Search for the insertions and chromosomal rearrangements affecting changes in gene expression in D. melanogaster strains with impaired transposition control of the gypsy retrotransposon

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Abstract

Transposable elements (TE) increase the frequency of spontaneous mutations in the genome and are also capable of altering function and affecting gene expression, so it is important to have an idea of their activity and position in the genome. The paper demonstrates the advantage of combining the analysis of two sequencing methods of searching TE insertions and chromosomal rearrangements: full-genome nanopore sequencing allows the detection of TE insertions, and the use of transcriptome sequencing evaluates the effect of insertions on gene expression. The results are presented using SS (w1, flamenco mutant) and MS (w1, flamenco mutant, active copy of gypsy) strains with the flamenco phenotype as an example to investigate the causes of impaired control of TE activity. The laboratory wild type strain D32 was used as a control. Insertions and deletions of TE into the euchromatin regions of the genome and into the introns of genes relative to the reference genome were found in the studied strains and wild-type strains. In the analyzed genomes, a search for insertions and deletions in RNA interference system genes and in differentially expressed genes in SS and MS strains with flamenco phenotype was performed. We have detected TE insertions in various structures of AGO3, CG17147, Su(var)3-3, Gasz, CG43348, moody, CG17752 genes. For most of the analyzed genes, no correlation between a change in the TE position and a decrease or increase in gene transcription was found. A chromosomal rearrangement affecting the 3’-untranslated region has been detected for the vig gene. Based on the results of long-read sequencing, a de novo genome assembly for the MS strain was obtained. The increased expression in SS and MS strains for CR45822 and pst genes was found to be associated with triplication, but not with changes in gene regulatory sequences or TE insertion.

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About the authors

I. V. Kukushkina

Lomonosov Moscow State University

Author for correspondence.
Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234

A. R. Lavrenov

Lomonosov Moscow State University; Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences

Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234; Moscow, 119071

P. A. Milyaeva

Lomonosov Moscow State University; Shenzhen MSU-BIT University

Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234; Longgang District, Shenzhen, 518172

A. I. Lavrenova

Lomonosov Moscow State University

Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234

I. V. Kuzmin

Lomonosov Moscow State University

Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234

L. N. Nefedova

Lomonosov Moscow State University

Email: nefedova@mail.bio.msu.ru

Faculty of Biology

Russian Federation, Moscow, 119234

A. I. Kim

Lomonosov Moscow State University; Shenzhen MSU-BIT University

Email: vladimirova-bph@yandex.ru

Faculty of Biology

Russian Federation, Moscow, 119234; Longgang District, Shenzhen, 518172

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2. Additional materials for the article by I. V. Kukushkina et al.
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3. Fig. 1. Scheme of alignment of reads with the reference genome, including the sequence of the MGE, for long-read sequencing and short-read sequencing. The MGE in the studied genome is shown in red, the reference genome with the position of the insertion (purple) is shown in black. When aligning long reads, including the sequence of the MGE, surrounded by genomic sequences, the data will be interpreted as a new insertion (a). Short fragments of the MGE will be erroneously transferred to a random sequence of this MGE in the reference genome (б, в).

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4. Fig. 2. Scheme of amplification of LTR of LTR retrotransposons, which have two co-directed LTRs in their structure. During PCR, products are synthesized that contain two homologous and co-directed LTRs at the 3'-ends. The fragment is completed by overlapping homologous regions.

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5. Fig. 3. Visual display of the results of nanopore sequencing analysis in the IGV program. Reads corresponding to the reference genome are shown in gray. Single nucleotide polymorphisms corresponding to the substitution of a certain nucleotide are marked in red, yellow, and green. Black bars in the read are undefined nucleotides. Reads have boundaries and reading direction. The purple rectangle with numbers in the reads corresponds to insertions in the AGO3, CG43348 genes and in the region between the CG17147 and Su(var)3-3 genes with the insertion length indicated (а, б, в). Long black lines with numbers correspond to deletions in the Mur2B gene intron in the lines with the length indicated (г).

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6. Fig. 4. Triplication of the locus between the CR45822 and CR45835 genes. A 2-3-fold increase in read coverage between the CR45822 and CR45835 genes is observed (a). On the de novo assembled region of chromosome 3L, the sequences of the CR45822, CR45835, pst, Sec63, and CTCF genes are compared based on nanopore sequencing data. Triplication of the CR45822, CR45835, pst genes, and fragments of the CTCF and Sec63 genes is visible, and it is possible to determine proper polymorphic variants of some genes (б).

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