Psyllid samples used in molecular procedures

Twelve C. pruni samples (i.e., insects from the same locality) used in this study, mostly from France, are described in Sauvion et al. (2007) (Table 1, Figure 1). These samples constitute 310 psyllids (males or females) genotyped at nine microsatellite loci and previously assigned to groups referred to as A and B (Table 1). We aimed to increase our dataset with 14 additional samples in order to cover the known distribution area of C. pruni to the greatest extent possible [14]. The individuals of seven of these 14 samples were genotyped: three samples from France and four samples from Serbia, Italy, Germany and Spain (samples 13 to 19, Table 1). All microsatellite genotypes were assigned to a group using the Bayesian admixture procedure described in Sauvion et al. [15]. In all the 19 genotyped samples, two individuals assigned to either group were selected for ITS2 sequencing (detailed below). As outgroups, we sequenced one individual from Cacopsylla fulguralis (Kuwayama, 1908) and one individual from Psylla alni (Linné, 1758). Eight individuals of the 19 previously described samples plus seven other samples were analyzed at the diagnostic PCR (see below) to test the reliability of this test on individuals of diverse origins. Our aim was to test four individuals from each group.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Origin of C. pruni individuals used in this work.

The 12 samples previously genotyped and assigned to groups A or B in Sauvion et al. [15] are shown in black. The 14 samples added to cover the known distribution area of C. pruni are shown in gray. See Table 1 for details on each location.

https://doi.org/10.1371/journal.pone.0072454.g001

Finally, in order to ascertain that the diagnostic PCR amplified only C. pruni DNA, we performed it on individuals of 18 species of Cacopsylla, including all those known to transmit phytoplasma to Rosaceae, one species of Psylla, one species of Triozidae, and one species of Aphalaridae (Table 2), according to the recent classification of Psylloidea [10,11]. Four to five individuals per species were first amplified at the ITS2 locus using generic primers (see below), to ascertain DNA quality at this locus. For each species, one individual that yielded good amplification was then tested with the diagnostic PCR.

All psyllids were stored in absolute ethanol until DNA extraction. DNA of individual psyllids was purified from whole bodies using either the CTAB (cetyltrimethylammonium bromide) method [20] in individual 1.5 ml tubes, or the High-Salt method based on TNES (Tris, NaCl, EDTA, SDS) buffer [21] in semi-deep well plates. DNA was diluted in 60 µL of water.

Internal Transcribed Spacer 2 (ITS2) DNA sequencing and phylogenetic analysis

The ITS2 spacer and parts of the rRNA 28S and 5.8S genes were amplified with primers CAS5p8sFcm (5’-CGAACATCGACAAGTCGAACGCACA-3’), adapted by E. Jousselin for aphids (com. pers.) from Ji et al. [22], and CAS28sB1d (ID: S50426), specific for Hemiptera (5’- TTGTTTTCCTCCGCTTATTAATATGCTTAA-3’) [22]. The PCR was performed in a final volume of 25 µL containing: 2.5 µL of QIAGEN Coraload buffer (containing 45 pmol MgCl₂), 0.05 mM of each dNTP, 3 mM of MgCl₂ (Qiagen), 0.7 µM of each primer, 0.625 U of Taq DNA Polymerase (Qiagen) and 2 µL of DNA extract. The PCR cycles were as follows: initial denaturation at 94°C for 3 min; followed by 40 cycles at 92°C for 30 s, 48°C for 1 min and 72°C for 1 min, with a final 10-min extension period at 72°C. Purification of PCR products and Sanger sequencing with both primers were performed by the GENOSCOPE (Évry, France).

Sequences were analyzed with Geneious Pro 5.4 [23]. Chromatograms from DNA sequences were assembled for each individual and then aligned with the Muscle algorithm and visually inspected. A neighbor-joining tree [24] was constructed using the PAUP* 4.0b10 [25] plugin in Geneious. We used the unequal-frequency Kimura 3-parameter + gamma model of nucleotide evolution [26], as suggested by MODELTEST [27]. Node support was calculated from 1,000 bootstrap replicates.

Primers for diagnostic PCR

The reciprocal monophyly of the two C. pruni groups (see results) permitted the development of specific primers. We used the Primer3 web interface (http://bioinfo.ut.ee/primer3/ Accessed 2013 July 19) to design three sets of primers, each comprising a “universal” primer binding the sequence of both groups, and two specific primers, each binding the ITS2 of one group only (Figure 2). This specificity was ensured by the presence of indels that differentiated groups at the primer binding sites. These binding sites were chosen at distant positions so that a triplet of primers made it possible to identify groups by sizing the amplicon produced in a single PCR. In the following, the name of a group-specific primer comprises the letter A or B to specify the genetic group that it targets. For the first set, the universal primer was Cp480R (5’-TACATCCGAGGGTCGGTATC-3’), and the specific primers were CpA300F (5’- GGCCAGTAGTTAAACCGGACT-3’) and CpB120F (5’-TCCACGGGGTCCGCGATA-3’). For the second set, the same universal primer Cp480R was used with the specific primers CpA50F (3’-TTGTGTCTGTGTTTCGAGAGC-5’) and CpB350F (3’-AATCCAAACCCCGCGATG-5’). For the third set, the universal primer was Cp135F (3’- ATACGCGCTCGATCTGACAT-5’), and the two specific primers were CpA425R (3’- TCGACTCTCTCGCCTCTCTT-5’) and CpB315R (3’-TTAACCGCTGGGGCTAGG-5)’.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Positions of the primer binding sites in the internal transcribed spacer 2 (ITS2) of C. pruni groups A and B.

Primers are shown in black for set 1, gray for set 2 and white for set 3.

https://doi.org/10.1371/journal.pone.0072454.g002

PCR was performed in a final volume of 12 µL containing: 1.1 µL of QIAGEN Coraload PCR buffer (containing 45 pmol MgCl₂), 0.05 mM of each dNTP, 2.5 mM of MgCl₂ (Qiagen), 0.2 µM of each primer (10 µM), 0.625 U of Taq DNA Polymerase (Qiagen) and 2 µL of DNA extract. Due to the high yield of this PCR, DNA template was diluted 30 times before being added to the mix. The PCR cycles were as follows: initial denaturation at 94°C for 5 min; followed by 30 cycles at 94°C for 30 sec, 65°C for 20 sec and 72°C for 30 sec, with a final 5-min extension period at 72°C. Amplicons were submitted to a 100V, 40 min electrophoresis in 2% agarose gels stained with ethidium bromide for visualization under UV light.

Phylogeny of C. pruni at the ITS locus

Figure 3 shows the neighbor-joining tree of ITS sequences from C. pruni individuals with different origins (two to four individuals tested in 18 samples, Table 1), and from C. fulgaris and P. alni (the latter used as the outgroup). The sequencing was unsuccessful for only eight individuals out of the 58 tested, three from Tordera (sample 1, two individuals from group A and one from group B), three from Romette (sample 10, two individuals from group A and one from group B), and two individuals from group A from Montgamé (sample 12). Two highly divergent clades strictly correspond to previously characterized microsatellite genotype clusters, so that each cluster is monophyletic at that locus (Figure 3). Sequence divergence within a clade is low in comparison to divergence between clades. Sequences are available under Genbank accession numbers KF305120 to KF305171.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Neighbor-joining phylogeny of ITS2 sequences from individuals of C. pruni.

Tip labels indicate the sample number of each insect (as defined in Table 1), followed by its sampling locality, individual number, sex (m for males) and assignment to genetic group corresponding to microsatellite genotype cluster A or B, according to Sauvion et al. [15]. Bootstrap support is indicated next to nodes when it exceeds 95%.

https://doi.org/10.1371/journal.pone.0072454.g003

Diagnostic PCR

Figure 4A shows typical bands obtained by electrophoresis of PCR products from each C. pruni group using the three primer sets designed to amplify the ITS2 region. For primer set 1, the expected PCR products are 172 base pairs (bp) for group A and 377 bp for group B. For primer set 2, the expected PCR products are 421 bp (A) and 151 bp (B), and for primer set 3, the expected PCR products are 293 bp (A) and 177 bp (B). The mixtures of group A DNA and group B DNA (holes A+B on Figure 4A) resulted in two bands of the expected sizes.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Amplicons from PCRs using the ITS2 primer sets designed for the characterization of C. pruni individuals.

Panel A: typical bands obtained from single individuals belonging to groups A or B, and of pools of two individuals of each group (A+B) with each set of primers. The rightmost and leftmost lanes used a 100 bp DNA ladder. Numbers indicate the expected sizes of the amplicons. Panels B and C: PCRs using generic ITS2 primers FCM/B1D and the primer set 3 (Cp135F / CpB315R / CpA425R), respectively, on single individuals from the 24 psyllid species described in Table 2.

https://doi.org/10.1371/journal.pone.0072454.g004

Eight individuals per sample (males or females assigned to groups A and/or B based on their microsatellite genotype) were tested with the three primers sets. All amplicons obtained were of the expected sizes (Table 1). Seven, eight and two individuals resulted in no band for the primer sets 1, 2 and 3, respectively. For each sample, the remaining individuals (178 from group A and 103 from group B) were tested with at least one primer set. Only 2% produced no fragment in one PCR, but all of the 799 individuals produced expected amplicons in at least one PCR. None produced two bands. The amplification with primer set 2 seems less efficient for the individuals in group B: the fragments appeared to be the faintest with set 2 than with set 1 or 3 for almost all of the individuals that we tested. Amplification of DNA from C. pruni males and females revealed no difference.

The specificity of the three primer sets for C. pruni was evaluated by performing PCR assays on individuals of 22 other psyllid species. All were amplified at the ITS2 region with the generic primers (Figure 4B), but none showed amplification with any of the primer sets designed for C. pruni. Indeed, amplicons were obtained only for the C. pruni individuals, corresponding to lanes 14 and 15 in Figure 4C, which shows results for primer set 3 (results not shown for primer sets 1 and 2).