|Home | About | Journals | Submit | Contact Us | Français|
The most common type of coniferous mobile genetic elements are retrotransposons. Despite of their early positive impact on evolution of modern coniferous species they can have a significant negative impact for Forestry and breeding. Breaking genomic structural integrity mobile elements can cause phenotypic defects of plants. In this regard, the study of the diversity of coniferous mobile genetic elements is particularly interesting. In the present paper, we describe mobile genetic elements in dsRNA fraction of Pinus sylvestris having witches broom abnormalities. In result of assembled contigs analysis by RepeatMasker 70 mobile genetic elements were identified. A 68 of that were retroelements. Most of elements represented by Gypsy (16 contigs) and Copia (48 contigs). In 4 cases retroelements specific to Pinus taeda were identified. In most cases fragments of integrase (24), reverse transcriptase (22) and RNaseH (15) were identified. Results of the study may be of interest for coniferous breeding and genetic specialists.
The raw data of these experiments have been deposited at NCBI under the accession number SAMN06185845.
In coniferous plants retrotransposons make up a large part of the genome , . It is estimated that 70% of the genetic material is repetitive genetic elements. About 58% among it of the genome constitute LTR-containing retrotransposons (35% Group Ty3/Gypsy, 16% a group of Ty1 / Copia, 7% is not classified), 1% - the LINE-elements, 1% - DNA transposons, 10% - unclassified high copy number DNA . Mobile genetic elements played an important role in the evolution of conifers genomes , . However, the virus-like genetic elements (or retrotransposons) have a negative value for forestry, because of their displacement and insertions they cause different disturbances to structural and functional organization of the genome . On the other hand, virus-like mobile genetic elements are a reservoir of natural variation and play an important role in macroevolution processes . Studies of retrotransposons could shield light to complexity of its regulation to develop methods of transposon\destabilisation depending on breeding program needs.
Samples of the Scots pine (Pinus sylvestris L.) were collected in forest stands around the Homiel town (52°26′43″N and 30°59′03″E), which is located about 40 km from the southeastern border of the Republic of Belarus. Pine samples were green shoots (with needles) of witch’s brooms (21 pcs. from individual trees), spruce samples were green shoots of pineapple galls, produced by Adelges abietis L. (6 pcs. from individual trees). Samples were dissected with thin scalpels under a stereomicroscope (Leica M50, Leica Microsystems GmbH, Germany). Inner tissues was carefully separated from the coating surrounding tissues, and quickly transferred to microtubes containing RNAlater solution (Thermo Scientific, U.S.A.).
Total RNA was prepared using the GeneJet Plant RNA Purification Mini Kit (Thermo Scientific, U.S.A.), which is based on a combination of guanidine-isothiocyanate lysis and silica-membrane purification in a mini-spin column. The procedures were performed according to the manufacturer's instructions. The quality of the RNA was analyzed with denaturating RNA electrophoresis in 1 × TAE 1% agarose gel and quantified by UV absorption using Implen P330 (Implen GmbH, Germany).
The ds cDNA pool was prepared using Maxima H Minus Double-Stranded cDNA Synthesis Kit (Thermo Scientific, U.S.A.). Briefly, the synthesis of first strand cDNA began with 1 μg of total RNA and 0.5 μg random hexanucleotide primers. The resulting first strands of cDNA (RNA-DNA hybrid molecule) were processed with enzyme mix of E. coli RNase H, DNA polymerase I and DNA ligase immediately. E. coli RNase H inserted nicks into the RNA, providing 3′ OH-primers for DNA polymerase I. The 5′–3′ exonuclease activity of E. coli DNA polymerase I removed the RNA strand in the direction of synthesis, while its polymerase activity replaced the RNA with deoxyribonucleotides. DNA ligase linked the gaps to complete the ds cDNA strand. The quality of the ds DNA was analyzed with electrophoresis in 1 × TBE 1.5% agarose gel and quantified by UV absorption using Implen P330 (Implen GmbH, Germany).
A fragment library was generated with an input of 100 ng ds cDNA using the Ion Xpress Plus Fragment Library Kit (Thermo Scientific, U.S.A.). Next, DNA fragmentation, adaptor ligation, fragment size selection and library amplification were carried out according to manufacturer's instructions. The targeting of fragments of approximately 330 bp was performed with 1.5 × TBE 2% agarose gel (Helicon, Russia). Prior to emulsion PCR, the size distribution were assessed with 1.5 × TBE 2% agarose gel (Helicon, Russia); concentrations of the libraries were normalized with Ion Library Equalizer Kit (Thermo Scientific, USA). The fragment library was adjusted to approximately 26 pM and amplified with Ion Sphere particles™ (ISPs) by emulsion PCR using the Ion OneTouch™ Instrument with the Ion OneTouch™ 200 Template Kit (Thermo Scientific, USA) and template-positive ISP enrichment according to the manufacturer's protocol (Thermo Scientific, USA). About 50% of the ISPs were and then sequenced on an Ion 314™ chip using the Ion Torrent Personal Genome Machine (PGM™) (Thermo Scientific, USA) for 130 cycles (520 flows) with the Ion PGM™ 200 Sequencing Kit (Thermo Scientific, USA). Initial processing of ION PGM data carried out in an automatic mode using Ion Torrent Suite (Thermo Scientific, USA) software.
Ion Torrent Personal Genome Machine (Thermo Scientific, USA) sequencing can generate processed reads 200–260 base length for analyzed cDNA fragments.
Initial data had been cleared off from sequences with a grade lower than Q < 20 via FASTQ filter quality (http://hannonlab.cshl.edu/fastx_toolkit). Since RNA sequencing data contained rRNA sequences it was necessary to conduct the removal of rRNA sequences from the raw data using riboPicker tool. The resulting data set was assembled with Trinity (version 20130814) in accordance with the guidance provided by the developers, and the default settings. To identify retroelements in assembled sequences RepeatMasker program was used . The program uses Repbase the repetitive elements library .
After quality filtration and rRNA cleaning a 110,746 high quality reads were assembled to 1558 contigs. Length of contigs varied from 201 to 11,902 bp. Average length was 421 bp and N50 was 442 bp. In result of 1558 contigs analysis by RepeatMasker, 70 mobile genetic elements were identified (Table 1).
A 68 of that were retroelements (Table 2). Most of elements represented by Gypsy (16 contigs) and Copia (48 contigs). In 4 cases retroelements specific to Pinus taeda were identified. It is worth nothing that in two cases DNA-transposons were also identified. In general this data comply previously published where both Gypsy and Copia were shown to be dominant .
Studies of contigs showed that in most of cases fragments of integrase (24), reverse transcriptase (22) and RNaseH (15) were identified (Table 3). Results of the study may be of interest for coniferous breeding and genetic specialists.
This study was funded by Joint Grant of Russian (RFBR No. 15-54-04004) and Byelorussian (BRFFR No. B15RM-007) Foundations for Fundamental Research.
The authors declare that they have no competing interests.
The authors acknowledged the Russian and Byelorussian Foundations for Fundamental Research for the supporting of the study.