Electronic Supplementary Materials For

Electronic Supplementary Materials for

In silico mining of microsatellite markers for the Red Palm Weevil as contribution to its invasion management

Silvia Belvedere1, Alessandra La Marca2, Veronica Marcari1, Gabriele Senia1, Alessio De Biase1*

1Department of Biology and Biotechnologies “Charles Darwin”, Sapienza Rome University, Viale dell'Università 32, 00185 Rome, Italy

2 BBCA-onlus, Via del Bosco 10, 00060 Sacrofano (Rome), Italy

* corresponding author:

Table S1 Rhynchophorus ferrugineus samples information
Country / Collection date / No. of genotyped individuals
Italy / November 2009-January 2014 / 22
Greece / June 2011-December 2012 / 17
Spain / June 2013 / 6
Saudi Arabia and Qatar / March-June 2012 / 23
Vietnam / December 2012-June 2013 / 17
Malaysia / April 2013 / 17
Table S2 Criteria for bioinformatic isolation of SSR loci on 454 output by QDD software
Min no. of repeats for dibase repetitions / 6
Min no. of repeats for tribase repetitions / 5
Min no. of repeats for tetrabase repetitions / 5
Min no. of repeats for pentabase repetitions / 4
Min no. of repeats for hexabase repetitions / 4
Min length of flanking regions / 50 bp
Final product length / 75-320 bp
Primer length / 8-27 bp
Max length of homopolymers within the primer sequence / 4 bp
Max difference in Tm between paired primers / 3 °C
Tm range of the primer pair / 57-63 °C
GC content / > 20%
Table S3Nucleotidesequences and GenBank accession numbers of 14 microsatellite loci designed on 454 sequence data from R. ferrugineus (individual sampled at Rome, Italy)
Locus / Length (bp) / Sequence / GenBank accession no.
RPW02 / 300 bp / TCCGATTCTGTTCAGACCAAATAACTGGGCAAAAGAAACTTTAAATGAACTATTAGATTTACTGACATTAAATTTGACAATATAAACGGCATTAAACACCATATCAATAAATTTACGACCAATGGATGACACTAATAGAAATTAATTCGTTATATATATATATATATATATATTGTTACAATGTTGCCAAATTGTACCGTAAATTACTATCTTTCACGTAAACCGTCAAGATAAGCGTTACCTCTATACTTTAAGGAAACGAATTTAATTAATTTATTAGCACTTCCGCGGTATAAAGGA / LK392620
RPW03 / 212 bp / TCCCATGCCATTGCTATTTTGAATTGCTATATATATATATATATATATATTGATGACGTACATTGATTGTTTTATTGGAAGTGCGATATGAACGTAGATTTTGTTGCTATGGATAAATTAGTTGAATGAAGAGGTTTCAGAAGACATGGACATGATTTTCTTTGAAGCCTTTAAGATCAATTTCAAATATTTTGGTAGTCGTTTCCCGTTTC / LK392621
RPW06 / 80 bp / AACCTCCGGAATCGTACCTTGTATGGTTCTATATATATATATATATAAATATTGTTCGACCCGAATGGACGATGGATGAG / LK392622
RPW11 / 159 bp / TCGTCAGTTAGATCCCATGATATTTTNGAAACGATATTATTGGTTATAGAATGAGAATTATTATTATTATTATTATTATTAAACGCTATTAAAGGTTACTAACTACTGGTGAATTTTATTATTGTGTATTTTAGCGTGTATGATAAATTAAAAGCTTTC / LK392623
RPW13 / 170 bp / GTCTGTTTGGTGGAGCGATGGGCCGGGGTTGGTCGGATGGTCCGATAGTGGTGGTGGTGGTGGTGGTGGAGATGGTGGCGGGAGGAAAAGGCGACGGGGCGGCGAAGGGCGGTCGCGTCTACTGACGGCCGCCGAACAGCTGGTACTGAAACAGCAGAAGCAATGACAGG / LK392624
RPW16 / 228 bp / AAGGGCTTCGAAAGAGGAACTTTACTTTGGCGTCGGAAGTTTTCGTGTCCTGGCGCGGATTAATACGAAAGTTCTATCAGGCAGGCGGCGGCGGCGGCGGCGGTGGCGGATGGGTAAGCCTGTAGTTTTGTTGTGGAGATTTATTGGGGAATACGGATTGTTTAAAGTCAAATATTAACCGAGGGAGGCGCGAAAGGAATGGAATGGCGCTGTGGGAATTAGAGCAGG / LK392625
RPW17 / 222 bp / AAACGGCTCGATATACGTGGAGGATTTATTTATAACCAATAAGTAACATTTCAATCTTTACGGGCCGTTGTTGTTGTTGTTGTTCCCGGTTAGAAACCAGAAAATCCTGGTCGATCCCTGTTCCAGAAATAGGCTGACTAGCGTGAGGATACTGATTGAGAAGCACAGCACGGCGCAGGAGTTGAGCAGGGGCGTACAGGTAGTCGTTCTCCGACCATTTGT / LK392626
RPW20 / 90 bp / GCCGTTGATGTTGATACACGACGGAGTGTATCAGATAGATAGATAGATAGATCTCCAGTATTTCAGATATGACGAAACAAAAGTCTTGCA / LK392627
RPW22 / 163 bp / AAGCGTTCCCGGTTTACTTTGGCGTTTCGGAAGCCGGAAGGCAAATGTTTGCCCGGATAAAGTTGTCCGATGCGGCGACTTATCCCTGGGCGTTTGTTCGGTTCCGCTCGCTACTTGTTTATTTGTTTGTTTGTTTGTTTGTCGTTCAACACTTGCATCATCA / LK392628
RPW24 / 99 bp / GCCTATGCTACACAAAATAACAGCTTTTCTAACTAACTAACTAACTAACTTTGTAACTTTACTAGCTAAAATAACATTTCCTGCATTCATAAGTTTGCC / LK392629
RPW25 / 98 bp / TGATATTCGAAGACTGGTATAACAAAGTTATACTGAATGAATGAATGAATGAATAAGTGAATGAATTACCAATTGCTGTTGGTTTGTTGATACGACGC / LK392630
RPW26 / 141 bp / CAACTGATGTGGCCGATAGAAGAATCTGTGATGTTAAAAGTCATATTCCCAGACGTAGCACAAGGCATTTGTAAGGTTGGGTTTTGAAGGATTCATTCATTCATTCATTCATAATATACAGAAAATTGATTGCTTAGGGTC / LK392631
RPW32 / 239 bp / ACGACGACATTCATCGAACAGAAGTCGCAATCATAATGATTTGATTTCAGCAATCATAATATAATCATATATATATATATCATAATAAGATCTTTCAATTCTTTCTATAACGTTAGCACAGAAAATGAGAAGTACACATTTTCACCAAGTACAACAAAACAGTTTCAGTACCCATGACTTATTACTATTTACTGCAACAAAACCAAAGTACTTCATACGCATCTTTGTTCCTACTGATG / LK392632
RPW36 / 146 bp / CAGCTACAAACAGTGCCTTTATCTAAGTGTATAAGATATTAAATAAGTTATATCTATTTTNNTATATATATATATANAAANNAAAAGAGCATGTAAAGCAACGATAACAATAACATTATTGAAATTATTTCCTTGCCTTTTCCAGC / LK392633
Table S4 Characteristics of 14 microsatellite markers screened on 102 individuals of R. ferrugineus collected from different regions of the primary and secondary distribution range
Italy (n=22) / Greece (n=17) / Spain (n=6) / Saudi Arabia/Qatar (n=23) / Vietnam (n=17) / Malaysia (n=17)
Locus / Repeat / NA / Ar / He/Ho / NA / Ar / He/Ho / NA / Ar / He/Ho / NA / Ar / He/Ho / NA / Ar / He/Ho / NA / Ar / He/Ho
RPW02 / (TA)11 / 3 / 2.23 [0.30] / 0.43/0.32 / 2 / 1.24 [0.18] / 0.06/0.06 / 2 / 1.67 [0.22] / 0.17/0.17 / 6 / 3.71 [0.59] / 0.73/0.61 / 7 / 4.60 [0.75] / 0.83/0.47 / 6 / 3.89 [0.70] / 0.74/0.47
RPW03 / (TA)11 / 4 / 1.45 [0.25] / 0.45/0.41 / 2 / 1.17 [0.14] / 0.17/0.18 / 3 / 1.67 [0.22] / 0.67/0.33 / 6 / 1.62 [0.24] / 0.62/0.20 / 9 / 1.82 [0.15] / 0.82/0.47 / 4 / 1.44 [0.25] / 0.44/0.13
RPW06 / (TA)9 / 7 / 3.57 [0.72] / 0.77/0.62 / 7 / 3.64 [0.70] / 0.78/0.76 / 4 / 3.23 [0.38] / 0.74/0.67 / 8 / 3.81 [0.81] / 0.79/0.68 / 10 / 4.42 [0.76] / 0.87/0.44 / 6 / 3.64 [0.73] / 0.76/0.56
RPW11 / (ATT)8 / 2 / 1.72 [0.20] / 0.24/0.18 / 2 / 1.94 [0.06] / 0.40/0.29 / 2 / 1.67 [0.22] / 0.17/0.17 / 2 / 1.17 [0.14] / 0.04/0.04 / 5 / 3.03 [0.74] / 0.53/0.59 / 3 / 2.48 [0.30] / 0.49/0.59
RPW13 / (GTG)7 / 1 / 1.00 [0.00] / - / 2 / 1.24 [0.18] / 0.06/0.06 / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 3 / 1.66 [0.41] / 0.17/0.18 / 1 / 1.00 [0.00] / -
RPW16 / (CGG)6 / 2 / 1.98 [0.02] / 0.46/0.59 / 2 / 1.96 [0.04] / 0.43/0.47 / 2 / 1.67 [0.22] / 0.17/0.17 / 2 / 1.32 [0.22] / 0.09/0.09 / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / -
RPW17 / (GTT)6 / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 2 / 1.94 [0.06] / 0.40/0.41 / 2 / 1.97 [0.03] / 0.45/0.41
RPW20 / (AGAT)5 / 2 / 1.99 [0.01] / 0.50/0.86 / 2 / 1.99 [0.01] / 0.49/0.76 / 2 / 2.00 [0.00] / 0.53/0.83 / 2 / 1.99 [0.01] / 0.50/0.70 / 2 / 1.83 [0.14] / 0.30/0.12 / 1 / 1.00 [0.00] / -
RPW22 / (TTTG)5 / 2 / 1.72 [0.20] / 0.24/0.27 / 2 / 1.24 [0.18] / 0.06/0.06 / 1 / 1.00 [0.00] / - / 2 / 1.98 [0.02] / 0.46/0.26 / 2 / 1.57 [0.25] / 0.17/0.06 / 2 / 2.00 [0.00] / 0.51/0.41
RPW24 / (CTAA)5 / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 1 / 1.00 [0.00] / - / 2 / 1.76 [0.18] / 0.26/0.13 / 3 / 2.25 [0.36] / 0.39/0.24 / 2 / 1.24 [0.18] / 0.06/0.06
RPW25 / (TGAA)5 / 2 / 1.94 [0.06] / 0.41/0.27 / 2 / 1.68 [0.22] / 0.21/0.24 / 2 / 1.91 [0.08] / 0.30/0.33 / 3 / 2.17 [0.15] / 0.53/0.52 / 2 / 1.76 [0.18] / 0.26/0.18 / 2 / 1.94 [0.06] / 0.40/0.41
RPW26 / (ATTC)5 / 2 / 1.92 [0.07] / 0.38/0.32 / 2 / 1.83 [0.14] / 0.30/0.24 / 3 / 2.65 [0.23] / 0.53/0.50 / 2 / 1.76 [0.18] / 0.26/0.13 / 3 / 1.80 [0.41] / 0.22/0.24 / 1 / 1.00 [0.00] / -
RPW32 / (AT)7 / 2 / 1.96 [0.04] / 0.43/0.32 / 2 / 1.99 [0.01] / 0.49/0.29 / 2 / 2.00 [0.00] / 0.53/0.83 / 2 / 1.71 [0.21] / 0.23/0.26 / 2 / 1.83 [0.14] / 0.30/0.35 / 3 / 2.90 [0.09] / 0.67/0.71
RPW36 / (TA)7 / 2 / 1.92 [0.07] / 0.38/0.41 / 2 / 1.99 [0.01] / 0.49/0.29 / 2 / 1.98 [0.02] / 0.41/0.50 / 3 / 2.59 [0.29] / 0.52/0.39 / 4 / 3.26 [0.34] / 0.70/0.41 / 3 / 2.64 [0.25] / 0.57/0.47
NA, number of alleles; Ar, allelic richness (in square brackets variance values); He, expected heterozygosity; Ho, observed heterozygosity are listed for each marker

Acknowledgements

Authors are grateful to all colleagues and friends that helped in some way our effort; many of them by collecting samples to be analysed and many others in supporting our activities in various way.

They are: S.A. Aldosari (King Saud University, Saudi Arabia), S. Arnone (Enea Casaccia, Rome, Italy), L. Bartolozzi (University of Florence, Italy), R. Cervo (University of Florence, Italy), A. Cline (California Department of Food & Agriculture, Sacramento, USA), E. Colonnelli (Rome, Italy), F. Di Cristina (BBCA Onlus, Rome, Italy), M. Cristofaro (Enea Casaccia, Rome, Italy), M. Di Giovanni (Bioparco, Rome, Italy), M. Guedj (BBCA Onlus, Rome, Italy), J. Jones (Flowers of Crete, Greece), F. Lecce (Enea Casaccia, Rome, Italy), S. Longo (University of Catania, Italy), G. Mazza (CRA, Florence, Italy), S. Musmeci (Enea Casaccia, Rome, Italy), A. Paolini (BBCA Onlus, Rome, Italy), P. Roversi (CRA, Florence, Italy), R. Sasso (Enea Casaccia, Rome, Italy), M. Montagna (University of Milan, Italy), J. Ng (Kota Baru, Malaysia), L. Ren (Chinese Academy of Sciences, China), P.S.P.V. Vidyasagar (King Saud University, Saudi Arabia), L.V. Vu (Vietnam National Museum of Nature, Hanoi, Vietnam).

This work was carried out with the financial support of Sapienza University of Rome (Italy) within the Project FARI 2010 prot. C26I10SBMC "Bioinformatica, ecologia molecolare e specie invasive: in silico mining di librerie genomiche da pirosequenziamento 454 per l'individuazione di loci microsatelliti" and the University Project 2012 prot. C26A12KRHP "Tagged Next Generation Sequencing e in silico mining di polimorfismi nucleotidici (SNP) nello studio delle invasioni biologiche".