DOI: 10.2478/dna-2014-0002
DNA Barcodes 2014; Volume 2: 10–16
Research Article
Open Access
Lu Qian, Yulin An, Junxian Song, Mei Xu, Jianlin Ye, Cuiping Wu, Bin Li, Dejun Hao
COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay Abstract: Gypsy moth, an important forest/urban pest worldwide, is separated into the European and Asian subspecies, and has important quarantine significance. Diagnostic technique that can accurately and quickly distinguish subspecies is lacking. This study aimed to evaluate genetic difference between the subspecies, and subsequently to develop a reliable and high throughput molecular based diagnostic tool for distinguishing the subspecies. COI genes of 25 gypsy moth samples from China, Russia, Mongolia, Japan and the United States were sequenced. DNASTAR analysis revealed that gypsy moth COI gene was 1531bp long. The UPGM phylogenetic tree constructed based on the COI gene indicated that European subspecies (U.S. population) and Asian subspecies were distinctively divided into two branches. Japanese populations had a far distantly relationship with other Asian populations forming a separate branch. There was a single base substitution (base transformation only) at 14 consistent locations between Asian and American populations, but 13 of them coded the same amino acid. A MGB proper and TaqMan assay was designed base on the base substitution at 406th bp that coded a different amino acid. This allele typing assay took only 4 hours and could accurately distinguish gypsy moth subspecies of Europe and Asia. The study enriches the knowledge basis of genetic differentiation of gypsy moth subspecies. And more importantly the TaqMan assay is the first report of such diagnostic tool that could deliver rapid and accurate results and suitable for routine quarantine inspections to distinguish Asian and European gypsy moth subspecies. Keywords: Gypsy moth subspecies; COI gene; molecular marker; TaqMan assay *Corresponding author: Lu Qian: Nanjing Forestry University, Nanjing, China; E-mail:
[email protected] Lu Qian, Yulin An, Mei Xu, Jianlin Ye, Cuiping Wu, Bin Li: Jiangsu Entry-Exit Inspection & Quarantine Bureau, Nanjing, China; Junxian Song: Liyang Plant Protection and Quarantine Station, Changzhou, China; Dejun Hao: Nanjing Forestry University Nanjing, China
This study was supported by the Ministry of Science and Technology of the People’s Republic of China (Science and technology supporting project: 2012BAK11B03; International cooperation project: 2009DFA31950) and Jiangsu Entry and Exit Inspection and Quarantine Bureau (2014KJ45). The gypsy moth, Lymantria dispar (L.) (Lepidoptera: Erebidae) is a polyphagous forest/urban pest, known to feed on foliage of hundreds of trees, shrubs and plants species causing devastating damage when outbreaks occur [1]. Three sub-species have been designated based on differences in geographical distribution, behavioral and ecological characteristics. They are Asian gypsy moth, L. dispar asiatica Vnukovskii, European gypsy moth (also referred as North American gypsy moth), L. dispar dispar (L.), and Japanese gypsy moth, L. dispar japonica (Motschulsky) [2]. In earlier classification Asian gypsy moth and Japanese gypsy moth was grouped together as Asian gypsy moth (AGM) with distributions in Asia and some European areas [3-7]. European gypsy moth (EGM) is native to Europe, and was introduced into North America in 1869 [2]. The major differences between AGM and EGM are female flight capability and broader host range of AGM (500 vs. 250 host species) [8,9]. For this reason AGM is considered to be more threatening globally than EGM. Since 1991, the USDA-APHIS has implemented surveillance programs at ports and surrounding areas, and performed special quarantine inspections for vessels from Japan and Russia to prevent EGM entering North America. In 2009, North American Plant Protection Organization (NAPPO) issued a specific inspection guideline for vessels from gypsy moth infested areas in Asia (RSPM 33, www.nappo.org) Detection, rapid and accurate species identification, and source determination are important steps for any invasive species prevention effort. Gypsy moth subspecies molecular differentiation has attracted substantial research effort due to their high invasive potential and undesirable life history characteristics [10]. Allozyme
© 2014 Lu Qian, et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay
analysis was used to reveal genetic variation among populations of Japan, Europe and the U. S. [3]. Variations of microsatellite DNA and mitochondrial DNA (mtDNA) were used to track gypsy moth sources [3-5]. Asian and North American populations were differentiated by an amplified ribosomal DNA restriction analysis [11]. More recent, RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) technology were used as molecular markers for gypsy moth population diagnostics [7]. mtDNA is the DNA contained in mitochondria outside of cell nucleus. The properties of mtDNA include maternal inheritance, relatively fast mutation rate and lack of recombination, which make them very useful molecular markers for studies of population genetic structure, geographical variation, species characteristics and phylogeny [12,13]. The mitochondrial COI gene is easy to be amplified and sequenced, and was successfully used in investigations of phylogenetic relationships of closely related species and became an efficient and widely used genetic marker [14,15]. In this study, we used COI gene sequence analysis to investigate genetic differences between AGM and EGM, to identify single nucleotide polymorphisms (SNPs), and to develop a TaqMan allelic discrimination assay. The study established a scientific basis for genetic differentiation of gypsy moth subspecies. The developed assay could be used for quarantine purpose to distinguish gypsy moths from different geographical locations, and could have a significant impact on port inspection policies.
Materials and methods Insect samples Specimens from Russian Far East, Mongolia, Japan, the United States, and China collected between 2007 and 2009 were used in this study, including 25 gypsy moth samples (female moths or eggs) and 1 Lymantria xylina sample (Table 1). All samples were donated by Dr. Baode Wang (U.S. Department of Agriculture Animal and Plant Health Inspection Service) except for specimen from Heilongjian, China, which was collected by the authors. The samples were stored in 95% ethanol under -70°C before use.
DNA extraction Genomic DNA was extracted using an animal tissue/cell genomic DNA extraction kit (GenMagBio, BeiJing, China)
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following the manual instruction. All buffers and reagents used came with the kit. Adult moth thorax muscles or 5 eggs (from a single egg mass) were pulverized in 2.0 mL Eppendorf tubes with a stainless steel pestle, then 180 μL of lysis buffer and 20 μL of proteinase K were pipetted into each tube. The tubes were incubated at 55°C for 3 h. After tissue digestion, the supernatant was transferred into a new tube, and sequentially 200 μL binding buffer, 200 μL ethanol and 20 μL of magnetic beads were added into the tubes. The mixture was stirred and left at room temperature for 10 min for cell lysis and ensuring that DNA was absorbed by the magnetic beads. The tube was then placed in a magnetic separator for 60 s. Excess fluid was removed and 500 μL of washing buffer was added and mixed for 10 min. The tube was returned to the separator for another 30 s and the supernatant was discarded. This washing step was repeated twice to remove any residual contaminants. The remaining DNA-magnetic bead complex was resuspended in 20 μL of elution buffer and vortexed to obtain a homogenous suspension. DNA was eluted by incubation at 55°C for 10 min followed by magnetic separation for 30 s. The DNA supernatant was transferred to a new tube and preserved at -20°C.
PCR amplification and sequencing According to published Lepidoptera COI sequences, a pair of universal primers at both ends of COI gene, COIF (5’-CTTAAAATTTGCAATTTTATATC-3’) and COIR (5’TTAAATCCATTACATATAGTCTG-3’), was designed (synthesized by Takara Bio., Dalian, China) and used to amplify a 1685 bp fragment of the COI gene. Each PCR reaction of 50 μL contained 5 μL of 10x PCR buffer (Takara Bio), 4 μL dNTPs (2.5 mmol/L each), 4 µL MgCl2 (25 mmol/L), 2 µL of 10 pmol/L of each primer, 0.5 U Taq polymerase (Takara Bio), and 1 μL of DNA template. The mixture was heated at 94°C for 2 min followed by 30 cycles of 30 s at 94°C denaturation, then 40 s at 45°C annealing, and 2.5 min at 72°C extension and a final extension at 72°C for 10 min. Each PCR product was checked by 1.5% agarose gel. Purified DNA was sequenced from both directions and performed by Nanjing Genscript Biological Technology Co. Ltd. (Nanjing, China). The obtained COI gene sequences have been submitted to GenBank (accession # HM013726 - HM013745).
Sequence and computational analysis The COI gene sequences were aligned using DNASTAR software. MEGA 4.0 was used for base pair and polymorphic loci analysis. UPGMA (unweighted paregroup method with arithmetic mean) method was used
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Lu Qian, Yulin An, Junxian Song, Mei Xu, Jianlin Ye, Cuiping Wu, Bin Li, Dejun Hao
Table 1: Gypsy moth samples tested in the study Sample code (26)
Collection site
Year
GenBank accession no.
Haplotype
RVO1
Vostochnyy, Russia
2007
HM013724
Hap1
RVO2
Vostochnyy, Russia
2007
HM013725
Hap1
RV
Vladivostok, Russia
2007
HM013726
Hap1
RFV1
Far East Port Vladivostok
2007
HM013727
Hap1
RFV2
Far East Port Vladivostok
2007
HM013728
Hap1
RN1
Nahodka, Russia
2007
HM013729
Hap1
RN2
Nahodka, Russia
2007
HM013730
Hap1
RS
Slavyanka, Russian
2007
HM013731
Hap1
MG1
Mongolia
2007
HM013732
Hap2
MG2
Mongolia
2007
HM013733
Hap1
MG3
Mongolia
2007
HM013734
Hap1
MG4
Mongolia
2008
HM013735
Hap2
MG5
Mongolia
2008
HM013743
Hap1
JAP1
Japan
2007
HM013736
Hap3
JAP2
Japan
2007
HM013737
Hap4
JAP3
Japan
2007
HM013738
Hap3
HLJ
Hei longjiang, China
2009
HM013741
Hap1
WV
West Virginia, USA
2008
HM013745
Hap5
ME
Maine, USA
2008
-
Hap5
MA1
Massachusetts, USA
2008
HM013739
Hap5
MA2
Massachusetts, USA
2008
HM013742
Hap5
MA3
Massachusetts, USA
2008
-
Hap5
NJ1
New Jersey, USA
2008
HM013740
Hap5
NJ2
New Jersey, USA
2008
HM013744
Hap5
PA
Pennsylvania, USA
2008
-
Hap5
LX (L. xylina)
Xiamen, China
2009
-
Hap6
to construct phylogenetic tree. Dnasp 5.0 was used to calculate haplotype diversity (HD) and nucleotide diversity (Pi).
TagMan PCR assay Based on an identified single nucleotide polymorphism at 406 bp of the COI gene sequence, two MGB (minor groove binding molecule) probes were designed to amplify a 100 bp conservative segment of the COI gene. One probe with the same sequence as EGM was labeled with VIC at 5’end, and the other with the same sequence as AGM was labeled with FAM at 5’end (carboxyfluorescein) as fluorophore markers. The sequences of the primers and probes were: primer forward: 5’-GGATGAACTGTTTACCCTCCTCTATCT-3’; primer
reverse: 5’-TGATGAAATACCAGCTAAGTGAAGAGAAAA-3’; 406 VIC: 5’-ACAGATCTACCTCTATGAGCA-3’; and 406 FAM: 5’-AGATCTACCTCCATGAGCA-3’. They were synthesized by ABI (Applied Biosystems, Inc., California, USA) in a 40x pre-mix with the primer concentration of 36 µmol/L and probe concentration of 8 µmol/L), and diluted to 10x with sterilized dd H2O and stored at -20°C before use. PCR reaction system in total of 25 µL contained 1 µL of DNA template (~50 ng), 2.5 µL of above 10x primer and probe pre-mix solution, 12.5 µL of 2xTagMan buffer, and 9 µL of dd H2O. The detection run included a start at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. FAM (483-533 nm) and VIC (523-568 nm) signals were read before and after the reaction using an ABI 7500 PCR system.
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COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay
Results
COI gene based phylogeny
COI gene amplification, sequence characteristics and geographic differences
UPGMA analysis of the obtained COI gene sequences divided the 25 gypsy moth populations and one L. xylina population into six groups (Figure 1). Gypsy moth samples from Russia, Mongolia, Japan, China and the U.S. were clearly clustered into 3 groups, i.e. Asian subspecies, Japanese subspecies and European subspecies, matched with the geographic distributions of those subspecies. Asian and European subspecies were clearly delineated with 100% support rate.
PCR amplification using isolated genomic DNA as a template and COIF/COIR primers generated a specific product with a length of 1531 bp coding for 510 amino acids. Taking RV01 as an example, the T, C, A and G contents were 39.1, 15.7, 31.6 and 13.5%, respectively. A+T content was 70.7%, much higher than that of G+C content at 29.3%. Those measurements were applicable to other tested populations as the sequences were very similar between each other. Examination of the COI gene sequences revealed that all geographic differences were concentrated between 78-1357 bp (Table 2). A total of 17 base pairs were discovered to be different and all were transitional substitution. Between U.S. and Asian populations, single-nucleotide polymorphisms (SNPs) were detected at 14 consistent sites. Within Asian populations, four SNPs were detected with three found among Japanese populations and one found among Mongolia populations. Among the 509 amino acids coded by the COI gene of all 25 samples only two positions were different. They were G at position 135 bp in the 17 Asian samples and S in the six U.S. samples; S at position 388 bp in MG1 and MG4 and G in the rest of the samples. Five haplotypes representing single-nucleotide polymorphisms were identified in the 25 tested samples with only one of them (Hap 4) was unique and not shared among different populations (Table 1). Twelve samples from Russia, China and Mongolia (MG2, MG3 and MG5) shared the exact same sequence (Hap 1). Two other Mongolia samples (MG1 and MG4) had the same sequence (Hap 2). Two of the three Japanese samples (JAP1 and JAP3) had the same sequence (Hap 3). All six U.S. samples shared the same sequence (Hap 5). The site differences of the haplotypes were list in Table 2. Haplotype diversity HD was estimated at 0.672, and nucleotide diversity Pi was 0.00429 only.
TaqMan-MGB diagnostics Gene divergent plot was constructed based on the signals of FAM and VIC (Figure 2). The 25 tested samples clustered into two groups in accordance to their geographic distribution. RV01, RFV2, RV, RN1, RN2, JAP1-3, MG1-3 and HLJ populations were designated as Asian subspecies with G at 406 bp (marked as blue diamonds), while MA1-3, HE, NJ1, WV, PA and NJ2 were designated as European subspecies with A at 406 bp (marked as red circles). All samples contained either G or A at 406 bp, indicating that they are homozygous. The diagnostics completely agreed with full sequencing, indicating that the method could successfully distinguish Asian and European subspecies of gypsy moths. These results confirmed that the designed probes were specific to the gypsy moth subspecies, and might be very well suited for a high throughput SNP assay in population diagnostics.
Discussion The genetic variation of gypsy moth has been investigated with various genetic marker technologies in an effort of evaluating levels of variability and relatedness within the species, and developing diagnostic tools at the subspecies level. For example, North American gypsy moths seem more closely related to European gypsy moths than to Asian
Table 2: COI gene sequence polymorphisms of gypsy moth geographic populations* Haplotype Hap1 Hap2 Hap3 Hap4 Hap5
Polymorphic positions 78
234
381
406
600
666
669
684
786
855
864
993
1053
1122 1165 1338 1357
A G
G A
T C -
G A
A G -
A G
G A
C T
A G
G A A A
C T
C T
T C
C T
* ‘-‘ indicates same nucleotide as haplotype 1.
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G A -
A G
T C
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Lu Qian, Yulin An, Junxian Song, Mei Xu, Jianlin Ye, Cuiping Wu, Bin Li, Dejun Hao
RV RS RFV 2 RV O 1 RN2 51 H LJ
RV O 2 MG2
Asian
MG3 59
MG5 RFV 1
100
RN1 MG1
66 M G 4
JA P 2 35
JA P 1
Japanese
81 JA P 3
MA1 N J2 MA2 100 M E
European
WV N J1 L. x y lina 0.020
0.015
0.010
0.005
0.000
Figure 1: Phylogenetic tree constructed by UPGMA method based on the entire COI gene sequence showing genetic relationships of tested geographic populations. Bootstrap support rates are listed above the corresponding node.
or Japanese gypsy moths [3,16]. Pfeifer et al. [5]employed a specific primer ITS22 of nDNA that revealed fragment length polymorphisms among geographical populations of gypsy moths. A RAPD-PCR marker (FS21) was designed to distinguish the North American and Asian gypsy moth populations [11]. Similarly Schreiber et al. [6] used 3 RAPDPCR markers (FS22, FS23 and FS24) for the same purpose. More recently, COI, COII and NDI genes of mtDNA of gypsy moth pupations from various regions worldwide were sequenced and compared [17]. The results revealed that the tested gypsy moth populations resolved into four
groups with populations from Okinawa and Hokkaido of Japan in two separate groups, and populations of Honshu and Kyushu of Japan and mainland Asia in one group, and populations from Europe, Tunisia and North America in one group. In our study, only 5 unique haplotypes were observed among the tested 25 populations and the division corresponded to their geographic distributions. Between AGM and EGM, 14 SNPs were observed indicating clear divergence. Two haplotypes were observed in populations from Japan, but the divergence could not be defined further
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COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay
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Figure 2: Gene divergent plot of European and Asian gypsy moth subspecies based on the signals of FAM and VIC. The 25 tested samples clustered into two groups in accordance to their geographic distribution. RVO1, RVO2, RV, RFV1, RFV2, RN1, RN2, RS, MG1-5, JAP1-3 and HLJ populations were designated as Asian subspecies with G at 406 bp (marked as blue diamonds); WV, ME, MA1-3, NJ1, NJ2 and PA were designated as European subspecies with A at 406 bp (marked as red circles).
due to limited samples and lack of detailed collection site information. The UPGMA generated phylogenetic tree revealed that the tested gypsy moth populations were divided into three clades: mainland Asia, Japan, and North America according to their perspective geographic distributions. This division agrees with Pogue and Schaefer‘s [2] three subspecies division based on morphological and biological characteristics, and is also similar to divisions based on mtDNA variations by Bogdanowicz et al. [17]. The genetic differences between AGM and EGM were noticed by researchers previously [3-7]. The clear division (100% support) between the two in this study confirmed the complete divergence. MGB probe is mainly used in detection of SNPs and has been widely used in clinical studies [18-20]. Because of its high specificity and reliability, and time saving properties, the technology has become popular in distinguishing similar or related species. After intensive sequencing and identification of conservative SNPs of the COI gene we have successfully developed a TaqMan MGB assay for distinguishing Asian and European/North American gypsy moth populations. The diagnostic results of the assay completely agreed with that of full sequencing. The present study is the first to report such application of TaqMan MGB probe in gypsy moth geographic population diagnostics. MGB-DNA probes are extremely stable duplexes with single-stranded DNA targets through binding at DNA minor
grooves, which allows for shorter probes, higher melting temperature (Tm), and improved sequence specificity, especially for high A+T content probes. Those properties are very well suited for COI based gypsy moth population diagnostics as the COI gene is high in A+T content and sequence variations are relative small among different populations, which makes the diagnostics are difficult to achieve with regular primer markers or regular TaqMan probes. The TaqMan-MGB assay reported here can rapidly distinguish Asian and European gypsy moth populations (in 4 h) and significantly reduce quarantine inspection related costs compared with other diagnostic tools. Conflict of interest: Dr. Qian has a patent ‘A method of distinction type of Asian or European gypsy moth’ with royalties paid to The department of Animal, Plant and Food Inspection Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau. Received: November 27, 2013; Accepted: February 23, 2014.
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Lu Qian, Yulin An, Junxian Song, Mei Xu, Jianlin Ye, Cuiping Wu, Bin Li, Dejun Hao
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