2023 Volume 73 Issue 4 Pages 382-392
The brown planthopper (BPH: Nilaparvata lugens Stål) is one of the most destructive insects in rice production. The use of host plant resistance has potential to reduce damage caused by BPH. The heat tolerance japonica rice ‘Sagabiyori’, with superior grain quality and high soluble starch in the stem, is highly susceptible to damage by BPH. Here, to enhance its BPH resistance, we developed seven near-isogenic lines (NILs) carrying BPH2, BPH17-ptb, BPH32, BPH3, BPH17, BPH20, and BPH21 through marker-assisted selection and evaluated resistance to two BPH populations. Most lines were more resistant to the Hadano-1966 BPH population than Sagabiyori but were less effective against the highly virulent Koshi-2013 population. Nevertheless, in antixenosis tests, Koshi-2013 settled less on all NILs than on Sagabiyori. In addition, adult mortality and the percentage of fresh weight loss of lines carrying BPH17 and BPH3 indicated that these lines have higher resistance to Koshi-2013 than Sagabiyori. Current study revealed that BPH resistance of Sagabiyori became stronger by transferring BPH3 and BPH17 genes. Thus, BPH3 and BPH17 might be valuable for breeding programs to enhance BPH resistance of high grain quality rice varieties with heat tolerance.
Rice (Oryza sativa L.) is one of the world’s most important food crops, feeding more than half of the population. The brown planthopper (BPH), Nilaparvata lugens Stål, is a destructive pest that directly sucking phloem sap and vectors Rice grassy stunt virus (RGSV) and Rice ragged stunt virus (RRSV) diseases (Bottrell and Schoenly 2012). Heavy infestation by BPH results in the complete death of rice plants. In East Asia, BPH migrates from northern Vietnam to southern China and is carried from China to Japan by strong southwesterly winds during the rainy season (Hu et al. 2017). Between 2005 and 2008, a total of 2.7 million t of rice yield was lost over four years in China (Hu et al. 2016a) due to direct damage from BPH, while 0.4 million t was lost in Vietnam, mainly to RGSV and RRSV (Brar et al. 2009). Furthermore, in 2013, there was outbreaks of BPH occurred in western and southwestern Japan, resulting in a loss of 10.5 billion yen in rice production. In addition, outbreaks of BPH in 2019 caused almost the same tremendous economic losses to Japanese rice farmers as in 2013 (Sanada-Morimura 2020).
The improvement of host plant resistance in rice is used to reduce BPH damage (Dyck and Thomas 1979, Khush 1995) and is considered one of the most cost-effective and environmentally friendly strategies for BPH management. To date, more than 45 loci for BPH resistance (designated BPH1 to BPH45) have been identified in rice (Du et al. 2020, Wang et al. 2022, Yang et al. 2019, Zhang et al. 2020). Among genes mapped to specific locations, most are clustered on chromosomes 12L (cluster A), 4S (cluster B), 6S (cluster C), 4L (cluster D), and 3L (cluster E) (Fujita et al. 2013, Hu et al. 2016a). Among these, nine genes BPH6, BPH9, BPH14, BPH17, BPH18, BPH26, BPH29, BPH32, and BPH30 have been cloned and characterized for BPH resistance (Du et al. 2009, Guo et al. 2018, Ji et al. 2016, Ren et al. 2016, Shi et al. 2021, Tamura et al. 2014, Wang et al. 2015, Zhao et al. 2016).
Monogenic resistance is vulnerable to rapid adaptation by pest populations. BPH populations have sufficient genetic variability to enable them to overcome specific resistance genes in a resistant cultivar over several generations (Zhao et al. 2016). In the late 1970s, BPH populations adapted to cultivars with the BPH1 and BPH2 genes (IR26, IR36, etc.), after these cultivars were widely adopted across Asia (Saxena and Barrion 1985). In addition, continuous rearing of BPH populations on resistant rice cultivars for 7 to 15 generations under controlled conditions allowed adaptation to resistance to BPH1, BPH2, BPH3, BPH8, BPH9, BPH10, and BPH32 (Claridge and Den Hollander 1982, Ferrater et al. 2015, Ketipearachchi et al. 1998). A recent multinational study indicated that BPH populations across Asia have adapted to rice cultivars carrying BPH1, BPH2, BPH5, BPH7, BPH8, BPH9, BPH10, and BPH18 (Horgan et al. 2015). In long-term monitoring of BPH from 2001 to 2019 in Japan, the survival rates of immigrant populations of BPH collected during 2016–2018 on the highly resistant ‘Rathu Heenati’ and ‘Balamawee’ were 11%–20% and 22%–64%, respectively (Fujii et al. 2021). Natural BPH populations are becoming progressively more virulent to resistant cultivars through adaptation.
The effects of any single resistance gene may be revealed in detail by using near-isogenic lines (NILs) that carry the gene in the genetic background of a susceptible cultivar. At least 16 NILs with BPH resistance genes (BPH3, BPH4, BPH6, BPH9, BPH10, BPH12, BPH14, BPH15, BPH17, BPH18, BPH20, BPH21, BPH25, BPH26, BPH30, and BPH32) have been developed in the genetic backgrounds of several susceptible indica and japonica cultivars (Hu et al. 2016b, Jena et al. 2017, Myint et al. 2012, Nguyen et al. 2019, Qiu et al. 2012, Shi et al. 2021). The resistance of cultivars with single genes is weaker and less durable than that of cultivars with multiple resistance genes. Researchers have proposed the pyramiding of two or more genes to enhance resistance and thereby avoid pest adaptation (Horgan et al. 2015). Combinations of multiple BPH resistance genes have increased resistance to BPH. For instance, a pyramided line (PYL) with BPH14 and BPH15 had greater resistance to BPH from China than NILs with either gene alone (Hu et al. 2016a). Similarly, pyramiding of BPH25 and BPH26 had positive epistatic effects against BPH populations collected in Vietnam, the Philippines, and Japan (Myint et al. 2012). Therefore, the development of cultivars carrying multiple BPH resistance genes might be an effective way to enhance BPH resistance.
The japonica cultivar ‘Sagabiyori’ has a “special A” rating for its high eating quality and grain quality, and is grown in more than 20% of rice fields in Saga Prefecture, Japan. Moreover, Sagabiyori has heat tolerance: high grain quality under more than 30°C at maturity stage, due to high content of nonstructural carbohydrate (NSC) in the stem as soluble sugars and starch (Tanamachi et al. 2016). As a phloem sucking insect, BPH which takes sucrose from leaf sheath, consequently reduce NSC accumulation in the stem and disrupts translocation of assimilate may eventually lead to hopper burn (Watanabe and Kitagawa 2000). Sagabiyori is susceptible to damage from BPH, which is presumably related to its high NSC content. It is necessary to enhance BPH resistance in Sagabiyori with high NSC content but we did not know effectiveness of BPH resistance genes in Sagabiyori. Therefore, this study was conducted to identify factors that inhibit BPH multiplication in existing BPH-resistant genetic resources under Sagabiyori genetic background. Here, we developed seven NILs with BPH resistance genes (BPH2, BPH3, BPH17-ptb, BPH32, BPH17, BPH20, and BPH21) in the Sagabiyori genetic background to evaluate the effects of each gene against two BPH populations collected in Japan, one in 1966 (before resistant cultivars were widely released) and one in 2013 (recently arrived from China). To understand factors for suppressing BPH multiply in Sagabiyori background, the NILs for BPH resistance with Sagabiyori background were evaluated detailed.
To develop NILs carrying BPH resistance genes in the Sagabiyori background, we used seven NILs with BPH resistance genes in the ‘Taichung 65’ (T65) genetic background as donor parents (Nguyen et al. 2019). BPH2, BPH17-ptb, and BPH32 were derived from the broad-spectrum BPH-resistant cultivar ‘PTB33’ (acc. no. IRGC19325) (Angeles et al. 1986, Nguyen et al. 2021); BPH3 and BPH17 from Rathu Heenati (acc. no. IRGC11730) (Jairin et al. 2007a, Sun et al. 2005); and BPH20 and BPH21 from ‘IR-71033-121-15’, which was derived from Oryza minuta (Rahman et al. 2009). Sagabiyori was crossed with each T65-NIL, and three backcrosses to Sagabiyori generated BC3F1 plants (Fig. 1). In each backcross generation, plants carrying the BPH resistance genes from the donor parent were selected by marker-assisted selection (MAS) using simple sequence repeat (SSR) markers linked to the target genes (Supplemental Table 1). Self-pollination produced BC3F2 and BC3F3 plants with resistance genes. Finally, seven BC3F4 Saga-BPH pre-NILs were developed, one per gene (Table 1). Further backcrossing to Sagabiyori generated BC5F1 plants, which were self-pollinated to produce BC5F2 lines as advanced Saga-BPH NILs (Table 1). In this study, the lines with BC5 generation were denoted as NILs because most of genetic background excepting target gene region from donor was theoretically substituted by recurrent parent. The lines with BC3 generation, those were lower background recovery rate of Sagabiyori, designated as pre-NILs. BPH resistance was evaluated in the pre-NILs, and the genetic background was surveyed and agronomic traits were evaluated in the NILs.
Breeding scheme for development of pre-NILs and NILs carrying BPH resistance genes in the Sagabiyori genetic background.
| Line | Gene | Chromosome | Donor | Pre-NIL generation | NIL generation |
|---|---|---|---|---|---|
| Saga-BPH2 | BPH2 | 12 | PTB33 | BC3F4 | BC5F2 |
| Saga-BPH17-ptb | BPH17-ptb | 4 | PTB33 | BC3F4 | BC5F2 |
| Saga-BPH32 | BPH32 | 6 | PTB33 | BC3F4 | BC5F2 |
| Saga-BPH3 | BPH3 | 6 | Rathu Heenati | BC3F4 | BC5F2 |
| Saga-BPH17 | BPH17 | 4 | Rathu Heenati | BC3F4 | BC5F2 |
| Saga-BPH20 | BPH20 | 4 | IR71033-121-15 | BC3F4 | BC5F2 |
| Saga-BPH21 | BPH21 | 12 | IR71033-121-15 | BC3F4 | BC5F2 |
We collected ~2 cm of leaves of 14-day-old seedlings and freeze-dried them for 48 h. Total DNA was extracted by the potassium acetate method (Dellaporta et al. 1983). The genotypes of SSR markers in plants in each generation were determined by polymerase chain reaction (PCR) and electrophoresis. The PCR amplification mix (8 μL) contained 3 μL of 2× GoTaq Green Master Mix (pH 8.5), 1 μL of 0.25 μM primers, and 4 μL of 1:20-diluted DNA. PCR amplification comprised an initial 96°C for 5 min; 35 cycles at 96°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final step at 25°C for 1 min. PCR products were separated by electrophoresis at 200 V in 4% agarose gel with 0.5 μg/mL ethidium bromide in 0.5× TBE buffer for 1 h and photographed under ultraviolet light.
For MAS of BPH resistance genes on chromosome (Chr.) 4S, plants with BPH17 and BPH17-ptb were selected using markers RM1305 and B40, and plants with BPH20 were selected using RM1305 and RM16531. For MAS of genes on the short arm of Chr. 6, plants with BPH3 and BPH32 were selected using markers RM508 and RM588. For MAS of genes on the long arm of Chr. 12, plants with BPH2 and BPH21 were selected using markers RM28404 and RM28493 (Supplemental Table 1).
To confirm the substitution of chromosomal segments around resistance genes in each NIL, we investigated the genotypes using 65 SSR markers distributed on target segments carrying Chrs. 4, 6, and 12. 26 markers on Chr. 4, 21 on Chr. 6, and 18 on Chr. 12 (Fig. 2). The graphical genotype of each NIL was determined by the polymorphism between Sagabiyori and each NIL in the SSR marker regions using the method of Young and Tanksley (1989). A map was constructed in GGT v. 2.0 software (Van Berloo 2008, Young and Tanksley 1989).
Graphical genotypes of NILs for BPH resistance genes in Sagabiyori genetic background. A. NILs for BPH17-ptb, BPH17, and BPH20 on chromosome 4. B. NILs for BPH3 and BPH32 on chromosome 6. C. NILs for BPH2 and BPH21 on chromosome 12. The map position of each marker is based on physical distance. Circles indicate the approximate position of resistance genes.
We surveyed whole genome background of each NIL using genotyping by random amplicon sequencing-direct (GRAS-Di) technology. Total DNA of Sagabiyori and each NIL were extracted from young seedlings by DNeasy Plant MiniKit (Qiagen, Germany). GRAS-Di libraries were constructed (Hosoya et al. 2019) and sequencing of the libraries was performed using NovaSeq6000 by Eurofins Genomics, Tokyo. Genotyping was performed by GRAS-Di software (TOYOTA, Aichi, Japan) which are commercially available. The marker quality that was empirically determined based on the number of reads and reproducibility of genotyping were evaluated by GRAS-Di software and the GRAS-Di software ranked markers using A, B, C, D, and E in descending order of quality. Markers of qualities E-ranked was less reliable than those of qualities A, B, C, and D. The quality A, B, C, and D among the GRAS-Di markers were used for estimating substituted chromosomal segments from donor parents. Among GRAS-Di markers, polymorphic and co-dominant markers between each NIL and Sagabiyori were selected to estimate locations of substituted chromosomal segments from donor parents (Supplemental Fig. 1).
BPH populations used for resistance testsWe used two BPH populations to evaluate the BC3F4 Saga-BPH pre-NILs for resistance. The Hadano-66 population was collected in Hadano City, Kanagawa Prefecture, in 1966 (Myint et al. 2009b). This population, which has weakest virulence to rice plant, is crucial for evaluating the effect any BPH resistance gene because it was collected before releasing BPH resistance varieties. Second, Koshi-2013 population was collected in Koshi City, Kumamoto Prefecture, in 2013 (Fujii et al. 2021). Both strains were maintained on the susceptible japonica cultivar ‘Reiho’ at 25°C under 16 h light/8 h dark at National Agriculture and Food Research Organization (NARO), JAPAN. Both were maintained on T65 under the same conditions at Saga University.
Modified seedbox screening testThe resistance of all pre-NILs and Sagabiyori to each BPH strain was evaluated by the modified seedbox screening test at 25°C (Horgan et al. 2015). We sowed 30 seeds of each line and Sagabiyori in single rows in a plastic tray (23.0 cm × 30.0 cm × 2.5 cm) with 2.5 cm between rows, with three replicates. At 7 days after sowing (DAS), the plants were thinned to 20 plants per row. The plants were then infested by second- and third-instar nymphs at a density of ~20 BPH per plant. When all plants of Sagabiyori were dead, all lines were scored for damage on a scale of 0 to 9 following the standard damage score (DS) evaluation system for rice (IRRI 2014).
Antibiosis testAntibiosis tests were conducted at 25°C as described by Myint et al. (2009b). Ten seeds of each pre-NIL, Sagabiyori (susceptible control), and Rathu Heenati (resistant control) were individually sown in 215-mL plastic cups. At 4 weeks after sowing, plants were trimmed to 15 cm height and covered with a plastic tube with windows for ventilation. Each tubes were infested by five small-abdomen brachypterous female BPHs. At 5 days after infestation (DAI), the adult mortality (ADM) were calculated as the rate of dead BPH among infested BPH.
The feeding rates of BPH on the pre-NILs were determined as described by Heinrichs et al. (1985) with minor modifications. Ten seeds of each pre-NIL and the parents were individually sown in 215-mL plastic cups. When plants were 30 days old, the plants in each cup were covered with an inverted transparent plastic cup with ventilators for keeping insects at the base of plants. A filter paper treated with 0.1% bromocresol green in ethanol was placed inside the inverted transparent plastic cups to absorb honeydew excreted by the insects. The yellow-orange filter papers turned blue when honeydew was absorbed. Before infestation, the insects of both strains were starved for 2 h in a plastic box with a paper towel saturated with distilled water to maintain hydration. Each plant was infested by two large-abdomen brachypterous adult female BPHs. At 24 h, the filter papers were collected and the area of honeydew was measured in ImageJ software (v. 1.53e; NIH, USA; https://imagej.nih.gov/ij).
Antixenosis testOne plant each of a pre-NIL and Sagabiyori were sown together in a 215-mL plastic cup, with five replicates. At 30 DAS, the plants in each cup were covered with plastic tubes with ventilators. Into each tube, we placed 20 second-instar BPH nymphs. The number of insects that settled on each plant was recorded every day until 5 DAI. The antixenosis level was calculated as the percentage of insects settled on each plant per total in each tube.
Tolerance testThe tolerance of each line was tested as described by Heinrichs et al. (1985). Individual plants of pre-NILs, Sagabiyori, and Rathu Heenati were sown in 1-L plastic cups with three replications. At 45 DAS, each plant was covered with a plastic tube with ventilation, and 100 second- and third-instar BPH nymphs were infested into each tube. Another three cups were covered with a plastic tube were maintained without BPH as controls. When the susceptible control cultivar died, the plants were cut at the soil surface and weighed. The percentage of plant fresh weight loss (PFWL) was used as an inverse measure of tolerance. PFWL is calculated as:
The BC5F2 Saga-BPH NILs and Sagabiyori were grown in a paddy field at Saga University in 2021, and their agronomic traits were characterized. Seedlings were transplanted at 30 DAS at one plant per hill, with 20 cm between hills and 25 cm between rows. Each line was planted in at least three rows (8 plants per row). We measured six agronomic traits (six plants per trait) in the same row: days to heading (DTH), culm length (CL), panicle length (PL), leaf length (LL), leaf width (LW), and panicle number (PN). DTH was assessed as the days from sowing until 50% of panicles flowered. CL was measured from the soil surface to the panicle neck. PL is the length from the panicle neck to the tip of the longest panicle. Flag leaf length (LL) and flag leaf width (LW) were measured on the longest flag leaf of each sampled plant. Panicle number (PN) is the number of reproductive panicles of each plant at maturity.
Statistical analysisMean values of BPH resistance (DS and ADM) of the NILs and agronomic traits were compared by one-way ANOVA. For multiple comparisons we used Tukey–Kramer test for BPH resistance and the Dunnett’s test for agronomic traits in R v. 4.1.2 software.
Seven pre-NILs and seven NILs with BPH resistance genes were developed through MAS and backcrossing (Table 1; Fig. 1). Using 26 markers on Chr. 4, we detected the donor BPH17-ptb chromosomal segment between RM518 and RM401 (2.0–13.2 Mbp) in Saga-BPH17-ptb; BPH17 between RM518 and RM5749 (2.0–20.1 Mbp) in Saga-BPH17; and BPH20 between RM518 and RM1205 (2.0–19.6 Mbp) in Saga-BPH20 (Fig. 2A). Using 21 markers on Chr. 6, we detected the donor BPH3 segment between RM6775 and RM588 (0.2–1.6 Mbp) in Saga-BPH3; and BPH32 between RM6775 and RM204 (0.2–3.2 Mbp) in Saga-BPH32 (Fig. 2B). Using 18 markers on Chr. 12, we detected the donor BPH2 segment between RM28404 and S12091B (21.9–23.7 Mbp) in Saga-BPH2, in addition to a long heterozygous region at RM101–RM1246 (8.8–19.2 Mbp); and BPH21 between RM1986 and S12091B (21.3–23.7 Mbp) in Saga-BPH21 (Fig. 2C). These results show that the targeted resistance genes from the donor parents were introduced into Sagabiyori.
For surveying genetic background of each NIL, 25575 GRAS-Di markers those were distributed on 12 rice chromosomes were used for genotyping. Based on genotyping by GRAS-Di, Saga-BPH2 had the substituted chromosomal segments of donor from 19.65 Mbp to 23.96 Mbp on chromosome 12 and from 2.85 Mbp to 5.27 Mbp on chromosome 6. Saga-BPH17-ptb had the substituted chromosomal segments of donor from 1.12 Mbp to 14.10 Mbp on chromosome 4. Saga-BPH32 had the substituted chromosomal segments of donor from 0.13 Mbp to 6.26 Mbp on chromosome 6. Saga-BPH3 had the substituted chromosomal segments of donor from 0.11 Mbp to 1.65 Mbp on chromosome 6. Saga-BPH17 had the substituted chromosomal segments of donor from 1.23 Mbp to 20.26 Mbp on chromosome 4. Saga-BPH20 had the substituted chromosomal segments of donor from 0.14 Mbp to 7.98 Mbp on chromosome 4. Saga-BPH21 had the substituted chromosomal segments of donor from 19.19 Mbp to 24.55 Mbp on chromosome 12 and from 7.65 Mbp to 20.35 Mbp on chromosome 2 (Supplemental Table 2). Additionally, the genetic background recovery of Sagabiyori was estimated based on genotypes of GRAS-Di markers: 98.2% for Saga-BPH2, 96.52% for Saga-BPH17-ptb, 98.36% for Saga-BPH32, 99.59% for Saga-BPH3, 94.9% for Saga-BPH17, 97.9% for Saga-BPH20, and 95.16% for Saga-BPH21, respectively (Supplemental Table 2).
Screening test with Hadano-1966 and Koshi-2013 populationsSagabiyori was highly damaged by both the Hadano-1966 and Koshi-2013 BPH populations (DS ≈ 8; Fig. 3A, 3B, Supplemental Fig. 2). Across pre-NILs, DSs against Hadano-1966 ranged from 3.7 to 7.0. DSs of Saga-BPH2 (4.2), Saga-BPH17-ptb (5.3), Saga-BPH3 (4.2), Saga-BPH17 (5.0), and Saga-BPH21 (3.7) were significantly lower than that of Sagabiyori (8.0; Fig. 3A). Against the more virulent Koshi-2013, DSs of Saga-BPH32 (6.0) and Saga-BPH17 (6.9) were marginally lower than that of Sagabiyori (8.5). DSs of the other pre-NILs were almost the same as that of Sagabiyori (Fig. 3B). Thus, most of the pre-NILs were more resistant than Sagabiyori to Hadano-1966, although less resistant to Koshi-2013 than to Hadano-1966.
Results of screening test of pre-NILs carrying BPH resistance genes with two BPH populations: Damage score (DS) against A. Hadano-1966 and B. Koshi-2013 populations. Bars with the same letter are not significantly different at P < 0.05 by Tukey–Kramer test.
At 5 DAI, the ADM was significantly higher on Rathu Heenati (100%) than on Sagabiyori (12% against Hadano-1966, 6% against Koshi-2013; Fig. 4A, 4B). Against Hadano-1966, the ADMs on most of the NILs (Saga-BPH17-ptb, 96%; Saga-BPH3, 66%; Saga-BPH17, 98%; Saga-BPH20, 96%; Saga-BPH21, 86%) were significantly higher than that on Sagabiyori (Fig. 4A). Against Koshi-2013, however, the ADMs on most of the pre-NILs were the same as that on Sagabiyori, and only that on Saga-BPH17 (44%) was significantly higher and that on Saga-BPH3, 22%, was marginally higher than that on Sagabiyori (6%; Fig. 4B).
Antibiosis test of pre-NILs carrying BPH resistance genes against two BPH populations. A. and B. Adult mortality against A. Hadano-66 and B. Koshi-2013 populations. C. and D. Honeydew area of C. Hadano-66 and D. Koshi-2013 populations at 24 h after infestation. Bars with the same letter are not significantly different at P < 0.05 by Tukey–Kramer test.
Honeydew excretion was measured as an indicator of BPH feeding on the pre-NILs. At 24 h after infestation, the honeydew excretion areas were significantly smaller on Rathu Heenati (11.3 mm2 against Hadano-1966, 12.5 mm2 against Koshi-2013) than those on Sagabiyori (98.2 and 88.3 mm2, respectively; Fig. 4C, 4D). Against Hadano-1966, those on Saga-BPH17-ptb (20 mm2), Saga-BPH32 (41 mm2), Saga-BPH17 (28.8 mm2), and Saga-BPH21 (20.6 mm2) were significantly smaller than that on Sagabiyori (98.2 mm2; Fig. 4C). Against Koshi-2013, those on most of the pre-NILs were same as that on Sagabiyori (88.3 mm2), but those on Saga-BPH3 (66.8 mm2) and Saga-BPH17 (52.4 mm2) were marginally smaller (Fig. 4D).
Thus, the Koshi-2013 BPH population had higher virulence than the Hadano-1966 BPH population on the pre-NILs. Nevertheless, Saga-BPH3 and Saga-BPH17 showed antibiosis effects against the Koshi-2013 population (Fig. 4B, 4D).
Antixenosis test with Hadano-1966 and Koshi-2013 populationsWe compared the degrees of antixenosis of the seven BPH resistance genes by comparing the numbers of BPH that settled on pairs of each NIL and Sagabiyori at 5 DAI (Fig. 5). The number of BPH was always lower on the NILs than on Sagabiyori (means: Hadano-1966, 20% vs. 57%; Koshi-2013, 31.8% vs. 52.5%). BPH settling behavior differed significantly between Rathu Heenati (11.3% of Hadano-1966; 9.5% of Koshi-2013) and Sagabiyori (66.4% and 75.3%, respectively; Fig. 5A, 5B). Against Hadano-1966, BPH settling percentages were significantly lower on Saga-BPH2 (25.5%), Saga-BPH17-ptb (13.5%), Saga-BPH32 (20.9%), Saga-BPH17 (24.9%), Saga-BPH20 (13.5%), and Saga-BPH21 (14.7%) than on Sagabiyori (56.8%), and marginally lower on Saga-BPH3 (31.0%) (Fig. 5A). Against Koshi-2013, BPH settling percentages were significantly lower on Saga-BPH2 (28.6%), Saga-BPH17-ptb (31.8%), Saga-BPH32 (28.6%), Saga-BPH3 (27.5%), and Saga-BPH17 (22.9%) than on Sagabiyori, and slightly lower on Saga-BPH20 (39.1%) and Saga-BPH21 (43.8%) (Fig. 5B). Thus, pre-NILs with a single BPH resistance gene could reduce the settling of both populations.
Antixenosis test of pre-NILs carrying BPH resistance genes against two BPH populations. BPH settling against A. Hadano-1966 and B. Koshi-2013 populations. Asterisks indicate significant differences between pre-NILs and Sagabiyori: * P < 0.05, ** P < 0.01, *** P < 0.001 by t-test.
Against the Koshi-2013 population, Saga-BPH20 (60.2%), Saga-BPH21 (68.0%), and Sagabiyori (57.3%) had the highest PFWL, significantly higher than that of Rathu Heenati (–2.1%; Fig. 6). Saga-BPH17 (–1.0%) had the lowest PFWL among the pre-NILs and thus the highest tolerance. The PFWLs of Saga-BPH2 (38.9%), Saga-BPH17-ptb (29.4%), Saga-BPH32 (25.4%), and Saga-BPH3 (25.1%) were marginally lower than that of Sagabiyori. Thus, most of the pre-NILs had a higher tolerance than Sagabiyori.
The percent of fresh plant weight loss on Pre-NILs carrying BPH resistance genes against Koshi-2013 BPH population through tolerance test. Bars with the same letter are not significantly different at P < 0.05 by Tukey–Kramer test.
We evaluated morphological differences between Sagabiyori and the seven NILs in six agronomic traits (Table 2). PL, LW, and PN were not significantly different from those of Sagabiyori. DTH for NILs were similar to that of Sagabiyori, except that those of Saga-BPH17 and Saga-BPH20 were slightly and significantly shorter. CLs and LLs of most of the NILs were not significantly different from those of Sagabiyori, but CLs of Saga-BPH2 and Saga-BPH21 were longer and that of Saga-BPH17 was shorter, and LLs of Saga-BPH3 and Saga-BPH20 were shorter and that of Saga-BPH-17ptb was longer. Overall, the agronomic characteristics of most of the NILs were similar to those of Sagabiyori.
| Line | Agronomic trait (mean ± SD) | |||||
|---|---|---|---|---|---|---|
| DTH | CL (cm) | PL (cm) | LL (cm) | LW (cm) | PN | |
| Saga-BPH2 | 75.0 ± 0.5 | 90.8 ± 4.7** | 17.5 ± 0.5 | 33.6 ± 6.6 | 1.2 ± 0.0 | 15.7 ± 2.1 |
| Saga-BPH17-ptb | 74.5 ± 1.1 | 83.6 ± 4.9 | 18.6 ± 1.0 | 36.2 ± 3.2* | 1.3 ± 0.1 | 16.6 ± 3.5 |
| Saga-BPH32 | 74.0 ± 1.0 | 83.3 ± 2.4 | 18.3 ± 0.9 | 34.0 ± 2.8 | 1.3 ± 0.0 | 13.5 ± 1.5 |
| Saga-BPH3 | 74.0 ± 1.2 | 82.9 ± 3.3 | 17.8 ± 0.9 | 23.4 ± 2.4* | 1.3 ± 0.1 | 17.7 ± 3.9 |
| Saga-BPH17 | 72.0 ± 0.8* | 72.8 ± 3.7** | 19.0 ± 1.1 | 26.2 ± 5.3 | 1.3 ± 0.1 | 15.1 ± 2.5 |
| Saga-BPH20 | 67.0 ± 0.5* | 80.5 ± 2.4 | 18.0 ± 1.3 | 22.6 ± 1.9** | 1.3 ± 0.1 | 11.8 ± 2.4 |
| Saga-BPH21 | 76.0 ± 1.3 | 89.9 ± 4.4** | 19.7 ± 0.4 | 27.3 ± 4.1 | 1.3 ± 0.1 | 14.4 ± 3.8 |
| Sagabiyori | 75.3 ± 1.2 | 79.2 ± 3.3 | 19.0 ± 1.2 | 30.6 ± 3.9 | 1.3 ± 0.1 | 15.5 ± 2.9 |
DTH, days to heading; CL, culm length; PL, panicle length; LL, flag leaf length; LW, flag leaf width; PN, panicle number per plant. * P < 0.05, ** P < 0.01 (Dunnett’s test with Sagabiyori as the control).
Outbreaks of BPH in western and southwestern Japan in 2013 and 2019 cost Japanese rice farmers greatly (Fujii et al. 2021). As the commercial varieties grown in these regions do not have BPH resistance genes, it is essential to introduce BPH resistance into them. Since the early 1970s, resistant varieties have been used extensively in breeding for BPH resistance in japonica varieties, such as ‘Norin-PL4’ (Murata et al. 1998), ‘Saikai 190’ (Kobayashi et al. 2014), ‘Kanto BPH1’, and ‘Akiharuka’. However, these varieties are not widely grown in Japan. Therefore, the improvement of the BPH resistance of the commercial variety Sagabiyori is necessary for future rice production. The seven NILs for BPH resistance in the Sagabiyori background that we bred can be used as commercial cultivars or breeding materials.
In general, NILs are used for genetic studies such as the characterization of gene effects, development of molecular markers linked with target genes, comparisons of gene expression, and isolation of target genes (Fujita et al. 2010, Xiao et al. 2016). In the context of BPH, they are important for monitoring virulence among differential BPH populations in Japan (Myint et al. 2009b, Nguyen et al. 2019), East Asia (Fujita et al. 2009), and the Philippines (Jena et al. 2017). By monitoring BPH virulence using our NILs, we can determine the effectiveness of resistance genes against specific BPH populations. Here, almost all of the NILs were resistant to Hadano-1966 but less effective against Koshi-2013 (Figs. 3–6). The virulence of Koshi-2013 BPH was significantly greater than that of Hadano-1966, and the single genes in the pre-NILs were less effective (Figs. 3, 4). These tendencies of BPH virulence correspond with studies monitoring the virulence of BPH populations in Japan (Fujii et al. 2021, Nguyen et al. 2019).
The virulence of BPH on differential cultivars fluctuates by location and year. BPH has adapted to resistant cultivars containing BPH1, BPH2, BPH3, BPH5, BPH7, BPH8, BPH9, BPH10, and BPH18 across Asia. In Japan, resistance in differential cultivars with BPH1 and BPH2 broke down during the 1990s (Tanaka and Matsumura 2000). The virulence of BPH populations collected in 1999 (Isahaya-99), 2005 (Nishigoshi-05), and 2013 (Koshi-2013) was significantly greater than that of Hatano-1966 (Myint et al. 2009b, Nguyen et al. 2019). Here, most of the NILs were resistant to Hadano-1966 but weak against Koshi-2013 (Figs. 3–5). These results suggest that BPH virulence has increased year by year, and that BPH2, BPH17-ptb, BPH32, BPH20, and BPH21 are now less effective against Koshi-2013. The BPH settling test showed that the pre-NILs carrying single resistance genes are more resistant than Sagabiyori to Koshi-2013, although their antibiosis effects were less effective against Koshi-2013 (Fig. 5). Although all of the pre-NILs were weak against Koshi-2013, most of them deterred it.
Among the seven NILs, Saga-BPH3 and Saga-BPH17 (from Rathu Heenati) were resistant to the migrant Koshi-2013 population. The resistance of Saga-BPH17 to Koshi-2013 was significantly higher than that of Sagabiyori in antibiosis, antixenosis, and tolerance tests (Figs. 4–6). The resistance of Saga-BPH3 was marginally higher than that of Sagabiyori in antibiosis and tolerance tests (Figs. 4, 6). BPH3 and BPH17 in Rathu Heenati were reported as offering a broad spectrum of resistance to various BPH populations in Asia (Fujii et al. 2021, Horgan et al. 2015, Myint et al. 2009b). The BPH3 gene in Rathu Heenati, mapped on the short arm of Chr. 6 between markers RM19291 (1.2 Mbp) and RM8072 (1.4 Mbp), conferred strong resistance to Thai BPH populations (Jairin et al. 2007a, 2007b). It also conferred strong resistance in the genetic background of indica cultivars ‘Hemeizhan’ and ‘9311’ to field populations in China (Hu et al. 2016b, Xiao et al. 2016). Our results agree with those studies. In contrast, the resistance of BPH3 to Koshi-2013 was lower in the T65 genetic background (Nguyen et al. 2019). Therefore, the resistance effect of BPH3 might depend on the genetic background or the BPH population. BPH17 encodes plasma membrane–localized lectin receptor kinases OsLecRK1–3; (Liu et al. 2015), while most BPH resistance genes, such as BPH14, BPH18, and BPH26, encode coiled-coil nucleotide-binding-site leucine-rich repeats (CC-NBS-LRR). The functions of three genes in a cluster of four at BPH17 contribute to broad-spectrum durable BPH resistance effects (Liu et al. 2015). This could explain the stronger effects of BPH17 against Koshi-2013.
Resistant cultivars carrying multiple BPH resistance genes had greater or more durable resistance (Jena et al. 2017, Myint et al. 2009a, Nguyen et al. 2019, Xiao et al. 2016). For example, PYLs BPH6 + BPH12 (Qiu et al. 2012), BPH3 + BPH27 (Liu et al. 2016), BPH25 + BPH26 (Myint et al. 2012), and BPH17 + BPH21 (Jena et al. 2017) had higher resistance than monogenic lines with each of these genes alone. Here, the resistance of Saga-BPH3 and Saga-BPH17 was higher than that of Sagabiyori (Figs. 4–6). From the DS and ADM results, the resistance of BPH17 was stronger than that of BPH3, indicating that BPH17 would be a useful gene resource for improving BPH resistance in rice breeding programs. Through pyramiding BPH3 and BPH17, lines carrying both resistance genes might have stronger antibiosis, antixenosis, and tolerance according to resistance level of Saga-BPH3 and Saga-BPH17 in this study. In Nguyen et al. (2019), PYL carrying BPH3 and BPH17 with T65 genetic background had been developed and characterized ADM against Koshi-2013. The ADM of PYL carrying BPH3 and BPH17 with T65 genetic background against Koshi-2013 was higher than that of corresponding NILs but was lower than that of Rathu Heenati. The difference in ADM between PYL and Rathu Heenati might be due to BPH resistance genes in Rathu Heenati except BPH3 and BPH17 such as BPH14 (Pannak et al. 2023), Qbph3, and Qbph10 (Sun et al. 2005).
Moreover, our new NILs will facilitate the development of PYLs carrying BPH3 and BPH17, which will be important for increasing resistance to the Koshi-2013 population. Although most of the NILs developed here have less resistance to Koshi-2013, pyramiding of BPH resistance genes might enhance resistance. In future research, it will be essential to develop PYLs as possible resources for breeding programs. Recently, the development of heat tolerance rice varieties become important because of climate changes. The rice varieties with heat tolerance such as ‘Nikomaru’ and ‘Genkitsukushi’ has been developed in Japan and there was tendency that heat tolerance rice varieties have high NSC content (Tanamachi et al. 2016). Current study revealed that the Sagabiyori background with high NSC content became stronger resistance by transferring BPH3 and BPH17 genes. These BPH resistance genes would be effective on the other rice varieties with high NSC content.
SBDS, SZ, HY, and DF designed the study. SBDS, CDN, and DF developed the plant materials. SSM provided BPH populations. SBDS and DF performed the experiments and wrote the 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. This work was supported by JSPS KAKENHI grants JP17K07606 and JP21K05527. We also thank the Project for Science and Technology Research Partnership for Sustainable Development (SATREPS) by JST/JICA. Department of Agricultural Research, Ministry of Agriculture, Livestock and Irrigation, Myanmar, for the doctoral fellowship granted to SBDS. This research was part of the dissertation submitted by the first author in partial fulfilment of a Ph.D. degree. All authors provide consent for publication of this manuscript.
