAC84K7, BAC56H24, and BAC48P7) were distributed in, at least, 5 chromosomes in the species studied, and a group of four genes from BAC53B20 (grsf1, rufy3, slc4a4 and npffr2) and genes from BAC48K7 (dmrt2, dmrt3, dmrt1, c9orf117, kank1 and fbp1) formed a conserved cluster in all species. The analysis of repetitive sequences showed that the number of retroelements and simple repeat per BAC showed its highest value in the subcentromeric region where 53B20, 16E16 and 48K7 BACs were localized. This region contains all the dmrt genes, which are associated with sex determination in some species. In addition, the presence of a satellite “chromosome Y” (motif length: 860 bp) was detected in this region. These findings allowed to trace an evolutionary trend for the large metacentric chromosome of S. senegalensis, throughout different rearrangements, which could be at an initial phase of differentiation as sex chromosome.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics

ABSTRACTThis study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Analyzing and Characterizing the Chloroplast Genome of Salix wilsonii.

Salix wilsonii is an important ornamental willow tree widely distributed in China. In this study, an integrated circular chloroplast genome was reconstructed for S. wilsonii based on the chloroplast reads screened from the whole-genome sequencing data generated with the PacBio RSII platform. The obtained pseudomolecule was 155,750 bp long and had a typical quadripartite structure, comprising a large single copy region (LSC, 84,638 bp) and a small single copy region (SSC, 16,282 bp) separated by two inverted repeat regions (IR, 27,415 bp). The S. wilsonii chloroplast genome encoded 115 unique genes, including four rRNA genes, 30 tRNA genes, 78 protein-coding genes, and three pseudogenes. Repetitive sequence analysis identified 32 tandem repeats, 22 forward repeats, two reverse repeats, and five palindromic repeats. Additionally, a total of 118 perfect microsatellites were detected, with mononucleotide repeats being the most common (89.83%). By comparing the S. wilsonii chloroplast genome with those of other rosid plant species, significant contractions or expansions were identified at the IR-LSC/SSC borders. Phylogenetic analysis of 17 willow species confirmed that S. wilsonii was most closely related to S. chaenomeloides and revealed the monophyly of the genus Salix. The complete S. wilsonii chloroplast genome provides an additional sequence-based resource for studying the evolution of organelle genomes in woody plants.

Identity determination in diagnostic surgical pathology.

From a technical perspective, specimen identity determination in surgical pathology over the last several decades has primarily focused on analysis of repetitive DNA sequences, specifically microsatellite repeats. However, a number of techniques have recently been developed that have similar, if not greater, utility in surgical pathology, most notably analysis of single nucleotide polymorphism (SNPs) and gene panels by next generation sequencing (NGS). For cases with an extremely limited sample or a degraded sample, sequence analysis of mitochondrial DNA continues to be the method of choice. From a diagnostic perspective, interest in identity determination in surgical pathology is usually centered on resolving issues of specimen provenance due to specimen labeling/accessioning deficiencies and possible contamination, but is also frequently performed in cases for which the patient's clinical course following definitive therapy is remarkably atypical, in cases of an unexpected diagnosis, and by patient request for "peace of mind". However, the methods used for identity determination have a much broader range of applications in surgical pathology beyond tissue provenance analysis. The methods can be used to provide ancillary information for cases in which the histomorphology is not definitively diagnostic, as for example for tumors that have a virtually identical microscopic appearance but for which the differential diagnosis includes synchronous/metachronous tumors versus a metastasis, and for the diagnosis of hydropic early gestations versus hydatidiform molar pregnancies. The methods also have utility in several other clinical settings, for example to rule out a donor-transmitted malignancy in a transplant recipient, to monitor bone marrow transplant engraftment, and to evaluate natural chimerism.

The complete chloroplast genome sequence of the endangered species Syringa pinnatifolia (Oleaceae)

Syringa pinnatifolia is an endangered endemic species in China with important ornamental and medicinal value, and it needs urgent protection. Here, we report the complete chloroplast (cp) genome structure of S. pinnatifolia and its evolution is inferred through comparative studies with related species. The S. pinnatifolia cp genome was 155 326 bp and contained a large single copy region (LSC) of 86 167 bp and a small single copy region (SSC) of 17 775 bp, as well as a pair of inverted repeat regions (IRs) of 25 692 bp. A total of 113 unique genes were annotated, including 79 protein‐coding genes, 30 tRNA genes and four rRNA genes. The GC content of the S. pinnatifolia cp genome was 37.9%, and the corresponding values in the LSC, SSC and IR regions were 36.0, 32.1, 43.2% respectively. Repetitive sequences analysis revealed that the S. pinnatifolia cp genome contained 38 repeats. Microsatellite marker detection analysis identified 253 simple sequence repeats (SSRs), which provides opportunities for future studies of the population genetics and phylogenetic relationships of Syringa. Phylogenetic analysis of 29 selected cp genomes revealed that S. pinnatifolia is closely related to Syringa vulgaris and all 27 Lamiales species formed a clade separate from the two outgroup species. This newly characterized S. pinnatifolia chloroplast genome will provide a useful genomic resource of phylogenetic inference and the development of more genetic markers for species discrimination and population studies in the genus Syringa.