The authors have declared that no competing interests exist.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) using new designed primers pair for Heat Shock Protein70 homologue (HSP70h) of
Viruses represent an important threat to plant production worldwide. In spite of numerous efforts for their elimination, the number of these viruses is still noticeably limited. Their eradication is being counterbalanced by emergence of new viruses which induce severe economic losses. These emerging plant viruses highlight the importance of studies of plant virus evolution. Much of adaptive potential of viruses stems from their large population sizes, and their high degrees of variability. The use of error-prone RNA polymerase during replication by RNA viruses is the primary driver of high mutation rates. Arising mutations can become fixed within virus populations, and are subject to the effects of processes such as natural selection. Selection is a directional process by which the frequencies of variants that are the fittest in a given environment will increase in the population (positive selection) whereas those of less fit variants will decrease (negative selection). At the molecular level, selection detection relies on comparisons of the relative numbers of non-synonymous and synonymous substitutions. Silent substitutions, resulting in no change in the amino acid encoded by the codon, are assumed to arise
Olive is one of the most widely grown fruit tree in Tunisia. It plays a major social, economical and cultural role. Olive trees cover an estimated area of 1.7 million hectares out of which 20,000 are irrigated. El Air et al.
In spite of widely provided efforts to characterize OLYaV at molecular level, studies about molecular evolution of this virus are nowadays still lacking. The objective of this work was to give a preliminary idea on evolutionary strategies employed by this virus to survive by searching for the occurrence of potential recombination events and evaluate selection pressure exerted on amino acids even though using a limited genomic region of OLYaV (partial sequences of HSP70h-coding gene).
Shoot samples from 37 cultivars showing yellowing symptoms on leaves were collected from two mother block stands Institut de l’Olivier of Sfax (southern Tunisia) and Gatrania (central Tunisia), and from a nursery Nour located at Karma (central Tunisia).
Newly designed specific primers by using Primer3 software
Phloem tissue, scraped from 12 shoots (two-year old) per tree collected in spring of 2013, was powdered in liquid nitrogen. Then, 100 mg of each sample was used for total RNA extraction using PureLink RNA™ Mini Kit (Life technologies, Carlsbad, CA-USA) according to the manufacturer’s protocol. RNA was finally eluted with 50 µl of RNase/DNase-free distilled water.
Purified total RNA (5µl) were mixed with 1 µl random hexamer primer (Bioron GmbH, Ludwigshafen, Germany) (0.5 µg.µl-1), 1 µl 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP at neutral pH), and 13 µl RNase/DNase-free distilled water. The reaction mixture was incubated at 65°C for 5 min and chilled on ice for 5 min. After a brief centrifugation, a second reaction mixture containing 6 µl 5X First-Strand Buffer [250 mM Tris-HCl (8.3 at room temperature), 375 mM KCl, 15 mM MgCl2], 3 µl 0.1M DTT, and 1 µl RNaseOUT™ (40 U.µ-1) (Life technologies, Carlsbad, CA-USA), was added to the former. The reaction mixture was incubated at 37°C for 2 min and chilled on ice for 5 min. A third mixture containing 1 µl M-MLV (200 U.µl-1) (Life technologies, Carlsbad, CA-USA), 3 µl 10 mM dNTP mix, 4 µl 5X First-Strand Buffer, and 12 µl RNase/DNase-free distilled water, was added to the former two. The reaction mixture was incubated successively at 25°C for 10 min, 37°C for 50 min, and 70°C for 15 min. Incubation was done in a thermal cycler (Multigene optimax, Labnet, Edison, USA).
The PCR reaction mixture using the newly designed specific primer was prepared by using 5 µl of the resulting cDNA. The OLYaV cDNA was transferred to a tube containing 5 µl 10X PCR buffer minus Mg++ 200 mM Tris, 2 µl 10 mM dNTPs, 3 µl 50 mM MgCl2, 15 pmol of each forward and reverse primers, 2 U
Amplicons of successfully amplified isolates were cloned into pCR2.1 vector using a TOPO TA cloning kit (Life technologies, Carlsbad, CA-USA) and used to transform DH5α cells of
Databank searches for homologies to OLYaV were performed using the FASTA
The nucleotide sequences of PCR products along with the reference sequence AJ440010 were aligned using CLUSTALW 2.1, CLUSTALX 2.1
Occurrence of potential recombination events between nucleotide sequences was explored with SBP (Single breakpoint), GARD
Dna
RT-PCR successfully amplified the targeted genome portion of 10 out of the 37 collected accessions. The size amplicon obtained (
The sequences of the amplicons obtained from the isolate genome candidates as well as the sequence reference AJ440010 were aligned and showed a divergence of sequences ranging roughly from 86.06 to 99.40, and from 91.89 to 99.55 for the nucleotide and amino acid residues, respectively (
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99.10 | 95.05 | 96.85 | 98.20 | 96.85 | 97.30 | 95.05 | 95.05 | 93.24 | 97.75 | |
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96.70 | 95.95 | 97.75 | 99.10 | 97.30 | 97.75 | 95.50 | 95.50 | 93.24 | 98.65 | |
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92.80 | 93.55 | 94.59 | 95.95 | 93.69 | 94.14 | 95.50 | 94.59 | 92.79 | 95.50 | |
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94.45 | 94.90 | 90.85 | 98.65 | 95.05 | 95.50 | 95.05 | 95.05 | 94.59 | 96.85 | |
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94.90 | 95.50 | 91.15 | 93.70 | 96.40 | 96.85 | 95.50 | 95.50 | 94.14 | 97.75 | |
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94.45 | 95.95 | 90.70 | 93.25 | 93.85 |
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94.14 | 94.14 |
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95.95 | |
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94.45 | 95.95 | 90.70 | 93.25 | 94.15 |
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94.59 | 94.59 | 92.34 | 96.40 | |
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86.81 | 88.31 | 88.46 | 86.96 | 86.81 | 87.56 | 87.56 | 98.20 | 95.05 | 95.05 | |
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87.26 | 88.76 | 88.31 | 87.11 | 87.26 | 88.16 | 88.01 | 98.50 | 96.85 | 95.05 | |
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87.56 | 87.11 | 87.11 | 85.46 | 86.36 | 86.21 | 91.75 | 92.65 | 93.24 | |
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95.05 | 96.85 | 92.20 | 94.00 | 94.15 | 94.00 | 94.00 | 86.96 | 87.41 | 86.96 |
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To detect potential recombination breakpoint in aligned sequences of 11 accessions, three methods were used: DSS (Differences of Sums of Squares) implementend in TOPALi v2.5, Single-Breakpoint (SBP) and the genetic algorithm GARD incorporated in HyPhy package. DSS statistics (window size: 500, step size: 10) revealed a major peak strongly supporting the presence of recombination signal in aligned sequences having the position 380 (
On phylogeny, using MEGA6 which did not take into account the presence of recombination signals when reconstructing trees, inferred isolates split into two major groups. While the former encompassed the accessions KP143750, KP143751, KP143752, KP143753, KP143754, KP143755, KP143756, and AJ440010, the latter comprised the accessions KP143757, KP143758, and KP173759 (
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HSP70h | 4330.24 | 3.76926 | 99 | 0.16140 | 0.00660 | N.S. |
375 | 0.00060 | 0.05400 | * | |||
585 | 0.01080 | 0.00060 | ** |
To examine whether the number of segregating sites in the sequences departs from the neutral expectation, the software Dna
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Tajima’s statistics | Fu and Li’s |
Fu and Li’s |
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11 | 175 | 0.09316 | 0.08867 | 1.00 | 0.00150 | 59.145 | -0.23234 | -0.14339 | -0.08937 | 0.0238 | 0.3978 | 0.0598 |
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d |
p |
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Negatively | 10 | -1.97201 | -4.92061 | 0.0913056 |
selected | 15 | -2.95801 | -7.38092 | 0.0275897 |
sites | 23 | -1.97201 | -4.92061 | 0.0913056 |
30 | -1.97201 | -4.92061 | 0.0913056 | |
35 | -1.97201 | -4.92061 | 0.0913056 | |
44 | -3.57527 | -8.92111 | 0.0123457 | |
55 | -1.97201 | -4.92061 | 0.0913056 | |
59 | -2.68145 | -6.69084 | 0.037037 | |
62 | -3.94402 | -9.84122 | 0.00833671 | |
67 | -3.83349 | -9.56544 | 0.07772 | |
75 | -3.57527 | -8.92111 | 0.0123457 | |
91 | -2.26985 | -5.66379 | 0.0741817 | |
110 | -2.68145 | -6.69084 | 0.037037 | |
126 | -1.97201 | -4.92061 | 0.0913056 | |
138 | -3.57527 | -8.92111 | 0.0123457 | |
153 | -1.97201 | -4.92061 | 0.0913056 | |
155 | -2.68145 | -6.69084 | 0.037037 | |
188 | -1.97201 | -4.92061 | 0.0913056 | |
192 | -2.32631 | -5.80467 | 0.069136 | |
195 | -2.68145 | -6.69084 | 0.037037 | |
201 | -1.97201 | -4.92061 | 0.0913056 | |
204 | -1.97201 | -4.92061 | 0.0913056 | |
206 | -1.97201 | -4.92061 | 0.0913056 |
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Negatively | 7 | 0.376525 | 0.0482881 | -0.328237 | 0.91319 |
Selected | 10 | 1.01519 | 0.0456949 | -0.969495 | 0.967528 |
Sites | 11 | 0.455078 | 0.0448951 | -0.410183 | 0.935291 |
15 | 2.61849 | 0.0595076 | -2.55898 | 0.991581 | |
20 | 0.492294 | 0.047063 | -0.445231 | 0.932668 | |
23 | 0.895002 | 0.0503206 | -0.844682 | 0.962082 | |
25 | 0.521667 | 0.058624 | -0.463043 | 0.900604 | |
26 | 0.369219 | 0.0503314 | -0.318888 | 0.914685 | |
30 | 0.544195 | 0.0447714 | -0.499424 | 0.955598 | |
32 | 0.375424 | 0.0564002 | -0.319023 | 0.900405 | |
33 | 0.922784 | 0.0518401 | -0.870944 | 0.962434 | |
35 | 1.36873 | 0.0460378 | -1.3227 | 0.980008 | |
41 | 1.15635 | 0.0532465 | -1.1031 | 0.985075 | |
44 | 1.62073 | 0.0536177 | -1.56711 | 0.996202 | |
50 | 0.315945 | 0.0503845 | -0.26556 | 0.902535 | |
54 | 0.683067 | 0.049905 | -0.633162 | 0.958644 | |
55 | 1.35989 | 0.0524097 | -1.30748 | 0.970511 | |
59 | 0.588387 | 0.049929 | -0.538458 | 0.9555 | |
60 | 1.15795 | 0.0505032 | -1.10745 | 0.978787 | |
62 | 2.17435 | 0.0554162 | -2.11894 | 0.995938 | |
67 | 0.800283 | 0.0418359 | -0.758447 | 0.959242 | |
75 | 2.93152 | 0.0573228 | -2.8742 | 0.999472 | |
76 | 0.812665 | 0.0414693 | -0.771196 | 0.973929 | |
77 | 0.403859 | 0.0576326 | -0.346226 | 0.927378 | |
80 | 0.350879 | 0.0464663 | -0.304412 | 0.914935 | |
87 | 3.47983 | 0.0775901 | -3.40224 | 0.985108 | |
89 | 0.873739 | 0.0518223 | -0.821917 | 0.940992 | |
90 | 0.897773 | 0.0531118 | -0.844661 | 0.942366 | |
91 | 1.85109 | 0.0552167 | -1.79587 | 0.983054 | |
93 | 0.756641 | 0.0420503 | -0.714591 | 0.970106 | |
94 | 0.424848 | 0.0467753 | -0.378072 | 0.947464 | |
95 | 1.84316 | 0.0460251 | -1.79713 | 0.983607 | |
97 | 0.607864 | 0.0514706 | -0.556393 | 0.93042 | |
99 | 0.453983 | 0.0469972 | -0.406986 | 0.930995 | |
102 | 0.870746 | 0.0623283 | -0.808418 | 0.905165 | |
105 | 0.430765 | 0.0521587 | -0.378606 | 0.938245 | |
106 | 1.27912 | 0.0532653 | -1.22586 | 0.996594 | |
109 | 0.923667 | 0.0540133 | -0.869654 | 0.935368 | |
110 | 1.09897 | 0.0385232 | -1.06045 | 0.991653 | |
111 | 0.428189 | 0.0411385 | -0.38705 | 0.957295 | |
Negatively | 112 | 0.366705 | 0.0510693 | -0.315636 | 0.904948 |
Selected | 115 | 0.443421 | 0.05616 | -0.387261 | 0.935545 |
Sites | 116 | 0.410196 | 0.0436296 | -0.366566 | 0.933641 |
118 | 0.338126 | 0.060822 | -0.277304 | 0.911567 | |
122 | 0.366705 | 0.0510693 | -0.315636 | 0.904948 | |
123 | 0.643768 | 0.0536651 | -0.590103 | 0.954154 | |
124 | 0.923667 | 0.0540133 | -0.869654 | 0.935368 | |
125 | 1.32003 | 0.0492819 | -1.27074 | 0.973421 | |
126 | 1.1893 | 0.0483109 | -1.14099 | 0.968835 | |
129 | 0.577151 | 0.0491697 | -0.527982 | 0.920347 | |
135 | 0.919261 | 0.0524697 | -0.866792 | 0.938291 | |
137 | 0.518636 | 0.0521278 | -0.466508 | 0.914708 | |
138 | 1.31968 | 0.049073 | -1.27061 | 0.994411 | |
141 | 0.651402 | 0.0480894 | -0.603313 | 0.964091 | |
143 | 0.36261 | 0.0466886 | -0.315921 | 0.916419 | |
146 | 0.384867 | 0.0510885 | -0.333779 | 0.911193 | |
148 | 0.386554 | 0.0471416 | -0.339413 | 0.920311 | |
151 | 0.518636 | 0.0521278 | -0.466508 | 0.914708 | |
152 | 0.437827 | 0.047596 | -0.390231 | 0.927345 | |
153 | 1.93543 | 0.0506751 | -1.88476 | 0.97741 | |
155 | 1.91207 | 0.0446616 | -1.86741 | 0.997526 | |
157 | 0.368715 | 0.0475331 | -0.321182 | 0.917491 | |
158 | 1.09207 | 0.0458234 | -1.04625 | 0.98401 | |
161 | 0.380027 | 0.0468234 | -0.333203 | 0.922408 | |
162 | 0.557184 | 0.0525124 | -0.504672 | 0.928791 | |
163 | 0.418799 | 0.0530538 | -0.365746 | 0.913946 | |
164 | 0.369178 | 0.0481584 | -0.321019 | 0.913452 | |
169 | 0.876945 | 0.0533388 | -0.823606 | 0.938637 | |
171 | 0.40689 | 0.0495071 | -0.357383 | 0.921078 | |
175 | 0.894541 | 0.0515968 | -0.842944 | 0.944597 | |
176 | 0.57468 | 0.0506531 | -0.524027 | 0.921218 | |
177 | 0.386554 | 0.0471416 | -0.339413 | 0.920311 | |
179 | 0.983744 | 0.0424601 | -0.941284 | 0.975106 | |
180 | 0.386554 | 0.0471416 | -0.339413 | 0.920311 | |
182 | 0.428362 | 0.0428762 | -0.385486 | 0.954511 | |
183 | 0.923667 | 0.0540133 | -0.869654 | 0.935368 | |
184 | 0.473183 | 0.0553586 | -0.417824 | 0.913902 | |
186 | 0.45382 | 0.0466407 | -0.40718 | 0.926122 | |
187 | 0.36261 | 0.0466886 | -0.315921 | 0.916419 | |
188 | 0.64281 | 0.0462455 | -0.596565 | 0.953451 | |
191 | 0.469009 | 0.0455844 | -0.423425 | 0.925376 | |
192 | 5.86336 | 0.0718329 | -5.79153 | 0.998808 | |
195 | 0.966169 | 0.0410356 | -0.925133 | 0.988749 | |
196 | 1.03519 | 0.0549933 | -0.980194 | 0.921603 | |
197 | 0.436485 | 0.0492842 | -0.387201 | 0.923837 | |
199 | 0.366705 | 0.0510693 | -0.315636 | 0.904948 | |
201 | 1.25258 | 0.0516082 | -1.20097 | 0.972552 | |
204 | 1.96 | 0.0546179 | -1.90538 | 0.974545 | |
206 | 4.03781 | 0.0649555 | -3.97286 | 0.983705 | |
207 | 0.592155 | 0.0427802 | -0.549375 | 0.962824 |
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d |
d |
d |
p |
|
Negatively | 10 | 2.87875 | 0 | 0.000 | -7.18315 | 0.0950597 |
selected | 15 | 8.86782 | 0 | 0.000 | -22.1273 | 0.0381136 |
sites | 35 | 4.55354 | 0 | 0.000 | -11.3621 | 0.056537 |
41 | 4.21022 | 0 | 0.000 | -10.5055 | 0.0800715 | |
44 | 5.81412 | 0 | 0.000 | -14.5076 | 0.0333948 | |
55 | 3.80051 | 0 | 0.000 | -9.48315 | 0.0904535 | |
60 | 4.38885 | 0 | 0.000 | -10.9512 | 0.0660975 | |
62 | 5.40726 | 0 | 0.000 | -13.4924 | 0.0399924 | |
67 | 6.00319 | 0 | 0.000 | -14.9794 | 0.0504539 | |
69 | 4.95666 | 0 | 0.000 | -12.368 | 0.0388506 | |
75 | 10.4746 | 0 | 0.000 | -26.1365 | 0.00356791 | |
76 | 3.47216 | 0 | 0.000 | -8.66386 | 0.0659542 | |
87 | 10.8158 | 0 | 0.000 | -26.9879 | 0.0466957 | |
90 | 4.59962 | 0 | 0.000 | -11.4771 | 0.0986656 | |
91 | 7.03144 | 0 | 0.000 | -17.5451 | 0.0378984 | |
93 | 3.31213 | 0 | 0.000 | -8.26452 | 0.0775172 | |
95 | 8.17657 | 0 | 0.000 | -20.4024 | 0.045526 | |
106 | 5.26575 | 0 | 0.000 | -13.1393 | 0.0526288 | |
110 | 4.462 | 0 | 0.000 | -11.1337 | 0.0355557 | |
125 | 8.24749 | 0 | 0.000 | -20.5794 | 0.0564762 | |
138 | 4.67957 | 0 | 0.000 | -11.6766 | 0.0477805 | |
153 | 11.3135 | 0 | 0.000 | -28.2299 | 0.0474348 | |
155 | 8.39498 | 0 | 0.000 | -20.9474 | 0.00765174 | |
158 | 5.69615 | 0 | 0.000 | -14.2132 | 0.0320832 | |
175 | 4.59949 | 0 | 0.000 | -11.4768 | 0.0922158 | |
179 | 3.8005 | 0 | 0.000 | -9.48314 | 0.0601061 | |
192 | 78.0962 | 0 | 0.000 | -194.868 | 0.00173726 | |
195 | 3.81155 | 0 | 0.000 | -9.51071 | 0.0561057 | |
201 | 3.99332 | 0 | 0.000 | -9.96426 | 0.0836751 | |
204 | 8.2942 | 0 | 0.000 | -20.696 | 0.0623541 | |
206 | 5314.18 | 0 | 0.000 | -13260.1 | 0.0118554 | |
Positively selected site | 82 | 0 | 6.06023 | Infinite | 15.1217 | 0.0519717 |
|
|
d |
dN | dN |
p |
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Negatively | 10 | 2.87884 | 0 | 0.000 | -7.18338 | 0.0276303 |
selected | 11 | 2.30241 | 0 | 0.000 | -5.74504 | 0.0589999 |
sites | 15 | 8.86782 | 0 | 0.000 | -22.1273 | 0.00718757 |
20 | 2.23505 | 0 | 0.000 | -5.57697 | 0.0663179 | |
23 | 2.91352 | 0 | 0.000 | -7.26992 | 0.0362612 | |
26 | 1.57283 | 0 | 0.000 | -3.92457 | 0.0954015 | |
30 | 1.89029 | 0 | 0.000 | -4.71672 | 0.0421214 | |
33 | 3.78636 | 0 | 0.000 | -9.44785 | 0.0327252 | |
35 | 4.55342 | 0 | 0.000 | -11.3618 | 0.0131776 | |
41 | 4.21022 | 0 | 0.000 | -10.5055 | 0.0162333 | |
44 | 5.81392 | 0 | 0.000 | -14.5071 | 0.00422218 | |
54 | 2.82908 | 0 | 0.000 | -7.05921 | 0.0381106 | |
55 | 3.80051 | 0 | 0.000 | -9.48315 | 0.0256626 | |
59 | 2.20792 | 0 | 0.000 | -5.50928 | 0.0470725 | |
60 | 4.38873 | 0 | 0.000 | -10.9509 | 0.0151289 | |
62 | 5.40726 | 0 | 0.000 | -13.4924 | 0.00554271 | |
67 | 6.00319 | 0 | 0.000 | -14.9794 | 0.0199245 | |
75 | 10.4746 | 0 | 0.000 | -26.1365 | 0.000184154 | |
76 | 3.47209 | 0 | 0.000 | -8.66366 | 0.0170004 | |
77 | 1.83673 | 0 | 0.000 | -4.58307 | 0.0799872 | |
87 | 10.8158 | 0 | 0.000 | -26.9879 | 0.0112877 | |
89 | 4.12611 | 0 | 0.000 | -10.2956 | 0.0449104 | |
90 | 4.59949 | 0 | 0.000 | -11.4768 | 0.0407129 | |
91 | 7.03144 | 0 | 0.000 | -17.5451 | 0.00834995 | |
93 | 3.31213 | 0 | 0.000 | -8.26452 | 0.0215954 | |
94 | 1.68988 | 0 | 0.000 | -4.21665 | 0.0522862 | |
95 | 8.17657 | 0 | 0.000 | -20.4024 | 0.0121216 | |
97 | 2.98447 | 0 | 0.000 | -7.44694 | 0.0608662 | |
99 | 2.30234 | 0 | 0.000 | -5.74488 | 0.0622687 | |
102 | 3.18138 | 0 | 0.000 | -7.93829 | 0.0544263 | |
105 | 1.69934 | 0 | 0.000 | -4.24024 | 0.0706894 | |
106 | 5.26575 | 0 | 0.000 | -13.1393 | 0.00637464 | |
109 | 3.62226 | 0 | 0.000 | -9.03838 | 0.0569133 | |
110 | 4.46188 | 0 | 0.000 | -11.1334 | 0.00555655 | |
111 | 1.68988 | 0 | 0.000 | -4.21665 | 0.0405256 | |
115 | 1.97076 | 0 | 0.000 | -4.91751 | 0.0662502 | |
116 | 1.90796 | 0 | 0.000 | -4.76082 | 0.0674488 | |
123 | 2.73036 | 0 | 0.000 | -6.81288 | 0.0462351 | |
124 | 3.62226 | 0 | 0.000 | -9.03838 | 0.0569133 | |
125 | 8.24749 | 0 | 0.000 | -20.5794 | 0.0170062 | |
126 | 2.85992 | 0 | 0.000 | -7.13616 | 0.0294044 | |
129 | 1.63291 | 0 | 0.000 | -4.0745 | 0.0988515 | |
135 | 3.74326 | 0 | 0.000 | -9.34031 | 0.0519666 | |
138 | 4.67971 | 0 | 0.000 | -11.677 | 0.00694664 | |
141 | 3.16258 | 0 | 0.000 | -7.89138 | 0.0282591 | |
148 | 1.4131 | 0 | 0.000 | -3.52601 | 0.097974 | |
152 | 2.08777 | 0 | 0.000 | -5.20947 | 0.0694962 | |
153 | 11.3135 | 0 | 0.000 | -28.2299 | 0.0131378 | |
155 | 8.39498 | 0 | 0.000 | -20.9474 | 0.000766624 | |
Negatively | 158 | 5.69615 | 0 | 0.000 | -14.2132 | 0.00631352 |
selected | 161 | 1.45007 | 0 | 0.000 | -3.61826 | 0.091037 |
sites | 162 | 3.1211 | 0 | 0.000 | -7.78787 | 0.0600393 |
163 | 2.02455 | 0 | 0.000 | -5.05174 | 0.0872654 | |
169 | 4.126 | 0 | 0.000 | -10.2953 | 0.0482113 | |
171 | 1.90796 | 0 | 0.000 | -4.76082 | 0.0796031 | |
175 | 4.59962 | 0 | 0.000 | -11.4771 | 0.0378602 | |
177 | 1.4131 | 0 | 0.000 | -3.52601 | 0.097974 | |
179 | 3.8005 | 0 | 0.000 | -9.48314 | 0.0151491 | |
180 | 1.4131 | 0 | 0.000 | -3.52601 | 0.097974 | |
182 | 1.72055 | 0 | 0.000 | -4.29317 | 0.0428165 | |
183 | 3.62226 | 0 | 0.000 | -9.03838 | 0.0569133 | |
184 | 2.41038 | 0 | 0.000 | -6.01445 | 0.0854211 | |
186 | 1.75413 | 0 | 0.000 | -4.37695 | 0.0814891 | |
188 | 1.79075 | 0 | 0.000 | -4.46834 | 0.0481462 | |
191 | 1.52175 | 0 | 0.000 | -3.79712 | 0.0936881 | |
192 | 78.0962 | 0 | 0.000 | -194.868 | 0.000186554 | |
195 | 3.81146 | 0 | 0.000 | -9.51048 | 0.010199 | |
197 | 2.0912 | 0 | 0.000 | -5.21802 | 0.07389 | |
201 | 3.99332 | 0 | 0.000 | -9.96426 | 0.0226521 | |
204 | 8.2942 | 0 | 0.000 | -20.696 | 0.0186686 | |
206 | 1457.28 | 0 | 0.000 | -3636.26 | 0.00354566 | |
207 | 2.31414 | 0 | 0.000 | -5.77433 | 0.0317939 | |
PositivelySelected site | 220 | 1e-06 | 1.28249 | 1282490.000 | 3.20011 | 0.0905593 |
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Positively | 3 | 0.693586 | 0.664224 | -0.0293626 | 0.933881 | 123.309 |
selected | 4 | 0.768286 | 0.664586 | -0.1037 | 0.884521 | 66.8707 |
site | 82 | 0.775199 | 0.664852 | -0.110348 | 0.88026 | 64.1806 |
220 | 0.633177 | 0.664177 | 0.0310005 | 0.974124 | 328.664 | |
Negatively selected site | 75 | 2.0235 | 0.0224856 | -2.00102 | 0.998862 | 100.564 |
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p |
q |
27 | 0.0463014 | 0.0463014 | 0.895957 | 10000 | 0.104043 | 0.0232567 | 1 | |
82 | 0 | 0 | 0.932169 | 105.292 | 0.0678312 | 0.00336169 | 0.746294 | |
220 | 0 | 0 | 0.910884 | 29.2189 | 0.0891156 | 0.0121083 | 1 |
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Coding region | Inferred Rate distributions | Rate Class | 1 | 2 | 3 | Rate class | 1 | 2 | 3 |
HSP70 | Null Model (M1) | 0.59 | 1.65 | 2.28 | ω | 0.01 | 1.00 | - | |
Probability | 0.613 | 0.387 | 0.000 | Probability | 0.873 | 0.127 | - | ||
Alternative Model (M2) | 0.59 | 1.64 | 1.68 | ω | 0.01 | 1.00 | 4.84 | ||
Probability | 0.613 | 0.243 | 0.144 | Probability | 0.873 | 0.127 | 0.000 |
Populations of plant viruses are genetically heterogeneous, and the frequency distribution of genetically varied entities in the population may fluctuate with time. This process is known as evolution. The variability of virus population led to the notion of quasispecies which assumes high mutation rates for RNA viruses. Since the presence of high mutation rates of RNA viruses and high accumulation levels in plant cells, RNA viruses have large and highly diverse populations. Consequently, viral populations would easily respond to changing selection pressure, and the evolution of high mutation rates would have an adaptive behaviour, allowing the virus to survive in changing environments. Since not accounting for other evolution forces such as recombination which can mislead not only phylogenetic analyses, but can increase the false positive error rate in positive selection inference. In this study, the author sought after recombination in the sequence of a portion (667 bp) of HSP70h-coding gene of OLYaV of 10 Tunisian accessions along with a reference sequence extracted from GenBank (Accession number AJ440010). In fact, three methods were used: DSS implemented in TOPALi v2.5 program, SBP, and GARD, both incorporated in HyPhy package. While the two former detected only a single breaking point, i.e. positions 380, and 369, respectively, the latter attempted to find all recombination signals in the genomic segment 667 bp. The breakpoints that were found in GARD analysis corresponded to hot spots particularly in positions 99, 375, and 585 bp. GARD, a powerful tool screened multiple-sequence alignments for recombination, determined all possible locations of breakpoints, inferred phylogenies for each putative non recombinant fragment and assessed goodness of fit by an information-based criterion such as small sample Akaike Information Criterion (AIC)
Nowadays, 15 different viruses infecting olive with diverse taxonomic allocation are described
The author is grateful to Ministry of Higher Education and IRESA (Institution de la Recherche et de l’Enseignement Supérieur Agricoles) of Ministry of Agriculture in Tunisia for providing funds to carry out this work.
The author declares that he has no conflict of interest