Large scale rice germplasm screening for identification of novel brown planthopper resistance sources

Rice (Oryza sativa L.) is a staple food crop globally. Brown planthopper (Nilaparvata lugens Stål, BPH) is the most destructive insect that threatens rice production annually. More than 40 BPH resistance genes have been identified so far, which provide valuable gene resources for marker-assisted breeding against BPH. However, it is still urgent to evaluate rice germplasms and to explore more new wide-spectrum BPH resistance genes to combat newly occurring virulent BPH populations. To this end, 560 germplasm accessions were collected from the International Rice Research Institute (IRRI), and their resistance to current BPH population of China was examined. A total of 105 highly resistant materials were identified. Molecular screening of BPH resistance genes in these rice germplasms was conducted by developing specific functional molecular markers of eight cloned resistance genes. Twenty-three resistant germplasms were found to contain none of the 8 cloned BPH resistance genes. These accessions also exhibited a variety of resistance mechanisms as indicated by an improved insect weight gain (WG) method, suggesting the existence of new resistance genes. One new BPH resistance gene, Bph44(t), was identified in rice accession IRGC 15344 and preliminarily mapped to a 0–2 Mb region on chromosome 4. This study systematically sorted out the corresponding relationships between BPH resistance genes and germplasm resources using a functional molecular marker system. Newly explored resistant germplasms will provide valualble donors for the identification of new resistance genes and BPH resistance breeding programs.


Introduction
Rice (Oryza sativa L.) is one of the most significant cereal crops in the Asian-Pacific region, providing a reliable food resource for more than one-half of the world's population (Chen et al. 2022;Hu et al. 2016).Meanwhile, rice is vulnerable to threats from insect pests, which substantially contribute to yield losses.Among rice insects, the brown planthopper (BPH; Nilaparvata lugens Stål) is the most devastating pest throughout Asia, as well as some South Pacific islands and Australia (Sogawa et al. 2003).BPH severely damages rice plants by sucking the phloem sap through its piercing mouthparts (stylet), and it also causes indirect damage through transmitting several viral diseases such as rice grassy stunt virus, rice ragged stunt virus, and rice wilted stunt virus (Cheng et al. 2014;Heinrichs 1979;Susanto et al. 2020).Heavy infestation of BPH can cause the wilting and dying of rice plants, a symptom acknowledged as "hopperburn" (Watanabe and Kitagawa 2000).In China, it is estimated that over 25 million hectares of rice fields have been damaged by BPH, resulting in a loss of 2.7 million tons of production from 2005 to 2008 (Hu et al. 2016;Qiu et al. 2012).The economic losses caused by BPH in Asia have been estimated at more than 300 million dollars annually (Zhang et al. 2016).The most commonly practiced method of controlling BPH is the use of pesticides or insecticides, which is expensive and harmful to human health and the environment.Moreover, overapplication of pesticides not only causes the evolutionary resistance of BPH to the pesticides, but it also indiscriminately kills predatory insects and leads to BPH resurgence (Ling et al. 2011).Compared with conventional chemical control, the development of BPH-resistant varieties is the most economically effective and environmentally friendly approach to control BPH (Kim et al. 2022;Matsumura et al. 2009).
During the course of its co-evolution with BPH, rice has evolved complex systems of resistance to BPH (Zhou et al. 2021).Resistance to BPH was first reported in 1969 in the variety Mudgo (Zhao et al. 2016).Up to now, more than 40 BPH resistance genes have been identified in cultivated rice and wild species (Du et al. 2020).Among them, seventeen BPH resistance genes (Bph6, Bph14, Bph18/ Bph1, Bph26/bph2, Bph3/Bph15, bph29, Bph32, Bph7/Bph9/Bph10/Bph21, Bph30/Bph40, and Bph37) have been cloned by a map-based cloning approach (Cheng et al. 2013;Du et al. 2009;Guo et al. 2018;Ji et al. 2016;Jing et al. 2017;Liu et al. 2015;Ren et al. 2016;Shi et al. 2021;Tamura et al. 2014;Wang et al. 2015;Zhao et al. 2016).Meanwhile, BPH biotypes have evolved in virulence mechanisms that enable them to overcome BPH resistance in rice (Jing et al. 2014).For instance, the BPH-resistant variety IR26 containing the single resistance gene Bph18 was released by the International Rice Research Institute (IRRI) in 1973 and saved rice production in large areas with BPH damage.Whereas, the resistance of Bph18 broke down by a new BPH biotype in 1976 in Indonesia, Sri Lanka, Solomon Islands, and Vietnam (Khush and Coffman 1977).IR36 and other varieties carrying Bph26 were then released by the IRRI and showed effective resistance to the new biotype.But again, Bph26-derived resistant varieties were adapted by another new BPH population (Alam and Cohen 1998;Ketipearachchi et al. 1998).BPH populations throughout Asia were largely virulent to the derived varieties containing BPH18 and BPH26 in the 1990s (Ito et al. 1994;Myint et al. 2009;Thwin et al. 2003).So far, more than 10 BPH biotypes have been reported (Hong and Jun 2003;IRRI 1982;Li 1994;Saxena and Barrion 1985;Shrestha and Adhikary 1987).In addition, Horgan et al. (2015) examined the virulence of 12 BPH populations from South and Southeast Asia against resistant rice varieties, and the increasing virulence of BPH populations was demonstrated.Therefore, it is still urgent to identify novel broad-spectrum and durable BPH resistance genes to achieve stable rice production.
Most BPH-resistant varieties are from South Asia (Khush and Coffman 1977).Kaneda et al. (1981) screened about 3300 cultivars and breeding lines worldwide and found that most of the resistant indigenous cultivars originated from Sri Lanka and southern India.Similar results have been reported by the IRRI, which evaluated a large number of germplasm accession reactions to three BPH biotypes (Heinrichs et al. 1985).In the present study, 560 cultivars originating from South Asia (Sri Lanka, India, and Bangladesh) with resistance to at least one BPH biotype were collected from the IRRI to evaluate their resistance to the more-destructive Bangladesh type, a predominant BPH biotype in China (Lv et al. 2009).These cultivars were also genotyped for the existence of cloned BPH resistance genes by using functional dominant molecular markers.These candidate accessions can be deployed as valuable donors in the breeding of BPH-resistant rice cultivars and new sources for the isolation of novel BPH resistance genes.

Plant materials and mapping populations
A total of 560 rice accessions resistant to at least one BPH biotype were collected from the IRRI (Supplementary Table S1).IRGC 15344, a Sri Lankan indica rice cultivar resistant to BPH biotype III (Heinrichs et al. 1985), was crossed with the Chinese indica BPH-susceptible variety 93-11 (Supplementary Fig. S1).The resulting F 1 was selfed to generate F 2 mapping populations and the corresponding F 2:3 families for genetic analysis and gene mapping.Meanwhile, an F 1 plant of 93-11/IRGC 15344 was backcrossed with 93-11 to generate the BC 1 F 1 population.The heterozygous BC 1 F 1 plants resistant to BPH were selected to generate BC 1 F 2 populations for further mapping.The gene-linked markers 344-0-7 and 344-1-1 flanking the Bph44(t) locus were used to choose plants with heterozygous Bph44(t) from each backcross population for the subsequent backcrossing.

BPH insect source and resistance evaluation
The BPH populations obtained from a paddy field were reared on susceptible variety TN1 in a greenhouse of Wuhan University.BPH resistance was evaluated by the seedling bulk test, according to Huang et al. (2001) and Du et al. (2009).The seeds of each cultivar were pregerminated for the same developmental stage.Seeds with the identical developmental stage were selected and sown in plastic boxes.Thirty seeds of resistance-improved individual plants were sown in rows that were flanked by rows of seeds of the susceptible lines (TN1 or 93-11).When the material grew to the three-leaf stage, the seedlings were inoculated with 2nd-to-3rd instar BPH nymphs at a rate of 10 insects per seedling and covered with nylon net.When the susceptible variety plants completely died, the BPH resistance was assessed using the following six-scale grading system: 0, no harm; 1, very minor damage; 3, 1st and 2nd leaves of the majority of the plants were partially yellowed; 5, there was evident yellowing and stunting; 7, the majority of the plants were wilted or dead; 9, all plants were dead.A lower score indicates that the plants are more resistant to BPH, while a higher score indicates that the plants are more susceptible to BPH.The resistance scores of each F 2 plant was calculated as the weighted average scores of corresponding F 3 seedlings.The assessment trials were conducted three times.
In the weight gain (WG) assay, the newly emerged BPH female adults were weighed and placed separately in a preweighed parafilm sachet and affixed to the rice plants' leaf sheaths.(Shi et al. 2021).After 2 days, the body weight of BPH was recalculated again; the difference in weight of the insect was recorded as WG of BPH.The analysis involved at least 15 individuals.In order to facilitate comparison, rice variety G509 was used as the resistant control, whereas Nipponbare and Zhenshan 97 were used as the susceptible materials.

DNA extraction and PCR-based genotyping
To prevent contamination from polysaccharide and polyphenol components, the whole genomic DNA of fresh rice leaves at the three-leaf stage was extracted by a modified CTAB procedure (Porebski et al. 1997).The extracted DNA was dissolved in 1× Tris-EDTA buffer.Nine functional dominant molecular markers were designed with Primer3.0 (http:// prime r3.ut.ee/) based on specific sequences of cloned BPH resistance genes.The primer sequences and PCR product sizes are listed in Table 1.The target sequences were amplified using the PCR protocols described by Yang et al. (2002), with minimal adjustments for various primers.The PCR products were checked by 1% agarose gel electrophoresis and staining with 2 μL of ethidium bromide (EB), respectively.

Gene mapping
Equal amounts of DNA from 12 exceptionally resistant F 2 plants and 10 extremely susceptible F 2 plants were mixed to form resistant and susceptible bulks, respectively, according to the phenotypes of the F 2 mapping population.The BPH resistance gene was mapped by the green super rice chip (BSA-chip).
The resistant and the susceptible bulks as well as the DNAs of the two parents (93-11 and IRGC 15344) were detected in the green super rice chip GSR 40K array at the Greenfafa Institute (Wuhan, China), according to the Infinium HD Assay Ultra Protocol (http:// www.illum ina.com/).The BPH resistance gene was mapped using bulked segregant analysis based on the green super rice chip (BSA-chip).
The DNAs of both parents (93-11 and IRGC 15344) were deep (~30×) sequenced on an Illumina HiSeq X Ten platform for the insertion-deletion (InDel) calling and development.According to a base-quality Q score using the Phred scale from the Annotation Project website, low-quality readings were removed or clipped using Burrows-Wheeler Aligner software (version 0.5.8c)(Li and Durbin 2010).Using SAMtools (version 0.1.12a),InDels between IRGC 15344 and 93-11, ranging in size from 20 to 40 bp, were retrieved (Li et al. 2009).Primer3 was used to create PCR primers from the upstream and downstream sequences of the putative InDel sites on the reference genome Nipponbare.The PCR products were between 160 and 250 bp in size to enable simple identification of the polymorphism between IRGC 15344 and 93-11 on a 1.5% agarose gel.The InDel molecular markers are listed in Supplementary Table S13.

Results
Evaluation of rice germplasms resistance to BPH Due to their rapid adaptation, BPH populations have become increasingly virulent against resistance genes (Horgan et al. 2015).Identifying new broad-spectrum and durable BPH resistance genes is an inevitable trend for stable rice production.To this end, we obtained 560 traditional cultivars or landraces from the IRRI (Supplementary Table S1), which mainly originated from South Asia and had at least one BPH biotype resistance (Heinrichs et al. 1985).We re-evaluated their resistance to the more-destructive BPH Bangladesh type, a predominant BPH biotype in China (Lv et al. 2009).Accessions with a mean rating of 0-3.49, 3.50-5.49,or 5.50-9.00were designated as resistant (R), moderately resistant (MR), or susceptible (S) to BPH, respectively (Heinrichs et al. 1985;Wu et al. 2022).As shown in Supplementary Table S2, 176 accessions (31.4 %) were resistant to the Bangladesh type with the rating scores between 0 and 3.49, 138 accessions (24.6%) exhibited moderate resistance with scores ranging from 3.50 to 5.49, and a total of 246 accessions (43.9%) were susceptible to the Bangladesh type with scores between 5.50 and 9.00.Additionally, 105 accessions scored as highly resistant with the rating scores ranging from 0 to 2, accounting for 60.2% of the 176 BPH-resistant accessions screened (Table 2).As exemplified by Fig. 1,

Bph14
Bph14 was the first BPH resistance gene isolated through map-based cloning (Du et al. 2009).By comparing the Bph14 sequence with the corresponding regional sequences of 93-11, Nipponbare, Shuhui498, and RP Bio-226, we found a specific fragment in Bph14 promoter region, which was missing in other rice varieties.We designed the Bph14-specific dominant marker B14D according to the unique fragment of Bph14 promoter (Table 1) to screen the rice accessions for the Bph14 locus.
As shown in Fig. 2, a 543-bp fragment was specifically amplified by B14D from RI35, a recombinant inbred line containing the Bph14 locus, while no fragment was amplified in other cultivars containing Bph3, Bph18, Bph26, Bph9, Bph7, Bph32, and Bph6, respectively.The results suggested that the dominant marker B14D can be used to detect the Bph14 locus in the rice germplasm.We further genotyped these 560 rice accessions using B14D (Fig. 3).The specific fragment of Bph14 was amplified in 69 cultivars of the 560 rice accessions assayed (Supplementary Table S3), indicating that these accessions harbor the Bph14 locus.As Bph14 carries a unique leucine-rich repeat (LRR) domain with a high nucleotide diversity (Du et al. 2009), we thus obtained the LRR sequences of 30 accessions randomly selected from the 69 accessions identified by B14D with direct sequencing of the PCR products amplified with primers designed for the Bph14 LRR region.The sequence comparison revealed that these accessions shared 100% sequence identity with Bph14 (Supplementary Table S4).These results further confirmed that B14D is a dominant Bph14-specific marker that can effectively identify rice materials carrying the Bph14 locus.

Bph3/Bph15
We designed the Bph3-dominant functional marker B3D with continuous polymorphic sites at amino acids 88, 89, and 92 of OsLecRK3 (Table 1) to distinguish cultivars carrying Bph3 from the rice germplasm.We identified 88 rice accessions that carry the Bph3 locus using the B3D marker (Supplementary Table S5).Thirty-two accessions carrying the Bph3 locus were randomly selected for amplification with B3D (Supplementary Table S4).The nucleotide sequences of the B3D-amplified PCR products from these 32 accessions were identical to Bph3, indicating that the dominant functional marker B3D is a reliable marker to identify accessions harboring Bph3.

Bph1/9 locus
The eight BPH resistance genes clustered on the long arm of chromosome 12 are actually allelic and divided into four allelic types (Bph7, Bph18, Bph26, and Bph9) (Zhao et al. 2016).We compared the genomic sequences corresponding to the Bph1/9 locus from these representative haplotype rice varieties using Clustalw software 1.83 to develop the Bph1/9 locus-specific dominant functional marker B1279D that distinguishes the four resistance allelotypes and other susceptible allelotypes (Table 1).
70 Page 6 of 14 Vol:. (1234567890) Among them, 109 accessions carried Bph18, 189 accessions harbored Bph26, 2 accessions contained Bph7, and 20 cultivars harbored Bph9 (Supplementary Table S6).We also identified a few accessions containing two BPH resistance genes in the Bph1/9 locus.As nucleotide diversity was much higher in the LRR domain of the Bph1/9 locus than in the coiledcoil and nucleotide-binding site domains (Zhao et al. 2016), we compared the LRR sequences of representative accessions harboring Bph18, Bph26, Bph7, and Bph9.The LRR sequences of accessions identified by the gene-specific dominant functional marker were identical to the BPH resistance gene used to design the dominant functional marker (Supplementary Table S4).These results demonstrated that the specific dominant functional marker of the Bph1/9 locus can effectively identify rice varieties carrying Bph18, Bph26, Bph7, and Bph9, respectively.

Bph32
We designed the dominant functional marker B32D targeting its specific sequence in the 517-538 bp region of the open reading frame to differentiate Bph32 from other susceptible alleles (Table 1).Among 560 rice accessions, we identified 105 that carry Bph32 with unique amplicons of B32D (Supplementary Table S7).
The corresponding sequences of 36 randomly selected accessions identified by B32D were identical to Bph32 (Supplementary Table S4), further confirming that they carry Bph32.

Bph6
High levels of nucleotide variations were observed among the Bph6 alleles, particularly in the N-terminal region of 1-1533 bp (Guo et al. 2018).Based on sequence alignments of Bph6 haplotypes, nucleotide polymorphic regions in the N-terminus (73-360 bp) were targeted for designing the functional dominant marker B6D to distinguish Bph6-harboring cultivars from the rice germplasm (Table 1; Fig. 3).In total, 79 accessions were found to contain Bph6 (Supplementary Table S8).Sequences of the N-terminal region of the Bph6 locus in 35 randomly selected accessions identified by B6D were analyzed and found to be identical to the nucleotide sequences of Bph6.Among these 40 accessions, 7 varieties carry heterozygous Bph6 (Supplementary Table S4).These results further validated the accuracy of B6D for screening rice germplasms harboring Bph6.

Relationship between resistant germplasm and BPH resistance genes
The above genotyping analysis for the presence of BPH resistance genes with distinct functional molecular markers has shown that a total of 447 accessions contain one to five of the cloned BPH resistance genes (Supplementary Table S9).Among them, 302 varieties carried a single resistance gene, 94 varieties contained two resistance genes, 34 contained three resistance genes, 16 contained four resistance genes, and 1 accession harbored five resistance genes (Supplementary Fig. S2A, B, C, D).
Similarly, the resistance levels of 305 accessions carrying any of Bph18, Bph26, Bph7, and Bph9 to Bangladesh type BPH were also significantly different, ranging from extremely resistant to highly sensitive; in particular, 23, 30, 90, and 162 accessions were highly resistant (HR), resistant (R), moderately resistant (MR), and susceptible (S) to BPH, respectively (Supplementary Table S6).Among them, Bph18 and Bph26 in the Bph1/9 locus account for the largest proportion of phenotypic variation, with 133 resistant materials and 151 susceptible materials in 284 accessions (Supplementary Table S6).We further randomly selected 39 accessions containing Bph18 and evaluated their resistance levels to the BPH biotypes I, II, and III.These results demonstrated that the 23 rice accessions conferred strong resistance to BPH biotype I and that all accessions were susceptible to the more virulent biotypes II and III (Supplementary Table S11), consistent with the relationship between resistance genes and biotypes (Alam and Cohen 1998;Zhou et al. 2021).Vol:.( 1234567890) New BPH-resistant germplasm resources in rice Intriguingly, based on stringent phenotyping coupled with genotyping using functional markers, 46 accessions were demonstrated to be resistant to Bangladesh type BPH and contained none of the 8 cloned BPH resistance genes (Supplementary Table S12).These accessions may carry other cloned BPH resistance genes or other new BPH resistance genes that have been mapped but yet to be published.We further studied the performance of BPH on 23 resistant germplasms by using the WG as a phenotypic index: the lower the insects' WG rate after feeding on rice plants, the greater the plants' resistance (Zhou et al. 2021).The average WG rate of BPH female adults feeding on susceptible control Nipponbare for 48 h was 0.91.Meanwhile, the body WG of 23 rice accessions varied, ranging from −0.09 to 0.59.Twentythree rice varieties were divided into resistant (R; WG < 0.3), moderately resistant (MR; 0.3 <WG< 0.6), moderately susceptible (MS; 0.6 <WG< 0.9), or susceptible (S; WG>0.9).Among the 23 rice accessions tested, 15 and 8 rice accessions were divided into R, and MR, respectively (Fig. 4).The results demonstrated that these 23 rice accessions had strong antibiosis effects towards the BPH.According to the variety of resistance mechanisms as indicated by the WG rate of these accessions, we speculate that there might be different underlying resistance genes.

Molecular mapping of Bph44(t)
In this study, IRGC 15344 was further selected to explore the new BPH resistance locus.We assessed the BPH resistance scores of the F 2 mapping population in order to investigate the genetic causes of BPH resistance in IRGC 15344.The proportion of resistant to susceptible plants was consistent with a 3:1 ratio (118:38; c2c = 0.034 c20.05,1 = 3.84), according to the previously reported method (Qiu et al. 2010), showing that a strong dominant gene influenced the resistance of IRGC 15344.We next performed a BSA analysis using the green super rice chip GSR 40K.An adjacent region on chromosome 4 was obviously distinguished between the resistant and susceptible pools (Fig. 5A), indicating that the BPH resistance gene was located in this region.This gene was named Bph44(t).To detect the accurate location of Bph44(t), we developed polymorphic InDel markers to genotype 156 F 2 plants with the evaluated BPH resistance scores.Therefore, Bph44(t) was initially mapped to the 0-2 Mb region of chromosome 4 (Fig. 5B).Further analysis of 400 BC 1 F 2 plants using the flanking markers identified 13 recombinants.
According to the genotype of the recombinant and the resistance phenotype of BPH, Bph44(t) was narrowed to the 0.6-1.2Mb region of chromosome 4 flanked by 344-0-6 and 344-1-2 (Fig. 5C and D).A total of sixty-six genes were predicted in the corresponding region of Nipponbare reference genome by using a rice genome annotation project database.Most of them are annotated as zinc finger protein, 3-ketoacyl-CoA synthase, amidase family proteins, retrotransposons proteins, expressed proteins, oxidoreductases, or amidase family proteins.Bph30 with a leucine-rich repeat (LRR) domain spanning Chr4 from 921,713 to 929,966 (LOC_Os04g02520, Shi et al. 2021), and two genes encode the nucleotide-binding (NB-ARC) (LOC_Os04g02450 and LOC_Os04g02860) were contained in Bph44(t) region.As LOC_Os04g02520 was identified as the most possible candidate of Bph33 in KOLAYAL and POLIYAL (Hu et al. 2018), along with most BPH resistance genes isolated to date encode NBS-LRR proteins, we also considered LOC_Os04g02520 as candidate for Bph44(t).To test if Bph44(t) is Bph30, we designed primers in the promoter and terminator region of Bph30.We obtained the full-length genome sequence corresponding to Bph30 in IRGC 15344 and found that it was significantly different from that of Bph30 in AC-1613 with more amino acid shift in the coding region.These results demonstrated that Bph44(t) might be a novel major BPH resistance gene, or a superior allelic variation of Bph30.

Discussion
BPH is the most harmful pest of rice, causing 30% of the total loss of rice crops, and has become a serious threat to Asian rice production (Noda 2016;Bhatt et al. 2018).Exploring BPH resistance genes in rice germplasm and breeding BPH-resistant rice varieties are the most economical, practical, and environmentally sustainable strategy to control BPH (Chen et al. 2022).To date, 17 BPH resistance genes have been isolated from about 40 identified genes by map-based cloning (Chen et al. 2022).Most of the BPH resistance genes have been found to be concentrated in the same or similar chromosomal segments.For example, eight resistance genes are clustered on the long arm of chromosome 12 (Zhao et al. 2016), twelve genes are clustered in three regions on chromosome 4 (Huang et al. 2013;Kumar et al. 2018;Li et al. 2019;Li et al. 2010;Qiu et al. 2010;Rahman et al. 2009;Ram et al. 2010), and five genes are located on the short arm of chromosome 6 (Jairin et al. 2007;Jairin et al. 2010;Myint et al. 2012;Yang et al. 2012).It has been shown that eight BPH resistance genes clustered on chromosome 12L are actually multiple alleles of the same gene (Zhao et al. 2016).In addition, it is still unclear whether other BPH resistance genes located in the same chromosomal region are really different genes or different alleles of the same gene.On the other hand, the virulence mechanisms of BPH populations have evolved, thus enabling them to overcome BPH resistance in rice (Jing et al. 2014), and they have become increasingly virulent against resistance genes (Horgan et al. 2015).In order to cope with the biotype variation of BPH, it is still necessary to further explore new BPH resistance genes with a specific resistance spectrum in the rice germplasm for the sustainable control of BPH.
In the 1980s, the IRRI identified hundreds of BPH-resistant germplasm resources showing resistance to different BPH biotypes (Heinrichs et al. 1985).Because the virulence of BPHs may have changed greatly over decades, whether these resistant sources can be applied to BPH resistance breeding programs needs to be further clarified.At the same time, although 40 BPH resistance genes have been identified in rice, the number of nonallelic resistance genes may be relatively small.So, it is of great value to systematically investigate the distribution of cloned BPH resistance genes and to explore new BPH resistance genes in these germplasm accessions identified by the IRRI.In this study, we obtained 560 traditional cultivars or landraces, mainly with South Asian origin, from the IRRI and evaluated their resistance to the predominant BPH Bangladesh type in China (Lv et al. 2009).A total of 105 extremely resistant (resistance score: 0-2.00) materials, 71 resistant (resistance score: 2.01-3.49)materials, and 138 moderately resistant (resistance score: 3.50-5.49)materials were identified (Supplementary Table S2).Further molecular screening of these germplasms was carried out by developing dominant functional molecular markers of cloned BPH resistance genes, including Bph18/Bph1, Bph26/bph2, Bph15/Bph3, Bph6, Bph7, Bph9, Bph14, and Bph32, showing that a total of 447 accessions contain one to five of the cloned BPH resistance genes (Supplementary Table S9).Among the 314 resistant varieties, 31 contained Bph18, 105 contained Bph26, 77 contained Bph6, 2 contained Bph7, 8 contained Bph9, 53 contained Bph14, and 61 contained Bph3.In addition, 75 samples harbored Bph32 (Supplementary Table S2).Furthermore, 46 accessions did not carry any of the eight cloned BPH resistance genes (Supplementary Table S12).These results systematically determined the corresponding relationship between BPH-resistant germplasm resources and BPH resistance genes, providing the application guidance of these germplasms in a BPH resistance breeding program.
When multiple resistance genes exist in the same plant, it generally provides more protection for rice to enhance resistance (Divya et al. 2018).Consistently, most of the extremely resistant accessions contain two or more BPH resistance genes (Table 2).Notably, 179 accessions identified to carry cloned BPH resistance genes were susceptible to the predominant BPH Bangladesh type in China (Supplementary Table S10).Among them, 61, 56, and 7 accessions carried Bph18, Bph26, and Bph9, respectively, while 6 accessions carried Bph18 and Bph26.These 130 accessions accounted for 72.6% of 179 susceptible accessions.Another 38 susceptible materials containing two or more resistance genes also carried one of Bph18, Bph26, and Bph9.This finding might be reasonable as the resistance of Bph18 and Bph26 was broken down or adapted by the more-destructive Bangladesh type (Alam and Cohen 1998;Ketipearachchi et al. 1998;Zhou et al. 2021).The resistance of a major BPH resistance gene is also affected by minor QTLs.For example, the rice variety IR64 along with the Bph18 gene detected multiple minor QTLs on several chromosomes, showing durable and stable resistance even after the spread of the Bph18destroyed BPH population (Alam and Cohen 1998).Another possibility is that some resistance genes may produce antagonism when aggregated or coexpressed, resulting in reduced or even lost resistance (Sundaram et al. 2009).
Based on strict phenotypic identification and genotyping of functional markers, 23 accessions identified in this study may contain new BPH resistance genes as they exhibited a variety of resistance mechanisms in terms of the insect WG rate.In fact, Bph33 in IRGC 36295 (KOLAYAL) and IRGC 36352 (POLI-YAL) and Bph43 in IRGC 8678 were recently identified and fine-mapped to chromosomal regions, respectively (Hu et al. 2018;Kim et al. 2022).In our study, the BPH resistance gene Bph44(t) in IRGC 15344 was identified and narrowed to the region similar to the location of Bph30 in AC-1613 and Bph33 in KOLAYAL and POLIYAL (Hu et al. 2018;Shi et al. 2021).Sequence comparison revealed that Bph44(t) is a new major BPH resistance gene, or the allele of Bph30.Like Bph30, Bph44(t) will also play an important role in BPH resistance breeding programs for sustaining the control of BPH.

Conclusion
To combat the emerging high virulence BPH populations, the resistance of 560 rice accessions from the International Rice Research Institute (IRRI) to the predominant BPH Bangladesh type in China was evaluated.The corresponding relationship between BPH-resistant germplasm resources and cloned BPH resistance genes was determined using developed distinct functional markers.Twenty-three resistant accessions were identified to be novel brown planthopper resistance sources.A novel BPH resistance gene Bph44(t) was explored and mapped to a region on chromosome 4. Newly explored resistant germplasms will provide valuable donors for the isolation of novel BPH resistance genes and BPH resistance breeding programs.

Fig. 1 Fig. 2
Fig. 1 BPH resistance assay of representative resistant germlasm after BPH infestation.The seedling plants were exposed to BPH insects and photographed after 5 days.LuoY-ang69, Bph6, and Bph9 pyramided line in 93-11 genetic background, was used as resistant control.Resistant control was Luoyang 69 which carried BPH6 and BPH9 under the genetic background of 93-11.TN1 was used as susceptible control.The resistant LuoYang69 plants and representative resistant germplasm IRGC 36352, IRGC 20263, IRGC 15427, IRGC 36246, and IRGC 8670 grew well after BPH attack, while the TN1 plants died.Scale bars = 5 cm

Fig
Fig.3Molecular screening of BPH resistance genes in rice germplasms using developed functional dominant markers.The genomic DNA of 17 representative rice germplasms was amplified by PCR using functional dominant markers B14D, B3D, B1279D, B18D, B26D, B7D, B9D, B32D, and B6D.The corresponding rice materials, functional markers, and DNA marker are indicated at the top, right, and left, respectively

Fig. 5
Fig. 5 Molecular mapping of Bph44(t).A Rice chip GSR 40K analysis based on bulked segregant analysis (BSA) mapping of Bph44(t) on chromosome 4 of rice.White lines represent the genetic background of 93-11, while red lines represent that of IRGC 15344.The heterozygous genomic segments between IRGC 15344 and 93-11 are denoted by blue lines.S means susceptible; R means resistant.B The genomic area delimited by the markers 344-0 and 344-2 is where Bph44(t) was mapped.C Physical map showing the location of markers 344-0-6 and

Table 1
Functional dominant markers developed in this study Vol:. (1234567890)

Table 2
Summary of resistance of rice accessions to current BPH populations of China

Table 3
Distribution of different resistance genes and resistance phenotypes in 560 germplasm resources