Quantitative trait loci identification, fine mapping and gene expression profiling for ovicidal response to whitebacked planthopper (Sogatella furcifera Horvath) in rice (Oryza sativa L.)
- Yaolong Yang†1,
- Jie Xu†1,
- Yujia Leng1,
- Guosheng Xiong2,
- Jiang Hu1,
- Guangheng Zhang1,
- Lichao Huang1,
- Lan Wang1,
- Longbiao Guo1,
- Jiayang Li2,
- Feng Chen3,
- Qian Qian1 and
- Dali Zeng1, 2Email author
© Yang et al.; licensee BioMed Central Ltd. 2014
Received: 17 January 2014
Accepted: 22 May 2014
Published: 28 May 2014
The whitebacked planthopper (WBPH), Sogatella furcifera Horváth, is a serious rice pest in Asia. Ovicidal resistance is a natural rice defense mechanism against WBPH and is characterized by the formation of watery lesions (WLs) and increased egg mortality (EM) at the WBPH oviposition sites.
This study aimed to understand the genetic and molecular basis of rice ovicidal resistance to WBPH by combining genetic and genomic analyses. First, the ovicidal trait in doubled haploid rice lines derived from a WBPH-resistant cultivar (CJ06) and a WBPH-susceptible cultivar (TN1) were phenotyped based on the necrotic symptoms of the leaf sheaths and EM. Using a constructed molecular linkage map, 19 quantitative trait loci (QTLs) associated with WLs and EM were identified on eight chromosomes. Of them, qWL6 was determined to be a major QTL for WL. Based on chromosome segment substitution lines and a residual heterozygous population, a high-resolution linkage analysis further defined the qWL6 locus to a 122-kb region on chromosome 6, which was annotated to encode 20 candidate genes. We then conducted an Affymetrix microarray analysis to determine the transcript abundance in the CJ06 and TN1 plants. Upon WBPH infestation, 432 genes in CJ06 and 257 genes in TN1 were significantly up-regulated, while 802 genes in CJ06 and 398 genes in TN1 were significantly down-regulated. This suggests that remarkable global changes in gene expression contribute to the ovicidal resistance of rice. Notably, four genes in the 122-kb region of the qWL6 locus were differentially regulated between CJ06 and TN1 in response to the WBPH infestation, suggesting they may be candidate resistance genes.
The information obtained from the fine mapping of qWL6 and the microarray analyses will facilitate the isolation of this important resistance gene and its use in breeding WBPH-resistant rice.
KeywordsFine mapping Gene expression profiling Ovicidal response qWL6 Sogatella furcifera Horváth Whitebacked planthopper
Rice (Oryza sativa L.) is one of the world’s most important crops, providing a staple food for nearly half of the global population. In Asia, Africa, and Latin America, the demand for rice is expected to increase due to the steadily increasing population . In China, for example, rice production will need to increase by approximately 20% by 2030 to meet the domestic demand if rice consumption per capita remains at its current level . Yet rice production is continually threatened by insects, diseases, and other stresses. In recent years, rice infestations by insects have intensified across Asia, resulting in heavy yield losses .
The whitebacked planthopper (WBPH), Sogatella furcifera Horváth, is a serious rice pest in Asia. It damages the plants by sucking sap from the phloem and transmitting viruses, which lead to reductions in plant height, number of productive tillers, filled grains, and yield [4, 5]. During the tillering stage, a heavy WBPH infestation results in the complete necrosis of rice plants, a condition commonly known as hopper burn [6–8]. The permanent breeding areas for the WBPH are in the tropics, where the population is maintained in the paddy field throughout the year. As an insect that can travel long distances, WBPH migrates from northern Vietnam to southern China, and then to central China and Japan, depending on the southwest monsoon in the rainy season. In temperate regions, WBPHs cannot live through the winter, and they are replaced each year by immigrants from southern regions . In rice production practices, WBPH infestation is managed primarily by the use of chemical pesticides, which are both economically and environmentally costly. Moreover, the pesticides kill WBPH predators, and the overuse of pesticides prompts the evolution of resistance in the insects, which in turn leads to a pest resurgence. Some groups have produced rice plants transformed with Bacillus thuringiensis (Bt) genes for protection against WBPHs . However, the potential ecological risks of transgenic plants may limit the deployment of Bt rice . Thus, the exploitation of host plant resistance has generally been considered one of the most economical and environmentally friendly approaches for the management of WBPHs.
Classical genetic analysis of selected rice accessions has led to the identification of six major WBHP-resistance genes, Wbph1 to Wbph6 . Wbph1 is located on the short arm of chromosome 7 near the RFLP marker, RG146A . Wbph2 is on the short arm of chromosome 6 in ARC10239 , Wbph6 is on chromosome 11 and flanked by RM167 and RM267 . The other three WBHP resistance genes, Wbph3–5, have not yet been mapped to the rice genetic map. In addition to these major WBPH-resistance genes, a number of quantitative trait loci (QTLs) have been identified that are associated with the quantitative resistance of rice to WBPHs. These QTLs were identified by analyzing various rice lines, including recombinant inbred lines (RILs) , doubled haploid (DH) populations , introgression lines using wild rice species as the resistance donor , and backcross-inbred lines (BILs) derived from interspecific crosses involving wild rice species . Despite the lack of molecular identity for any of the WBPH resistance genes, some have been tentatively associated with either tolerance, antibiosis, or antixenosis, the three types of natural rice resistance mechanisms against WBPH .
One type of rice antibiosis resistance to WBPHs is ovicidal resistance, which is characterized by the formation of watery lesions (WLs) that result in the death of the WBPH eggs at those sites within 12 h of oviposition . The egg mortality (EM) depends on the rice developmental stage and is greatest at the maximum tillering stage. This ovicidal response to WBPHs is especially prominent in the japonica cultivars in Japan . In addition, Seino et al. (1996) found that benzyl benzoate was present in the watery lesions of some japonica rice, but was undetectable in the intact plant tissue and non-watery lesions , suggesting benzyl benzoate was the ovicidal substance in the watery lesions.
Regarding the genetic basis of the rice ovicidal response to WBPHs, a total of 15 QTLs have been identified using the rice RILs developed from a cross between the WBPH-resistant japonica variety Asominori and the WBPH-susceptible indica variety IR24 . Four of the 15 QTLs were further shown to be for the ovicidal trait based on the phenotyping for EM . Nevertheless, our understanding of the genetic basis of WL induction for WBPH resistance is extremely limited.
In addition to the continued identification of major resistance genes and QTLs, our general understanding of plant resistance to insect herbivory has significantly improved with the employment of various genomic tools, one of which is global gene expression profiling . For rice, gene expression profiling has been performed to understand the defenses against chewing insects such as the fall armyworm , sap-sucking insect brown planthopper (BPH) , and water weevil . These analyses showed that the defenses against these insects involved global changes in rice gene expression and led to the identification of a large number of candidate defense genes.
Our study of rice resistance to WBPHs has focused on the Chinese japonica rice variety Chunjiang 06 (CJ06), which showed the strongest ovicidal response to WBPHs among the rice lines screened . In addition, CJ06 exhibited sucking-inhibitory resistance to the WBPH, a type of antixenosis resistance . This dual-mechanism of WBPH resistance in CJ06 makes this variety a unique genetic material for studying rice resistance to WBPHs. Based on a cross between CJ06 and TN1, a WBPH-susceptible indica rice, we previously constructed a DH population containing 120 lines. Our previous characterization of the phenotypic expression of WBPH resistance in this DH population  suggested that the combined functions of both the major resistance genes and QTLs affected the host-plant response to infestations by WBPHs.
Building on our previous work, this study had two objectives. The first was to improve our understanding of the genetic basis of rice resistance to WBPHs. We aimed to identify the QTLs associated with the ovicidal response, especially those for WLs, using our CJ06/TN1 DH population. Once identified, these QTLs were mapped to the rice genetic maps through fine mapping. The second objective was to improve our understanding of the molecular basis of rice resistance to the WBPH. To this end, we conducted a microarray analysis to compare the gene expression changes in WBPH-infested and uninfested CJ06 and TN1 plants.
TN1 and CJ06 exhibited distinct ovicidal responses to WBPH feeding
Ovicidal response in the parents and doubled haploid population
2.6 ± 0.55
0.2 ± 0.45**
94.2 ± 13.0
19.2 ± 16.4**
2.8 ± 0.45
0.4 ± 0.55**
93.8 ± 10.8
11.8 ± 14.7**
The distribution of the ovicidal response in the DH population revealed a major locus and multiple minor loci for WBPH resistance
The detection of quantitative trait loci associated with watery lesions and egg mortality
Quantitative trait loci identified in the doubled haploid population for the ovicidal response
Variance explained (%)
Of the identified QTLs, most showed negative additive effects, suggesting the CJ06 alleles may increase the ovicidal response to WBPHs. The loci associated with the WLs that were flanked by RM6176 and RM539 on chromosome 6 presented the largest explained variance and showed additive effects. The LOD (Logarithm of Odds) scores were 12.23 and 12.14 in 2006 and 2007, respectively. Their proportion of the phenotypic variation was over 30%, with the CJ06 allele on chromosome 6 increasing the phenotypic grade for the WLs to approximately 1.6. This major QTL was named qWL6. The QTLs for WBPH EM were also found in this region both years, and the explained EM variation was nearly 25%. The CJ06 alleles on chromosomes 2, 5, and 7 and the long arm of chromosome 1 may strengthen the ovicidal response. We also found that some alleles from the TN1 variety may increase the resistance to WBPH, such as the loci on chromosomes 3, 4, and 10 and the short arm of chromosome 1.
Development of the chromosome segment substitution lines for the major quantitative trait locus qWL6
Fine mapping of qWL6to a 122-kb region
For fine mapping of the qWL6 gene, the SSR marker RM8258 on one side of the qWL6 target region and the Indel marker AP4687 on the other side were used to identify recombination break points in the segregating progeny derived from the residual heterozygous lines. Seedlings (n = 216) selected from 1,440 BC6F2 progeny were transplanted into greenhouse conditions to evaluate their ovicidal resistance to WBPHs. Of these, 41 displayed no visible WLs and were used for further fine mapping. The analysis of RM8258 identified 10 recombination events between it and qWL6 on one side, and the analysis of AP4687 detected 31 recombination events between the Indel marker and qWL6 on the other side. Eight polymorphic markers were available to narrow down the region of the qWL6 locus. AP4280 detected 7 recombinants, whereas Indel marker M4 co-segregated with qWL6. The markers M1, M2, and M3 revealed 4, 2, and 1 recombinants, respectively, on one side, while markers M5, M6, M7, and M8 indicated 1, 2, 5, and 9 recombinants, respectively, on the other side (Figure 5C). Therefore, the qWL6 locus was finally delimited to an approximately 122.1-kb DNA region between the two Indel markers, M3 and M5.
This 122.1-kb region of the Nipponbare rice genome retrieved from the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml) encoded 20 genes in its annotation (gene IDs from LOC_Os06g09910 to LOC_Os06g10109). Two candidate genes, LOC_Os06g09910 and LOC_Os06g09930, were predicted to be phosphopantothenoylcysteine decarboxylase (PPCDC) and a G protein coupled receptor (GPCR), respectively; the others are unknown proteins.
Differentially expressed genes between CJ06 and TN1
Genes differentially expressed in the whitebacked planthopper-infested CJ06 and TN1 varieties
Probe set ID
Defense and stress related genes
Pathogenesis-related thaumatin-like protein
Germin-like protein subfamily 1 precursor
Pathogenesis-related protein precursor
Pathogenesis-related protein PRB1precursor
Anthranilate N-benzoyltransferase protein 1
Multicopper oxidase family protein
Genes involved in cell signaling
S-locus-like receptor protein kinase
ATP binding protein
Leucine Rich Repeat family protein
Transcription factors and genes involved in RNA processing
NAC domain-containing protein 18
Myb-like DNA-binding domain containing
Helix-loop-helix DNA-binding domain
NAC-domain containing protein 90
Transcription factor MYC7E
Genes implicated in hormone signaling
Auxin efflux carrier component 4
Auxin-responsive protein IAA14
Auxin responsive protein, expressed
1-aminocyclopropane-1-carboxylate oxidase 1
VQ motif family protein
Two-component response regulator ARR8
Polymorphic sites of candidate genes and the putative amino acid differences between CJ06 and TN1
Expression patterns of the candidate genes in the 122-kb region
Expression of candidate genes in the interval between the two Indel markers M3 and M5
cDNA/ homologous EST
Probe Set ID
G protein coupled receptor
The WBPH Sogatella furcifera Horváth is a serious insect pest throughout the rice-growing regions of the world, and it has become one of the major threats to rice crops throughout Asia, damaging plants both through its feeding behavior and as a viral vector . The production of resistant varieties is an ecologically sound approach to prevent WBPH infestations . In an effort to use host-plant defenses, attempts to resolve the genetic basis of WBPH resistance in rice have resulted in the identification of the six primary genes Wbph1 to Wbph6 . Sidhu et al. described new sources of major genes conferring resistance to WBPHs in a population prevalent in northern India . However, information regarding the genetic and molecular mechanisms of WBPH resistance in rice is scarce. The major obstacle is in evaluating the resistance to WBPHs; in addition, WBPH resistance in rice has earned a reputation as being difficult to investigate. Nevertheless, QTL analyses using genetic populations derived from crosses of NILs or CSSLs have proven to be powerful tools for investigating the genetic and molecular basis of such quantitative traits [30–32]. Several successful examples of QTL cloning resulted primarily from the development of the corresponding NILs or CSSLs [33–35]. Bph14 is the first rice insect resistance gene to be cloned as the result of a map-based cloning approach . Bph14 encodes a coiled-coil, nucleotide-binding, and leucine rich repeat (CC-NB-LRR) protein and confers resistance to BPHs, another major insect rice pest in Asia.
In this study, we performed QTL mapping for the rice ovicidal response that causes the death of WBPH eggs and finely mapped a major QTL for WL production that was delimited to an approximately 122.1-kb DNA region. Yamasaki et al.  identified a major QTL for the ovicidal response in the interval between R1594 and L688 on the short arm of chromosome 6, which also contains the qWL6 region in our study.
There are two different resistance mechanisms to WBPH in some japonica rice, namely ovicidal resistance and sucking-inhibitory resistance [17, 25]. Ovicidal resistance gives rise to egg mortality in the watery lesions induced at the oviposition sites, while sucking-inhibitory resistance restricts planthopper feeding and colonization on the rice plants . In on our previous work, we identified the Chinese japonica variety CJ06 that has these two independent mechanisms, and subsequently constructed a DH population to examine their performance . In this study, we identified a total of 19 QTLs associated with the WLs and EM on eight chromosomes. The expression of the ovicidal response is somewhat suppressed in the sucking inhibitory variety under natural WBPH infestations in the field, because the strong antixenosis against WBPH females in these lines reduces oviposition rates. Nevertheless, the CSSLs used in the fine mapping could lack the sucking inhibitory antixenotic effects.
Among the identified QTLs, some of the loci have been reported in earlier studies in different populations. The QTLs on the long arms of chromosomes 1, 3, 4, 5, and 6 were reported by Yamasaki [4, 8]. The loci associated with the WLs and EM were flanked by R1954 and L688 in a near-isogenic line (NIL) population , and the QTLs on chromosomes 2 and 7 were identified by Geethanjali . Some QTLs co-localized with those for BPH resistance, such as the locus on chromosome 4 (RM401–RM6997) described by Huang . Tan et al.  also showed that two WBPH-resistance genes in rice share the same loci with those for BPH resistance, suggesting the possibility of common loci conferring resistance to both WBPH and BPH in rice. An analysis of the QTL information for both planthoppers in the same mapping population would help to verify this hypothesis.
It is now known that the responses by rice to BPH feeding are most likely similar to pathogen-defense responses [36, 40]. For example, Bph14 is a member of the CC-NB-LRR disease resistance gene family, and it provides resistance to BPH in a mechanism that is fundamentally similar to defense mechanisms against pathogens that activate a salicylic acid-dependent pathway .
In this study, qWL6 was delimited to a 122.1-kb DNA region which contains 20 open reading frames. Two candidate genes, LOC_Os06g09930 and LOC_Os06g09910, were annotated to encode PPCDC and GPCR, respectively. PPCDC belongs to the lyase family, specifically the carboxy-lyases, and catalyzes the decarboxylation of 4′-phosphopantothenoylcysteine to form 4′-phosphopantotheine. In addition, it can act as an inhibitory subunit of the protein phosphatase Ppz1, which is involved in many cellular processes such as the G1-S phase transition and salt tolerance . GPCRs are found only in eukaryotes and are involved in signal transduction. Interestingly, there were no significant differences in the transcript levels of LOC_Os06g09930 and LOC_Os06g09910 between CJ06 and TN1. Nine other unknown genes also had no significant differences in expression. However, using an Affymetrix microarray, four of the unknown genes in the chromosome region containing qWL6 were found to be differentially expressed between CJ06 and TN1, and real-time RT-PCR authenticated that their differential expression was induced by WBPH feeding. Whether the mapped qWL6 gene is a homologous gene of known function or a new gene encoding a protein of unknown function should be determined by a genomic sequence analysis and functional complementation assays.
We examined the differential expression of genes responding to a WBPH infestation between the highly resistant CJ06 variety and the susceptible TN1 variety. The gene expression in CJ06 showed more overall activity than in TN1, irrespective of the presence or absence of WBPHs (Additional file 2: Figure S1), and a higher number of differentially induced genes (up- and down-regulated) were found in CJ06 than in TN1. In addition, the expression of genes related to secondary metabolism, defense, and cell and hormone signaling showed prominent differences between CJ06 and TN1, whether infested WBPHs or not. An Asian rice gall midge infestation also elicited diverse responses in rice that involved the induction of genes in primary metabolism, nutrient metabolism and transport, DNA synthesis, defense, and secondary metabolism . Many genes implicated in defense and stress had higher expression levels in the resistant CJ06 variety, even absent a WBPH infestation, and these genes were found to be even more strongly expressed after feeding by WBPH. The results for genes related to secondary metabolism, cell signaling, translation, and protein turnover were similar.
Some differentially expressed genes were anchored in the identified QTL regions. For example, LOC_Os01g64310 and LOC_Os10g11500 localized to the QTL region for WLs between RM5389 and RM401 on chromosome 1 and the region for EM between RM216 and RM467 on chromosome 10, respectively. Some differentially expressed genes related to secondary metabolism and defense were also anchored in the QTL regions (Additional file 3: Figure S2). However, among the genes that were highly differentially induced, none were located in the qWL6 interval. In addition, some nonsynonymous nucleotide substitutions were identified between the resistant CJ06 and susceptible TN1 varieties, implying a functional difference in the resulting amino acid sequences may play an important role in WBPH resistance.
These results provide important genetic information for improving rice in that the markers tightly linked to qWL6 could facilitate the incorporation of ovicidal alleles into rice breeding lines and the selection of plants with the ovicidal response. The WBPH populations on some of the varieties derived from the crosses between the indica and japonica varieties were six times higher than those on the ovicidal japonica variety Reiho , demonstrating that qWL6 is crucial for japonica varieties. In future breeding, when transferring useful indica genes into japonica varieties, the improved lines must retain the japonica ovicidal allele at qWL6 to suppress the proliferation of WBPHs. It would also be feasible to transfer qWL6 into the non-ovicidal indica varieties by marker-assisted selection as a means of suppressing the WBPH population in those cultivated areas. The identification of qWL6 and the ovicidal QTLs is an initial step facilitating the positional cloning of a gene and QTLs that confer resistance to insect oviposition in rice by utilizing the Indel markers linked to qWL6 and the ovicidal QTLs as the starting points. This positional cloning would also clarify the molecular and genetic mechanisms of the ovicidal response to WBPH.
We identified 19 QTLs associated with WLs and EM in two different years, and qWL6 was identified as a major QTL in the rice response to infestation by WBPH. Based on the CSSLs and residual heterozygous population, qWL6 was delimited to a 122-kb region on chromosome 6. An Affymetrix microarray analysis showed that the resistant-CJ06 and susceptible-TN1 varieties had different responses to WBPH feeding. In addition, four genes in the 122-kb region of the qWL6 locus were differentially regulated in CJ06 and TN1 in response to WBPH infestation, suggesting they may be candidate resistance genes. These results will facilitate isolating this important resistance gene and its use in breeding WBPH-resistance rice.
The WBPH-resistant japonica CJ06 and WBPH-susceptible indica TN1 varieties were used as parents to make hybrids. The anthers from the F1 plants were collected and cultured on the inducing medium SK3. Doubled plants were obtained through natural doubling or by treatment with colchicine . This DH population consisted of 170 lines, 120 of which were used for constructing a linkage map and evaluating the ovicidal response at random. This population has also been used for studies on ligule length and leaffolder resistance [42, 43].
Evaluation of the ovicidal response
The evaluation for WBPH resistance was performed as described by Sogawa  with minor modifications. The ovicidal response of the rice leaf sheaths at WBPH oviposition sites leads to necrotic discoloration, and the intensity of necrotic symptoms was visually rated from 0 to 3 based on the following categories: 0, no visible necrotic symptoms; 1, brownish oviposition damage, but no WLs; 2, discontinuous WLs; 3, conspicuous, vertically elongated WLs. An average score below 1.0 was defined as the non-ovicidal response, while scores above 1.0 were regarded as an ovicidal response.
EM was measured by using young plants at the early tillering stage, which were grown individually in disposable plastic cups (7 cm diameter, 9 cm high). Gravid females were individually confined on the upper portion of the leaf sheath of each plant with parafilm sachets (2 cm × 2 cm) and permitted to feed and lay eggs for two days at room temperature (26–30°C). Approximately fifteen plants were used for each line. The EM was calculated by counting the live and dead eggs 5–6 days after oviposition by dissecting the leaf sheath tissues at the oviposition sites. Eggs with reddish eye-spots were recorded as developing live eggs, while white opaque eggs were recorded as dead. A 60% EM was defined as the cut-off value to distinguish resistance from non-resistance.
Development of chromosome segment substitution lines
CSSLs were developed to obtain a pure genetic background and lessen the effect of other minor QTLs. The plants carrying the TN1 genotype flanking qWL6 were crossed with CJ06 followed by five successive backcrosses. Simultaneously, the SSR markers RM6176 and RM539 were used to initially identify plants having the TN1 genotype in the backcrossed progeny. A set of 74 uniformly distributed SSR markers (Additional file 4: Table S1) on a previous linkage map  were used to select individuals in the BC5F1 families containing as little TN1 DNA in the genetic background as possible.
DNA extraction and PCR analysis
The preparation of genomic DNA for the large screen to select recombinants was performed as previously described . The refined DNA was extracted from fresh rice leaves with the CTAB method . PCR was performed in 20-μl reactions containing 0.2 mM of each primer; 200 mM dNTP mix; 50 mM KCl; 10 mM Tris–HCl, pH 8.3; 1.5 mM MgCl2; 0.1% Triton X-100; and 1 unit of Taq polymerase. The PCR program began with an initial denaturation at 94°C for 5 min, which was followed by 40 cycles of 94°C for 1 min, 55°C for 45 s, and 72°C for 50 s with a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis on 3.0–5.0% (w/v) agarose gels and stained with ethidium bromide.
Primers were designed around qWL6 on chromosome 6 to distinguish the CJ06 and TN1 alleles (Additional file 4: Table S1). The Indel markers were developed based on the sequence differences between indica var. 93–11 and japonica var. Nipponbare (http://blast.ncbi.nlm.nih.gov/. nih.gov). The sequences were aligned using the SeqMan program of DNAstar (Gene-Codes) to identify the insertions/deletions, and primers flanking the Indels were designed using the Primer Premier 5.0 program and tested on the parents.
RNA extraction for the GeneChip analysis
Five CJ06 plants and five TN1 plants at the active tillering stage were transplanted into small separate cages enclosed with white nylon mesh, and approximately 100 gravid WBPH females were put into each cage to infest the plants. After 5 days, the upper portion of the leaf sheaths was sampled, pooled, and stored in liquid nitrogen. The leaf-sheath samples from the uninfested rice plants were collected and used as controls. Total RNA was extracted using the TRIzol® reagent and high quality RNA was sent to CapitalBio Corporation for Affymetrix GeneChip Expression Analysis. A total of four chips were used in the experiment, representing both the infested and uninfested parents, CJ06 and TN1.
Quantitative real-time RT-PCR
The expression levels of four genes (LOC_Os06g09960, LOC_Os06g09970, LOC_Os06g10000, and LOC_Os06g10109) were analyzed using real-time quantitative RT-PCR as a validation of the microarray results. The primers designs are listed in Additional file 5: Table S2. The quantitative assay of the transcript abundance was performed with l μl of each cDNA diluted with SYBR Green Master mix (USA) and assayed with an ABI 7900 sequence detection system according to the manufacturer protocol (Applied Biosystems, USA). Actin mRNA was used as an internal control. The relative quantification method (∆∆CT) was used to evaluate the relative abundance of the transcripts .
Data analysis and QTL mapping
An analysis of variance of all phenotypic characters was performed with the JMP statistical package, version 7.0 for Windows (SAS Institute Inc., Cary, NC, USA). Interval QTL mapping was performed for the WLs and EM with MapMaker/QTL 1.1 software. The presence of a QTL was defined as an LOD score larger than 2.0. The genetic variance explained by each QTL and the QTL additive effects were calculated, and the identified QTLs were named according to the standard nomenclature .
Availability of supporting data
The data sets supporting the results of this article are available in the qWL6_FineMapping_DataSets repository in http://www.cnrri.org/upload/2014/qWL6_FineMapping_DataSets.rar.
Coiled-coil, nucleotide-binding, and leucine rich repeat
G protein coupled receptor
Logarithm of Odds
Quantitative trait locus
Recombinant inbred line
Reverse transcription polymerase chain reaction
Simple sequence repeat
We thank Pro Kazushige Sogawa (Japan International Research Center for Agricultural Sciences, Japan) for helping to evaluate the ovicidal response. This work was supported by grants from the Ministry of Agriculture of China for transgenic research (No.2013ZX08009003-001), the National Natural Science Foundation of China (31221004, 31171531) and the State Key Basic Research Program (2013CBA01403).
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