Characterization of the Rhipicephalus (Boophilus) microplus Sialotranscriptome Profile in Response to Theileria equi Infection

This study intends to characterize the sialotranscriptome profile of Rhipicephalus (Boophilus) microplus in response to Theileria equi and identify genes of interest with differential genomic expression, indicating relevant targets in the tick–protozoan interactions. The experimental design consisted of RNA sequencing from uninfected and T. equi-infected R. microplus salivary glands (SGs) to obtain transcriptomic profiles for characterization and comparison. A total of 288,952 transcripts were obtained from both tick profiles, 3456 transcripts (p < 0.05) differentially expressed in response to T. equi infection. The uninfected SGs’ registered 231,179 transcripts, of which 155,359 were annotated. The most transcribed sequences were female-specific histamine binding protein and lipocalins. Regarding the T. equi-infected SGs, from the 238,964 assembled transcripts, 163,564 were annotated. The most transcribed sequences were histone demethylase JARID1 and Y-box-binding protein. Five transcripts (cystatin, arginase, nuclear factor κB kinase inhibitor subunit β (IκB), IκB delta, lysosomal-trafficking regulator, and reeler protein) presented the gene ontology (GO) category “response to protozoan” and were exclusively displayed in the T. equi-infected profile. The transcriptome of T. equi was also analyzed, registering 4728 hits. The study’s genetic and molecular information would be of great value for future studies and biotechnological applications envisaging disease control.

New validated Eucalyptus SSR markers located in candidate genes involved in growth and plant development

Aim of study: To validate and characterize new microsatellites or Simple Sequence Repeats (SSR) markers, located within genomic transcribed sequences related to growth and plant developmental traits, in Eucalyptus species.Area of study: Eucalyptus species from different Australian origins planted in Argentina.Materials and methods: In total, 134 SSR in 129 candidate genes (CG-SSR) involved in plant development were selected and physically mapped to the E. grandis reference genome by bioinformatic tools. Experimental validation and polymorphism analysis were performed on 48 individuals from E. grandis and interspecific hybrids (E. grandis x E. camaldulensis; E. grandis x E. tereticornis), E. globulus, E. maidenii, E. dunnii and E. benthamii.Main results: 131 out of 134 CG-SSR were mapped on the 11 chromosomes of E. grandis reference genome. Most of the 134 analyzed SSR (> 75%) were positively amplified and 39 were polymorphic in at least one species. A search of annotated genes within a 25 kbp up and downstream region of each SSR location retrieved 773 genes of interest.Research highlights: The new validated and characterized CG-SSR are potentially suitable for comparative QTL mapping, molecular marker-assisted breeding (MAB) and population genetic studies across different species within Symphyomyrtus subgenus.Keywords: CG-SSR; cross-transferability; EST; eucalypts; microsatellite.

Transcribed sex-specific markers on the Y chromosome of the oriental fruit fly, Bactrocera dorsalis

Background The Oriental fruit fly, Bactrocera dorsalis, is a highly polyphagous invasive species with a high reproductive potential. In many tropical and subtropical parts of the world it ranks as one of the major pests of fruits and vegetables. Due to its economic importance, genetic, cytogenetic, genomic and biotechnological approaches have been applied to understand its biology and to implement the Sterile Insect Technique, currently a part of area-wide control programmes against this fly. Its chromosome complement includes five pairs of autosomes and the sex chromosomes. The X and Y sex chromosomes are heteromorphic and the highly heterochromatic and degenerate Y harbours the male factor BdMoY. The characterization of the Y chromosome in this fly apart from elucidating its role as primary sex determination system, it is also of crucial importance to understand its role in male biology. The repetitive nature of the Y chromosome makes it challenging to sequence and characterise. Results Using Representational Difference Analysis, fluorescent in situ hybridisation on mitotic chromosomes and in silico genome resources, we show that the B. dorsalis Y chromosome harbours transcribed sequences of gyf, (typo-gyf) a homologue of the Drosophila melanogaster Gigyf gene, and of a non-LTR retrotransposon R1. Similar sequences are also transcribed on the X chromosome. Paralogues of the Gigyf gene are also present on the Y and X chromosomes of the related species B. tryoni. Another identified Y-specific repetitive sequence linked to BdMoY appears to be specific to B. dorsalis. Conclusions Our random scan of the Y chromosome provides a broad picture of its general composition and represents a starting point for further applicative and evolutionary studies. The identified repetitive sequences can provide a useful Y-marking system for molecular karyotyping of single embryos. Having a robust diagnostic marker associated with BdMoY will facilitate studies on how BdMoY regulates the male sex determination cascade during the embryonic sex-determination window. The Y chromosome, despite its high degeneracy and heterochromatic nature, harbours transcribed sequences of typo-gyf that may maintain their important function in post-transcriptional mRNA regulation. That transcribed paralogous copies of Gigyf are present also on the X and that this genomic distribution is maintained also in B. tryoni raises questions on the evolution of sex chromosomes in Bactrocera and other tephritids.

PHYLOGENETIC ASSESSMENT OF TREE SPECIES OF ACONITUM L. FROM KAZAKHSTAN BY USING ITS AND MATK MARKERS

The Aconitum L. is a diverse genus consisting of more than 300 species all over the World, including 11 species grown in Kazakhstan. The phylogeny of the genus was mostly studied by using internal transcribed sequences (ITS) of the nuclear genome and maturase K (matK) of the chloroplast genome. Therefore, in this study it was decided to assess the phylogenetic position of three local species A. leucostomum, A. soongoricum and A. apetalum, by using ITS and matK. The application of Maximum-Likelihood (ML) method suggested that the A. soongoricum belong to subgenus Aconitum, and A. leucostomum and A. apetalum to the subgenus Lycoctonum, which was congruent to previous taxanomic studies for this genus. The Median-Joining network using ITS suggested that A. sachalinense belongs to the group of processors of the genus among species that were involved in the analysis. The study is the first attempt to understand phylogenetic relationship of three species of Aconitum grown in Kazakhstan.

Broad noncoding transcription suggests genome surveillance by RNA polymerase V

Eukaryotic genomes are pervasively transcribed, yet most transcribed sequences lack conservation or known biological functions. InArabidopsis thaliana, RNA polymerase V (Pol V) produces noncoding transcripts, which base pair with small interfering RNA (siRNA) and allow specific establishment of RNA-directed DNA methylation (RdDM) on transposable elements. Here, we show that Pol V transcribes much more broadly than previously expected, including subsets of both heterochromatic and euchromatic regions. At already established RdDM targets, Pol V and siRNA work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to siRNA but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo if a transposon is reactivated. We propose a model where Pol V surveils the genome to make it competent to silence newly activated or integrated transposons. This indicates that pervasive transcription of nonconserved sequences may serve an essential role in maintenance of genome integrity.

Resolving a Systematic Error in STARR-seq for Quantitative Enhancer Activity Mapping

STARR-seq assesses millions of fragments in parallel measuring enhancer activity quantitatively. Here we show that STARR-seq is critically flawed with a systematic error in the cells of Arabidopsis thaliana (A. thaliana). Large amount of self-transcripts (STs) is lost during reverse transcription because these STs are polyadenylated after alternative polyadenylation sites (APAS) inside the test sequences. We solved this problem by using specially designed primer and recovered self-transcribed sequences independent from the PAS usage. In A. thaliana, we identified active enhancers and also enhancers quiescent in their endogenous genomic loci. Different from traditional STARR-seq identified enhancers, enhancers identified by new method are highly enriched in sequences proximal to the 5’ and 3’ ends of genes, and their epigenetic states correlate with gene expression levels. Our solution applies to methods based on self-transcript quantification. In addition, our results provide an invaluable functional enhancer activity map and insights into the functional complexity of enhancers in A. thaliana.

Genomic Organization and Generation of Genetic Variability in the RHS (Retrotransposon Hot Spot) Protein Multigene Family in Trypanosoma cruzi

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS protein family in T. cruzi was demonstrated by phylogenetic and recombination analyses. Transcribed sequences carrying the RHS domain were classified into ten distinct groups of monophyletic origin. We identified numerous recombination events among the RHS and traced the origins of the donors and target sequences. The transcribed RHS genes have a mosaic structure that may contain fragments of different RHS inserted in the target sequence. About 30% of RHS sequences are located in the subtelomere, a region very susceptible to recombination. The evolution of the RHS family has been marked by many events, including gene duplication by unequal mitotic crossing-over, homologous, as well as ectopic recombination, and gene conversion. The expression of RHS was analyzed by immunofluorescence and immunoblotting using anti-RHS antibodies. RHS proteins are evenly distributed in the nuclear region of T. cruzi replicative forms (amastigote and epimastigote), suggesting that they could be involved in the control of the chromatin structure and gene expression, as has been proposed for T. brucei.

Contribution of retrotransposition to developmental disorders

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient’s symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.

An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana

Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions. The activity profile of this intron at different locations was similar to that of a natural intron from the UBQ10 gene, suggesting that the motif increases mRNA accumulation by the same mechanism. A series of introns with different numbers of this motif revealed that the effect on expression is linearly dependent on motif copy number up to at least 20, with each copy adding another 1.5-fold increase in mRNA accumulation. Furthermore, 6 copies of the motif stimulated mRNA accumulation to a similar degree from within an intron or when introduced into the 5′-UTR and coding sequences of an intronless construct, demonstrating that splicing is not required for this sequence to boost expression. The ability of this motif to substantially elevate expression from several hundred nucleotides downstream of the transcription start site reveals a novel type of eukaryotic gene regulation.