2025 Volume 75 Issue 5 Pages 369-377
The brown planthopper (Nilaparvata lugens (Stål); BPH) is a serious insect pest of rice (Oryza sativa L.). Host plant resistance is an effective means of controlling this pest; up to date at least 46 BPH resistance genes have been identified. However, BPH can overcome resistance conferred by single resistance genes. Therefore, it is necessary to detect new and durable resistance genes. Here, we identified quantitative trait loci (QTLs) for BPH resistance from the wild rice Oryza nivara (Sharma et Shastry) IRGC 89073. Using backcrossed populations derived from IRGC 89073 × the susceptible ‘Taichung 65’, we detected two resistance QTLs, qBPH4 on chromosome 4 and qBPH11 on chromosome 11. qBPH11 was validated in the BC3F2 population. A near-isogenic line carrying qBPH11 showed significant resistance to BPH in antibiosis, antixenosis, MSST, and honeydew tests. These results suggest that IRGC 89073 harbors valuable genetic resources that could enhance BPH resistance in rice breeding programs, particularly through pyramiding strategies using marker-assisted selection.
Rice (Oryza sativa L.) is a staple food for more than half of the world’s population. However, its production is threatened by the brown planthopper (BPH), Nilaparvata lugens (Stål), a major insect pest that feeds on the phloem sap and significantly reduces grain yield. In severe infestations, “hopperburn” occurs, when rice plants dry out and die. BPH can also transmit Rice grassy stunt virus (RGSV) and Rice ragged stunt virus (RRSV), which can further decrease yield (Cabauatan et al. 2009). Severe yield losses caused by BPH outbreaks have been reported in recent years in several rice-producing countries, including India, Indonesia, China, Japan, Vietnam, Korea, and Bangladesh (Sogawa 2015). While chemical pesticides are the primary method used to control BPH, their use poses both economic and environmental challenges. Excessive use of insecticides not only increases production costs but also harms natural enemies, leading to ecological imbalances and the resurgence of more virulent biotypes of BPH (Tanaka et al. 2000). In contrast, using host plant resistance provides a more sustainable and environmentally friendly strategy for effective and long-term BPH management.
Since the late 1960s, scientists have been screening and identifying BPH resistance germplasm from wild rice species and cultivars across Southeast Asia (Brar and Khush 1997). At least 46 genes associated with BPH resistance (designated BPH1 to BPH46) have been identified (Fujita et al. 2013, Hu et al. 2016, Yan et al. 2023). The derivation of over half of these genes from wild rice species suggests their rich genetic diversity and potential as a valuable source of new alleles for BPH resistance (Brar and Khush 1997). However, only a small number confers broad-spectrum effectiveness in monogenic rice lines due to the emergence of new BPH biotypes in Asia (Fujii et al. 2021, Nguyen et al. 2019). This evidence highlights the vulnerability of single resistance genes to BPH. Therefore, identifying new durable resistance genes and using them in pyramiding breeding programs are necessary to ensure stable rice production.
O. sativa was domesticated from Oryza rufipogon in Asia. O. rufipogon includes both perennial and annual forms. The annual form was previously classified as a separate species, Oryza nivara (Sharma et Shastry). In this study, we continue to use the name O. nivara to refer to the annual form, in line with earlier classifications and to maintain consistency with previous studies. Both forms are distributed mainly across South and Southeast Asia (Grillo et al. 2009). Several studies have identified BPH resistance genes derived from O. rufipogon, namely BPH27, BPH29, BPH35, BPH36, BPH38, and BPH41 (Huang et al. 2013, Li et al. 2019, Wang et al. 2015, 2022, Yang et al. 2020, Zhang et al. 2020) —and from O. nivara: BPH34, BPH39(t), BPH40(t) and BPH45 (Akanksha et al. 2019, Kumar et al. 2018, Li et al. 2023). These findings highlight the importance of these wild species as valuable sources of genetic resistance.
In this study, we focused on the resistant QTLs, qBPH11 and qBPH4, in an accession of O. nivara, IRGC 89073 and validated their resistance through QTL analysis, providing theoretical insights for BPH resistance breeding. We previously found that IRGC 89073 has strong resistance to the highly virulent Koshi-2013 BPH population. To reveal the genetic basis of this resistance, we carried out quantitative trait locus (QTL) analysis using backcrossed populations derived from a cross between O. sativa ‘Taichung 65’ (T65) and IRGC 89073. We validated and characterized one QTL in a near-isogenic line (NIL). The findings provide valuable genetic information for breeding BPH-resistant rice cultivars through marker-assisted selection.
We crossed O. nivara IRGC 89073 from Lao PDR (provided by IRRI) with the susceptible T65, and backcrossed the F1 plants to T65. We evaluated BC1F1 plants for resistance to the highly virulent Koshi-2013 BPH population (Fujii et al. 2021). Resistant plants were further backcrossed to T65 to generate a BC2F1 population. We evaluated 47 BC2F1 plants (from the BC1F1 plant with the highest resistance level) for resistance to BPH and used them for QTL analysis.
We crossed nine BC2F1 plants with BPH resistance in the antibiosis test with T65 to produce BC3F1 plants. Among these plants, we selected one plant carrying qBPH11 (named BC3F1-11) by marker-assisted selection to generate a BC3F2-11 population for validating the QTL. From this population, we selected a plant homozygous for qBPH11 to develop a NIL at BC3F3-11 and characterized it. To detect any additional loci associated with BPH resistance, we selected a BC3F1 plant without the qBPH11 (named BC3F1-4) and self-pollinated it to produce BC3F2-4 and BC3F3-4 populations for QTL analysis (Fig. 1).
Breeding schemes for development of segregating populations and near-isogenic line derived from ‘Taichung 65’ × IRGC 89073. We named the populations for qBPH11 BC3F1-11, BC3F2-11 and BC3F3-11; and for qBPH4 BC3F1-4, BC3F2-4 and BC3F3-4.
Total DNA of the backcrossed populations and parents was extracted by using the potassium acetate method (Dellaporta et al. 1983). We used polymerase chain reaction and agarose gel electrophoresis, as described in a previous study (Nguyen et al. 2019) to genotype the backcrossed populations using SSR markers. We tested 384 SSR markers distributed across all 12 rice chromosomes for polymorphism between donor parent IRGC 89073 and recurrent parent T65 (Supplemental Table 1). We genotyped 47 BC2F1, 154 BC3F2-11, and 132 BC3F2-4 plants by markers polymorphic between parents (McCouch et al. 2002, Temnykh et al. 2001).
BPH populations used to evaluate plant resistanceWe evaluated resistance to two BPH populations. The Hadano-1966 population, collected from Hadano City in Kanagawa Prefecture in 1966, was captured before the release of rice cultivars with BPH resistance and has weak virulence. The Koshi-2013 population, collected from Koshi City in Kumamoto Prefecture in 2013, has overcome the BPH1 and BPH2 resistance genes (Fujii et al. 2021). Both populations were maintained on the susceptible japonica ‘Reiho’ at 25°C under a 16-h light/8-h dark cycle at the National Agriculture and Food Research Organization. Both populations have been maintained on T65 under the same conditions at Saga University.
Modified seedbox screening testTo evaluate BPH resistance in the NIL and BC3F3-4 populations, we conducted a modified seedbox screening test (MSST) following the method of Horgan et al. (2015). We sowed 25 seeds in a single row within a plastic tray (23.0 cm × 30.0 cm × 2.5 cm), along with three rows of the susceptible T65 and one row of the resistant IRGC 89073, at a row spacing of 2.5 cm. The positions of all rows were randomized. Seven days after sowing, seedlings in each row were thinned to 20 plants and then infested with second- and third-instar BPH nymphs at a density of 20 insects per plant. When all T65 plants had died, the MSST damage score was recorded using the standard evaluation system for rice (IRRI 2014).
Honeydew testWe performed the honeydew test of Heinrichs et al. (1985) with modifications to evaluate the effects of the NIL and the BC3F2-11 population. We grew lines individually in 215-mL plastic cups with five replications. At 30 days, each plant was enclosed in an inverted transparent plastic cup containing filter paper stained with 0.1% bromocresol green. The filter paper changes from yellow-orange to blue upon contact with honeydew excreted by BPH. Before infestation, Hadano-1966 BPHs were starved for 1 h. Each plant was then infested with two medium-abdomen brachypterous adult female BPHs. After 24 h, the filter papers were collected and the honeydew area was measured in ImageJ v. 1.53a software (National Institutes of Health, Bethesda, MD, USA; https://rsb.info.nih.gov/ij).
Antibiosis testWe conducted antibiosis tests following the method of Tanaka (2000) to see the effects of QTLs associated with BPH resistance. Lines carrying a resistance QTL, along with the parental lines, were individually sown in 215-mL plastic cups, with five replications each. At 30 days, the plants were enclosed in transparent plastic cups and infested with five thin-abdomen brachypterous female BPHs. Adult mortality was determined by calculating the percentage of dead BPHs at 5 days after infestation (DAI).
Antixenosis testOne plant of NIL and one T65 plant were grown together in a 215-mL plastic cup, with five replications. At 30 days, the plants were enclosed in plastic tubes fitted with ventilators. Inside each tube, 20 second-instar BPH nymphs were released. The number of insects that settled on each plant was recorded daily up to 5 DAI. The percentage of insects that settled on each plant was used to determine the level of antixenosis (Nguyen et al. 2021).
Tolerance testWe used the tolerance test of Heinrichs et al. (1985). Individual plants of NIL, T65, and IRGC 89073 were sown in 1-L plastic cups with three replications. At 45 days, each plant was enclosed in a plastic tube with ventilation, and 100 second- and third-instar BPH nymphs were placed in each tube. Another plants of NIL, T65, and IRGC 89073 plants covered with a plastic tube were maintained as controls without infestation. When the susceptible control T65 was completely wilted, the plants were cut at the soil surface and weighed. The percentage of plant fresh weight loss (PFWL), used as an inverse measure of tolerance, was calculated as:
QTL analysis of BC2F1, BC3F2-11, and BC3F2-4 populations using genotypic and phenotypic data in Windows QTL Cartographer v. 2.5 software estimated QTLs by interval mapping (IM) and composite interval mapping (CIM) methods (Wang et al. 2012). The optimal logarithm of odds (LOD) threshold was used to determine the presence of QTLs at threshold values of 2.5 for BC2F1, 2.6 for BC3F2-11, and 3.9 for BC3F2-4.
Statistical analysisThe mean values of BPH resistance of the NIL were compared by one-way ANOVA. The MSST damage score, honeydew area, adult mortality, plant tolerance and antixenosis effect were compared among the NIL and parental lines by Dunnett’s test and the Tukey–Kramer test in R v. 4.3.2 software.
IRGC 89073 exhibited strong resistance to Koshi-2013 population with 100% adult mortality of BPH similar to the known resistant cultivar ‘Rathu-Heenati’ (Fig. 2). In contrast, the T65 was susceptible with 6.7% adult mortality of BPH. This significant difference between T65 and IRGC 89073 confirms the strong antibiosis in IRGC 89073. To identify the genetic basis of its BPH resistance, we evaluated the BC2F1 population by antibiosis test. The frequency distribution of adult mortality at 5 DAI was continuous, ranging from 0% to 100% (Fig. 3, Supplemental Table 2). This distribution suggests that IRGC 89073 has multiple QTLs contributing to BPH resistance. CIM identified qBPH11 between markers RM5582 and RM5349 on chromosome (chr.) 11, with phenotypic variance explained (PVE) = 21.3%. The IRGC 89073 allele reduced adult mortality and increased BPH resistance (Table 1).
Adult mortality of BPH (%) on O. nivara IRGC 89073 infested by Koshi-2013 BPH population at 5 DAI.
Frequency distribution of adult mortality of BPH (%) in the BC2F1 population derived from ‘Taichung 65’ × O. nivara IRGC 89073 infested by Hadano-1966 BPH population. Bars indicate means of parents with SD.
| QTL | Chr. | Marker interval | Physical location (Mb) | LOD score | Phenotypic variance (%) | Dominance effecta |
|---|---|---|---|---|---|---|
| qBPH11 | 11 | RM5582–RM5349 | 17.7–19.0 | 2.5 | 21.3 | 31.1 |
a Positive effect indicates the effect of the allele from O. nivara IRGC 89073.
To confirm qBPH11, we conducted QTL analysis of the BC3F2-11 population. The IRGC 89073 was strong resistance with a honeydew area of 0.0 mm2, whereas T65 was susceptible with a honeydew area of 87.1 mm2. The distribution of honeydew area in the BC3F2-11 population was continuous, ranged from 0.0 to 258.9 mm2 (Fig. 4A, Supplemental Table 3). A single QTL, qBPH11, was detected between RM3083 and RM5582 on chr. 11 (PVE = 41.8%). The IRGC 89073 allele decreased honeydew area and increased BPH resistance (Table 2, Supplemental Table 4).
Frequency distribution of (A) honeydew area in the BC3F2-11 population and (B) damage scores in modified seedbox screening test (MSST) in the BC3F3-4 population derived from ‘Taichung 65’ × O. nivara IRGC 89073 infested by Hadano-1966 BPH population. Bars indicate means in parents with SD.
| QTL | Chr. | Marker interval | Physical location (Mb) | LOD score | Phenotypic variance (%) | Additive effecta | Dominance effecta |
|---|---|---|---|---|---|---|---|
| qBPH11 | 11 | RM3083–RM5582 | 16.4–17.7 | 17.3 | 41.8 | –42.5 | –29.5 |
a Negative effect indicates the effect of the allele from O. nivara IRGC 89073.
To detect any additional QTL in IRGC 89073, we conducted QTL analysis using BC3F3-4 populations that did not carry qBPH11. IRGC 89073 exhibited resistance with a low damage score, whereas T65 was susceptible with high damage score. The distribution of MSST damage scores in BC3F3-4 population was continuous, ranged from 3 to 9 (Fig. 4B). The BC3F1-4 are heterozygous on chrs. 3, 4, 5, and 7 and the genotypes of these regions on BC3F2-4 were investigated for QTL analysis (Fig. 5A). A single QTL for BPH resistance, qBPH4, was identified between RM8213 and RM1305 on chr. 4, with PVE = 68.7%. The IRGC 89073 allele decreased damage score and increased BPH resistance (Table 3, Supplemental Table 5).
Graphical genotypes of (A) qBPH11-NIL at BC3F3-11 and (B) BC3F1-4 population (qBPH4 QTL analysis). The 12 bars indicate the 12 rice chromosomes. Horizontal lines across the chromosomes indicate the positions of polymorphic SSR markers.
| QTL | Chr. | Marker interval | Physical location (Mb) | LOD score | Phenotypic variance (%) | Additive effecta | Dominance effecta |
|---|---|---|---|---|---|---|---|
| qBPH4 | 4 | RM8213–RM1305 | 4.4–5.6 | 33.7 | 68.7 | –2.0 | –0.6 |
a Negative effect indicates the effect of the allele from O. nivara IRGC 89073.
We selected a NIL carrying qBPH11 with the lowest number of IRGC 89073 segments from the BC3F3-11 population (Fig. 5B, Supplemental Table 6). The NIL carrying qBPH11 has introgressed chromosomal segments of IRGC 89073 on chrs. 2, 3, 8, 11, and 12 and size of introgressed chromosomal segment on chr. 11 was 13.4 Mb from RM441 to RM1341. To characterize the resistance mechanism, we conducted honeydew, antibiosis, MSST, tolerance, and antixenosis tests (Fig. 6). In tests using the weakly virulent Hadano-1966 population, the honeydew area was 14.9 mm2 on qBPH11-NIL and 6.3 mm2 on IRGC 89073, both significantly lower than the 72.4 mm2 on T65 (Fig. 6A). Adult mortality was significantly higher on both qBPH11-NIL (62%) and IRGC 89073 (96%) than on T65 (12%) (Fig. 6B). MSST damage scores were significantly lower on qBPH11-NIL (4.5) and IRGC 89073 (3.0) than on T65 (8.38) (Fig. 6C). And significantly fewer BPH settled on qBPH11-NIL (33%) than on T65 (54%) (Fig. 6D). In the tolerance test using the highly virulent Koshi-2013 population, PFWL was nearly equivalent between qBPH11-NIL and T65, and was significantly lower on IRGC 89073 than on T65 (Fig. 6E). The results suggested qBPH11-NIL conferring antibiosis and antixenosis to Hadano-1966 population. However, the NIL had no significant resistance to the Koshi-2013 population in any test, but IRGC 89073 remained significantly more resistant to both BPH populations (Fig. 6, Supplemental Fig. 1).
Effects of qBPH11 in (A) honeydew test, (B) antibiosis test, (C) damage score in modified seedbox screening test (MSST), (D) antixenosis test (Hadano-1966 BPH), and (E) tolerance test (Koshi-2013 BPH). Bars indicate SD. (A, B, C, E) Bars with the same letter are not significantly different between genotypes by Tukey–Kramer multiple comparison test (P < 0.05). (D) Asterisks indicate significant difference between the indicated line and T65: * P < 0.05, *** P < 0.001 by t-test.
To date, at least 46 BPH resistance genes have been identified (Fujita et al. 2013, Yan et al. 2023). However, many known resistance genes have lost their effectiveness against the recent Koshi-2013 BPH population (Nguyen et al. 2019), and more are likely to do so. Therefore, preserving existing resistance gene resources and identifying new BPH resistance genes are necessary for the sustainable long-term control of BPH.
Many O. nivara accessions carry strong resistance to BPH. BPH34, a resistance gene identified from IRGC 104646, confers resistance to the “biotype 4” BPH population collected in Punjab, India (Kumar et al. 2018). Madurangi et al. (2010) found several O. nivara accessions with resistance to BPH populations collected in Sri Lanka. Here, IRGC 89073 had strong resistance to the highly virulent Koshi-2013 BPH population in antibiosis testing (Fig. 2). QTL analysis revealed two QTLs associated with BPH resistance: qBPH4 on chr. 4 and qBPH11 on chr. 11, and we validated qBPH11 (Tables 2, 3).
Most identified BPH resistance genes have been detected on chrs. 3, 4, 6, and 12 (Fujita et al. 2013), and only three genes have been identified on chr. 11 (Supplemental Fig. 2). BPH28(t), from ‘DV85’, was fine-mapped between Indel55 and Indel66 (16.90 Mb–19.96 Mb) (Wu et al. 2014). BPH43, from ‘IRGC 8678’, was mapped between indel markers 16–22 and 16–30 (16.79 Mb–16.90 Mb) (Kim et al. 2022). qBPH11.3, from landrace CL48, was fine-mapped between indel markers 11M16.781 and 11M16.896 (16.75 Mb–16.90 Mb) (Li et al. 2024). Here, we validated qBPH11 between RM3083 and RM5582 (16.43 Mb–17.71 Mb) (Table 2), which overlaps with BPH28(t), BPH43, and qBPH11.3 (Supplemental Fig. 2). qBPH11 may correspond to one of those genes. Fine mapping will be necessary to determine the exact location of qBPH11 and to clarify its allelic relationship with known BPH resistance genes. Several other BPH resistance genes have been identified on the short arm of chr. 4 (Supplemental Fig. 2): BPH12 from B14 (introgression line of O. officinalis) between RM16459 and RM1305 (5.21–5.62 Mb) (Qiu et al. 2012); BPH15 from B5 (introgression line of O. officinalis) between RG1 and RG2 (6.90–6.95 Mb) (Yang et al. 2004); BPH17 from ‘Rathu Heenati’ between RM16493 and RM16531 (6.37–7.93 Mb) (Shar et al. 2024); BPH17-ptb from PTB33 between RM1305 and RM6156 (5.62–7.86 Mb) (Nguyen et al. 2021); BPH35 from RBPH660 between RM3471 and PSM20 (6.31–3.94 Mb) (Zhang et al. 2020); BPH36 from GX2183 between S13 and X48 (6.46–6.49 Mb) (Li et al. 2019); BPH40 from SE232, SE67, and C334 at 4.48 Mb (Shi et al. 2021); and BPH41-2 from GXU202 between W4-4-3 and W1-6-3 (4.68–4.78 Mb) (Wang et al. 2022). In the same region, we identified qBPH4 between RM8213 and RM1305 (4.42–5.62 Mb) (Table 3). qBPH4 overlaps with BPH12 (5.21–5.62 Mb) and the position of BPH40 (cloned at 4.48 Mb), and partially overlaps with BPH17-ptb (5.62–7.86 Mb) and BPH41-2 (4.68 and 4.78 Mb). Other genes, such as BPH15, BPH17, BPH35, and BPH36, are located adjacent to qBPH4. Therefore, qBPH4 may correspond to those BPH resistance genes. Fine mapping is needed to determine its precise position and its relationship with these known resistance genes.
Plant resistance to insects works through three main mechanisms: antibiosis, antixenosis, and tolerance. Antibiosis affects insect survival or development, antixenosis deters insects, and tolerance allows the plant to endure damage with minimal cost (Kogan and Ortman 1978, Painter 1951). Understanding the resistance mechanisms associated with individual resistance genes is important and can be useful in combining or pyramiding genes, improving the durability and effectiveness of resistance to BPH (Du et al. 2020). Several genes have been reported in the same region on the long arm of chr. 11: BPH28(t) confers resistance mainly through tolerance (Wu et al. 2014), and BPH43 and qBPH11.3 both function through antibiosis and antixenosis (Kim et al. 2022, Li et al. 2024). Our results indicate that qBPH11 contributes to BPH resistance through antibiosis by inhibiting BPH sucking and antixenosis (Fig. 6A–6D), but not through tolerance (Fig. 6E). This suggests that qBPH11 corresponds to BPH43 or qBPH11.3. The possible candidate gene of BPH43 was nucleotide-binding LRR receptor (NLR) family protein and BPH43 was associated with rapid defense activation, including the induction of defense response pathways, hydrogen peroxide catabolism and hypersensitive response (Kim et al. 2022). Similarly, qBPH11.3 was mapped to a region containing a cluster of disease resistance genes and candidate genes of qBPH11.3 were NLR family protein (Li et al. 2024). Based on previous study, the biological functions of NLR family proteins related to BPH resistance suggested that these genes may inhibit feeding, growth and reproduction of BPH through callose deposition and cell wall thickening (Yan et al. 2023). The observed phenotypic effect and its overlap with known genes suggest that qBPH11 may function through inhibiting the sucking of phloem sap of BPH from the plant, by structural reinforcement of cell walls. While qBPH11-NIL showed strong resistance to BPH in different evaluation tests, agronomic traits such as yield components and heading date on qBPH11-NIL were not evaluated in this study. Agronomic traits for qBPH11-NIL will be characterized in future study. The qBPH4 is adjacent to BPH15 and BPH17, which are reported to encode lectin receptor kinases (OsLecRKs) involved in pattern-triggered immunity and downstream defense activation (Cheng et al. 2013, Liu et al. 2015). Additionally, BPH40 encodes leucine-rich domains (LRDs) proteins that increase cell wall thickness, physically restricting BPH feeding (Shi et al. 2021). We did not directly evaluate the resistance mechanism of qBPH4 other than MSST and it will be necessary to evaluate in detail.
IRGC 89073 proved resistant to both Hadano-1966 and Koshi-2013 BPH populations (Fig. 6, Supplemental Fig. 1). Several studies have found that highly resistant rice cultivars often possess not only major resistance genes but also minor genes, which together can enhance the durability of resistance (Hu et al. 2016). Here, O. nivara IRGC 89073 showed strong resistance to the highly virulent Koshi-2013 BPH population. qBPH11 alone appeared to be ineffective and did not confer the same resistance level as in the donor parent (Fig. 6, Supplemental Fig. 1). These findings suggest the need to develop a pyramid line combining qBPH4 and qBPH11. The combination will likely to be additive, its evidence has been demonstrated in combinations such as BPH3 + BPH17-ptb, BPH2 + BPH32, and BPH20 + BPH32, which improved resistance against both weak and virulent BPH populations like Hadano-1966 and Koshi-2013 (Nguyen et al. 2019). Further research is necessary to identify the precise locations of these QTLs and to explore their potential for use in breeding programs focused on developing durable, BPH-resistant rice cultivars.
NHN, NDC, and DF designed the study. NHN, NDC, and DF developed the plant materials. SSM provided BPH populations. SZ supported the research and writing. NHN, NDC, and DF performed experiments and wrote this paper.
We thank the staff of the Insect Pest Management Research Group, Kyushu Okinawa Agricultural Research Center, NARO, for rearing and providing the insect populations. Seeds of wild accession O. nivara IRGC 89073 were provided by IRRI. This work was supported by JSPS KAKENHI Grant Number JP24KK0123. We also thank the Government of Japan for the doctoral fellowship (MEXT) granted to NHN. This research was part of the dissertation submitted by the first author in partial fulfilment of a Ph.D. degree. All authors have contributed to the paper and have provided consent for its publication.
