Dodatne informacije o plavom trkaču, Caranx crysos (Mitchill, 1815), u sjevernom Jadranu: meristička i molekularna karakterizacija

In October 2014, a blue runner (Caranx crysos) was caught by a professional fisherman in waters off St. Eufemija Point (west Istrian Coast, northern Adriatic Sea, Croatia). This is the second recorded blue runner in the northern Adriatic Sea, which represents the northernmost biogeographic sector of the Mediterranean Sea. Recently, blue runners were recorded in the southern and central Adriatic Sea, suggesting that the central Mediterranean C. crysos is expanding its area of distribution throughout the Adriatic Sea. As a point of interest, this most recent specimen presented an unusual meristic feature: the number of spines of its first dorsal fin was VI, whereas it is generally recognised that the number of such spines characteristic for this species is VIII. Species identity of the specimen was confirmed by DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene. In addition, we provided the first report of C. crysos mitochondrial 16S ribosomal RNA (16S rRNA) partial sequence.U listopadu 2014. godine, ribu plavi trkač (Caranx crysos) je ulovio profesionalni ribar nekoliko stotina metara van rta Sv. Eufemija (zapadna obala Istre, sjeverni Jadran, Hrvatska). Ovo je drugi po redu nalaz ribe plavi trkač koja je ulovljena u sjevernom Jadranu, što predstavlja najsjevernije biogeografsko područje sredozemnog mora. Nedavno ova vrsta je zabilježena u južnom i srednjem Jadranu što ukazuje da se populacija C. crysos iz središnjeg Sredozemnog mora proširuje u Jadranskom moru. Razmatrani primjerak je pokazao neobičnu merističku značajku: broj bodlji u prvoj leđnoj peraji je VI, dok je općenito za ovu vrstu prihvaćeno (priznato) VIII bodlji. Identitet vrste potvrđen je analizom mitohondrijske DNA sekvence za pod-jedinicu I gena citokrom c oksidaze (COI). U ovom radu također je po prvi put analiziran dio mitohondrijske DNA sekvence odgovorne za kodiranje 16S ribosomske RNA (16S rDNA) kod vrste C. crysos

Reference-Guided De Novo Genome Assembly of the Flour Beetle Tribolium freemani

The flour beetle Tribolium freemani is a sibling species of the model organism and important pest Tribolium castaneum. The two species are so closely related that they can produce hybrid progeny, but the genetic basis of their differences has not been revealed. In this work, we sequenced the T. freemani genome by applying PacBio HiFi technology. Using the well-assembled T. castaneum genome as a reference, we assembled 262 Mb of the T. freemani genomic sequence and anchored it in 10 linkage groups corresponding to nine autosomes and sex chromosome X. The assembly showed 99.8% completeness of conserved insect genes, indicating a high-quality reference genome. Comparison with the T. castaneum assembly revealed that the main differences in genomic sequence between the two sibling species come from repetitive DNA, including interspersed and tandem repeats. In this work, we also provided the complete assembled mitochondrial genome of T. freemani. Although the genome assembly needs to be ameliorated in tandemly repeated regions, the first version of the T. freemani reference genome and the complete mitogenome presented here represent useful resources for comparative evolutionary studies of related species and for further basic and applied research on different biological aspects of economically important pests

CenH3 distribution reveals extended centromeres in the model beetle Tribolium castaneum

Centromeres are chromosomal domains essential for kinetochore assembly and correct chromosome segregation. Inconsistent in their underlying DNA sequences, centromeres are defined epigenetically by the presence of the centromere-specific histone H3 variant CenH3. Most of the analyzed eukaryotes have monocentric chromosomes in which CenH3 proteins deposit into a single, primary constriction visible at metaphase chromosomes. Contrary to monocentrics, evolutionary sporadic holocentric chromosomes lack a primary constriction and have kinetochore activity distributed along the entire chromosome length. In this work, we identified cCENH3 protein, the centromeric H3 histone of the coleopteran model beetle Tribolium castaneum. By ChIP-seq analysis we disclosed that cCENH3 chromatin assembles upon a repertoire of repetitive DNAs. cCENH3 in situ mapping revealed unusually elongated T. castaneum centromeres that comprise approximately 40% of the chromosome length. Being the longest insect regional centromeres evidenced so far, T. castaneum centromeres are characterized by metapolycentric structure composed of several individual cCENH3-containing domains. We suggest that the model beetle T. castaneum with its metapolycentromeres could represent an excellent model for further studies of non-canonical centromeres in insects

Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle Tribolium and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles

Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs

Background: Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences that belong to the most abundant and the fastest evolving parts of the eukaryotic genome. A satellitome represents the collection of different satDNAs in a genome. Due to extreme diversity and methodological difficulties to characterize and compare satDNA collection in complex genomes, knowledge on their putative functional constraints and capacity to participate in genome evolution remains rather elusive. SatDNA transcripts have been detected in many species, however comparative studies of satDNA transcriptome between species are extremely rare. Results: We conducted a genome-wide survey and comparative analyses of satellitomes among different closely related Meloidogyne spp. nematodes. The evolutionary trends of satDNAs suggest that each round of proposed polyploidization in the evolutionary history is concomitant with the addition of a new set of satDNAs in the satellitome of any particular Meloidogyne species. Successive incorporation of new sets of satDNAs in the genome along the process of polyploidization supports multiple hybridization events as the main factor responsible for the formation of these species. Through comparative analyses of 83 distinct satDNAs, we found a CENP-B box-like sequence motif conserved among 11 divergent satDNAs (similarity ranges from 36 to 74%). We also found satDNAs that harbor a splice leader (SL) sequence which, in spite of overall divergence, shows conservation across species in two putative functional regions, the 25-nt SL exon and the Sm binding site. Intra- and interspecific comparative expression analyses of the complete satDNA set in the analyzed Meloidogyne species revealed transcription profiles including a subset of 14 actively transcribed satDNAs. Among those, 9 show active transcription in every species where they are found in the genome and throughout developmental stages. Conclusions: Our results demonstrate the feasibility and power of comparative analysis of the non-coding repetitive genome for elucidation of the origin of species with a complex history. Although satDNAs generally evolve extremely quickly, the comparative analyses of 83 satDNAs detected in the analyzed Meloidogyne species revealed conserved sequence features in some satDNAs suggesting sequence evolution under selective pressure. SatDNAs that are actively transcribed in related genomes and throughout nematode development support the view that their expression is not stochastic

Histone Modifications within the Human X Centromere Region

Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus. We used ChIP to examine the distribution of modified histones within centromere regions of multiple X chromosomes. Methylation of H3 at lysine 4 coincided with DXZ1 higher order alpha satellite, the site of CENP-A localization. Heterochromatic histone modifications were distributed across the 400–500 kb pericentromeric regions. The large arrays of alpha satellite and gamma satellite DNA were enriched for both euchromatic and heterochromatic modifications, implying that some pericentromeric repeats have multiple chromatin characteristics. Partial truncation of the X centromere resulted in reduction in the size of the CENP-A/Cenp-A domain and increased heterochromatic modifications in the flanking pericentromere. Although the deletion removed ∼1/3 of centromeric DNA, the ratio of CENP-A to alpha satellite array size was maintained in the same proportion, suggesting that a limited, but defined linear region of the centromeric DNA is necessary for kinetochore assembly. Our results indicate that the human X centromere contains multiple types of chromatin, is organized similarly to smaller eukaryotic centromeres, and responds to structural changes by expanding or contracting domains