Investigations of molecular evolutionary mechanisms in partially sequenced heat shock protein 70 homologue-coding gene of Olive leaf yellowing-associated virus isolates from Tunisia

Reverse Transcription Polymerase Chain Reaction (RT-PCR) using new designed primers pair for Heat Shock Protein70 homologue (HSP70h) of Olive leaf yellowing-associated virus revealed 667 amplified product of 10 olive accessions collected from various olive-growing regions in Tunisia. Amplicons were cloned and sequenced. The sequences were deposited in the international databases. Pairwise sequence comparisons among 10 Tunisian isolates along with a reference sequence (AJ440010) extracted from GenBank revealed a nucleotide identity of 86.06-99.40 and an amino acid similarity of 91.89-99.55. Sequence multiple alignments were searched for evidence of recombination using three methods, ie. Differences of Sums of Squares (DSS) implemented in TOPALi v2.5 software and Single Breakpoint (SBP) along with GARD, a genetic algorithm, both incorporated in HyPhy package. All used methods pointed out the presence of putative breaking points in partially sequenced HSP70h-coding gene. Since failing to account for recombination can mislead the phylogeny inference and can elevate the false positive error rate in positive selection assessment, the use of GARD resulted in the reconstruction of different phylogenies on the left as well as on the right sides of putative recombination breaking points, and the 11 accessions were distributed into at least three clusters compared to MEGA6 software which delineated only two clades. Nonetheless, by dividing the aligned sequences at breakpoints into separate sequence sets, MEGA6 delineated a clustering pattern different from the former two. As a result, recombination reshuffled the affiliation of the different accessions to the clusters. Analysis of selection pressures exerted on HSP70h encoded protein using different models (SLAC, IFEL, FEL, REL, PARRIS, FUBAR, MEME, GA Branch, and PRIME) taking into account recombination, and implemented in HyPhy package, revealed that it underwent predominantly purifying selection as confirmed by Tajima’s D, Fu and Li’s D and F tests, and SNAP algorithm. However, a few sites were also under positive selection as assessed by various models such as FEL, IFEL, REL, MEME, and PRIME. DOI : 10.14302/issn.2575-7881.jdrr-15-849 Corresponding author: Dr. Moncef Boulila, Professor, Institut de l’Olivier B.P. 14; 4061 Sousse Ibn-Khaldoun, Tunisia E-mail: boulila.moncef@yahoo.fr


Introduction
Viruses represent an important threat to plant production worldwide. In spite of numerous efforts for their elimination, the number of these viruses is still  [1]. However, replaced substitutions that lead to resultant changes in amino acids can often affect tertiary structure and function, and are typically either under positive (adaptive) or negative (purifying) selection. Nonsynonymous substitutions will be mildly deleterious and will typically be removed from populations under negative selection unless they directly impose some fitness advantage [1]. The ratio of dN/dS (rate of nonsynonymous substitutions per non-synonymous site/ rate of synonymous substitutions per synonymous site) can suggest whether a gene has had a variable rate of non-synonymous changes than would be expected from random neutrality and is potentially undergoing positive or negative selection for amino acid changes in that region. Thus, the value of dN/dS more than 1 suggests that the gene is under positive selection. A value close to 1 suggests that a gene is under neutral selection and is experienced neutral evolution. However, a value of less than 1 indicates that a gene is under the influence of negative or purifying selection. Recombination is another driving force of plant RNA virus evolution. In addition to increasing sequence variability, RNA recombination can be an efficient tool for viruses to repair viral genome, thus contributing to viral fitness [2] [3] [4] [5] [6] [7]. It may also play a role in the formation of subviral RNAs that include defecting interfering (DI) RNAs associated with many plant viruses, i.e. some members of the Tombusviridae [8].
DI-RNAs are mainly derived from the parent (helper) virus via sequence deletion. The ease of their genetic manipulation has resulted in rapid discoveries on cisacting RNA replication elements required for replication and recombination [4]. Additionally, these DI-RNAs played a major role in post-transcriptional gene silencing (PTGS). They could trigger potent gene silencing response against the helper virus without hurting themselves from the same response [9].
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 found in symptomless trees in numerous countries [11].
OLYaV is currently a member of the family Closteroviridae [13]; but it is not allocated to any of the genera composing this family because more biological and molecular data are likely to be needed for unequivocal classification. This virus has a monopartite positive-sense single-stranded RNA. Only part of the viral genome, comprising 4,605 nucleotides from ORFs 1b (RdRp), 2(21kDa), 3(7kDa), 4(HSP70h), and the 5' end of ORF 5 (HSP90h) has been sequenced [13] [14].
These sequences have been deposited in GenBank under the accession number AJ440010. Later, genome sequencing has been extended towards the 3' terminus of ORF 5 (HSP90h) giving rise to a segment having a size of 854 nucleotides [15]. This RNA is protected by a coat protein having a molecular weight of 24 kDa. No seed and vector transmission are recorded so far [11].
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).

Oligonucleotide primers
Newly designed specific primers by using Primer3 software [16], were used for molecular studies of HSP70h-coding gene, and having the following sequences: sense primer : 5'-ATC ATG AAC GAG CCT TCA GC-3' ; antisense primer : 5'-CGG CAG CGA CTA TAA TAC GA-3'. These primers should be amplifying a DNA copy of 667 base pairs. The virus sense and antisense primers correspond to nucleotides (nt) positions 2618-2637, and 3265-3284, respectively, of the sequence submitted to GenBank (AJ440010) by Saponari et al. [14].

Recombination analyses
Occurrence of potential recombination events between nucleotide sequences was explored with SBP

RNA polymorphism and evolution
DnaSP version 5.10.01 [33] was used to estimate Tajima's D [34] and Fu and Li's D and F [35] statistical tests to examine the hypothesis of neutrality operating on the OLYaV (partial HSP70h gene) sequences. An estimation of several population genetic
The GA-Branch program utilizes a genetic algorithm to test an extensive number of models of codon evolution based on small sample AIC score. This analysis is able to classify each branch to a specific dN/dS rate class. α 2 , secondary structure factor; α 3 , volume; α 4 , refractivity/heat capacity; α 5 , charge/iso-electric point).     and four ( Fig.4c) clusters, those of the segments 100-375 bp (Fig.4d), and 376-585 bp (Fig.4e) were identical to the tree reconstructed without taking into account recombination (Fig.4a) (Table 3) as clearly evidenced by GARD plots (Fig.6). GARD     (Fig. 5a), 100-375 bp ( Fig. 5b), 376-585 bp (Fig. 5c), and 586-666 bp (Fig. 5d), respectively. In each tree, three even four clusters with different topologies were delineated. Scale bar indicates the number of substitutions per nucleotide.             Criterion (AIC) [46]. AICc derived from a maximum likelihood model fit to each segment [47]. Thus, since putative recombination events occurred, inferred phylogeny produced by MEGA6 software does no longer reflect the real evolutionary process of the analyzed sequences (Fig. 4a). Therefore, different tree topologies were necessary. Using MEGA6 software, only two separated sequence sets, i.e., segments 1-99 bp and 586-667 bp gave rise to two different tree topologies ( Fig.4b, 4c) compared to the segments 100-375 bp for all phylogenetic branches that were under negative selection (Fig. 7). PRIME program, however, indicated that purifying and adaptive selection signatures prevailed as well.

Discussion
Nowadays, 15 different viruses infecting olive with diverse taxonomic allocation are described [11].