Molecular Ecology Notes (2005)
doi: 10.1111/j.1471-8286.2005.01149.x
PRIMER NOTE
Blackwell Publishing, Ltd.
Isolation and characterization of microsatellite loci from the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae) S . V E L E Z ,*‡ M . S . T A Y L O R ,* M . A . F . N O O R ,† N . F . L O B O * and J . L . F E D E R * *Department of Biological Sciences, 107 Galvin Life Science Bldg., University of Notre Dame, Notre Dame, Indiana, 46556, USA, †Department of Biological Sciences, Life Sciences Bldg. Annex, Louisiana State University, Baton Rouge, LA 70803, USA
Abstract We report the isolation and development of 81 novel primers for amplifying microsatellite loci in the Rhagoletis pomonella sibling species complex, and the sequencing, characterization and analysis of basic population genetic parameters for nine of these genes. We also report the successful cross-species amplification of several of these loci. The R. pomonella sibling species complex is a textbook example of genetic differentiation in sympatry via host-plant shifting. Microsatellite markers can be useful for mapping host-plant-associated adaptations in Rhagoletis that generate reproductive isolation and facilitate speciation, as well as for resolving the genetic structure and evolutionary history of fly populations. Keywords: apple maggot fly, cross-species amplification, genetic population structure, microsatellite, Rhagoletis Received 7 June 2005; revision accepted 18 July 2005
The Rhagoletis pomonella (Diptera: Tephritidae) sibling species complex is a textbook example of sympatric speciation (Bush 1969). As early as 1864, Walsh hypothesized that certain phytophagous insect specialists could speciate in the absence of geographical isolation in the process of shifting and adapting to new host plants (Walsh 1864). In particular, Walsh (1867) cited the shift of R. pomonella from its native host hawthorn (Crataegus spp.) to introduced domesticated apple (Malus pumila), as an example of sympatric speciation in action. Bush (1969) subsequently proposed that the ≥ 5 sibling species comprising the R. pomonella complex also arose via sympatric host shifts. Characterization of the genetics of the R. pomonella group therefore holds great potential for understanding how barriers to gene flow form in sympatry and lead to speciation. The aim of this study was to expand the molecular genetics of R. pomonella for mapping and phylogenetic analysis by isolating and characterizing a set of variable microsatellite loci. We isolated DNA using PUREGENE DNA PurificaCorrespondence: Sebastian Velez, ‡Present address: Museum of Comparative Zoology, Harvard University, 26 Oxford St., Cambridge MA, 02138, USA. Fax: 617-496-5949; E-mail:
[email protected] © 2005 Blackwell Publishing Ltd
tion kits (Gentra Systems) from eight flies sampled from apple and hawthorn trees located in Grant Michigan, cut the strands with NheI and RsaI restriction enzymes (Promega Corp.) and ran the products on agarose gels. We then excised the band in the 200–600 bp range and constructed a subgenomic DNA library following Hamilton et al. (1999), using the TOPO TA Cloning Kit (Invitrogen Corp.). Our target sequence for enrichment was (TG)n. Plasmids were isolated using the Eppendorf Perfectprep Plasmid 96 Vac Direct Bind Kit (Brinkmann Instruments), and inserts were sequenced on an ABI PRISM 3700 automated DNA analyser with the BigDye Terminator version 3.1 kits (Applied Biosystems). For sequencing the inserts, we used the standard plasmid-specific M13fwd (5′-GTAAAACGACGGCCAGTG) and M13rev (5′-GGAAACAGCTATGACCATG) primers. Bases were automatically called by sequencing analysis version 3.7 software (Applied Biosystems). To help eliminate duplicate clones, we clustered insert sequences in sequencher (Gene Codes). A total of 129 unique clusters emerged from the analysis, of which 81 were chosen for primer development using primer version 3.0 software (Rozen & Skaletsky 2000). A list of these primers
2 PRIMER NOTE Table 1 Rhagoletis pomonella microsatellite primers screened for polymorphism. Given are locus names (with GenBank Accession nos in parentheses), repeat motifs, and forward (F) and reverse (R) primer sequences in 5′–3′ direction. Also shown for each locus is the allele size range for the microsatellite (in bp), number of alleles resolved, mean observed heterozygosity (HO) averaged across populations, FIS, FST and if the primers successfully amplified fragments from the three other Rhagoletis species (Cross amp.). Mean observed heterozygosity was obtained by averaging the observed heterozygosity of each population (eight individuals each from Grant apple, dogwood, Michoacan and Piletas; seven individuals from Grant hawthorn; see text for details of these locations). An asterisk after FIS or FST indicates significant genetic structure (P < 0.05)
Locus (GenBank)
Repeat motif
Primer sequence (5′−3′)
P2 (AY734886)
(AT)0 −2(GT)0 −31
P4 (AY734888)
P6 (AY734890)
[(GT)3(A)]0 −1 (GT)0 − 48 (GT)(AT)0 −1(GT)5 −11(GG) (GT)2(TT)0 −1[(TA)(GT)3]0 −1 (G)2− 6(GT)4 −19
P9 (AY734893)
(GT)7−11
P11 (AY734895)
(GTAT)1−3 (GT)4 −21 (GT)2(G)3 (GT)10 −14 [(GT)2(GG)]2 (AC)0 − 4(T)(GT)4 (GT)5 −27
F: TCCACTCAAATACGGCAACA R: AGAGATCCCGGTGTCGTTC F: GCAAGCGAGTCGTAATCACA R: CCCTCATCATTGTGGTCCTC F: GAGCAGCAGAGGAAAAAGGA R: TGCACTGGTGTATTCCAAGG F: AGTCAGAGTGCGGCAAAAGT R: CGGTAGACCTCAGGCTGATAG F: CGGCAGGTAAATGACCAAAA R: GCAATGACCGTTGGCTATTA F: ATGCAGCCATGACTGAGATG R: TGGAAAGTAATTTCACAAAGGCTA F: GGGTGTTCATGGTAGTTGTAGAT R: ACTAGTAAAGGAAAGGCGCAAT F: GACATCAACTGGTGGTACGC R: ACCAGCCACCGATCATATTT F: CGCGAGAATTTAGTTGAGCA R: TGCCAAGAAGTGTTGTTTCC
P5 (AY734889)
P12 (AY734896) P13 (AY734897) P15 (AY734899)
is available from the first author and all 81 sequences were submitted to GenBank (Accession nos AY734885 to AY734965). Rhagoletis pomonella specimens came from two Mexican hawthorn-infesting populations in Piletas, state of Veracruz (eight flies used for analysis), and Tancitaro, state of Michoacan (eight flies), and two sites in the United States (Grant, Michigan and Granger, Indiana). In Grant, we collected flies infesting either hawthorn (seven flies) or apple (eight flies); in Granger, the undescribed sister species to R. pomonella that infests flowering dogwood Cornus florida (eight flies). Cross-species amplification was tested on Rhagoletis electromorpha, Rhagoletis suavis and Rhagoletis cingulata (Table 2). Polymerase chain reaction (PCR) was performed using HotMaster Taq kits (Brinkmann Instruments), with final concentration of reagents as follows: Mg2+ at 2.5 mm, dNTP mix at 0.25 mm, primers at 0.5 µm and 50 ng of template DNA. Reactions were run on a RoboCycler 96 thermal cycler (Stratagene) under the following protocol: 3 min at 94 °C, 1 min at 56 °C and 1 min at 68 °C for one cycle; 1 min at 94 °C, 1 min at 56 °C, 1 min at 68 °C for 35 cycles; and 10 min at 68 °C final extension cycle. Allele size from PCR amplifications were determined with ceq 8000 genetic analysis system software version 9.0.25 (Beckman Coulter
Size range (bp)
No. alleles (n)
Mean HO
196–238
21
0.86
160–198
17
211–231
FST
Cross amp.
0.033
0.061*
Y
0.89
−0.024
0.044
Y
10
0.54
0.036
0.361*
Y
142–172
14
0.76
0.062
0.106*
N
152–160
4
0.32
− 0.101
0.247*
N
268–314
17
0.82
− 0.002
0.114*
N
239–269
13
0.81
0.003
0.090*
N
364–404
11
0.67
0.101
0.219*
N
271–309
16
0.74
0.144*
0.081*
N
FIS
Inc.), using WellRED D2, D3 and D4 fluorescence-labelled 5′ primers (Proligo), using the CEQ DNA Size Standard Kit 400 (Beckman-Coulter) as a reference. Negative controls with no DNA were included in all experiments. Population genetics parameters were calculated with arlequin version 2.001 (Schneider et al. 2000). Nine primer pairs were selected from the initial survey to directly score individual flies from the four R. pomonella and dogwood populations for allelic variation. The results indicated substantial genetic variation both within and among populations. Loci generally did not deviate from Hardy– Weinberg equilibrium within populations, as indicated by only one of the nine microsatellites having a significant FIS value (Table 1). However, eight of the nine loci displayed significant FST values across the five populations, suggesting genetic subdivision (Table 1). We tested for linkage disequilibrium using arlequin version 2.001 (Schneider et al. 2000) with 16 000 permutations and 100 initial conditions for all loci pairs and found no significant deviations. The primers here reported should be of great utility for further studies of the population structure, mapping and quantitative trait loci (QTL) for this important taxonomic group. Our successful cross-species amplification of several loci makes them potentially useful for examining interspecific differentiation and systematics in the genus Rhagoletis.
© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 10.1111/j.1471-8286.2005.01149.x
PRIMER NOTE 3 Table 2 Microsatellite primers developed from Rhagoletis pomonella flies, which successfully amplified fragments in Rhagoletis electromorpha, Rhagoletis cingulata and/or Rhagoletis suavis. Given are loci names (with GenBank Accession nos in parentheses; sequences and motifs are the same as in Table 1), the number of sequences recovered from cloning, the number of different alleles detected and their size range Locus (GenBank) P1 (AY734885)
P2 (AY734886)
P4 (AY734888)
P5 (AY734889)
Species
Clones recov.
No. of alleles
Size range
R. electromorpha R. cingulata R. suavis R. electromorpha R. cingulata R. suavis R. electromorpha R. cingulata R. suavis R. electromorpha R. cingulata R. cingulata
5 5 3 2 5 2 4 5 6 5 0 0
1 2 3 1 2 1 1 2 1 1 — —
236 218–232 230–236 391 405 –407 386 168 166–168 168 274 — —
Acknowledgements We thank Martín Aluja, Juan Rull and Martín Pale for collection efforts in Mexico (supported by Campaña Nacional Contra Moscas
de la Fruta (DGSV-SAGARPA-IICA) through the Instituto de Ecología, A.C., Xalapa, Mexico. Andrew Forbes, Ken Filchack, Hattie Dambroski and Xianfa Xie kindly provided flies from their collections. This project was funded by a National Science Foundation grant and state of Indiana 21st Century Fund awards to J.L.F., and a National Science Foundation Graduate Research Fellowship to S.V.
References Bush GL (1969) Sympatric host race formation and speciation in frugivorous flies of genus Rhagoletis (Diptera, Tephritidae). Evolution, 23, 237–251. Hamilton MB, Pincus EL, Di Fiore A, Fleischer RC (1999) Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. BioTechniques, 27, 500 –507. Rozen SH, Skaletsky HJ (2000) primer 3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology (eds Krawetz S, Misener S), pp. 365–386. Humana Press, Totowa, NJ. Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN: A Software for Population Genetics Data Analysis. Version 2.000. Genetics and Biometry Laboratory, Department of Anthropology, University of Geneva, Geneva, Switzerland. Walsh BD (1864) On phytophagic varieties and phytophagic species. Proceedings of the Entomological Society of Philadelphia, 3, 403–430. Walsh BD (1867) The apple-worm and the apple maggot. Journal of Horticulture, 2, 338–343.
© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 10.1111/j.1471-8286.2005.01149.x
