Haplotype diversity of palm weevil in Saudi Arabia through ITS gene sequencing

2.1 Sample collection and preservation

Twenty samples of palm weevil, R.ferrugineus were collected across Saudi Arabia between June to October 2018. Sampling was done across multiple cities in 10 provinces of Saudi Arabia viz., Medina, Tabuk, Hail, Riyadh, Al-Qassim Region, Aljobail, Almoterfy in Al-Ahsa'a, Al-Shoiba, Al Aqiq and Bisha (Fig. 1). The exact geographical locations of individual samples with its corresponding geographical co-ordinates are given in Table 1. Adults of R. ferrugineus were hand picked from infested date palm plantations (P. dactylifera L.) and preserved directly into 70% ethanol (Scharlau, Barcelona) contained in 50 mL conical tubes. Coloured ethanol due to pigment diffusion was replaced with fresh 70% ethanol once reached the laboratory. Samples were kept at 4 °C until DNA extraction.

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Fig. 1

Map reveals sample collection sites across Saudi Arabia. The color integers represents sample number and the position of the integer indicate the approximate area of the respective collected samples. Total of 20 samples were collected from 20 different study area.

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2.2 DNA isolation and PCR

For DNA isolation, approximately 3 mm² pronotal muscle was exercised from the preserved specimen using sterilized fine forceps and placed inside 200 µl PCR tubes. Ethanol in the tissues were evaporated either in room temperature or by placing the tubes in concentrator plus (Eppendrof, AG) at 45 °C for 10 min. DNA extraction and purification was carried out using the manufacture’s protocol of DNeasy blood and tissue DNA isolation Kit (Qiagen, USA) excerpt only one by tenth of actual volume of the reagents given were used. The final elution was done in 50 µl elution buffer. The eluted DNA was rinsed trice and repeatedly eluted from the binding column to increase the DNA concentration. The isolation of DNA was confirmed by running 2 µl of elutant in 1.5% agarose gel along with Lamda DNA (Takara Bio) as standard.

During polymerase chain reaction (PCR), internal transcribed spacer (ITS) gene region was amplified using the primer pair; ITS-F: 5′-ATATGCTTAAATTCAGCGGG-3′ and ITS-R: 5′-GGGTCGATGAAGAACGCAGC-3′ (Navajas et al., 1998). 50 µl PCR mixture consisted of 1x PCR buffer (Promega), 15 mM MgCl₂, 0.2 mM dNTPs, 20 pM of each primers, 1u of Taq DNA polymerase (GoTaq, Promega), 40 ng DNA and ultra-pure water to a final volume of 50 µl. PCR condition was done with an initial denaturation for 5 min at 94 °C. The, 40 cycles consisted of a denaturation step at 94 °C for 30 sec., an annealing step at 50 °C for 30 sec. and an elongation step at 72 °C for 30 sec. The final extension step was for 7 min at 72 °C. The PCR amplicons were resolved by 1.5% agarose gel electrophoresis with ethidium bromide (0.5ug/ml) in 1X TBE buffer powered at 95 V for 30 min. A 100 bp DNA ladder (Thermo Fisher Scientific) was used as a standard to detect and assess the size of the amplicon. PCR products were visualized under UV light and photographed using a Gel Documentation System (BIO-RAD 2000). The PCR products were purified using QIAquick PCR purification kit (Qiagen, USA).

2.3 Sequencing and analysis

Sequencing was performed in ABI PRISM 3730XL Analyzer (Macrogen, South Korea) using Big Dye TM Terminator Cycle Sequencing kit following manufacture’s instruction. Two way sequencing was performed and the chromatograms were compiled using BioEdit ver. 7.2 (Hall, 1999). Chromas Pro ver. 2.6.6 was used to double check the quality of the sequence chromatograms before compiling. Primer sequences were trimmed off from the final sequences. The sequences were aligned in Clustal X ver. 2.0.6 (Thompson et al., 1997) and the alignment was submitted to GenBank. Sequences could be accessed through GenBank accession numbers: MW575619-MW575638. Kimura-2 parametric distance analysis (Kimura, 1980) was conducted in Molecular Evolutionary Genetic analysis (MEGA) ver. X (Kumar et al., 2018).We calculated Tajima’s Neutrality Test (Tajima, 1989) using segregation and nucleotide data using following equations; m = number of sequences, n = total number of sites, S = Number of segregating sites, ps = S/N, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima’s test statistic. Tajima’s test values reveals the difference between the number of segregation nucleotide sites and the average of pair-wise differences. The analysis was conducted in MEGA ver. X.

DNA sequences were compared with GenBank reference sequences through Basic local alignment search tool (BLAST) (Altschul et al., 1990) using default parameters for searching the entire nucleotide collection using the conditions optimized for highly similar sequences. First five reference sequences of closest match to the query sequences of the present study were extracted and the repeated sequences were removed before being used for the phylogenetic tree construction. Neighbor-Joining tree (Saitou and Nei, 1987) was drawn using Molecular Evolutionary Phylogenetic Analysis software version 10.1.7 (Kumar et al., 2018) to infer the phylogenetic relationship among the sequenced haplotypes.The evolutionary distances were computed using the Kimura 2-parameter method (Kimura et al., 1980). The percentage of replicate trees in which the associated taxa clustered together was calculated using the bootstrap test (100 replicates) (Felsenstein, 1985).Interactive Tree Of Life (iTOL) (Letunic and Bork, 2019) ( https://itol.embl.de/) was used for the better representation of constructed phylogenetic tree.

Median joining haplotype network diagram was drawn using Network ver 10.2. ( https://www.fluxus-engineering.com/sharenet.htm), developed by Fluxus Technology Ltd. Aligned DNA sequences in FASTA format was converted into RDF (Resource Description Framework) format using FAS to RDF convertor tool ( http://fas2rdf.herokuapp.com/) and output file is used as input data to draw the network diagram.

3.1 Sequence identification

DNA was recovered from an ethanol-preserved specimen at a concentration of 5.34 ± 2.43 μg/μl. About 700 bp PCR amplicon was produced from all specimens. All sequences shared on average of 98.7% identity (96.06 to 100%) with the ITS sequences of R. ferrugineus in the GenBank (Table 1). While few sequences closely resembles the other ITS sequences previously sequenced from R. ferrugineus captured in Saudi Arabia (n = 4), many (n = 16) shared close identity with R. ferrugineus collected from Greece, India and Israel (Table 1). For better interpretation all haplotypes were named as UT (University of Tabuk) 1–20 and correlated with the corresponding Genbank accession numbers in the Table 2.

3.2 Haplo-groups and distance analysis

We identified three haplogroups based on the NJ tree analysis which were highlighted through 3 different colors (red, orange and blue) (Fig. 2). The haplotype UT9 was a singleton haplotype in the analysis. The haplotypes UT16, UT2, UT6 UT10, UT14 and UT5 formed the second haplo-group whereas the remaining haplotypes were clustered as third group at the lower end of the phylogenetic tree. Interestingly all reference sequences extracted from GenBank was classified within the third cluster of the Haplo-group. Lack of reference ITS sequences for first and second haplo-groups may be due their new origin and may be specific to Saudi Arabia. Also the members of third haplo-group such as UT13, UT14, UT15, UT17, UT18, UT19, and UT20 did not had even single nucleotide variation and were exactly identical.

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Fig. 2

The phylogenetic tree constructed using Neighbor-Joining method for specimens under investigation.

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During K2P distance analysis, all nucleotide positions containing gaps and missing data in the alignment were eliminated and there were a total of 468 nucleotide positions in the final aligned dataset.K2P distance varied between 0 and 0.06 among the sequenced haplotypes (Table 3). Maximum distance was recorded between two sets of haplotypes; viz; UT4 and UT3, UT4 and UT8. The overall distance of all the sequenced haplotypes were 0.02.

Table 3 Kimura-2 distance data of ITS sequences of 20 haplotypes of H. ferrugineus. Greener the shades, lower the scores. Redder the shades higher the scores.

The optimal tree with the sum of branch length = 0.12658351 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. There were a total of 468 positions in the final dataset.

There were a total of 44 segregation sites with overall nucleotide diversity was 0.019 (Table 4). Tajima’s neutrality value was < 0 indicating population expansion in terms of haplotype diversity.

3.3 Haplo-type network

Median joining haplotype network diagram reveals the presence of 10 haplotypes (Fig. 3). The haplotypes UT16, UT12, UT10, UT5, UT4 UT1, UT16 and UT11 are unique haplotypes represented by single members in this study. UT2 and UT3 bubbles in the network diagram also contains UT6 and UT8 haplotypes respectively. All remaining haplotypes were aggregated in UT15 bubble of the network diagram and forms the largest haplotype group (n = 9). There were a total of 7 median vectors connecting triplet of the different haplotypes. In the ITS sequences aligned, 244th position was found to be most frequently mutated (n = 6) position followed by 381^st position (n = 4). Overall, total number of mutations leading to the evolution of these 10 haplotypes was 34.

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Fig. 3

Median joining network diagram showing 10 haplotypes (yellow circles) of R. ferrugineus in this study. Majority consensus generated sequences was indicated by red points (median vectors; mv) which connects triplet of sequences. Mutations in the position of the characters were indicated CH and most frequent mutation (n = 6) was at CH244 position (indicated by red text). Size of the circle corresponds to the number of the haplotypes present.

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