Microsatellite Markers for Cryptolestes Ferrugineus (Coleoptera: Laemophloeidae) and Other Cryptolestes Species
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Bulletin of Entomological Research, Page 1 of 7 doi:10.1017/S0007485315000899 © Cambridge University Press 2015 Microsatellite markers for Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) and other Cryptolestes species Y. Wu1,2,F.Li1,Z.Li2, V. Stejskal3,Z.Kučerová3, G. Opit4, R. Aulicky3, T. Zhang1,P.He1 and Y. Cao1* 1Academy of State Administration of Grain, No. 11 Baiwanzhuang Street, Beijing, China: 2Department of Entomology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, China: 3Department of Pest Control of Stored Products and Food Safety, Crop Research Institute, Drnovská 507, Prague, Czech Republic: 4Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, Oklahoma, USA Abstract Cryptolestes ferrugineus (Stephens, 1831) is an important insect pest of stored pro- ducts. Due to its broad host range, short life cycle, and high reproductive capacity, this species has rapidly colonized temperate and tropical regions around the world. In this study, we isolated 18 novel polymorphic microsatellite loci from an en- riched genomic library based on a biotin/streptavidin capture protocol. These loci will be useful tool to better understand the genetic structure and migration patterns of C. ferrugineus throughout the world. The genetic parameters were estimated based on 80 individual C. ferrugineus from two natural populations. The results revealed that 18 loci were different polymorphic levels. The numbers of alleles ranged from 3 to 12, and eleven loci demonstrated polymorphic information contents greater than 0.5. The observed (HO) and expected (HE) heterozygosities ranged from 0.051 to 0.883 and 0.173 to 0.815, respectively. Five locus/population combinations signifi- cantly deviated from Hardy–Weinberg equilibrium. We also demonstrated the po- tential utility of the C. ferrugineus microsatellites as population and species markers for four additional Cryptolestes species. Keywords: Cryptolestes ferrugineus, Cryptolestes microsatellites, primers, population genetics (Accepted 7 October 2015) Introduction grain, and creating favorable conditions for mold and fungus growth. Moreover, the presence of excrement and insect The rusty grain beetle Cryptolestes ferrugineus (Stephens, fragments contaminate the grain and food products 1831) is a major secondary stored-product pest that feeds on (Trematerra et al., 2011). Due to its broad host range, short cereal, wheat, legume and oil seeds, as well as their processed life cycle, and high reproductive capacity, this species has rap- products (Throne et al., 2002; Hagstrum & Subramanyam, idly colonized temperate and tropical regions around the 2009). C. ferrugineus causes major economic losses by reducing world (Freeman, 1952; Sinha, 1975; Banks, 1979; Thomas & the amount of grain available for sale, lowering the quality of Zimmerman, 1989;Kučerová & Stejskal, 2002; Hagstrum & Subramanyam, 2009). The C. ferrugineus population of China is the largest and most widespread and has caused serious eco- *Author for correspondence nomic losses in several southern provinces, including Hainan, Phone: + 86-10-58523665 Yunnan, Guangxi, Guangdong, Fujian, Zhejiang, Jiangxi, Fax: + 86-10-58523700 Sichuan and Chongqing. Recently, increases in the import Email: [email protected] and export trades of grain have enhanced the dispersal and 2 Y. Wu et al. invasion of this pest. Once this insect pest is established and These insects were identified to the species levels by morph- begins to spread in a new area, eradication and control become ology (Lefkovitch, 1962; Halstead, 1993) and mitochondrial important but difficult. Nevertheless, established population cytochrome c oxidase subunit I (COI) sequence as described genetic techniques (using markers such as microsatellites) by Wang et al. (2014). are now routinely used in the management of pests (Wares et al., 2005; Ascunce et al., 2011). Isolation and screening of microsatellites Nuclear microsatellite makers are tandem repeats of nu- cleotide sequences that are distributed throughout the gen- The enrichment method used to establish the micro- ome. These genetic markers offer an advantage over other satellite-rich genomic libraries was modified from the classes of molecular markers because high mutation rates FIASCO method (Zane et al., 2002). This method was based lead to high levels of allelic variability within populations on biotinylated oligonucleotide sequences bound to (Selkoe & Toonen, 2006). These markers have become one of streptavidin-coated magnetic particles. Genomic DNA was ex- the most popular types of molecular markers for investiga- tracted from ten whole C. ferrugineus adults (a pooled samples) tions of population structures, colonization processes, tem- from the Qingyuan granary in Hebei according to the protocol poral and spatial population dynamics, and evolutionary of the DNeasy Blood & Tissue Kit (Qiagen). Following Sau3AI trends (Ascunce et al., 2011;Wuet al., 2011). Microsatellite ma- (TaKaRa) digestion of the genomic DNA for 3.5 h, restriction kers are especially useful in studies of species invasions in fragments of 300–1200 bp were recovered and purified using which they can help to distinguish the magnitude, location the QIAquick Gel Extraction Kit (Qiagen). The purified frag- and frequency of colonization events as well as differences ments were then ligated to two adaptor oligonucleotides in the levels of diversity and adaptive potential in the intro- (Adaptor A: 5′-GGCCAGAGACCCCAAGCTTCG-3′; and duced populations relative to the native populations (Davies Adaptor B: 5′-phosphate-GATCCGAAGCTTGGGGTCTCT ′ et al., 1999; Wares et al., 2005). GGCC-3 ) with T4 DNA ligase (TaKaRa) overnight at 16°C. The key to applying this technology is to obtain the micro- The ligation products were amplified using the adapter A se- satellite loci and design primers according to the flanking se- quence as the forward and reverse primers. Polymerase chain quences of the loci. Microsatellite loci have been isolated and reaction (PCR) amplification in final reaction volume of 25 µl characterized for important insect pests, such as fruit flies in consisted of 12.5 µl MasterMix with dye (TIANGEN, China), the genus Bactrocera (Wu et al., 2009; Buahom et al., 2013), 9.5 µl ddH2O, 1 µl (10pm) of primer (adapter A), and 2 µl of thrips (Brunner & Frey, 2004;Wuet al., 2014), Leptinotarsa de- ligation products as template DNA. PCR cycler conditions cemlineata Say (Grapputo, 2006), and Oedaleus decorus Gevm were initial denaturation at 94°C for 3 min, followed by 30 cy- (Berthier et al., 2008). Regarding common stored-product cles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s with the pests, microsatellite loci have been reported for three species final extension at 72°C for 10 min. The recovered PCR pro- of Bruchidae (Sembene et al., 2003; Alvarez et al., 2003, 2004, ducts were denatured and hybridized to biotinylated (AG)15 2005; Aebi et al., 2004), three species of Liposcelididae and (TCA)10 probes (Sangon). These heteroduplexes were (Mikac, 2006; Mikac & Fitzstimmons, 2010; Wei et al., 2011), then captured using Dynabeads M-280 Streptavidin and Tribolium castaneum Herbst (Pai et al., 2003). To date, (Invitrogen) and eluted. The DNA was subsequently enriched there are no reports of microsatellite markers isolated from by PCR using adaptor A as the primer (the same as above). The C. ferrugineus. In the present study, we describe the develop- microsatellite-enriched DNA fragments were ligated into ment and characterization of polymorphic microsatellite mar- pGEM-T Easy vectors (Promega) and transformed into kers in C. ferrugineus and assess their utilities as genetic DH5α-competent cells (TAKARA BIO INC). The positive re- markers for four Cryptolestes species. combinant clones were screened by PCR using the adapter A and M13 + /M 13 – as the primers, single clone as template, PCR reaction mixture components and cycling conditions was Materials and methods the same as above. PCR products were confirmed by 1% agar- ose gel electrophoresis. The clone amplified at least two bands Specimen rearing and collection was sequenced on an ABI 3730xl DNA Analyser (Microread Cryptolestes ferrugineus adults from a laboratory strain that Company, China). Based on the sequence data, the clones was established in 2014 from specimens collected from a gran- that yielded suitable flanking sequences were selected for pri- ary in Hebei were used to create an enriched DNA library. A mer design by software. The primers were first screened in 10 laboratory colony was maintained on whole wheat at 24°C C. ferrugineus. and 65–70% relative humidity. The adult specimens of five Cryptolestes species, i.e., C. ferrugineus (Stephens), Cryptolestes Polymorphism testing and cross-species amplification capensis (Waltl), Cryptolestes pusilloides (Steel & Howe), Cryptolestes pusillus (Schönherr) and Cryptolestes turcicus The polymorphisms of the microsatellite loci were further (Grouvelle) were acquired from China, the Czech Republic, tested in 80 C. ferrugineus individuals. Genomic DNA was pre- and the USA. Altogether, 11 adult strains were used in this pared from whole individual according to the protocol of the study, and the number of individual was shown in table 1. Tissue/Cell DNA Mini Kit (Tiangen). Amplification was per- Two strains of C. ferrugineus were used to test the polymorph- formed in a 20 µl volume containing 100 ng of genomic DNA, isms which collected from Shandong, 35°3′N/118°20′E and 2µlof10×Taq DNA polymerase buffer [100 mM Tris-HCl ′ ′ Hainan, 18°15 N/109°31 E, 40 individuals each area. Cross- (pH 8.3), 15 mM MgCl2], 40 µM dNTP, 0.5 U Taq polymerase species amplifications were performed on one strain each of (Tiangen) and 6 µM of each primer (Sangon), one of which was C. capensis and C. pusilloides and three strains each of C. pusillus labeled with a fluorescent dye (6-FAM or 5-HEX). The PCR and C. turcicus. The samples were laboratory strains or were profile included an initial denaturing step at 95°C for 5 min, collected from grain storage facilities and were preserved in followed by 35 cycles of 94°C for 30 s, 54°C for 35 s and 95% ethanol and stored at −80°C prior to DNA extraction.