Introduction
Three species of rice planthoppers are economically important in rice production in Asia: the brown planthopper (BPH) Nilaparvata lugens, the white-backed planthopper (WBPH) Sogatella furcifera, and the small brown planthopper (SBPH) Laodelphax striatellus. These three species are vectors of rice plant viruses (Hibino, Reference Hibino1979; Nemoto et al., Reference Nemoto, Ishikawa and Shimura1994; Zhou et al., Reference Zhou, Wen, Cai, Li, Xu and Zhang2008). The former two species, BPH and WBPH, also cause sucking damage to rice plants; BPH, in particular, causes a severe form of damage, which is called hopperburn. Unlike SBPH, BPH and WBPH cannot overwinter in temperate Asian countries, including Japan, Korea and northern China. They annually migrate into Japan and Korea during Bai-u rainy season (June–July) after travelling long distances from the subtropical and tropical areas in lower altitudes, where rice is cultivated throughout the year (Kisimoto, Reference Kisimoto1976; Watanabe & Seino, Reference Watanabe and Seino1991; Sogawa, Reference Sogawa1992, Reference Sogawa1995; Kisimoto & Sogawa, Reference Kisimoto, Sogawa, Drake and Gatehouse1995; Otuka et al., Reference Otuka, Dudhia, Watanabe and Furuno2005a, Reference Otuka, Matsumura, Watanabe and Ding2008). The migration of the species has been studied by such methods as observation on the ships (Asahina & Turuoka, Reference Asahina and Turuoka1968), meteorological analyses (Kisimoto, Reference Kisimoto1976; Seino et al., Reference Seino, Shiotsuki, Oya and Hirai1987) and mark-recapture experiments (Kiritani & Hirai, Reference Kiritani and Hirai1987). At present, BPH and WBPH migrating from northern Vietnam or southern China are considered as the primary sources of planthoppers in northeastern China, Korea and Japan. Furthermore, on the basis of meteorological computer simulations, Otuka et al. (Reference Otuka, Watanabe, Suzuki, Matsumura, Furuno and Chino2005b, Reference Otuka, Matsumura, Watanabe and Ding2008) suggested that BPH also migrated from northern Philippines to Taiwan and Japan.
For planthopper management, it is very important to know the characteristics of various biotypes in insect populations, such as insecticide susceptibility levels (Matsumura et al., Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008; Matsumura & Sanada-Morimura, Reference Matsumura and Sanada-Morimura2010), tolerance profile against resistant rice varieties (Sogawa, Reference Sogawa1992; Myint et al., Reference Myint, Yasui, Takagi and Matsumura2009; Naeemullah et al., Reference Naeemullah, Sharma, Tufail, Mori, Matsumura, Takeda and Nakamura2009) and wing-dimorphic traits (Morooka & Tojo, Reference Morooka and Tojo1992). Matsumura et al. (Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008) reported substantial degradation of susceptibility to neonicotinoids and phenylpyrazole, which are the insecticides most commonly used against BPH and WBPH in paddy fields of East and Southeast Asia. Resistance to these modern insecticides is becoming a serious problem in East and Southeast Asia. To determine the insecticide susceptibility of various phenotypes, potency assays of various insecticides in each population are necessary. However, such assays are time-consuming and labour-intensive. The virulence tests of planthoppers against planthopper-resistant rice varieties are similarly tedious. Therefore, simple methods for differentiation of phenotypes or biotypes need to be developed. In addition, prediction of planthopper occurrence and timely measures for planthopper control in East Asian countries require rapid determination of biotypes of immigrant planthopper populations.
In this regard, molecular markers linked with biotypes or representing regional populations will be extremely useful for planthopper management. Genome sequences can be handled easily with recent technology, because DNA is stable and can be isolated from individual planthoppers with ease. We had previously studied the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of WBPH to distinguish regional populations in Southeast and East Asia (Fu et al., Reference Fu, Matsumoto, Matsumura, Hirai, Sato and Noda2012). However, we did not find population-specific nucleotide sequence features; heterogeneity was found among rRNA gene copies of even single planthoppers. Since the ITS region showed too high a variation to be used as a molecular marker to discriminate local populations, we selected mitochondrial gene sequences as alternative candidates. Mitochondrial DNA sequences are often used as molecular markers for inter-species and inter-population differences. The population genetic structure of various species has been revealed using mitochondrial DNA sequences (Yoshida et al., Reference Yoshida, Yoshioka, Shirakihara and Chow2001; Nobre et al., Reference Nobre, Nunes, Eggleton and Bignell2006; Cai et al., 2008; Meraner et al., Reference Meraner, Brandstätter, Thaler, Aray, Unterlechner, Niederstätter, Parson, Zelger, Dalla Via and Dallinger2008). Mun et al. (Reference Mun, Song, Heong and Roderick1999) reported genetic variation of BPH and WBPH populations among Asian populations within an 850 bp region of the mitochondrial cytochrome oxidase I (cox1). That pioneering study, involving 71 BPH and WBPH individuals, created the expectation that planthopper populations could be distinguished on the basis of mitochondrial molecular sequences by utilising longer sequences and larger samples from local populations.
In this paper, we extended the size of the mitochondrial sequence and increased the number of samples to further explore the utility of molecular markers in distinguishing sub-populations of BPH and WBPH, and furthermore, we analysed the genetic structure of East and Southeast Asian rice planthopper populations on the basis of their mitochondrial sequences.
Materials and methods
Insect samples
Thirty-one populations of N. lugens (Stål) (BPH) and 25 populations of S. furcifera (Horváth) (WBPH) were collected from ten areas [Japan, China, Taiwan, Laos, Thailand, northern and southern Vietnam (Vn and Vs), northern and southern Philippines (Pn and Ps), and Papua New Guinea (PNG)] from 1966 to July 2009 (fig. 1 and table 1). These populations were collected in the field or obtained from insects maintained in the laboratory for several generations (1 month to 40 years) after collecting adults (table 1).
Fig. 1. Sampling sites of rice planthoppers in East and Southeast Asia; Japan (Ja–Jd and J1–J6), Taiwan (T1–T5), China (C1–C3), Vietnam (Northern area: Vn1–Vn4 and Southern area: Vs1–Vs5), Laos, Thailand (Thai1–Thai3), the Philippines (Northern area: Pn1–Pn6, Southern area: Ps1 and Ps2) and Papua New Guinea (PNG).
Table 1. Sampling data for BPH and WBPH populations.
1 Population numbers are indicated in the populations studied in the previous report [table 1 of Matsumura et al. (Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008)].
2 Southern Fruit Research Institute in Vietnam.
3 The collected population was derived ca. 20 individuals.
DNA purification, polymerase chain reaction (PCR) and sequencing
Total DNA was extracted from individual insects by using DNeasy (Qiagen Inc., Valencia, CA, USA), and was eluted in 100–200 μl. Three to 21 individuals of respective populations were obtained for DNA extraction (table 1).
Mitochondrial genes were amplified by PCR using the primer pairs B_C1-J-1718 and B_C2-N-3665 for BPH, and C1-J-1718 and B_C2-N-3665 for WBPH (table 2). The basic PCR mixture (30 μl) consisted of 0.15 μM deoxynucleotides, 0.5 μM forward primer, 0.5 μM reverse primer, 1 μl of the PCR template, and 0.5 U of Taq DNA polymerase (TAKARA BIO Inc., Tokyo, Japan) in PCR buffer (TAKARA BIO Inc.). The PCR temperature profile was 94 °C for 2 min, followed by 94 °C for 30 s, 52 °C for 30 s, 72 °C for 2 min for 30 (for BPH) or 35 cycles (for WBPH), with a final extension at 72 °C for 5 min. Electrophoresis in 1% (w/v) agarose/TAE gel was used to confirm the identity of the PCR-amplified products. The amplified fragments were purified using Sephacryl S-300 HR (GE Healthcare UK Ltd, Buckinghamshire, England) spin columns.
Table 2. Primers list.
Using a sequencing kit (ABI PRISM Dye Primer Cycle Sequencing Kits; Applied Biosystems Division, Perkin-Elmer Inc.), we performed sequencing reactions with the DNA amplification products and the following primers designed for this study: B_f1, B_f2 and B_r1 for BPH, and W_f1, W_f2 and W_r1 for WBPH (table 2). The reaction products were sequenced using a DNA Sequence System (model 3700; Perkin-Elmer Inc.). Nucleotide sequences with lengths of 1,928 bp (BPH) or 1,927 bp (WBPH) encoding cytochrome oxidase I (cox1)–trnL2–cox2 were determined.
Data analyses
The trnL2 region was predicted using tRNAscan-SE 1.21 (Lowe & Eddy Reference Lowe and Eddy1997, http://lowelab.ucsc.edu/tRNAscan-SE/). The default search mode was used (cove score cut-off ≥15), specifying mitochondrial/chloroplast DNA as the source and using the invertebrate mitochondrial genetic code for tRNA isotype prediction.
Median-joining (MJ) was performed using Network 4.5.1.0 (Bandelt et al., Reference Bandelt, Forster and Röhl1999, http://www.fluxus-engineering.com/index.htm) with default conditions. The pairwise fixation index Fst, analysis of molecular variance (AMOVA) and Mantel test were estimated using ARLEQUIN 3.11 (Excoffier et al., Reference Excoffier, Laval and Schneider2005). To test significance, the number of permutations was 1000. Unrooted neighbour-joining (NJ) trees were constructed based on Fst values using TreeFit (Kalinowski, Reference Kalinowski2009).
Migration rate between areas (2NM) were calculated using Isolation-with-Migration model (IMa2) (Hey, Reference Hey2010). Mitochondrial mutation rate per locus per year were estimated 0.001434432 (BPH, 1,928 bp/locus) and 0.001433688 (WBPH, 1,927 bp/locus), under the conditions that mitochondrial mutation rate was 6.2×10−8/single site/fly generation (Haag-Liautard et al., Reference Haag-Liautard, Coffey, Houle, Lynch, Charlesworth and Keightley2008) and rice planthoppers would produce 12 generations per year. We used the HKY model, burnin period steps 100,000 run duration 150,000. Parameters were set based on population mutation rates (4Neu) estimated using Arlequin3.11 (Excoffier et al., Reference Excoffier, Laval and Schneider2005). Migration prior value was 0.56 and 0.76, maximum time of population splitting 2.24 and 3.02, and maximum for population size parameter 5.6 and 7.55 for BPH and WBPH (random number seed was 1234).
Results
Nucleotide sequences of the cox1–trnL2–cox2 region of the mitochondrial genome were determined in 31 BPH populations (579 individuals) and 25 WBPH populations (464 individuals). There were no insertions or deletions in the sequences; the sizes were 1,928 bp and 1,927 bp for BPH and WBPH, respectively. There were no unexpected stop codons in the mitochondrial cox1 and cox2 genes of all haplotypes obtained in this study. While all sequences were used for haplotype network analyses, the Ja, Jb, Jc and Jd populations (table 1) were excluded for analysis of pairwise Fst, AMOVA, Mantel test and IMa2 because they were reared in the laboratory for a long time (8–40 years).
BPH: genetic diversity
We detected 30 haplotypes of the mitochondrial cox1–trnL2–cox2 region in BPH (DNA database accession numbers AB572299–AB572328) (table 3; supplementary table 1). The most frequently identified haplotype (haplotype 1 of BPH) was detected in 58.9% of the individuals (341/579 individuals). Polymorphic nucleotide sites in the other haplotypes were denoted by comparing the sequences with that of haplotype 1. For example, when thymine at the 257th position in haplotype 1 was altered to adenine, the variation was shown as T257A (supplementary table 1). Haplotype 1 was shared by all populations except C2 and PNG (table 3). Population-specific haplotypes were observed in C2 (haplotype 12) and PNG (haplotype 16) populations, although the number was small in C2 (n=3). Among the other 29 populations, 26 populations showed two to six haplotypes, and three populations, Jb, Jc and Jd, which had been reared in the laboratory for a long time, showed only haplotype 1. The numbers of variable sites were 28 in cox1, 12 in cox2 and 0 in trnL2. Thirty-nine variable positions were binary, and only C103 was ternary (C103T and C103A). Four sites in cox1 and one in cox2 were non-synonymous (supplementary table 1).
Table 3. The number of haplotypes in each population of BPH.
Haplotypes 1–30 correspond to DNA accession numbers AB572299–AB572348, respectively.
BPH: haplotype networks
An MJ haplotype network was created for 30 BPH haplotypes (fig. 2). A star-like phylogeny was obtained in the BPH network, in which haplotype 1, the most frequent haplotype, was centred. Thus, in the star phylogeny, the ancestral haplotype was centred and the other derived haplotypes had close connections to the former, implying that the populations were subject to recent expansion and/or selective mutation, (Slatkin & Hudson Reference Slatkin and Hudson1991; Rogers & Harpending Reference Rogers and Harpending1992; Avise Reference Avise2000; Mousset et al., Reference Mousset, Derome and Veuille2004). Five haplotypes (haplotypes 8 and 13–16) are connected to haplotype 1 via median vectors (mv) 1, 2 and/or 3 that were not present in samples. BPH haplotypes 8, 13 and 15 were all detected only in the Philippines, except haplotype 15 that was found in one population from Vietnam (Vn2). Haplotype 16 was only detected from PNG populations, and haplotype 14 was observed in Ja and Ps2.
Fig. 2. Haplotype network for BPH (n=579). Circle sizes are proportional to frequencies of haplotype sequences (1–30). Single line shows a predicted one-nucleotide substitution. The median vectors (mv) were not present in the samples. Haplotypes 8 and 13–16 were specifically detected in the populations shown within dotted squares.
BPH: genetic structure
All pairwise Fst values (Wright, Reference Wright1978) were calculated for BPH populations in the ten areas (table 4). An unrooted NJ tree was constructed based on Fst values (fig. 3). The Fst values between populations from PNG and all other areas were significantly high (more than 0.579, P<0.0001). Significantly high Fst values were also shown between populations in Ps and all other areas (0.176–0.579). Although the analysis indicated differences in genetic structure among populations from other eight areas, including Japan, Taiwan, China, Vn, Vs, Laos, Thailand and Pn, Fst values were relatively low (<0.125, which was between Thailand and Pn). In the eight areas, a hierarchical AMOVA showed that the variation attributed much more within populations (ΦST, 79.7%), than among populations within areas (ΦSC, 19.6%) and among areas (ΦCT, 0.74%). ΦST and ΦSC were significant (P<0.00001), on the other hand, ΦCT was not significant (P=0.312) (supplementary table 3), indicating no genetic structure among the areas. Mantel test was not significant in 24 populations of the eight areas (P=0.663), showing no correlation between Fst values and geographic distance among the populations. Migration rate (2NM) among areas using IMa2 showed significant level of gene flow of BPH: Taiwan>Pn (2NM=0.873, P<0.01), Vn>Thai (2NM=0.708, P<0.05), Vs>Thai (2NM=0.739, P<0.05), Pn>Japan (2NM=0.853, P<0.05), Pn>China (2NM=0.812, P<0.05), Pn>Ps (2NM=0.692, P<0.05), Ps>Pn (2NM=1.098, P<0.001) (supplementary table 4).
Fig. 3. Unrooted NJ trees based on Fst values of ten areas for BPH.
Table 4. Pairwise fixation index (Fst) values in BPH populations in ten areas.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; NS, not significant; Fst>0.200 values are indicated in bold.
WBPH: genetic diversity
We found 20 haplotypes in the mitochondrial cox1–cox2 region in WBPH (DNA database accession numbers AB572329–AB572348) (table 5; supplementary table 2). The most frequent haplotype (haplotype 1 for WBPH) was found in 48.5% of all WBPH individuals tested (225/464 individuals). The WBPH haplotype 1 was observed in all populations, except Jb, Jc and Vs2. Population-specific haplotypes were shown in Jb and Jc, which have been reared in the laboratory for a long time. One to seven haplotypes were observed in each of the other 23 populations. Eighteen and two polymorphic sites were present in cox1 and in cox2, respectively. All the variable positions were binary. Amino acid variable sites were two and one in cox1 and cox2, respectively (supplementary table 2).
Table 5. The number of haplotypes in each population of WBPH.
Haplotypes 1–20 correspond to DNA accession numbers AB572329–AB572348, respectively.
WBPH: haplotype network
Three major haplotypes (haplotypes 1, 8 and 14) were found in the WBPH MJ network (fig. 4). There was a single substitution between haplotypes 1 and 8 and between haplotypes 8 and 14; the other haplotypes were their satellites. The WBPH haplotypes 8 and 14 were the second (16.2%) and third (14.4%) most prevalent haplotypes, and were as widely distributed as haplotype 1. Haplotype 8 was detected in all areas except Laos, and haplotype 14 was detected in all areas except Vn (table 5).
Fig. 4. Haplotype network for WBPH (n=464). Circle sizes are proportional to frequencies of haplotype sequences (1–20). Single line shows a predicted one-nucleotide substitution.
WBPH: genetic structure
Pairwise Fst values were calculated for all combinations of the ten areas with WBPH. Although some values were significant, the highest value was less than 0.235 (between Taiwan and Vs) (table 6). In contrast to the findings for BPH, WBPH populations PNG and Ps populations did not show high Fst values against those from other areas (table 6). An unrooted NJ tree was constructed based on Fst values (fig. 5). For WBPH ten areas, a hierarchical AMOVA showed that the variation attributed much more within populations (ΦST, 85.01%), than among populations within areas (ΦSC, 15.13%) and among areas (ΦCT, −0.13%) (supplementary table 3), ΦST and ΦSC were significant (P<0.00001), on the other hand, ΦCT was not significant (P=0.546) (supplementary table 3), indicating no genetic structure among the ten areas. Mantel test was not significant among 23 populations of the ten areas (P=0.384). IMa2 showed significant migration rate (2NM) among Japan>China (2NM=0.21, P<0.05) (supplementary table 4).
Fig. 5. Unrooted NJ trees based on Fst values of ten areas for WBPH.
Table 6. Pairwise fixation index (Fst) values in WBPH among ten areas.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; NS, not significant; Fst values >0.2000 are indicated in bold.
Discussion
Our aims were to distinguish regional populations of planthoppers that show different biological properties, for example, virulence against rice plants or insecticide resistance, and to estimate the annual migratory route of BPH and WBPH using simple molecular markers. An earlier study showed that the ITS region of rRNA genes was not a good candidate as a molecular marker, because it was highly variable among the rice planthopper individuals and did not contain features specific to populations (Fu et al., Reference Fu, Matsumoto, Matsumura, Hirai, Sato and Noda2012). In the present study, mitochondrial sequences were selected as alternative candidate molecular markers. The mitochondrial cytochrome oxidase region in BPH populations in southern Philippines showed differentiation from those in other areas in pairwise Fst values. However, no specific haplotypes were related to regional populations, except haplotype 16 in PNG.
No genetic structure was indicated among BPH population from eight areas, excluding Ps and PNG, and major part of variance was observed at the population level (supplementary table 3). In the case of WBPH, some differentiation was shown among areas (table 6), however, no genetic structure was indicated among ten areas, and most variation was present at population level (supplementary table 3). Northern Vietnam area is considered as the primary source region of rice planthopper migrants to East Asia, including Japan, Korea and Northern China (Kisimoto Reference Kisimoto1976; Watanabe & Seino, Reference Watanabe and Seino1991; Sogawa, Reference Sogawa1995; Otuka et al., Reference Otuka, Watanabe, Suzuki, Matsumura, Furuno and Chino2005b, Reference Otuka, Matsumura, Watanabe and Ding2008). Low genetic differentiation and the genetic structure among BPH and WBPH populations from most areas studied here are consistent with the hypothesis that East Asian planthoppers are derived from northern Vietnam; however, the data reported here did not conclusively support it. IMa2 analysis showed significant gene flow among some areas (supplementary table 4), however, it did not show clear migration route of rice planthoppers. To our knowledge, the present study is the first extensive study on mitochondrial sequences of Asian planthopper populations. Our analyses, which utilized a larger sample size and a longer mitochondrial genomic sequence, did not yield a novel molecular marker for discrimination of planthopper populations, similar to the previous report by Mun et al. (Reference Mun, Song, Heong and Roderick1999). The data indicate that respective BPH and WBPH partly share a gene pool in Asia, and that the mitochondrial cox1–trnL2–cox2 region did not provide sufficient resolution for planthoppers populations.
The insecticide resistance in BPH and WBPH populations collected from East and Southeast Asia (Japan, China, Taiwan, Vietnam and the Philippines) has been reported (Matsumura et al., Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008; Matsumura & Sanada-Morimura, Reference Matsumura and Sanada-Morimura2010). Examination of insecticide resistance levels in 16 populations of BPH revealed resistance against O-sec-butylphenyl methyl carbamate and less resistance against phenylpyrazole (fipronil). Twelve populations of BPH, collected in Japan, China, Taiwan and Vietnam, showed resistance against neonicotinoid insecticides (imidacloprid and thiamethoxam), and four populations in the Philippines were susceptible (Matsumura et al., Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008). Among the 16 populations of BPH previously tested for insecticide resistance (Matsumura et al., Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008), individuals of 11 (J1, T1, T3, C1, C2, Vn1, Vn2, Vs1, Pn1, Pn2 and Pn4) were used in the present study. We did not find a clear correlation between mitochondrial haplotypes and insecticide resistance level. For example, a majority of individuals from the Philippines populations, which showed a relatively high genetic distance from other populations (Table 4), were of haplotype 1, present also in most of the other populations. The level of insecticide resistance is usually related to the amount and/or duration for which the corresponding insecticide has been used, whereas the mitochondrial genetic structure of populations reflects genetic flow over a longer time scale. Differences in the temporal scale of development of the two biological traits might contribute to the discrepancy of their distributions within populations. An additional layer of complexity is added by the gene exchange brought about by the long-distance migration of BPH.
In WBPH, neonicotinoid resistance is not as prevalent, however, resistance to phenylpyrazole has been observed widely in East and Southeast Asia. The individuals of the same 11 populations that were used for the insecticide resistance study (J4, T1, T4, T5, C1, C2, Vn2, Vs2, Pn2, Pn3 and Pn4; Matsumura et al., Reference Matsumura, Takeuchi, Satoh, Sanada-Morimura, Otuka, Watanabe and Van Thanh2008) were examined in the present study. Our analysis revealed relatively low genetic distance among these populations, indicating that simple molecular markers, such as mitochondrial sequences, may not be useful for insecticide-resistance monitoring in WBPH.
Other important issues in planthopper management are rice resistance against planthoppers, and virulence of planthoppers against the resistant rice varieties (Sogawa, Reference Sogawa1992; Zhang, Reference Zhang2007). The populations of BPH Ja, Jc and Jd, which had been maintained in the laboratory, were tested for their virulence against rice varieties (Myint et al., Reference Myint, Yasui, Takagi and Matsumura2009; Naeemullah et al., Reference Naeemullah, Sharma, Tufail, Mori, Matsumura, Takeda and Nakamura2009). These studies reported that the BPH Ja population, which has been reared from 1966, showed avirulence against all the rice varieties examined. The Jc population, which was collected in 1989, was virulent against the Mudgo rice variety carrying the BPH-resistant gene BPH1, and Jd, which was collected in 1999, was virulent against the ASD7 rice variety carrying bph2. Myint et al. (Reference Myint, Yasui, Takagi and Matsumura2009) pointed out that virulence status is not affected substantially by long-term mass rearing in the laboratory. However, similar to the findings for insecticide resistance, mitochondrial haplotype analysis does not seem to be useful in estimating the biotypes for resistant rice varieties.
Mitochondrial gene sequences are useful molecular markers for distinguishing populations in some insect species (Pramual et al., Reference Pramual, Kuvangkadilok, Baimai and Walton2005; Cai et al., Reference Cai, Cheng, Xu, Duan and Kirkendall2008; Lohman et al., Reference Lohman, Peggie, Pierce and Meier2008; Meraner et al., Reference Meraner, Brandstätter, Thaler, Aray, Unterlechner, Niederstätter, Parson, Zelger, Dalla Via and Dallinger2008; Nolan et al., Reference Nolan, Dallas, Piertney and Mordue (Luntz)2008). However, in long-distance-migrating rice planthopper species, BPH and WBPH, mitochondrial DNA appears to be less useful. Our interests focus on aspects of planthopper biology important to agriculture, e.g., insecticide resistance or mechanisms of virulence against resistant rice varieties. Genes responsible for these biological phenomena and other genomic regions closely linked to these genes are expected to provide more informative markers for distinguishing resistant individuals and populations.
The supplementary material for this article can be found at http://www.journals.cambridge.org/BER
Acknowledgements
The present work was supported by Grants-in-Aid for Scientific Research (No. 21380039) from the Japan Society for the Promotion of Science.
