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Discovery and Characterization Of Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing Margarita López-Uribe, Christine Santiago, Steve Bogdanowicz, Bryan Danforth To cite this version: Margarita López-Uribe, Christine Santiago, Steve Bogdanowicz, Bryan Danforth. Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 py- rosequencing. Apidologie, Springer Verlag, 2013, 44 (2), pp.163-172. 10.1007/s13592-012-0168-3. hal-01201284 HAL Id: hal-01201284 https://hal.archives-ouvertes.fr/hal-01201284 Submitted on 17 Sep 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2013) 44:163–172 Original article * INRA, DIB and Springer-Verlag France, 2012 DOI: 10.1007/s13592-012-0168-3 Discovery and characterization of microsatellites for the solitary bee Colletes inaequalis using Sanger and 454 pyrosequencing 1 1 2 Margarita M. LÓPEZ-URIBE , Christine K. SANTIAGO , Steve M. BOGDANOWICZ , 1 Bryan N. DANFORTH 1Department of Entomology, Cornell University, Ithaca, NY 14853, USA 2Evolutionary Genetics Core Facilities, Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA Received 5 June 2012 – Revised 8 August 2012 – Accepted 18 September 2012 Abstract – The recent implementation of next-generation sequencing for the discovery of microsatellite markers has made this technology the most effective method for generating genetic markers in non-model organisms. Here, we report the de novo discovery of microsatellite markers for the solitary bee Colletes inaequalis using cloning/Sanger sequencing and direct 454 pyrosequencing from microsatellite-enriched genomic libraries. We identified and successfully multiplexed 18 highly variable microsatellite markers in 585 individuals. The number of alleles per locus ranged from 3 to 23, and the expected heterozygosity ranged from 0.056 to 0.912. These genetic markers will allow for the investigation of levels of inbreeding and fine-scale population structure in C. inaequalis. Our results contribute to the literature demonstrating that 454 sequencing is more time- and cost-efficient than cloning/Sanger sequencing at identifying a large number of genomic regions with microsatellite repeat motifs. SSRs / cloning / next-generation sequencing / Colletidae 1. INTRODUCTION co-dominant multi-allelic markers have been widely used in genome mapping, parentage Simple sequence repeats (SSRs) or micro- analysis, population structure, and phylogeo- satellites are short DNA sequences consisting of graphic studies, for organisms ranging from tandem repeated motifs that vary in length, bacteria to humans (Blair and McCouch 1997; typically from 1 to 6 bp long. SSRs are most Weber et al. 1991). commonly present in non-coding regions and In the past, one of the major limitations for are characterized by high levels of repeat length the use of microsatellite markers in non-model polymorphism that are the result of two muta- organisms was the time and cost of developing tion mechanisms, replication slippage and un- these markers de novo (Zane et al. 2002). When equal crossover (Schlötterer and Tautz 1992). microsatellite markers were first described Due to their high levels of polymorphism, these (Tautz 1989), their isolation and characteriza- tion were time-consuming and expensive be- cause protocols were based on a random Corresponding author: M.M. López-Uribe, screening of whole genome libraries. Currently, [email protected] there are several enrichment protocols for Manuscript editor: Marina Meixner microsatellite libraries (Zane et al. 2002), which 164 M.M. López-Uribe et al. can greatly accelerate the process of micro- nucleotides and (2) direct sequencing of satellite development by making the screening enriched libraries for microsatellites using process more targeted. Moreover, with the Roche 454 sequencing technology. We estimat- availability of next-generation sequencing tech- ed the variability of the identified SSRs markers nology, de novo isolation of microsatellite using four microsatellite multiplex sets in 585 markers has become routine for non-model C. inaequalis individuals. organisms (Mikheyev et al. 2010; Perry and Rowe 2011; Zalapa et al. 2012). A large number of microsatellite loci can be detected by direct 2. MATERIAL AND METHODS pyrosequencing of enriched libraries followed by bioinformatic detection of simple repeated 2.1. Enriched library preparation sequences in the assembled sequenced data (Andrés and Bogdanowicz 2011). Genomic DNA was extracted from two C. inae- Colletes inaequalis is a solitary bee, meaning qualis males collected in Ithaca (NY, USA, 42°26′ each nest is occupied by a single reproductively 39.8″N 76°29′32.4″W) using the Qiagen DNeasy active female, native to eastern North America. Blood and Tissue Kit. DNA quality and concentration This species is a wild pollinator of early spring were measured using a Nanodrop 1000 spectropho- wild plants and could become an important tometer. We digested the genomic DNA using two native pollinator for crops such as blueberries restriction enzymes, BsaA I and Hinc II, to minimize (Batra 1995) and apples (Gardner and Ascher problems of unequal sampling of genomic regions 2006). C. inaequalis has a solitary life cycle due to genome composition biases. During digestion, characterized by adults emerging from their double-stranded SNX linkers were ligated to the ends underground cocoons early in the spring. Both of the genomic fragments (Hamilton et al. 1999). An females and males have a strong tendency to stay aliquot of the digestion was incubated with synthetic, close to their emergence sites. Males are the first single-stranded, biotinylated oligonucleotides repre- to become active, and they remain patrolling the senting 12 repeat motifs (GT, TC, TA, TTA, GTT, emergence area waiting for females to mate with. TTC, GCT, TTTA, TTTG, TTTC, GATA, and GTAT) When females emerge, males actively search for that had streptavidin attached to the 3′ end. Genomic them to copulate, while females immediately DNA/biotinylated complexes were captured with start building and provisioning their nests in the streptavidin-coated magnetic beads. A PCR reaction natal area (Batra 1980). Observations from using a SNX forward primer was then used to transform behavioral studies indicate that nest aggregations the captured genomic fragments into double-stranded of C. inaequalis,andotherearlyspringColletes DNA (Andrés and Bogdanowicz 2011). spp., naturally experience philopatric behavior and high levels of inbreeding (Vereecken 2008). 2.2. Microsatellite discovery by cloning However, this question has not been investigated and Sanger sequencing due to the lack of species-specific hypervariable genetic markers. PCR products from the enrichment protocol were In this study, we describe and characterize 18 digested with Nhe I and ligated into pUC 19 plasmids hypervariable microsatellite markers for the previously digested with Xba I and dephosphorylated. ground-nesting bee C. inaequalis (Colletidae). Plasmids were introduced into Escherichia coli cells We enriched for microsatellite loci by hybridiz- through electroporation. Transformed bacteria were ing biotinylated nucleotide repeats to restriction cultured on plates containing ampicillin and X-gal, enzyme digested genomic DNA. We then then transferred onto nylon membranes and fixed by screened for microsatellites using two different autoclaving. These membranes were screened for methods: (1) the traditional cloning protocol microsatellites through hybridization to 33P- that selects positive clones containing micro- radiolabeled oligonucleotides with the same repeat satellites by hybridization of radiolabeled oligo- motifs used for the enrichment process. Plasmid Microsatellites for the solitary bee Colletes inaequalis 165 DNA from bacterial colonies that were found to be minimum of six perfect repeats using the software positive for the presence of microsatellite repeats were PrimerSelect (DNASTAR). We used the following extracted and Sanger sequenced using M13 primers criteria for primer design: maximum primer length at the vector/insert boundary. of 30 bp, melting temperature from 54 to 60 °C, maximum dimer duplexing of 3 bp, and maximum 2.3. Microsatellite discovery by 454 hairpin duplexing of 3 bp. For primer testing, we sequencing genotyped ~30 male individuals using the univer- sal tag three-primer method (Schuelke 2000)that Post-enrichment PCR products were purified includes: 5′ 6-FAM M13 primer, marker-specific with a Qiagen PCR purification kit and ligated to forward primer plus a long tag at the 5′ end that is 454 A and B adapters. The C. inaequalis library complementary to the M13 primer, and marker- was pooled with seven other libraries generated specific reverse primer plus a six base “pigtail” from other taxa for sequencing in half 454 run. Each (GTTTCT) attached to the 5′ end to reduce stutter library was identified with a unique multiplex peaks (Brownstein et al. 1996). Simplex
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