Research Article |
Corresponding author: Mauro Mandrioli ( mauro.mandrioli@unimo.it ) Academic editor: Vladimir Lukhtanov
© 2014 Mauro Mandrioli, Federica Zanasi, Gian Carlo Manicardi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mandrioli M, Zanasi F, Manicardi G (2014) Karyotype rearrangements and telomere analysis in Myzus persicae (Hemiptera, Aphididae) strains collected on Lavandula sp. plants. Comparative Cytogenetics 8(4): 259-274. https://doi.org/10.3897/CompCytogen.v8i4.8568
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Karyotype analysis of nine strains of the peach-potato aphid Myzus persicae (Sulzer, 1776), collected on Lavandula sp. plants, evidenced showed that five of them had a standard 2n = 12 karyotype, one possessed a fragmentation of the X chromosome occurring at the telomere opposite to the NOR-bearing one and three strains had a chromosome number 2n = 11 due to a non-reciprocal translocation of an autosome A3 onto an A1 chromosome. Interestingly, the terminal portion of the autosome A1 involved in the translocation was the same in all the three strains, as evidenced by FISH with the histone cluster as a probe. The study of telomeres in the M. persicae strain with the X fission evidenced that telomerase synthesised de novo telomeres at the breakpoints resulting in the stabilization of the chromosomal fragments. Lastly, despite the presence of a conserved telomerase, aphid genome is devoid of genes coding for shelterin, a complex of proteins involved in telomere functioning frequently reported as conserved in eukaryotes. The absence of this complex, also confirmed in the genome of other arthropods, suggests that the shift in the sequence of the telomeric repeats has been accompanied by other changes in the telomere components in arthropods in respect to other metazoans.
Aphids, Myzus persicae , holocentric chromosomes, karyotype rearrangements, de novo telomeres, shelterin
Karyotype features are usually stable within species, and chromosomal changes, if they occur, contribute to the formation of a post-zygotic barrier between biological populations causing the establishment of reproductive isolation and speciation as a possible consequence (
Despite these general rules, the speciation models were still problematic since numerous cases of intraspecific karyotype instability have been described in literature and at present the most extreme case was published by
Aside from special cases, such as polyploidy, chromosomal speciation remained a controversial mechanism, especially in animals other than mammals (e.g.,
The peach potato aphid Myzus persicae (Sulzer, 1776) is a good experimental model for the study of chromosome rearrangements since numerous variations regarding both chromosome number and structure have been reported (
The evolutionary history of the M. persicae group is marked with speciation events (for a review see
The frequent occurrence of different chromosome numbers and the inheritance of chromosomal fragments have been related to the holocentric structure of aphid chromosomes (
The spread of chromosomal rearrangements has also been favoured in M. persicae by the continuous expression of the telomerase gene, which allows a de novo synthesis of new telomeres at the chromosomal breakage sites (
As
In the present paper we analysed the presence of karyotype variants in nine M. persicae strains collected on Lavandula sp. plants. Moreover we verified if the synthesis of de novo telomeres is common in M. persicae populations with fragmented chromosomes and analysed the evolutionary conservation of the shelterin complex, a group of proteins generally associated with telomere functioning.
Specimens of M. persicae were obtained from 9 different aphid populations collected on Lavandula sp. plants. In particular, the strains labelled as Mo1, Mo2, Mo3 and Mo4 have been collected in Modena (Italy), whereas the strains Re1, Re2a, Re2b, Re3 and Re4 have been collected in Correggio (Reggio Emilia, Italy). Each population was established as a clone from a single female aphid originally collected from the field and thereafter maintained as a colony of parthenogenetic females on pea (Pisum sativum, Linnaeus, 1758) plants at 19 °C with a light-dark regime of 16 hours light and 8 hours darkness.
Chromosome preparations were obtained from parthenogenetic females by spreading embryo cells, as reported by
DNA extraction, following a standard phenol-chloroform protocol, and fluorescent in situ hybridization (FISH) have been described in
The 28S rDNA genes have been amplified using the primers F (5’-AACAAACAACCGATACGTTCCG) and R (5’-CTCTGTCCGTT TACAACCGAGC), designed according to the insect 28S rDNA sequences available in GenBank. Amplification was performed using a Hybaid thermal-cycler at an annealing temperature of 60 °C for 1 minute (min) with an extension time of 1 min at 72 °C.
In order to amplify a DNA sequence containing the complete aphid histone gene cluster, the primers HIS-CLUST-F (5’-cgaaaccgtaaagggtacga) and HIS-CLUST-R (5’-ggcggctttgactttattga) have been designed on the basis of the Acyrthosiphon pisum genomic scaffold 368 (NW_003383857.1, from base 259987 to 272662). The amplification of a 7379 bp fragment was carried out by an Hybaid thermal-cycler using the Fermentas Long PCR Enzyme Mix making annealing and extension at 68 °C for 8 min for 25 cycles, according to the manufacturer’s instructions.
PCR digoxigenin labelling of the subtelomeric repeat was performed with a PCR DIG labelling kit according to the Roche protocol using the specific oligonucleotide primers MpR-F (5’–TCAAAGTTCTCGTTCTCC–3’) and MpR-R (5’–GTTTTAACAGAGTGCTGG–3’), designed according to the subtelomeric repeat sequence available in the literature (
In order to localize the telomeric (TTAGG)n repeats, a probe was obtained by PCR amplification using the two primers F (TTAGG)5 and R (CCTAA)5 in the absence of temfig, as described by
Random priming probe biotin-labelling was performed with the Biotin High Prime (Roche), whereas the PCR digoxigenin labelling were performed using the Dig High Prime (Roche). Both labelling were done according to the Roche protocols.
Propidium-stained and FISH slides were observed using a Zeiss Axioplan epifluorescence microscope. Photographs of the fluorescent images were taken using a CCD camera (Spot, Digital Instrument, Madison, USA) and the Spot software supplied with the camera and processed using Adobe Photoshop (Adobe Systems, Mountain View, CA).
Bioinformatic analyses for homologous genes coding for the proteins POT1, TRF1, TRF2, RAP1, TPP1 and TIN2 in aphids and other arthropods have been performed by BLAST alignments in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) both at DNA and protein levels using different homologous genes as reference sequences (Table
Telomere-associated proteins | Orthologous proteins in GenBank |
---|---|
POT1 | Homo sapiens (AAH02923), Schizosaccharomyces pombe (CAB16192) |
TRF1 | H. sapiens (NP_059523), S. pombe (NP_595979) |
TRF2 | H. sapiens (NP_005643) |
RAP1 | H. sapiens (ABA64473), S. pombe (BAB70735) |
TPP1/TEBPα | Danio rerio (NP_001124265), Stylonychia lemnae (AAU95535) |
TIN2 | H. sapiens (AF195512) |
The standard karyotype of M. persicae parthenogenetic females consists of 12 chromosomes (five pairs of autosomes and two X chromosomes) (
Chromosomal figs (a–d) and karyotypes (e–h) obtained from specimens belonging to clones Mo1 (a, e) e Mo2 (b, f), Mo3 (c, g) and Mo4 (d, h). Simultaneous in situ hybridization with the histone (in blue) and subtelomeric DNA probes (in green) (i–j) revealed in both clones Mo3 (i) and Mo4 (j) that the A1–A3 translocation involved the autosome 1 telomere close to the histone probe. Arrows indicate X chromosomes; asterisks indicate rearranged autosomes. Bar = 10 mm.
Chromosomal figs (a, c–f) and karyotypes (g–k) obtained from specimens belonging to clones Re1 (a, g, e) Re2a (c, h), Re2b (d, i), Re3 (e, j) and Re4 (f, k). Fluorescent in situ hybridization with the 28S probe (in green) (b) revealed that the fragmentation at the X chromosome in clone Re1 occurred at the telomere opposite to the NOR (b). Simultaneous in situ hybridization with the histone (in blue) and subtelomeric DNA probes (in green) in clone Re2b (i) revealed that the A1–A3 translocation involved the autosome 1 telomere close to the histone cluster. Arrows indicate X chromosomes. Asterisks indicate rearranged autosomes. Bar = 10 mm.
Previous study showed that the histone cluster map eccentrically on the autosome 1 (
Interestingly, in the clone Re1 all telomeres resulted labelled by the (TTAGG)n telomeric probe including the X chromosome (and its fragment) involved in the fission suggesting that a de novo synthesis of telomeres occurred in this clone (Fig.
FISH with the telomeric (TTAGG)n probe showed evident telomeric repeats at each chromosomes in clones Mo3 (a), Mo4 (b), Re1 (c) and Re2b (d). No interstitial telomeric signals were present in clones Mo3 (a), Mo4 (b) and Re2b (d) possessing a chromosomes derived from an autosome A1–A3 fusion. All telomeres resulted labelled by the (TTAGG)n telomeric probe in clone Re1 including the X chromosome involved in the fission suggesting that a de novo telomere synthesis occurred in this clone (c). Arrows indicate X chromosomes. Bar = 10 mm.
According to previous studies (
Taking into account that the unique aphid protein studied regarding the telomere functioning has been the telomerase (
Holocentric chromosomes have been frequently described as a powerful tool to stabilize and inherit chromosomal mutations resulting in karyotype changes (
The identification of several M. persicae populations bearing rearranged karyotypes made this species a complex, but intriguing, model for the study of aphid cytogenetics (
In this paper we report the presence of rearranged karyotypes, including fissions and translocations, in M. persicae strains collected on Lavandula plants. The analysis of their karyotypes confirmed that autosomes 3 and 1 are the chromosomes mostly involved in changes in the M. persicae complement (
Previous literature data (
The fission of chromosomes by tobacco and lavender oil mutagens may be lethal in organisms with monocentric chromosomes (possessing a localized centromeres), since chromosomal fragments tend to be lost during mitosis and meiosis. By contrast, aphids can cope with this due to the holocentric nature of their chromosomes. As a consequence, chromosome fragments can move to the daughter cells at successive cell divisions.
Our results confirmed that some portions of the aphid chromosomes seem to be more prone to fragmentation than others in presence of potential genotoxic compounds. Indeed, a fragmentation of the X chromosome similar to that reported in the present paper has been described in other M. persicae strains and it was localized near (or within) the heterochromatic band enriched in satellite DNAs (
Our data showed that the A1-A3 fusion seems to involve always the same terminal end of the autosome 1. Previous experiments (
From a chromosomal point of view, the species M. persicae is the sum of populations that have different karyotypes. Interestingly, similar karyotypic variants have been identified on different host plants (
A further element of interest in the Lavandula clones is related to their ability to synthesize new telomeres after chromosomal breakages. In aphids, telomeres consist of stretches of the (TTAGG)n repeat. This simple sequence has been reported also in the majority of insects (
Differently from the extensive study of the telomere composition, few papers have been focussed on the proteins associated to the telomere functioning in insects, with the exception of D. melanogaster, where telomeres are capped by a complex of fast-evolving proteins, called terminin (
In mammals, telomeres are capped by different proteins that play vital roles in telomere length regulation and chromosomal end protection (
According to literature data, shelterin complex is essential in telomere capping so that telomeres that are severely or completely devoid of telomeric proteins are more prone to damages and/or become the target of frequent recombination (
Due to the importance of the shelterin complex in the telomere functioning, it is very intriguing that this important set of proteins is absent in the studied arthropod genomes, including the aphid one. According to different essays performed both in animal and plants, shelterin complex has a exquisite specificity for the telomeric TTAGGG repeats due to the presence of multiple TTAGGG recognition folds in the complex (
Exceptions to the presence of all the shelterin proteins have been already reported in literature since, for instance, the subunit TIN2 and TPP1 have been so far only found in vertebrates (
The absence of the whole shelterin complex is extremely interesting from a functional point of view since it is generally implicated in the generation of the t-loop and in the control of the synthesis of telomeric DNA by telomerase (
This work is supported by the grant “Experimental approach to the study of evolution” from the Department of Animal Biology of the University of Modena and Reggio Emilia (M.M.).