Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention belongs to the field of plant genetic engineering, and particularly relates to a rice brown planthopper resistance gene, Bph9, and also to molecular markers of the gene and use of the gene and molecular markers thereof in producing brown
• Rice is an important crop that is a staple food for more than half of the human population in the world.
• the fine genetic map and physical map of the rice genome have been completed.
• rice genome is collinear to genomes of other gramineous crops, making rice a good model monocot plant.
• the full-scale development of research on functional genomes has become the frontier field of life sciences.
• the research on functional genes of rice is of great importance to socio-economic development and biological studies.
• Brown planthopper is an epidemic and serious pest of rice.
• Adults and nymphae of brown planthopper pierce and suck rice sap to cause yellow leaf or death, and brown planthopper may spread or induce various rice diseases, leading to yield reduction or loss.
• brown planthopper appeared occasionally only in local rice planting areas in China. Thereafter, with the changes in climate, environment, planting structure, tillage system and cultivation mode, the range of brown planthopper damage spread from the south to the north, the frequency of occurrence increased and the degree of damage worsened.
• brown planthopper has become the number one pest in rice production in China, and poses a serious threat to food security in China.
• the control of brown planthopper has mainly depended on chemical pesticides.
• the application of large amounts of chemical pesticides year after year has caused pesticide resistance in brown planthopper to increase many times over, resulting in the limited effects of pesticide control.
• the applications of chemical pesticides for brown planthopper control on the one hand, increase the production cost for farmers, and on the other hand, chemical pesticides also cause environmental and ecological problems, such as poisoning of non-target organisms, environmental pollution and food contamination.
• Bphl (Athwal et al.,1971; Hirabayashi and Ogawa, 1995; Sharma et al, 2003; Cha et al, 2008), bph2 (Athwal et al, 1971; Murata et al, 1998; Murai et al., 2001), Bph3 (Lakshminarayana and Khush, 1977; Jairin et al., 2007), bph4 (Kawaguchi et al, 2001), bph5 (Khush et al.,1991), Bph6 (Kabir and Khush, 1988; Qiu et al, 2010), bph7 (Kabir and Khush, 1988), bph8 (Nemoto et al, 1989), Bph9 ((Nemoto et al, 1989; Muruta and Fujiwara, 2001), BphlO (Ishii et al, 1994), Bphll (Takita,
• Bphl4 gene has been successfully cloned, and it is the first rice resistance gene isolated by the map-based cloning technology (Du et al 2009).
• Map-based cloning also known as positional cloning, is a kind of gene cloning
• the map-based cloning method includes subjecting target genes to genetic mapping, physical mapping, sequence analysis and genetic transformation for functional verification. Theoretically, any gene which can be mapped can be isolated by the map-based cloning method.
• the map-based cloning method is generally suitable for species having relatively small genomes; for example, rice as a monocot model plant has the features of small genome, small genome physical distance/genetic distance ratio and sufficient markers.
• Rice as a graminaceous model plant has a genome at the center of concentric circles formed by the genomes of seven graminaceous plants including wheat and sorghum, and is one of the crops which are most suitable for target gene isolation through the map-based cloning method.
• Multiple genes that have been cloned in rice were all cloned by the map-based cloning method, for example, bacterial blight resistance genes Xa-21 (Song WY et al. 1995, A Receptor
• Xa-1 Yoshimura et al. 1998, Expression of Xa-1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. PNAS, 95: 1663-1668
• Xa-26 Xa-26 a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein. Plant Journal, 37: 517-527
• rice blast-resistance genes Pi-b Wang et al. 1999, The Pi-b gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes.
• An objective of the present invention is to provide a rice brown planthopper resistance gene Bph9 and use thereof.
• Another objective of the present invention is to provide molecular markers of the brown planthopper resistance gene Bph9 and use thereof.
• the present invention constructs segregation populations of brown planthopper-resistant rice by genetic methods and isolates the rice brown planthopper resistance gene Bph9 by means of map-based cloning.
• the co-segregation marker detection indicates co-segregation of the gene with brown planthopper resistance, and the phenotype of brown planthopper resistance is shown in susceptible rice by genetic transformation of the Bph9 gene, which verifies the function of the gene.
• the present invention provides an isolated polynucleotide having the sequence of a rice brown planthopper resistance gene Bph9, said sequence being a nucleotide sequence shown as SEQ ID NO: 1, or a nucleotide sequence formed by the substitution, deletion or insertion of one or more nucleotides in SEQ ID NO: 1 and encoding an amino acid sequence which is the same or similar and has the same function.
• the present invention provides an isolated polynucleotide having a cDNA sequence of a rice brown planthopper resistance gene Bph9, the sequence being a nucleotide sequence shown as SEQ ID NO: 2, or a nucleotide sequence formed by the substitution, deletion or insertion of one or more nucleotides in SEQ ID NO: 2 and encoding an amino acid sequence which is the same or similar and has the same function.
• the isolated polynucleotide provided in the present invention has a cDNA sequence shown as SEQ ID NO: 2.
• the nucleotide sequence of the Bph9 gene provided in the present invention is shown as SEQ ID NO: 1; the gene has a full length of 15628bp and has two introns and three exons, the CDSs of the gene are segments 2963-4073, 6373-6996 and 12663-14960 respectively, the full length of cDNA of the gene is 4042bp, and the gene encodes 1206 amino acids with a protein sequence shown as SEQ ID NO: 3.
• the protein belongs to the NBS-LRR family and has the following active centers: a 175-322 segment which is a conserved NB-ARC region, a 392-672 segment which is a conserved NB-ARC region (comprising P-loop NTPase, AAA ATPase domain), and an 822-873 segment which is a Leucine rich repeat (LRR).
• the polynucleotide sequence encoding the protein above can be modified in its coding region under a condition of not changing the amino acid sequence or in its non-coding region under a condition of not affecting the protein expression. Therefore, the present invention also contains a nucleotide sequence which results from substitution, insertion and/or deletion of one or more nucleotides in the polynucleotide sequence encoding the protein above, and encodes
• polynucleotide fragments above provided in the present invention are operably ligated to a homologous or heterologous promoter sequence.
• the present invention also comprises a sense or antisense sequence based on said polynucleotide, comprises a cloning vector or expression vector containing said polynucleotide sequence or its fragments, host cells containing said vector, transformed plant cells and transgenic plants containing said polynucleotide sequence or its fragments.
• Said transgenic plant is a monocotyledonous plant.
• Said monocotyledonous plant is rice.
• the present invention provides the use of a polynucleotide of the invention in selective breeding of rice.
• the present invention provides the use of a polynucleotide of the invention in improving brown planthopper resistance of rice.
• the present invention provides the use of a polynucleotide of the invention in producing transgenic brown planthopper-resistant rice.
• the present invention provides a method for cultivating a plant having brown planthopper resistance, including:
• the present invention also provides a method for generating a plant having brown planthopper resistance, said method includes hybridizing the plant having the brown planthopper resistance gene Bph9 with another plant to generate an offspring plant having brown planthopper resistance.
• the plant is a moncottyledonous plant.
• the monocotyledonous plant is a rice plant.
• the present invention provides molecular markers linked to the brown planthopper resistance major gene Bph9, which are
• forward primer sequence 5 ⁇ -GGCAAAGACAAGCCATAAGC-3 ⁇ (SEQ ID NO: 8) reverse primer sequence S -ATCCATCAGCAATGACACGA ⁇ (SEQ ID NO:9) or InD28432 marker primers:
• forward primer sequence S -AACAGACACGTTGCGTCTTG ⁇ (SEQ ID NO: 12) reverse primer sequence S -CTTGCCGCTTAGAGGAGATG ⁇ (SEQ ID NO: 13) or InD14 marker primers:
• forward primer sequence S -CCACTCTGAAAATCCCAAGC ⁇ (SEQ ID NO: 14) reverse primer sequence S -ACCAGTTAAGTCACGCTCAAA ⁇ (SEQ ID NO: 15) or RM28466 marker primers:
• the rice genomic DNA is amplified using the above marker primers; and if an amplified fragment of 213 bp can be amplified using the primers RM28438, or an amplified fragment of 221 bp can be amplified using the primers InD28450, or an amplified fragment of 323 bp can be amplified using the primers InD28453, or an amplified fragment of 320 bp can be amplified using the primers InD28432, or an amplified fragment of 241 bp can be amplified using the primer InD2, or an amplified fragment of 397 bp can be amplified using the primers InD14, or an amplified fragment of 85 bp can be amplified using the primers RM28466, or an amplified fragment of 2
• the molecular markers RM28438, InD28450, InD28453, InD28432, InD2, InD14, RM28466, RM28481 and RM28486 can be applied to screening of brown planthopper-resistant rice containing the brown planthopper resistance gene Bph9.
• the present invention also provides another molecular marker of the rice brown planthopper resistance gene Bph9, which is obtained by PCR using InDel molecular marker IR2 primers, and the primers are:
• the present invention also provides molecular markers related to rice brown planthopper resistance, which are
• forward primer sequence S -GTTCGTGAGCCACAACAAATCC ⁇ (SEQ ID NO:4) reverse primer sequence S -GTTAAATGCTCCACCAAACACACC ⁇ (SEQ ID NO:5), or InD28450 marker primers:
• forward primer sequence 5 ⁇ -GGCAAAGACAAGCCATAAGC-3 ⁇ (SEQ ID NO: 8) reverse primer sequence S -ATCCATCAGCAATGACACGA ⁇ (SEQ ID NO:9), or InD28432 marker primers:
• forward primer sequence S -AACAGACACGTTGCGTCTTG ⁇ (SEQ ID NO: 12) reverse primer sequence S -CTTGCCGCTTAGAGGAGATG ⁇ (SEQ ID NO: 13), or InD14 marker primers:
• forward primer sequence S -CCACTCTGAAAATCCCAAGC ⁇ (SEQ ID NO: 14) reverse primer sequence S -ACCAGTTAAGTCACGCTCAAA ⁇ (SEQ ID NO: 15), or RM28466 marker primers:
• IR2 primers and the primers are:
• the present invention also provides the use of the molecular markers in selective breeding of brown planthopper-resistant rice.
• the present invention provides a molecular marker method for the rice brown planthopper resistance gene bph9, which amplifies a rice genomic DNA to be detected using one of the following primer pairs and detects the product of amplification:
• forward primer sequence S -GTTCGTGAGCCACAACAAATCC ⁇ (SEQ ID NO:4) reverse primer sequence S -GTTAAATGCTCCACCAAACACACC ⁇ (SEQ ID NO:5),
• forward primer sequence 5 ⁇ -GGCAAAGACAAGCCATAAGC-3 ⁇ (SEQ ID NO: 8) reverse primer sequence S -ATCCATCAGCAATGACACGA ⁇ (SEQ ID NO:9),
• marker primers InD28432 marker primers: forward primer sequence S -TGCAGACACCACATGCATAA ⁇ (SEQ ID NO: 10) reverse primer sequence S -ACGCATACACACAGGGACAA ⁇ (SEQ ID NO: 11),
• forward primer sequence S -AACAGACACGTTGCGTCTTG ⁇ (SEQ ID NO: 12) reverse primer sequence S -CTTGCCGCTTAGAGGAGATG ⁇ (SEQ ID NO: 13),
• forward primer sequence S -CCACTCTGAAAATCCCAAGC ⁇ (SEQ ID NO: 14) reverse primer sequence S -ACCAGTTAAGTCACGCTCAAA ⁇ (SEQ ID NO: 15),
• forward primer sequence 5 -TTCTCTGAATGCCCTGTCTCTCC-3 , (SEQ ID NO:20) reverse primer sequence S -GGCAAATCAGAACAAGTCTCACC ⁇ (SEQ ID NO:21),
• an amplified fragment of 213 bp can be amplified using the primers RM28438, or an amplified fragment of 221 bp can be amplified using the primers InD28450, or an amplified fragment of 323 bp can be amplified using the primers InD28453, or an amplified fragment of 320 bp can be amplified using the primers InD28432, or an amplified fragment of 241 bp can be amplified using the primers InD2, or an amplified fragment of 397 bp can be amplified using the primers InD 14, or an amplified fragment of 85 bp can be amplified using the primers RM28466, or an amplified fragment of 237 bp can be amplified using the primers RM28481, or an amplified fragment of 161 bp can be amplified using the primers RM28486, or an amplified fragment of 228 bp can be amplified using the primers IR2, this
• the present invention also provides a method of screening brown planthopper-resistant rice, which amplifies a rice genomic DNA to be detected by PCR using one of the above primer pairs; and if an amplified fragment of 213 bp can be amplified using the primers RM28438, or an amplified fragment of 221 bp can be amplified using the primers InD28450, or an amplified fragment of 323 bp can be amplified using the primers InD28453, or an amplified fragment of 320 bp can be amplified using the primers InD28432, or an amplified fragment of 241 bp can be amplified using the primers InD2, or an amplified fragment of 397 bp can be amplified using the primers InD14, or an amplified fragment of 85 bp can be amplified using the primers RM28466, or an amplified fragment of 237 bp can be amplified using the primers RM28481, or an amplified fragment of 16
• the Bph9 gene provided in the present invention is obtained by cloning by the following steps:
• An F 2 population is obtained by hybridization of a brown planthopper-resistant rice variety and a common rice variety and inbreeding of the Fi generation, and is used as a mapping population for the brown planthopper resistance gene.
• the brown planthopper resistance of the mapping population is identified by the standard seedbox screening technique. Seeds are harvested from the F 2 -generation plants and the seeds harvested from each F 2 -generation plant are sowed in a seedling tray to generate 20 seedlings (called one line). At the two-euphylla one-apical bud stage, the lines are exposed to 2-4 years old brown planthopper nymphae (10 nymphae/plant), the damage condition of each line is recorded, and each material is tested with three replications. According to the results of resistance identification, the resistance scores of plant lines in the mapping populatoin are evaluated.
• a Fosmid genome library of a resistant parent rice material (Pokkali, IRGC 108921) containing the brown planthopper resistance gene Bph9 is constructed, the PCR screening is performed through mapping interval molecular markers to obtain a positive clone covering the Bph9 mapping interval, the large fragment sequencing is performed by the dideoxy chain-termination method (Sanger method) to obtain a genome sequence of the segment where Bph9 is located, and candidate genes of Bph9 are determined by gene prediction using the software RiceGAAS and comparative analysis of the sequences of the Japonica rice variety 'Nipponbare'and the variety 9311.
• the Bph9 gene can be amplified from the brown planthopper-resistant rice genome by designing appropriate PCR primers according to the nucleotide sequences of Bph9 disclosed by the invention.
• the full-length cDNA of the gene Bph9 is subjected to double digesion with Ncol and Xhol to obtain a 3346 bp fragment which comprises most of the ORF region of the Bph9 gene and also comprises a large part of the No. 3 exon (the Xhol site is located at the terminal of the No. 3 exon).
• the Bph9 genome has a sequence length of 15628 bp and has two introns and three exons.
• the ORF length of the gene is 3621 bp, there is an Xhol site at 3344 bp, and the Xhol site is located at the terminal of the No. 3 exon.
• the Fosmid clone where the Bph9 gene is located is subjected to double digestion with Xhol and EcoRI to obtain a 1291 bp fragment, and the fragment comprises the part behind the Xhol site of the No. 3 exon and the transcriptional termination region of the Bph9 gene.
• the above fragments are ligated to the Ncol and EcoRI sites of a pGEM T easy vector by means of three-fragment ligation.
• a 4637 bp fragment which is obtained by double digestion with Ncol and EcoRI comprises the complete ORE of the Bph9 gene and the transcriptional termination region of the gene.
• the Bph9 gene promoter region (3026 bp fragment) which is obtained by double digestion with Sail and Ncol and the complete ORE and transcriptional termination region of the Bph9 gene (4637 bp fragment) which is obtained by double digestion with Ncol and EcoRI are ligated to the Sail and EcoRI sites of pCAMBIA1301 by means of three-fragment ligation. After verification by sequencing, it is proven that the resulting vector is the Bph9 gene genetic transformation vector (using its promoter region, complete ORF and its transcriptional termination region), and the vector is transformed into Agrobacterium tumefaciens EHA105 through electroporation.
• the Bph9 gene vector (using its promoter region, complete ORF and its transcriptional termination region) is introduced into a normal Indica rice variety Kasalath, and finally 15 5/?/i9-positive plants are obtained.
• the resistance is identified by the standard seedbox screening technique using the Ti-generation Bph9 transgenic plants, and the result shows that all the plants of the control rice variety Kasalath die and the transgenic positive plants survive with a resistance score of 2-3, indicating that the Bph9 gene has a function of resistance to brown planthopper. Therefore, the brown planthopper resistance gene Bph9 can be applied in rice and can also be applied in rice seeds to cultivate rice varieties having brown planthopper resistance.
• the successful cloning of the gene further verifies the reliability of cloning important genes of rice by the map-based cloning method, and the gene cloned by the method has definite functions and good effects.
• hybridization which can improve brown planthopper resistance in rice varieties to reduce the pest damage of brown planthopper and achieve the purposes of increased yield and stable yield.
• Piercing-sucking insects are a broad category of pest insects in agricultural production, the success in Bph9 gene cloning and brown planthopper resistance verification provides an important reference for research on piercing-sucking insect resistance in other plants.
• SEQ ID NO: 1 is a Bph9 rice genomic sequence.
• SEQ ID NO: 2 is a Bph9 cDNA.
• SEQ ID NO: 3 is a Bph9 amino acid sequence.
• SEQ ID NO: 4-27 are primer sequences useful in the invention.
• Fig. 1 is an image showing the results of identifying resistant plants and susceptible plants in F 2 :3 lines by the standard seedbox screening technique.
• P09-1 to P09-16 refer to the corresponding F 2:3 lines obtained by inbreeding of each F 2 individual plant in the Bph9-containing F 2 population which is constructed by hybridization of a brown planthopper-resistant parent Pokkali (IRGC 108921, containing the brown planthopper resistance gene Bph9) and a brown planthopper-susceptible rice variety Yangdao No. 6 (93-11).
• IRGC 108921 containing the brown planthopper resistance gene Bph9
• Yangdao No. 6 93-11
• Fig. 2 is an electropherogram of detecting individual plants using SSR marker RM28486.
• the first two track lanes are of the resistant parent Pokkali (IRGC 108921) and the susceptible parent Yangdao No. 6 (93-11) respectively, and the following track lanes are of the F 23 lines constructed by hybridization of Pokkali and Yangdao No. 6.
• the No. 2, 3, 5, 6, 8, 10, 12, 14, 15, 18, 19, 20, 21 and 23 F 23 lines, from which a 161 bp fragment can be specifically amplified using the SSR marker RM28486 linked to the brown planthopper resistance gene Bph9 all show resistance to brown planthopper, while the No. 1, 4, 7, 9, 11, 13, 16, 17, 22 and 24 F 23 lines, from which the 161bp specific fragment cannot be amplified, all show susceptibility to brown planthopper.
• Fig. 3 is a mapping of Bph9 in a rice chromosome.
• A the result of Bph9 primary mapping.
• the marker names are shown above the chromosome, the numbers refer to the genetic distance (cM) between the markers, the result of QTL scanning shows that there is a maximum LOD value of 44.1 between the molecular markers RM28486 and RM28438, and the distance between RM28486 and RM28438 is 1.7cM.
• n represents the number of individual plants in the F 2 mapping population.
• B the screening result for individual plants with recombination between the molecular markers RM28486 and RM28438.
• Bph9 is finely mapped between the molecular markers InD2 and RM28466, and shows co-segregation with the marker InD14.
• the numbers below the molecular markers represent the quantity of recombinant individual plants between the two molecular markers, n represents the quantity of individual plants of BC 2 F 2 population for recombinant individual plant screening.
• C the physical map between InD2 and RM28466; Bph9 is located within a region of about 68 kb between InD2 and RM28466.
• 19 and 544-22 are Fosmid clones which are mutually overlapped and cover the interval of the markers InD2 and RM28466.
• Fig. 4 is an image showing the results of identifying brown planthopper resistance in transgenic plants containing a transformation vector comprising the promoter region, complete ORF and transcriptional termination region of the Bph9 gene.
• 1 and 9 represent the susceptible control variety TNI
• 2 represents the resistant parent Pokkali (IRGC 108921)
• 3 represent the susceptible control variety Kasalath
• 4 represent the transgenic plant lines POK1, POK2 and POK3 containing a transformation vector comprising the promoter region, complete ORF and transcriptional termination region of the Bph9 gene.
• Fig. 5 is an image showing the results of identifying brown planthopper resistance in transgenic plants containing a genome complementary vector of the Bph9 gene.
• the TO-generation transgenic plants and the susceptible control variety TNI are transplanted diagonally, the four plants forming a square-like shape after transplantation, in which 1 and 4 represent the susceptible control variety TNI, 2 represents the Bph9 gene genome
• Fig. 6 is an example of a functional molecular marker IR2 (belonging to the InDel markers) developed according to the Bph9 genome sequence, and the length of an amplified fragment is 228 bp.
• 1 represents the resistant parent Pokkali (IRGC 108921) carrying the brown planthopper resistance gene Bph9
• 2 represents the susceptible material Yangdao No. 6 (93-11)
• 3 represents a material which is screened out from the backcross offspring of Pokkali and Yangdao No. 6 using the functional molecular marker IR2 and carries the brown planthopper resistance gene Bph9 (the material is resistant to brown planthopper).
• Fig. 7 refers to brown planthopper-resistant rice having the Bph9 gene, which is obtained by means of molecular marker-assisted selective breeding.
• the brown planthopper resistance is identified at the seedling stage and the insect source is brown planthopper populations in the farm fields in Wuhan city.
• 1, 5 and 9 represent the susceptible control variety TNI
• 2 and 6 represent the resistant parent Pokkali (IRGC 108921)
• 3 and 7 represent the susceptible material Yangdao No. 6 (93-11)
• 4 and 8 represent a cultivar Luoyang No. 9 (having the genetic background of Yangdao No. 6 and carrying the brown planthopper resistance gene Bph9).
• Fig. 8 refers to brown planthopper-resistant rice having the Bph9 gene, which is obtained by means of molecular marker-assisted selective breeding.
• the brown planthopper resistance is identified at the seedling stage and the insect sources are brown planthopper biotype I, biotype II and biotype III.
• the figure above refers to the insect source brown planthopper biotype I
• the middle figure refers to the insect source brown planthopper biotype II
• the figure below refers to the insect source brown planthopper biotype III.
• 1 and 6 represent the susceptible control variety TNI
• 2 and 4 represent the cultivar Luoyang No. 9 (having the genetic background of Yangdao No. 6 and carrying the brown planthopper resistance gene Bph9)
• 3 and 5 represent the susceptible material Yangdao No.
• Fig. 9 refers to brown planthopper-resistant rice having the Bph9 gene, which is obtained by means of molecular marker-assisted selective breeding.
• the brown planthopper resistance is identified at the near-tillering stage and the insect sources are brown planthopper populations in the farm fields in Wuhan city.
• the susceptible recipient parent Yangdao No. 6 and the variety Taichung local No. 1 are obviously dead, while the cultivar Luoyang No. 9 (having the genetic background of Yangdao No. 6 and carrying the brown planthopper resistance gene Bph9) still survives and the plants grow healthily.
• Fig. 10 refers to brown planthopper-resistant rice having the Bph9 gene, which is obtained by means of molecular marker-assisted selective breeding.
• the brown planthopper resistance is identified at the maturity stage and the insect sources are brown planthopper populations in farm fields in Wuhan city, and after exposure to insects, the susceptible recipient parent Yangdao No. 6 is obviously dead and the stems are withered.
• the cultivar Luoyang No. 9 (having the genetic background of Yangdao No. 6 and carrying the brown planthopper resistance gene Bph9) still survives, the stems are upright and the plants grow healthily.
• a brown planthopper-resistant parent Pokkali (IRGC 108921, containing the brown planthopper resistance gene Bph9) and a brown planthopper-susceptible rice variety Yangdao No. 6 (93-11) are hybridized to construct a Bph9-containing F 2 population; the varities 93-11 and Pokkali (IRGC 108921) are both from the National Crop Germplasm Preservation Center of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and the
• genomic DNAs of individual plants of the parents and F 2 population are extracted by the CTAB method (Murray MG & Thompson, 1980 Rapid isolation of high-molecular- weight plant DNA. Nucleic Acids Res 8: 4321-4325). Each F 2 individual plant is inbred to harvest a corresponding F 2:3 line.
• the resistance performance of each individual plant in the F2:3 lines is observed by the standard seedbox screening technique (see Fig. l), and the brown planthopper resistance phenotypes of F 2 individual plants are represented by the resistance scores of the F 2:3 lines.
• each line in the parents and F 2:3 population all the tested materials are subjected separately to seed soaking for germination promotion before sowing.
• 60 seeds of each line (variety) are sowed in a bread box which is 58 cm long, 38 cm wide and 9 cm high and filled with nutrient soil to 7 cm thickness.
• Each material is re-sowed with three replications in each box, with three replications of random sowing for the parents and TNI (susceptible control) respectively.
• the seedlings are thinned 7 days after sowing to remove diseased and weak seedlings.
• Table 1 Scoring standards for identifying resistance and susceptibility to brown planthopper.
• the result of identification by the standard seedbox screening technique shows that the resistance scores of the varieties Pokkali (IRGC 108921) and 9311 are 1.9 and 8.8 respectively, indicating that the variety Pokkali (IRGC 108921) is resistant to brown planthopper but the variety 9311 is susceptible to brown planthopper.
• the frequency distribution of brown planthopper resistance scores of the 135 F 2: 3 lines is a continuous distribution, with the minimum value of 2.1 and maximum value of 9.0. According to the brown planthopper resistance scores, the F 2: 3 lines are classified as the following three phenotypes: resistance, resistance/susceptibility segregation and susceptibility; while the genotypes of the
• RR homozygous resistance
• Rr heterozygous resistance
• rr homozygous susceptibility
• F 2 genotype a Number of F 2 Phenotype of the
• a RR homozygous resistance
• Rr heterozygous resistance
• rr homozygous susceptibility
• SSR motifs are searched for using the SSR search tool SSRIT (http://www.gramene.org/db/markers/ssrtool), and primers are designed according to their flanking sequences and serve as alternative markers.
• the setting parameters of the SSRIT are that: the maximum motif length is a tetramer, the minimum repeat is 5, and all the SSR motifs are searched. All the SSR motifs having more than 15 bases (motif length x repeat) are selected.
• the inserted/deleted sites existing in the genome sequences are searched for by the software PowerBlast, and the InDel markers are developed by the software Primer Premier 5.0.
• the analysis of the SSR markers is done with reference to the method proposed by Temnykh (Temnykh S et al, 2000. Mapping and genome organization of microsatellite sequences in rice. Theor Appl Genet 100: 697-712).
• the 10 ⁇ reaction system comprises: 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 50 ⁇ dNTPs, 0.2 ⁇ primers, 0.5U Taq polymerase and 20 ng DNA template.
• the amplification reaction takes place on a PTC- 100 PCR instrument: 94°C 2 min, 94°C 15 sec, 55°C 30 sec, 72°C 1.5 min, 35 cycles, 72°C 5 min.
• the product of amplification is separated using 6% Native-PAGE and developed by silver staining (Zhu et al, 2004. Identification and characterization of a new blast resistance gene located on rice chromosome 1 through linkage and differential analyses. Phytipathology 94:515-519).
• the amplified DNA bands are observed using a lamp box equipped with a fluorescent lamp. The result is recorded, and the primers having a polymorphism between parents are analyzed in the F 2 population to obtain the population genotype data.
• DNAs of 10 extremely resistant individual plants and DNAs of 10 extremely susceptible individual plants are selected and hybridized to construct resistance/susceptibility pools.
• the resistance/susceptibility pools are separately screened using the primers having a polymorphism between parents and molecular markers having a polymorphism between resistance/susceptibility pools are obtained, and it is indicated that such polymorphic markers are linked to resistance.
• the primers having a polymorphism between parents on the chromosome are selected to screen individual plants of the F 2 segregating population, the PCR process being the same as above, and the population genotype data is obtained.
• the population genotype data is processed by the software JoinMap 3.0 to construct a partial genetic map of rice and to obtain the genetic distance between molecular markers.
• the QTL scan of the target chromosome is performed by composite interval mapping using the software MapQTL 5.0.
• the present invention adopts PCR (polymerase chain reaction) and polyacrylamide gel electrophoresis to screen 3000 individual plants of the BC 2 F 2 population using the SSR markers RM28486 and RM28438 on two sides, obtaining 32 individual plants with recombination between the two markers.
• primers are designed according to their flanking sequences and serve as alternative markers. Also, the inserted/deleted sites existing in the genome sequences are searched for by the software PowerBlast, and the InDel markers are developed for by the software Primer Premier 5.0.
• R R R R R R R R R 1.9 a 93-11 and Pokkali are two parent materials, and the others are parts of recombinant individual plants; b as shown in the table, the molecular markers InD14 are cosegregated with resistance phenotype.
• R represents the genotype and phenotype of the resistant parent Pokkali (IRGC 108921)
• S represents the genotype and phenotype of the susceptible parent 9311
• H represents the heterozygous genotype and phenotype.
• the use of the molecular markers in the saturated linkage map in Fig. 3B is the use of:
• forward primer sequence S -GTTCGTGAGCCACAACAAATCC ⁇ (SEQ ID NO:4) reverse primer sequence S -GTTAAATGCTCCACCAAACACACC ⁇ (SEQ ID NO:5), or InD28450 marker primers:
• forward primer sequence 5 ⁇ -GGCAAAGACAAGCCATAAGC-3 ⁇ (SEQ ID NO: 8) reverse primer sequence S -ATCCATCAGCAATGACACGA ⁇ (SEQ ID NO:9), or InD28432 marker primers:
• forward primer sequence S -AACAGACACGTTGCGTCTTG ⁇ (SEQ ID NO: 12) reverse primer sequence S -CTTGCCGCTTAGAGGAGATG ⁇ (SEQ ID NO: 13), or InD14 marker primers:
• forward primer sequence S -CCACTCTGAAAATCCCAAGC ⁇ (SEQ ID NO: 14) reverse primer sequence S -ACCAGTTAAGTCACGCTCAAA ⁇ (SEQ ID NO: 15), or RM28466 marker primers:
• RM284308 or an amplified fragment of 221 bp can be amplified using the primers InD28450, or an amplified fragment of 323 bp can be amplified using the primers InD28453, or an amplified fragment of 320 bp can be amplified using the primers InD28432, or an amplified fragment of 241 bp can be amplified using the primer InD2, or an amplified fragment of 397 bp can be amplified using the primers InD14, or an amplified fragment of 85 bp can be amplified using the primers RM28466, or an amplified fragment of 237 bp can be amplified using the primers RM28481 , or an amplified fragment of 161 bp can be amplified using the primers RM28486, this indicates the existence of a brown planthopper resistance major gene locus Bph9 in rice. Therefore, the identification of existence of the Bph9 resistance gene by the molecular marker method provided
• the genomic DNA is physically fragmented, the fragmented DNAs are separated by alternating pulsed field electrophoresis using a CHEF Mapper apparatus, and the gel is extracted to recover the DNA fragment between 38 kbp and 48 kbp.
• the recovered DNA fragment is subjected to end-blunting and phosphorylation and then recovered by pulsed field
• T4 DNA ligase is added to 250 ng recovered DNA fragment and 500 ng pCClFOS (manufactured by the EPICENTRE company) vector and the resulting mixture stands overnight for ligation at 4°C. 10 ⁇ ⁇ ligation reaction solution is added to 25
• Maxplax Lambda packaging Extracts manufactured by the EPICENTRE company molten on ice, followed by package at 30°C for 90 minutes. Then, 25 Maxplax Lambda packaging Extracts is added for packaging at 30°C for 90 minutes.
• a bacteriophage diluted buffer solution (Phage Dilution Buffer) is added until the total volume is 1 mL. The mixture is blended gently until uniform. 25 chloroform is added into each tube. The mixture is blended gently until uniform and then stored at 4°C. The successfully-packaged bacteriophage particles are diluted with the bacteriophage diluted solution and then transfected into the host strain
• EPI300-T1R manufactured by the EPICENTRE company
• EPICENTRE EPICENTRE company
• 16 white single bacterial colonies are randomly selected from the plate and cultured, followed by DNA extraction and Notl digestion, and then the lengths of the inserted fragments are determined by pulsed field electrophoresis, and the result shows that the average length of the 16 DNAs is more than 35 kbp and the insertion rate is 100%, which comply with the requirements of the library.
• a 96-well clon-pool plate is made such that about 100 single clones are randomly selected in each well, 12 plates are made in total and stored at -80°C, and the library construction is completed.
• the Fosmid genomic library is screened using the molecular markers specific to the Bph9 fine mapping interval, the positive clones obtained by screening are subjected to terminal sequencing to determine the overlapping relationship of the positive clones and to finally determine that the two positive clones 544-22 and 19 cover the whole Bph9 fine mapping interval (molecular markers InD2 and RM28466 segments).
• the complete sequences of the Fosmid clones 544-22 and 19 are subjected to sequencing analysis to finally obtain the complete sequence of the Bph9 mapping interval, i.e., the sequence between the molecular markers InD2 and RM28466, having a length of about 68 kb.
• the complete sequence is used as a target to search the NCBI database, so as to obtain a homologous sequence of the Japonica rice variety 'Nipponbare' genome within the segment.
• Gene prediction and annotation is performed by use of FGENESH, and the comparative analysis is performed by use of ClustalW (Table 4).
• the comparison of the predicted genes shows that the Bph9 mapping region in the resistant parent Pokkali (IRGC 108921) has a total of eight predicted genes, of which four genes are transposon-related proteins (Table 4 Pokkali predicted genes g3, g4, g6 and g7), one is a gene with short encoding sequence (Table 4 Pokkali predicted gene g5), one is a metabolism-related gene (Table 4 Pokkali predicted gene gl Lipoxygenase), and two are resistance genes (Table 4 Pokkali predicted genes g2 and g8). According to the gene nature, so far it is believed that the infection processes of piercing-sucking insects and pathogenic bacteria to rice are similar, and it is possible that the mechanism of resistance to
• primers TPT203, S -GATCTCAGTGTGGGGAATGG ⁇ (SEQ ID NO:26), RR376-1, 5 ⁇ -AGAGCGAC AAGGGC AGATAA-3 ⁇ (SEQ ID NO:27)
• Primers are designed according to the sequence, the 5 '-end and 3 '-end sequences of the candidate gene are obtained using a 573 '-end Full RACE kit from the TaKaRa company, the transcription initiation site and transcription termination site of the candidate gene are determined, and a full-length cDNA sequence of the gene is spliced.
• Primers are re-synthesized according to the full-length cDNA sequence, and the full-length cDNA of Bph9 is obtained by amplification and has a nucleotide sequence shown as SEQ ID NO: 2, in which the No. 1-64 bp are a 5UTR untranslated region and the No. 3686-4042 bp are a 3UTR untranslated region.
• the genomic DNAs of the resistant rice, susceptible varieties and offspring plants obtained by hybridization and backcross thereof are extracted and amplified by PCR using the primers designed based on the Bph9 gene sequence, and the molecular marker detection is performed by means of polyacrylamide gel electrophoresis.
• the plants having the same PCR bands as the resistant rice are the selected plants which contain the Bph9 gene, and the plants are subjected to continuous backcross and agronomic trait selection to breed rice with resistance to brown planthopper.
• pCAMBIA1301 (purchased from the Center for the Application of Molecular Biology to International Agriculture, Australia), and the vector pCAMBIA1301 is double-digested with enzymes Sail and EcoRI to ligate foreign fragments to the Sail and EcoRI sites of the vector pCAMBIA1301.
• the 3026 bp fragment which is obtained by double digestion of the Fosmid clone where the Bph9 gene is located with Sail and Ncol serves as the promoter region of the Bph9 gene (there is a translation initiation codon of the Bph9 gene at the Ncol site).
• a 3346 bp fragment is obtained by double digesion of the full-length cDNA clone of the Bph9 gene with Ncol and Xhol, which comprises most of the ORF region of the Bph9 gene and also comprises most of the No. 3 exon of the gene (an Xhol site is located at the terminal of the No. 3 exon).
• the Bph9 genome has a sequence length of 15628 bp and has two introns and three exons.
• the ORF length of the Bph9 genome is 3621 bp, there is an Xhol site at 3344 bp, and also the Xhol site is located at the terminal of the No. 3 exon.
• a 1291 bp fragment is obtained by double digestion of the Fosmid clone where the Bph9 gene is located with Xhol and EcoRI, and the fragment comprises the part behind the Xhol site of the No. 3 exon and the transcriptional termination region of the Bph9 gene.
• a 3346 bp fragment which is obtained by double digestion of the full-length cDNA clone of the Bph9 gene with Ncol and Xhol, and a 1291 bp fragment which is obtained by double digestion of the Fosmid clone where the Bph9 gene is located with Xhol and EcoRI are ligated to the Ncol and EcoRI sites of the pGEM T easy vector by means of three-fragment ligation.
• a 4637bp fragment which is obtained by double digestion with Ncol and EcoRI comprises the complete ORF and transcriptional termination region of the Bph9 gene.
• the Bph9 gene promoter region (3026bp fragment) obtained by double digestion with Sail and Ncol, and the Bph9 gene complete ORF and transcriptional termination region (4637bp fragment) obtained by double digesion with Ncol and EcoRI are ligated to the Sail and EcoRI sites of the vector pCAMBIA1301 by means of three-fragment ligation. After verification by sequencing, the resulting vector is the Bph9 gene genetic transformation vector (using the promoter region, complete ORF and transcriptional termination region of the gene), and is introduced into Agrobacterium tumefaciens EHA105 by electroporation.
• the monoclonal population is picked for expanding culture; and after verification by PCR using Bph9 gene-specific primers (RSP1 : S -AGGGCTACCTCATTGTGCTG ⁇ (SEQ ID NO:24), PCR1 : S -CCTTTTCGCTTCGGTAGACA ⁇ (SEQ ID NO:25), an equal volume of 50% glycerol is added and mixed thoroughly, and the resulting mixture is preserved at -70°C.
• RBP1 S -AGGGCTACCTCATTGTGCTG ⁇
• PCR1 S -CCTTTTCGCTTCGGTAGACA ⁇
• the backbone vector used is pCAMBIA1301 (purchased from the Center for the
• GFP green fluorescent protein
• pCAMBIA1301 GFP
• a 2366 bp fragment is obtained by double digestion of the Fosmid clone where the Bph9 gene is located with Sail and Kpnl
• a 13257 bp fragment is obtained by double digestion of the Fosmid clone where the Bph9 gene is located with Kpnl and EcoRI
• the two fragments comprise the genome sequence where the Bph9 gene is located (containing the promoter region, gene encoding region and transcriptional termination region), and the two fragments are ligated sequentially to the Sail and Kpnl sites and the Kpnl and EcoRI sites of the vector
• pCAMBIA1301 to generate a genome complementary vector of the Bph9 gene.
• the genome complementary vector is introduced into Agrobacterium tumefaciens EHA105 by electroporation.
• the monoclonal population is picked for expanding culture; and after verification by PCR using Bph9 gene-specific primers IR2 (forward primer sequence
• transcriptional termination region transformation vector and genome complementary vector
• brown planthopper-susceptible common rice variety Kasalath purchased from the National Rice Germplasm Bank or the International Rice Research Institute.
• 15 positive transgenic plants are obtained using the genetic transformation vector containing the promoter, complete ORF and transcriptional termination region of the Bph9 gene (named as POK1 , POK2, POK3, POK4, POK5, POK6, POK7, POK8, POK9, POK10, POK11 , POK12, POK13, POK14 and POK15 respectively).
• resistance identification is performed in the Ti-generation Bph9 transgenic plants by the standard seedbox screening technique. As shown in Fig. 4, the identification of brown planthopper resistance is performed in the positive transgenic lines POK1 , POK2 and POK3.
• the identification result shows that, 7 days after exposure to brown planthopper, the whole plants of the susceptible control variety TNI and susceptible transgenic recipient material Kasalath are dead, while the plants of the brown planthopper-resistant parent Pokkali grow healthily without leaf damage.
• the positive transgenic plants POK1, POK2 and POK3 obtained using the genetic transformation vector containing the Bph9 gene also grow healthily without leaf damage and have resistance scores of 2-3, which indicates that the Bph9 gene has a function of resistance to brown planthopper. Therefore, the brown planthopper resistance gene Bph9 can be applied in rice as well as rice seeds to breed rice varieties having brown planthopper resistance.
• 20 positive transgenic plants are obtained using the genome complementary vector of the Bph9 gene (named as PGK1, PGK2, PGK3, PGK4, PGK5, PGK6, PGK7, PGK8, PGK9, PGK10, PGK11, PGK12, PGK13, PGK14, PGK15, PGK16, PGK17, PGK18, PGK19 and PGK20 respectively).
• the identification of brown planthopper resistance is performed at the tillering stage.
• the materials PGK1, PGK2 and susceptible control variety TNI Taichung local No.
• brown planthopper-resistant parent Pokkali IRGC 108921 , containing the brown planthopper resistance gene Bph9
• brown planthopper-susceptible rice variety Yangdao No. 6 93-11
• F 23 lines constructed by Pokkali/Y angdao No. 6 hybridization, both 93-11 and Pokkali being from the National Crop Germplasm Preservation Center of the Institute of Crop Science, Chinese Academy of Agricultural Sciences.
• Molecular marker primers InD2, RM28466, RM28438, InD28450, InD28453, InD14, InD28432, RM28481 and RM28486, having nucleotide sequences shown as SEQ ID NOs: 4-21 respectively.
• Genomic DNAs of rice samples are extracted by the CTAB method.
• the sample DNAs are amplified using primers InD2, RM28466, RM28438, InD28450, InD28453, InD14,
• the 10 ⁇ reaction system comprises: lOxPCR buffer, 1.0 ⁇ ; 10 mM dNTPs, 0.1 ⁇ ; 10 ⁇ primers, 0.4 ⁇ ; 5 U/ ⁇ Taq DNA polymerase, 0.2 ⁇ and 50 ng DNA template.
• the amplification reaction takes place on a Bioer PCR instrument: 94°C 4 min, 94°C 30 s, 55°C 30 s, 72°C 40 s, 32 cycles, 72°C 5 min.
• the product of amplification is separated using 8% of Native-PAGE, and the map is read and analyzed by silver staining using silver nitrate after electrophoresis.
• the molecular marker-assisted selective breeding of the brown planthopper-resistant rice Luoyang No. 9 carrying the Bph9 gene is performed using the foregoing molecular markers, and specifically, it is implemented as follows: a brown planthopper-resistant parent Pokkali (IRGC 108921, containing brown planthopper resistance gene Bph9) is hybridized with a brown planthopper-susceptible rice variety Yangdao No. 6 (93-11) to obtain F ls followed by backcross using the variety Yangdao No.
• IRGC 108921 containing brown planthopper resistance gene Bph9
• the resulting BCiFi is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BCiFi containing the brown planthopper resistance gene Bph9 is back-crossed to the variety Yangdao No. 6 to obtain BC 2 Fi; the resulting BC 2 Fi is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BC 2 Fi containing the brown planthopper resistance gene Bph9 is back-crossed to the variety Yangdao No.
• the resulting BC 3 F 1 is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BC 3 F 1 containing the brown planthopper resistance gene Bph9 is back-crossed to the variety Yangdao No. 6 to obtain BC 4 F 1 ; the resulting BC 4 F 1 is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BC 4 F 1 containing the brown planthopper resistance gene Bph9 is back-crossed to the variety Yangdao No.
• BC 5 F 1 the resulting BC 5 F 1 is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BC 5 F 1 containing the brown planthopper resistance gene Bph9 is back-crossed to the variety Yangdao No. 6 to obtain BC 6 Fi; the resulting BC 6 Fi is screened using the Bph9-linked molecular markers (RM28438, RM28486), and the screened BC 6 Fi containing the brown planthopper resistance gene Bph9 is
• BC 7 F 1 back-crossed to the variety Yangdao No. 6 to obtain BC 7 F 1 ; and the resulting BC 7 F 1 is screened using the Bph9-linked molecular markers (RM28438, RM28486) to obtain BC 7 F 1 containing the Bph9 gene, genome -wide scanning is performed using the molecular markers (see Table 5), BC 7 F 1 which differs only in the brown planthopper resistance gene Bph9 locus from the variety Yangdao No. 6 and in which all other loci are replaced with the background of the variety Yangdao No. 6 are selected for generation-adding breeding, Yangdao No. 6/Pokkali (IRGC 108921)//Y angdao No.
• the banding pattern of individual plant is recorded as 1 if identical to that of the brown planthopper-resistant parent Pokkali (IRGC 108921), recorded as 2 if identical to that of the parent Yangdao No. 6 (93-11), and recorded as 3 if integrating the banding patterns of the two parents.
• the result of molecular marker screening shows that BC7F1 differs only in brown planthopper resistance gene Bph9 locus (interval of RM28481 and RM28438) from the variety Yangdao No. 6, and all other loci of BC7F1 are replaced by the genetic background of the variety Yangdao No. 6.

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