Brown planthopper resistant dominant complementary genes Bph50 and Bph51 as well as encoding protein and application thereof

Abstract

The invention discloses brown planthopper resistant dominant complementary genes Bph50 and Bph51 as well as an encoding protein and application thereof, and belongs to the technical field of biology. The amino acid sequence of the protein coded by the Bph50 gene is as shown in SEQ ID NO. 2, and the nucleotide sequence of the Bph50 gene is as shown in SEQ ID NO. 1. The amino acid sequence of the protein coded by the Bph51 gene is as shown in SEQ ID NO.4, and the nucleotide sequence of the Bph51 gene is as shown in SEQ ID NO.3. The invention provides a novel brown planthopper resistance gene and a brown planthopper resistance identification method aiming at the gene, and a foundation is laid for the cultivation of a novel brown planthopper resistant rice variety. According to the invention, the strain Xinxiangzhan No.1 which is high in yield, rich in fragrance and good in brown planthopper resistance is obtained by culturing BPHR96 containing dominant complementary genes Bph50 and Bph51 resistant to brown planthoppers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a dominant complementary gene Bph50 and Bph51 for resistance to brown planthopper, its encoded protein, and its application. Background Technology

[0002] Brown planthoppers are major migratory pests of rice, and Guangxi is a major source area for them in the rice-growing regions of the middle and lower reaches of the Yangtze River. Due to the high initial population of these pests from abroad, Guangxi is prone to frequent and large-scale outbreaks of brown planthopper infestations. For a long time, the control of brown planthoppers has relied primarily on chemical pesticides. However, the extensive use of chemical pesticides leads to pesticide residues, environmental pollution, and increased production costs. Furthermore, it can cause brown planthoppers to develop strong resistance to the most effective pesticides. In addition, the overuse of pesticides kills natural enemies of brown planthoppers in rice paddies, potentially triggering a resurgence of the infestation. Scientific research and production practices have proven that planting brown planthopper-resistant rice varieties is the most economical, effective, and environmentally friendly measure for controlling brown planthoppers. The discovery of brown planthopper-resistant genes and the study of resistance mechanisms will provide genetic resources and theoretical guidance for breeding brown planthopper-resistant varieties.

[0003] To date, 11 resistance genes have been cloned: Bph3, Bph6, Bph9 / Bph18 / Bph26, Bph14, Bph29, Bph30, Bph32, Bph37, and Bph40. Among them, Bph1, Bph2, Bph7, Bph10, Bph21, and Bph9 are multiple alleles at the same locus on chromosome 12 (Zhao et al., 2016). Bph3 consists of three clusters encoding lectin receptor kinases (OsLecRK1-OsLecRK3), and the combined action of these lectin receptor kinase genes regulates broad-spectrum and persistent resistance in rice to brown planthoppers. Bph6 is a novel insect-resistant gene whose protein is located in the exocyst complex and interacts with the exocyst complex subunit EXO70E1. The Bph9 gene encodes an NBS-LRR family protein with two NBS domains. Its sequence is of a rare type, causing cell death and activating the salicylic acid and jasmonic acid signaling pathways in rice plants to induce resistance to brown planthoppers. Bph14 and Bph26 both encode leucine-rich repeats (CC-NB-LRR), with the LRR domain playing a role in activating the rice defense response. Bph18 encodes a CC-NBS-NBS-LRR protein (containing two NBS domains), primarily located in the inner membrane of cells, and may be involved in recognizing brown planthopper feeding within the phloem cell membrane. Bph18 and Bph26 are alleles, containing three exons and two introns, but there are 195 single nucleotide polymorphisms between them, resulting in 105 differences in amino acid residues, 88 of which are located in the LRR domain. Bph29 contains a B3-binding domain, which can activate the salicylic acid signaling pathway and inhibit the jasmonic acid / ethylene pathway. The Bph30 gene encodes a protein containing two leucine-rich domains (LRDs), belonging to a novel planthopper resistance gene family. It provides broad-spectrum resistance to both brown and white-backed planthoppers by strengthening the thick-walled tissue of rice, forming a robust barrier that prevents brown planthoppers from feeding on phloem sap. Bph32 encodes a specific conserved domain protein (SCR), which is highly expressed in the rice leaf sheath 2 h and 24 h after brown planthopper feeding, inhibiting feeding. Zhou et al. (2021) identified 3502 associated SNPs and 59 sites associated with brown planthopper resistance using GWAS analysis; they cloned and verified the novel brown planthopper resistance gene Bph37, which encodes a nucleotide-binding site-rich leucine protein.

[0004] Although more than 50 brown planthopper resistance genes have been identified to date, and 11 of them have been cloned, only a very small number of these genes are currently available for effective breeding. Furthermore, the genetic diversity of resistance genes plays a crucial role in resisting the constantly evolving biotypes of brown planthoppers, necessitating the continuous discovery of new resistance genes to enrich the genetic resources for resistance breeding (Zhang et al., 2011). Du et al. (2018) argued that the insect resistance of rice and the pathogenicity of brown planthoppers are co-evolving, and that the frequency of brown planthopper resistance genes is relatively high in areas where brown planthoppers frequently occur. Guangxi is both a region with frequent brown planthopper occurrences and one of the areas with the richest genetic diversity of wild rice. Therefore, strengthening the identification of brown planthopper resistance in Guangxi wild rice germplasm resources can help screen for resistant germplasm resources with high utilization value. This invention cloned two dominant complementary genes, Bph50 and Bph51, against brown planthopper from the wild rice introduction line BPHR2170. The brown planthopper resistance is controlled by these two pairs of genes, which is a novel genetic mechanism for brown planthopper resistance. Summary of the Invention

[0005] The present invention provides dominant complementary genes Bph50 and Bph51 for resistance to brown planthopper, wherein the amino acid sequence of the protein encoded by the Bph50 gene is shown in SEQ ID NO.2; and the amino acid sequence of the protein encoded by the Bph51 gene is shown in SEQ ID NO.4.

[0006] In one embodiment of the present invention, the nucleotide sequence of the Bph50 gene is shown in SEQ ID NO.1.

[0007] In one embodiment of the present invention, the nucleotide sequence of the Bph51 gene is shown in SEQ ID NO.3.

[0008] This invention also provides the application of the above-mentioned dominant complementary genes Bph50 and Bph51 against brown planthopper in regulating brown planthopper resistance in rice.

[0009] This invention also provides a method for breeding rice with high yield, strong aroma, and good resistance to brown planthoppers, comprising the following steps:

[0010] (1) In the first year of early crop, the fragrant rice maintainer lines Manxiang B and Xinyinzhan were used as female parents and BPHR96 was used as male parent to obtain hybrid F1 seeds of Manxiang B×BPHR96 and Xinyinzhan×BPHR96 respectively.

[0011] (2) In the first year of late crop, 15 hybrid F1 plants of Manxiang B×BPHR96 and Xinyinzhan ×BPHR96 were planted. After removing impurities, the F1 plant seeds of Manxiang B×BPHR96 and Xinyinzhan ×BPHR96 were mixed and harvested to form the F2 population.

[0012] (3) In the second year, F2 populations were planted separately. The molecular markers PSM152, PSM226, and Y5-Y8 were used to detect the Bph50, Bph51, and aroma gene fgr in the F2 population of Manxiang B×BPHR96. Ten individual plants containing three Bph50, Bph51, and aroma gene fgr and with excellent main agronomic traits were selected and crossed with ten individual plants containing Bph50 and Bph51 and with excellent agronomic traits selected from the F2 population of Xinyinzhan×BPHR96 to obtain 10 hybrid F1 seeds. The primers corresponding to the molecular markers PSM152, PSM226, and Y5-Y8 are shown in SEQ ID NO.11-12, 15-16, and 7-10, respectively.

[0013] (4) In the second year, the late-season mixed sowing of hybrid F1 seeds was carried out. Molecular markers PSM152, PSM226, and Y5-Y8 were used to perform marker-assisted selection on Bph50, Bph51 and the aroma gene fgr. Several single plants containing three target genes and with excellent agronomic traits were selected and harvested separately to form F2 seeds.

[0014] (5) In the early third year, the F2 seeds of the single plants screened in step (4) are planted in plots. Molecular markers PSM152, PSM226, and YY5-YY8 are used to detect Bph50, Bph51 and the aroma gene fgr. Single plants with homozygous Bph50 and Bph51 genotypes, homozygous or heterozygous aroma gene fgr genotypes, and excellent agronomic traits are selected and harvested as single plants to form F3 lines.

[0015] (6) In the third year of late crop, the resistance of the F3 line to brown planthopper was identified by using molecular markers PSM152 and PSM226; when both PSM152 and PSM226 are highly resistant to brown planthopper, the plant is determined to be a highly resistant plant to brown planthopper.

[0016] (7) After the identification of resistance to brown planthopper, the aroma gene fgr was detected by YY5-YY8 marker. 31 single plants with homozygous genotype and excellent other major agronomic traits were screened out. At this point, the genotypes of the three target genes (Bph50, Bph51 and aroma gene fgr) were all homozygous. The single plants were harvested to form F4 lines.

[0017] (8) In the early fourth year, plant F4 lineage, select several single plants with excellent main agronomic traits, and harvest seeds from single plants to form F5 lineage;

[0018] (9) Subsequently, starting from the F5 generation of the fourth late crop, the plantation was continuously extended to accelerate the stabilization of agronomic traits. In the F7 generation of the fifth late crop, brown planthopper resistance was identified using the same method as step (6). The pedigree method was used to select up to the F10 generation of the seventh early crop. Several superior strains were selected from the F10 generation. In the seventh late crop, the selected strains were planted, and the overall stability and consistency of agronomic traits were observed. Yield comparison tests were also conducted, and several strains were eliminated. In the eighth early crop, brown planthopper resistance and aroma were identified for the selected superior strains. Finally, a strain with high yield, rich aroma, and good brown planthopper resistance was selected.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a novel brown planthopper resistance gene and a method for identifying brown planthopper resistance to this gene, laying the foundation for the breeding of new brown planthopper-resistant rice varieties. Attached Figure Description

[0021] Figure 1 This study analyzes the expression level of the Bph50 candidate gene LOC_Os04g12390 (Os04g0200400) in Example 1.

[0022] Figure 2 This study analyzes the expression level of the Bph51 candidate gene LOC_Os04g26320 (Os04g0330200) in Example 1.

[0023] Figure 3 The image shows the results of gene editing and identification of Bph50 and Bph51 using CRISPR / Cas9 in Example 1. A: Schematic diagram of the gene structures of Bph50 and Bph51 and the mutants generated by CRISPR / Cas9 targeted editing; gray rectangles, black rectangles, and black lines represent the UTR region, exons, and introns, respectively. B: Identification of the resistance of the brown planthopper in the CRISPR / Cas9-mediated Bph50 and Bph51 gene knockout mutants.

[0024] Figure 4 The breeding process of Xinxiangzhan No. 1 in Example 1. Detailed Implementation

[0025] The resistance source material BPHR2170: The F1 generation was obtained by crossing the high-quality restorer line Gui99, which is highly susceptible to brown planthopper, with the highly resistant Guangxi wild rice BPHR96 (see Chinese Patent, Application No.: 202110720600.5, Patent Name: A Breeding Method for a Rolled-Leaf Rice Restorer Line Resistant to Brown Planthopper). Subsequently, the F2 segregated population was subjected to brown planthopper resistance identification (insect population density of 60 insects, which is 5 times more than the standard resistance identification population). The resistant plants were then transplanted to the field for planting and self-pollination to obtain the F3 family. Plants with normal plant height, seed setting rate, growth period, and plant and leaf morphology were selected and retained. Starting from the F3 generation, the selected lines were subjected to brown planthopper resistance identification every other generation (insect population density of 60 insects). Pedigree selection was carried out up to F10, and finally, a wild rice introduction line material with uniform and stable plant and leaf morphology, growth period, and normal seed setting rate was obtained, which has a resistance level that can reach the immunity level. R4684 is a high-quality, multi-ear type restorer line bred by our laboratory through hybridization of Guanghui 998 and restorer line R470, followed by 10 generations of self-pollination and selection. It exhibits high susceptibility to brown planthopper.

[0026] The breeding process for R4684 is as follows:

[0027] In the early 2013 crop, Guanghui 998 was used as the female parent and restorer line R470 (see CN 113234850 B) as the male parent for hybridization, yielding 46 hybrid offspring seeds. All F1 seeds were sown in the late 2013 crop, and 30 plants were planted. In the early 2014 crop, 2500 F2 plants were planted, and 35 high-yielding individual plants with curled leaves, compact plant type, and multiple spikes were selected. In the late 2014 crop, F3 plants were planted according to their lines, eliminating inferior lines. Individual plants with inward-curling leaves, compact plant type, and multiple spikes were selected from lines exhibiting good main agronomic traits, and their rice quality (transparency, chalkiness, length-to-width ratio) was assessed before selection. Subsequent generations were selected using the pedigree method, with rice quality observed from individual plants exhibiting excellent main agronomic traits, inward-curling leaves, compact plant type, and multiple spikes. Poor-quality rice lines and individual plants were eliminated. By the late-season F9 generation in 2017, the F9 generation showed stable performance. Five superior lines with excellent main agronomic traits, inward-curling leaves, compact plant type, and multiple spikes were selected and testcrossed with the male-sterile lines Nafeng A, Longtepu A, and Tianfeng A. Heterosis was compared in the early-season 2018. In the experiment, one line, designated R4684, showed outstanding overall performance in hybrid combinations with Nafeng A, Longtepu A, and Tianfeng A. It exhibited good comprehensive agronomic traits, strong tillering ability, moderate plant type, vigorous growth, good ripening and color change, and better appearance and grain quality than the control variety, Teyou 7118. Its yield was 545.5-548.6 kg / mu, an increase of 5.8%-6.4% over Teyou 7118 (CK), and its growth period was 0.3 days shorter than Teyou 7118. Therefore, line R4684 was selected as the target line. This line has stable and consistent overall agronomic traits, with inward-rolling leaves, a compact plant type, and is a multi-ear type restorer line, and was named R4684.

[0028] Example 1

[0029] 1. Identify target candidate genes for Bph50 and Bph51.

[0030] Our research group previously conducted large-scale identification and evaluation of brown planthopper resistance in wild rice from Guangxi, screening out a common wild rice variety with immune resistance to brown planthoppers. Through hybridization and selection with cultivated rice and years of resistance identification, we obtained a wild rice introduction line, BPHR2170, with brown planthopper resistance levels comparable to the resistance source. Genetic analysis showed that the brown planthopper resistance of the wild rice introduction line BPHR2170 is controlled by two pairs of dominant complementary genes, Bph50 and Bph51, representing a novel genetic mechanism of brown planthopper resistance. Various genetic populations of the R4684 / BPHR2170 F1 generation were constructed using crosses between R4684 and BPHR2170 and continuous backcrosses. 2:3 Marker analysis of close linkage between pedigrees and immune cells and the use of QTLIciMapping to analyze their 192 F 2:3 QTL mapping was performed on the family, identifying one resistance locus each on the short and long arms of chromosome 4, named Bph50 and Bph51, which explained 33.1% and 55.9% of the phenotypic variation, respectively. Marker selection was used to construct single-gene lines of Bph50 and Bph51, as well as a double-gene line of Bph50+Bph51, and resistance was assessed. Results showed that the resistance level of the Bph50+Bph51 double-gene line was comparable to that of the resistance source BPHR2170, while both the Bph50 and Bph51 single-gene lines showed a resistance level of 9, indicating high susceptibility to brown planthoppers. Therefore, the mapping results and the resistance genetic analysis are consistent; the brown planthopper resistance of BPHR2170 is controlled by two pairs of dominant complementary genes, and resistance only occurs when both genes are present simultaneously.

[0031] By screening recombination exchange individuals from large backcross segregating populations (approximately 20,000 individuals in BC2F3, BC3F2, and BC5F2) across generations, and based on the corresponding family resistance identification results, Bph50 and Bph51 were finely mapped to ranges of 73kb and 30kb, respectively. Gene annotation of the Nipponbare reference genome sequences within the Bph50 and Bph51 mapping regions was performed using the websites http: / / rice.plantbiology.msu.edu / and http: / / rapdb.dna.affrc.go.jp / . The Bph50 mapping region contains 6 transposons and 2 annotation genes, R1 (LOC_Os04g12390) and R2 (LOC_Os04g12430). The R1 (LOC_Os04g12390) gene encodes the Jacalin lectin protein, and the R2 (LOC_Os04g12430) gene encodes an expressed protein. Studies have shown that plant lectins have a protective effect against piercing-sucking insects. Gene sequence analysis of the resistant and susceptible parents revealed that the coding region of the R2 (LOC_Os04g12430) gene showed no difference between the resistant and susceptible parents BPHR2170 and R4684. However, the R1 (LOC_Os04g12390) gene had a 10975 bp insertion downstream of the start codon at 31 bp in the susceptible parent R4684. This large insertion caused an interruption of the open reading frame, terminating transcription. Furthermore, qRT-PCR analysis was used to analyze the changes in the expression level of the R1 (LOC_Os04g12390) gene before and after feeding in the resistant and susceptible parents BPHR2170 and R4684. Figure 1 The results showed that the expression level of the R1 gene on the susceptible parent R4684 was very low or almost non-existent before and after feeding; while the expression level of the R1 gene on the resistant parent BPHR2170 was very high before and after inoculation, reaching its highest level 24 hours after inoculation, which was 1 times higher than the expression level at other time points. In summary, the expression level of the R1 gene on the resistant parent BPHR2170 was significantly or extremely significantly higher than that on R4684 before and after inoculation. Therefore, the R1 gene LOC_Os04g12390 (Os04g0200400) was finally identified as the target candidate gene.

[0032] Annotation of the Bph51 localization region revealed one transposon and one annotated gene, LOC_Os04g26320 (Os04g0330200). The LOC_Os04g26320 gene encodes Jacalin, a plant lectin known for its resistance to piercing-sucking insects. Sequence analysis of the resistant parental genes showed that in the susceptible parent R4684, a 428 bp insertion in the second exon of the LOC_Os04g26320 gene prematurely terminated protein translation, resulting in the loss of its resistance function. Furthermore, qRT-PCR analysis was used to examine the expression levels of the LOC_Os04g26320 gene in the resistant parents BPHR2170 and R4684 before and after feeding by brown planthoppers. The expression level of the LOC_Os04g26320 gene in BPHR2170 was induced by feeding on brown planthoppers, gradually increasing over time and reaching its highest level after 48 h. However, in the susceptible parent R4684, the expression level of the LOC_Os04g26320 gene was significantly or extremely significantly lower than that in BPHR2170 at all times except for 6 h, when there was no difference between the two genes. Figure 2 In conclusion, the LOC_Os04g26320 (Os04g0330200) gene is highly likely to be a target candidate gene for Bph51.

[0033] 2. Obtain full-length Bph50 and Bph51 DNA.

[0034] Using leaf DNA from the brown planthopper-resistant parent BPHR2170 as a template, primers were designed on NCBI using Primer-BLAST based on the Nipponbare reference genome sequences corresponding to the target candidate genes Bph50 and Bph51 and their promoters. This amplified multiple overlapping DNA sequences covering the target candidate genes Bph50 and Bph51 and their promoters, and then assembled the genome sequences covering the Bph50 and Bph51 genes and their promoters. Primers were resynthesized based on the spliced ​​genome sequences, and the full-length DNA and promoter sequences of Bph50 and Bph51 were amplified respectively. The full-length Bph50 gene and promoter sequence is 6490 bp, with the promoter sequence being 2007 bp and the gene sequence being 4483 bp. The Bph50 gene contains 2 exons and 1 intron, with a coding region (CDS) of 996 bp encoding 331 amino acids. Its nucleotide sequence and amino acid sequence are shown below. The full-length Bph51 gene and promoter sequence is 4418 bp, with the promoter sequence being 2320 bp and the gene sequence being 2098 bp. The Bph51 gene contains 2 exons and 1 intron, with a coding region (CDS) of 1119 bp encoding 372 amino acids. Its nucleotide sequence and amino acid sequence are shown below.

[0035] Bph50 full-length DNA sequence (SEQ ID NO.1):

[0036] ATGCACACATATTTGGGGACATTCATAGGTGTATTAA TGGTCTTGACACTTCAGCGTGTGAAGATAGGGCCGTGGGGTGGAACTGGAGGCCATGCTTGGGATGAGGGAGGCCA TGGTGCCAGTGCTGGCGGTTACACCGGTGTACGCCGAATGAGTATAGGGTCTTCCTGGTGTGTCAGCTCAATGTTG TTTGAATACGACGACAATGGCAAACGTGTGAAAGGTACCCCGCAAGGAGAGAGAGACAATGAAATACCCGAGGAGGAGCTCGACT TCCATGGGGAGGTGTTGACGCACATGTGCGGCTACCATGACAACCACCTCATCCGGTGGTTGCAGTTCAGGAGCAA CCGGAACAGGACGTTCGGACCATACGGCAATCTTGGAGAAGACAAAGCTGGATGGACGCGGTTCGAGGTCTCCATG GAGCACTCGGGGTCCATCGTCGGCTTCTGCGGCCGGAGTGGCAACTTCACGGACGCCATCGGCGTTTACGTCGCCG TCTGGAATCCCGAGAGGTTCTATGATAGCATGCGCAGGCAGGGCGTCCGCGTTTACCGGGCGTCGCCTCTGCGCAT GGACCTGCGTCAAATAGAAGAAGAGAAAAAGAAGGAAGAAGTGGAACGTGGGCGCCTACAGAAGGAGATCAAGGAG GGGCGAGAAAGTCTTCGGAAGTTACGATTGAAGTTTGGAGTGGATGTGCCGCAGCAGGATCAGGGAAAACGCCAGA CAATAGAGGAGCTGCAAGTGGAGCATGAGCAACTGGAACGGGAACGAGGGCGCTTGCTTCTACTGCTGAAAGTGGA GCATGAGCAATGGGAACGGGAACGAGGGCGCTTGCTTCGACAGAAACACCTAAGGGAGGAGTTACTCCAGTGGGAG GAGTTACTCCAGTGGTTTGACCCCAGACTTTTTGGTAGGAGCAGTCCCGGTTATTTTTTTTTCTCAAATAAATATC CCCGATCATATTCCCAAATCATCCCCAAATCAAAGTAG (The underlined part is the CDS sequence)

[0037] Bph50 protein sequence (SEQ ID NO.2):

[0038] MHTYLGTFIGVLMVLTLQRVKIGPWGGTGGHAWDEGGHGASAGGYTGVRRMSIGSSWCVSSMLFEYDDNGKRVKGTLQGERDNEIPEEELDFHGEVLTHMCGYHDNHLIRWLQFRSNRNRTFGPYGNLGEDKAGWTRFEVSMEHSGSIVGFCGRSGNFTDAIGVYVAVWNPERFYDSMRRQGVRVYRASPLRMDLRQIEEEKKKEEVERGRLQKEIKEGRESLRKLRLKFGVDVPQQDQGKRQTIEELQVEHEQLERERGRLLLLLKVEHEQWERERGRLLRQKHLREELLQWEELLQWFDPRLFGRSSPGYFFFSNKYPRSYSQIIPKSK*

[0039] Full-length DNA sequence of Bph51 (SEQ ID NO.3):

[0040] ATGGTCTTGACACTTCAGCGTGTGAAGA TAGGGCCGTGGGGTGGAACTGGAGGCCATGCTTGGGATGAAGGAGGCCATGGTGCCAGTGCTGGCAGTTACACCGG AGTACGCCGAATGAGTATAGGGTCTTCCTGGTGTGTCAGCTCGATGTTGTTTGAATACGACGACAATGGCAAACGT GTGAAAGGTACCCTGCATGGAGAGAGAGACAATGGAATACCCGAGGTATGTAGCAAGCAATACTTGATAGATTTGTGATCTTAGGATTTAGGAAGTGATTTTCCAAGTGAAAACAATTTCTCCACTCCTTATAGGATCTACTCTATCTTATTAGAAACTTATATTTATTACTGCCATGTCTACCCAAAATCTAAATATTTTTTGTTGAAAATTGATTAGGCGCCACCAACATCTGACGTCATCCTATTCGTCGCCCTCTCTGGGTGGCTTTTAGTTAACCGGTACCTATAAATTATAGGTACAAGTTTTTCTAAAAAAAACCGACACATATAGTATTTGTGTTGATTTTCCTCTACAACCAGCATATATAGTTACCCAAAGGTGACGTTTCTGTAACTTTTTTCATCCTGTGGAGGTAGGGAAAGGTTCATATGTGCCGGTTTTTAAATGAACGGACACCTATGAGGGTTTTGCAGTAATGATGCGCTAAGGGTTCAGTACTGTAGCTGTAGAGAAAGAAACTATTAACTTAATT ACCTTCAGTGGAAAGGTGGGTAAATTTTACAATTAATATACCATTTAAAGAAATGCCCACGCAAATGAATGGTTGGATTTTACTCACACCAGTGCCCCTTGTCACTTGTACCGTGTATCGTAAAACAACTCCTGGTAACGACGATGGGCACAGATCATGACCAATTTGTTAAAGCCCAAGGCCTCTGCTTCTTGCTTCTGACAATCTCATAAAGGTTTTCATGAGCGGTGAACCGGTGACTAGTTCATTTAATCATGATCTAATAGAACTCATCAACTAGTTGTATTATAGAATCAGTAGGGACTCTTCTTTAATCGTGGAGTTGTATTAATTTTTCCACTGTTTTAGTTATGTAAAAACACAGACAAATACTCAGTCGTCGATTGAACGGTTTAATTTGGGAGAAAATTAAAACAAACAGCTTGCAGAGAGATGCTTTGTACTAATTAATCACTGACTGACAAATAACCTGATGCATGGACGCACACAG GAGGAGCTCGACTTCCATGGGGAGGTGTTGACGCACATGTGCGGCTACCATGACAACCACCTCATCCGGTGGTTGCAGTTCAGGAGCAACCGGAACAGGACGTTCGGACCATACGG CAATCTTCTAGAAGACCAGGCTGGATGGACGCGGTTCGAGGTCTCCATGGAGCACTCGGGGTCCATCGTCGGCTTC TGCGGCCGGAGTGACGACTTCGTGGACGCCATCGGCGTCTACGTCGCCGTCTGGAATCCCGAGAGGTTCTATGATA GCATGCGCAGGCAGGGCGTCCGCGTTTACCGGGCGTCGCCTCTGCGCATGGACCTGCGTCAAATAGAAGAAGAGAA AAAGAAGGAAGAAGTGGAACGTGGGCGCCTACAGAAGGAGATCGAGGAGGGGCGAGAAAGTCTTCGGAACTTACGA TTGAAGCTTCGAGTGGATATGCCGCCGGATCAGAGAAAACGCCTGATACGACGCGACCTGCGAGTGGAGCATCAAG AAATCGAACGTCAGTTACAAGAGTTGCAGCAACTGGAACGCGGGCGTCAGGGAAAGCGCCAGACACTTGAGGAGCT GCAAGTGGAGCAACAAGAAATCGAACGTCAGTTACAAGAGATGCAGCAACTGGTACACGGGCGTCATTTACAGAAG ATGCAGCAAATGGTACGCAAGCGTCAGCAACTGGAGCAACAAGAAATCGAACGTCAGTTACAAGAGATGCAGCAAC TGGTACGCTCGCGTCAGCAACTGGAGCAACAAGAAATCAAACGTCAGTTACAAGAGATGCAGCAACTGGAACGCGA GCGTCAGCTACACCGGTTGAGAAAATTTGTAGTCCCGGTTCGTAACCCCCCTTTAGTCCCGGTTTCCAAACCGGGA CTACCAATCCGGGACTAA (The underlined part is the CDS sequence)

[0041] Bph51 protein sequence (SEQ ID NO.4):

[0042] MVLTLQRVKIGPWGGTGGHAWDEGGHGASAGSYTGVRRMSIGSSWCVSSM LFEYDDNGKRVKGTLHGERDNGIPEEELDFHGEVLTHMCGYHDNHLIRWLQFRSNRNRTFGPYGNLLEDQAGWTRFEVSMEHSGSIVGFCGRSDDFVDAIGVYVAVWNPERFYDSMRRQGVRVYRASPLRMDLRQIEEEKKKEEVERGRLQKEIEEGRESL RNLRLKLRVDMPPDQRKRLIRRDLRVEHQEIERQLQELQQLERGRQGKRQTLEELQVEQQEIERQLQEMQQLVHGRHLQKMQQMVRKRQQLEQQEIERQLQEMQQLVRSRQQLEQQEIKRQLQEMQQLERERQLHRLRKFVVPVRNPPLVPVSKPGLPIRD*

[0043] 3. Functional verification of Bph50 and Bph51 genes

[0044] To further verify the association between the Bph50 and Bph51 genes and resistance in brown planthoppers, we constructed knockout vectors pYLCRISPR-cas9-BPS for the Bph50 and Bph51 genes, respectively (target SEQ ID NO.5: ACCGGTGTAACCGCCAGCAC). TGG ) and pYLCRISPR-cas9-BPL (target SEQ ID NO.6: GCTTGGGATGAAGGAGGCCA TGG TGCCAGTGCTGG) Figure 3A), and respectively transformed into the insect-resistant parent BPHR2170. After the callus was cultured into seedlings, 25 and 21 transgenic lines were obtained, respectively. The brown planthopper resistance of the transgenic lines was identified by the seedling group method. The results showed that all BPHR2170 transgenic lines with Bph50 or Bph51 gene knocked out lost brown planthopper resistance (A). Figure 3 (B) This means that mutations caused by editing the Bph50 or Bph51 genes can lead to the loss of resistance function in BPHR2170. Therefore, it has been determined that both Bph50 and Bph51 genes are related to the control of resistance function in the BPHR2170 brown planthopper, and the two together control its resistance in the brown planthopper.

[0045] 4. Breeding utilization of brown planthopper resistance genes Bph50 and Bph51

[0046] The breeding process of Xinxiangzhan No. 1 (see...) Figure 4 ):

[0047] In the early 2011 crop, the fragrant rice maintainer lines Manxiang B and Xinyinzhan were used as female parents, and BPHR96, a highly resistant source of brown planthoppers, was used as the male parent for hybridization, resulting in F1 hybrids of Manxiang B × BPHR96 and Xinyinzhan × BPHR96, respectively. In the late 2011 crop, 15 F1 hybrids of Manxiang B × BPHR96 and Xinyinzhan × BPHR96 were planted. After weeding, the F1 hybrids of Manxiang B × BPHR96 and Xinyinzhan × BPHR96 were mixed and harvested to form F2 populations. In the early 2012 crop, F2 populations (1500 individual plants each) were planted, and the F2 populations were developed using the brown planthopper resistance genes Bph50 and Bph... 51 (Bph50 and Bph51 are resistance genes cloned from the highly resistant wild rice line BPHR2170 derived from BPHR96) and the aroma gene fgr were closely linked molecular markers PSM152, PSM226, and Y5-Y8. These markers were used to detect Bph50, Bph51, and the aroma gene fgr in the F2 population of Manxiang B × BPHR96. Ten individual plants containing all three genes (Bph50, Bph51, and the aroma gene fgr) and exhibiting excellent major agronomic traits were screened. This was in contrast to the screening of plants from the F2 population of Xinyinzhan × BPHR96 that simultaneously contained Bph50, Bph51, and the aroma gene fgr (genotype heterozygous or homozygous) and exhibited superior main agronomic traits. Ten individual plants with excellent agronomic traits were hybridized to obtain 865 F1 seeds from 10 hybrid (combinations). In the late 2012 crop, these 865 F1 seeds were mixed and sown to obtain 836 plants. Marker-assisted selection was performed using molecular markers PSM152, PSM226, and Y5-Y8 to target Bph50, Bph51, and the aroma gene fgr, selecting 33 individual plants containing all three target genes and exhibiting excellent agronomic traits. These individual plants were harvested to form F2 seeds. In the early 2013 crop, the 33 selected F2 plants were planted in plots of 50 plants each, and the molecular marker PSM152 was used again to further develop the F2 seeds. PSM152, PSM226, and YY5-YY8 were used to detect Bph50, Bph51, and the aroma gene fgr. Forty plants with homozygous Bph50 and Bph51 genotypes, and with excellent agronomic traits (homozygous or heterozygous for the aroma gene fgr), were selected and harvested individually to form F3 lines. In the late crop of 2013, the 40 F3 lines were tested for resistance to brown planthoppers. Forty plants from each line were sown. The results showed that no single plants with segregation of resistance to brown planthoppers appeared in the F3 plants of the 40 lines, proving the accuracy and efficiency of the PSM152 and PSM226 molecular markers in selecting Bph50 and Bph51 genes.After identifying brown planthopper resistance, normally growing plants were transplanted to the field and planted in plots of 30 individual plants per plot. The aroma gene fgr was then detected using the YY5-YY8 marker, and 31 homozygous individuals with excellent other major agronomical traits were selected to resist brown planthoppers. At this point, all three target genes (Bph50, Bph51, and the aroma gene fgr) were homozygous. Individual plants were harvested to form 31 F4 lines. In the early 2014 crop, these 31 F4 lines were planted, with each line planted in plots of 30 individual plants. 25 individuals with excellent major agronomical traits were selected, and seeds were harvested from these 25 F5 lines. Subsequently, from the late 2014 crop... Starting with the F5 generation, continuous planting was carried out to accelerate the stabilization of agronomic traits. In the late F7 generation of 2015, brown planthopper resistance was identified. The pedigree method was used to select up to the F10 generation of the early F10 generation in 2017, from which 5 superior strains were selected. In the late F10 generation of 2017, the 5 selected strains were planted in plots of 500 plants each with 3 replicates. The overall stability and uniformity of agronomic traits of the plants in each plot were observed, and yield comparison tests were conducted. Three strains were eliminated. In the early F18 generation of 2018, brown planthopper resistance and aroma were identified in the 2 selected superior strains. Finally, a strain C316 with high yield, strong aroma and good brown planthopper resistance was selected and named Xinxiangzhan No. 1. Xinxiangzhan No. 1 participated in the Guangxi Rice Consortium Variety Regional Trial in 2019, entered the Guangxi Rice Consortium Variety Regional Trial for retesting in 2020, and passed the Guangxi Rice Variety Approval in 2021 (Approval No.: Gui Shen Dao 2021034). Its resistance to brown planthopper in 2019 and 2020 was 1.83 and 1.89 respectively, and its resistance evaluation was high.

[0048] (1) fgr gene selection marker: Reference Yan Ying, Zhu Guangming, Zhang Lixia, et al. Development and application of molecular markers for rice aroma genes [J]. Northwest Botanical Journal, 2015, 35(02):269-274.

[0049] fgr gene selection marker YY5-YY8 primer sequence

[0050] YY5 CCGGTGCTCCTTTGTCATC (SEQ ID NO.7) YY6 TGAAACTGGTAAAAAGATTATGGC (SEQ ID NO.8) YY7 GAGCAGCTGAAATATATACC (SEQ ID NO.9) YY8 TTGCATCCTGCTCGTCTGG (SEQ ID NO.10)

[0051] (2) PSM152 primer sequence:

[0052] TCCAGAACAAGTAAAGACGA (SEQ ID NO. 11);

[0053] GAATTGAACCAGTTTCTACCT (SEQ ID NO. 12).

[0054] The PSM152 fragment amplified at BPHR2170 is 128 bp in length (SEQ ID NO.13), indicating high resistance to brown planthopper; the fragment amplified at R4684 is 158 bp in length (SEQ ID NO.14), indicating high susceptibility to brown planthopper.

[0055] TCCAGAACAAGTAAAGACGATTTTCAATAAAAGTGAATTCATTCGAGAGTTGTTAAGCAAACATACCTTTGATATAGTAACAGTAATTTCATACTGATCTCCATTATAGGTAGAAACTGGTTCAATTC (SEQ ID NO. 13).

[0056] TCCAGAACAAGTAAAGACGATTTTCAATAAAAGTGAATTCATTCGAGAGTTGTTAAGC AAACATACCT TTGATATTTTCAGTAAAAAA AAACATACCTTTGATATAGTAACAGTAATTTCATACTGATCTCCATTATAGGTAGAAACTGGTTCAATTC (SEQ ID NO. 14).

[0057] The BPHR2170 amplified fragment is missing (30bp) and the nucleotide sequence marked by the horizontal line is missing. The R4684 amplified fragment is 158bp long (SEQ ID NO.14) and has no missing fragment.

[0058] The resistance of the hybrid offspring of BPHR2170 and R4684 was also judged according to the above criteria.

[0059] (3) PSM226 primer sequence:

[0060] CATTAAAAACCGGGAATAAA (SEQ ID NO.15);

[0061] ATCTGACAGGAGATGAATCG (SEQ ID NO. 16).

[0062] The PSM226 fragment amplified by BPHR2170 is 113 bp in length (SEQ ID NO.17), indicating high resistance to brown planthopper; the R4684 fragment amplified by BPHR2170 is 98 bp in length (SEQ ID NO.18), indicating high susceptibility to brown planthopper.

[0063] CATTAAAAACCGGGAATAAAAACGATTTTTAGTCTCAGTTAAAAAAATTTTGATCTTTAGT CCCGGTTGGTTGGTA AAGATGATTTTTAGTCCCGATTCATCTCCTGTCAGAT (SEQ ID NO. 17).

[0064] CATTAAAAACCGGGAATAAAAACGATTTTTAGTCTCAGTTAAAAAAATTTTGATCTTTAGTAAGATGATTTTTAGTCCCGATTCATCTCCTGTCAGAT (SEQ ID NO. 18).

[0065] The BPHR2170 amplified fragment is 113 bp in length (SEQ ID NO.17), and the R4684 amplified fragment is missing (15 bp) marked with a horizontal line.

[0066] When both PSM152 and PSM226 are highly resistant to brown planthoppers, the plant is considered to be highly resistant to brown planthoppers.

[0067] The PCR amplification system and procedure for PSM152 and PSM226 markers were performed according to the method of Zhang Yuexiong et al. [Molecular Plant Breeding, 2018, 16(2): 460-465], as follows:

[0068] The total volume of the PCR reaction was 10 μL. The reaction mixture included: 1.0 μL DNA (50 ng / μL); 1.0 μL 10× buffer; 0.2 μL dNTPs (10 mmol / L); 1.0 μL each of forward and reverse primers (10 μmol / L); 0.1 μL Taq DNA polymerase (5 U / μL); and sterile double-distilled water (ddH2O) to a final volume of 10 μL.

[0069] The PCR reaction procedure was as follows: 94℃ for 5 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, for 30 cycles, with a final temperature of 72℃ for 5 min. The resulting amplified products were electrophoresed in a 7% acrylamide gel at 200 V for 40 min, detected by silver staining, and the band patterns of PSM152 and PSM226 markers were recorded.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A dominant complementary gene for resistance to brown planthopper, Bph50 and Bph51, characterized in that, The amino acid sequence of the protein encoded by the Bph50 gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the Bph51 gene is shown in SEQ ID NO.

4.

2. The dominant complementary genes Bph50 and Bph51 for resistance to brown planthoppers according to claim 1, characterized in that, The nucleotide sequence of the Bph50 gene is shown in SEQ ID NO.

1.

3. The dominant complementary genes Bph50 and Bph51 for resistance to brown planthoppers according to claim 2, characterized in that, The nucleotide sequence of the Bph51 gene is shown in SEQ ID NO.

3.

4. The application of the dominant complementary genes Bph50 and Bph51 against brown planthopper as described in any one of claims 1-3 in regulating resistance to brown planthopper in rice.

5. A method for breeding high-yielding, fragrant rice with good resistance to brown planthoppers, characterized in that, Includes the following steps: (1) In the first year of early crop, the fragrant rice maintainer lines Manxiang B and Xinyinzhan were used as female parents and BPHR96 was used as male parent to cross them, and the hybrid F1 seeds of Manxiang B × BPHR96 and Xinyinzhan × BPHR96 were obtained respectively. (2) In the first year of late crop, 15 hybrid F1 plants of Manxiang B×BPHR96 and Xinyinzhan ×BPHR96 were planted. After removing impurities, the F1 plant seeds of Manxiang B×BPHR96 and Xinyinzhan ×BPHR96 were mixed and harvested to form the F2 population. (3) In the second year, F2 populations were planted separately. The molecular markers PSM152, PSM226, and Y5-Y8 were used to detect the Bph50, Bph51, and aroma gene fgr in the F2 population of Manxiang B×BPHR96. Ten individual plants containing three Bph50, Bph51, and aroma gene fgr and with excellent main agronomic traits were selected and crossed with ten individual plants containing Bph50 and Bph51 and with excellent agronomic traits selected from the F2 population of Xinyinzhan×BPHR96 to obtain 10 hybrid F1 seeds. The primers corresponding to the molecular markers PSM152, PSM226, and Y5-Y8 are shown in SEQ ID NO.11-12, 15-16, and 7-10, respectively. (4) In the second year, the late-season mixed sowing of hybrid F1 seeds was carried out. Molecular markers PSM152, PSM226, and Y5-Y8 were used to perform marker-assisted selection on Bph50, Bph51 and the aroma gene fgr. Several single plants containing three target genes and with excellent agronomic traits were selected and harvested separately to form F2 seeds. (5) In the early third year, the F2 seeds of the single plants screened in step (4) are planted in plots. Molecular markers PSM152, PSM226, and YY5-YY8 are used to detect Bph50, Bph51 and the aroma gene fgr. Single plants with homozygous Bph50 and Bph51 genotypes, homozygous or heterozygous aroma gene fgr genotypes, and excellent agronomic traits are selected and harvested as single plants to form F3 lines. (6) In the third year of late crop, the resistance of the F3 line to brown planthopper was identified by using molecular markers PSM152 and PSM226; when both PSM152 and PSM226 are highly resistant to brown planthopper, the plant is determined to be a highly resistant plant to brown planthopper. (7) After the identification of resistance to brown planthopper, the aroma gene fgr was detected by YY5-YY8 marker. 31 single plants with homozygous genotypes and excellent other major agronomic traits were screened out. At this point, the genotypes of the three target genes Bph50, Bph51 and aroma gene fgr were all homozygous. The single plants were harvested to form F4 lines. (8) In the early fourth year, plant F4 lineage, select several single plants with excellent main agronomic traits, and harvest seeds from single plants to form F5 lineage; (9) Subsequently, starting from the F5 generation of the fourth late crop, the plantation was continuously extended to accelerate the stabilization of agronomic traits. In the F7 generation of the fifth late crop, brown planthopper resistance was identified using the same method as step (6). The pedigree method was used to select up to the F10 generation of the seventh early crop. Several superior strains were selected from the F10 generation. In the seventh late crop, the selected strains were planted, and the overall stability and consistency of agronomic traits were observed. Yield comparison tests were also conducted, and several strains were eliminated. In the eighth early crop, brown planthopper resistance and aroma were identified for the selected superior strains. Finally, a strain with high yield, rich aroma, and good brown planthopper resistance was selected.

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