CN109762929B - Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice - Google Patents
Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice Download PDFInfo
- Publication number
- CN109762929B CN109762929B CN201910234968.3A CN201910234968A CN109762929B CN 109762929 B CN109762929 B CN 109762929B CN 201910234968 A CN201910234968 A CN 201910234968A CN 109762929 B CN109762929 B CN 109762929B
- Authority
- CN
- China
- Prior art keywords
- rice
- bph9
- molecular marker
- brown planthopper
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 69
- 235000009566 rice Nutrition 0.000 title claims abstract description 69
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 63
- 241001556089 Nilaparvata lugens Species 0.000 title claims abstract description 54
- 239000003147 molecular marker Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 28
- 240000007594 Oryza sativa Species 0.000 title description 55
- 238000009395 breeding Methods 0.000 claims abstract description 22
- 230000001488 breeding effect Effects 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 238000012408 PCR amplification Methods 0.000 claims abstract description 8
- 241000209094 Oryza Species 0.000 claims abstract 19
- 238000006243 chemical reaction Methods 0.000 claims description 10
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 8
- 240000002582 Oryza sativa Indica Group Species 0.000 claims description 6
- 240000008467 Oryza sativa Japonica Group Species 0.000 claims description 6
- 229920003023 plastic Polymers 0.000 claims description 6
- 241000196324 Embryophyta Species 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 1
- 108700028369 Alleles Proteins 0.000 abstract description 10
- 239000003550 marker Substances 0.000 description 11
- 240000001606 Adenanthera pavonina Species 0.000 description 8
- 235000011470 Adenanthera pavonina Nutrition 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 3
- 102100039845 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-8 Human genes 0.000 description 3
- 101710112841 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-8 Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910000831 Steel Inorganic materials 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 238000007605 air drying Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000010959 steel Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 241001504226 Hoodia Species 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 235000017277 hoodia Nutrition 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 241001498622 Cixius wagneri Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000004416 odontoblast Anatomy 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of rice resistance breeding and molecular genetics, and particularly discloses a functional molecular marker of a brown planthopper resistant gene Bph9 of rice, an identification method and application. The molecular marker is Bph9-M, and the nucleotide sequence of the molecular marker is shown in a sequence table SIPOS sequence listing 1.0; the Bph9-M primer is as follows: forward primer TCATTAGGAACAGGCTATGCA, reverse primer TTCTTGTTGACACCGCTCAC. The application of the molecular marker Bph9-M in brown planthopper resistant breeding of rice comprises the following steps: extracting rice genome DNA; carrying out genotype identification by PCR amplification detection; and (4) carrying out phenotype identification on brown planthopper resistance. The molecular marker can effectively distinguish the Bph9 from other alleles Bph1, Bph2, Bph7, Bph10, Bph18, Bph21 and the like, is a PCR-type intra-gene functional molecular marker designed aiming at the Bph9 gene at present, and can be used for rice breeding practice simply, quickly and in a high-throughput manner.
Description
Technical Field
The invention belongs to the field of rice resistance breeding and molecular genetics, and particularly relates to a functional molecular marker of a brown planthopper resistant gene Bph9 of rice, an identification method and application.
Background
Brown planthopper is one of the most serious insect pests harming rice yield. In addition, the brown planthopper takes food and simultaneously spreads rice diseases, such as grassy bushy stunt, odontoblast and the like, so that the damage to rice is indirectly increased. At present, the prevention and control of the brown planthopper mainly comprise chemical pesticides, the harm of the brown planthopper is relieved to a certain extent by the wide use of the pesticides, but a series of problems such as environmental pollution, pesticide residue, ecological balance damage, drug resistance and the like are caused by the use of excessive pesticides. Many practices show that the cultivation of rice varieties with resistance to the brown planthopper by utilizing the self resistance of the rice is one of the most efficient and environment-friendly prevention and control methods for preventing and controlling the brown planthopper.
Since 1973 of International Rice institute, a series of rice varieties with brown planthopper-resistant genes such as Bph1, Bph2 and Bph3 are successively cultivated by using a conventional breeding means, but with the generation of new biotypes of brown planthopper, the resistance of the varieties is gradually lost, so that the use of new brown planthopper-resistant genes or the aggregation of a plurality of resistance genes becomes the main direction of next brown planthopper-resistant breeding. The identification of the resistance phenotype of the rice to the brown planthopper is complex and tedious, and the breeding process can be accelerated by using the molecular marker of the resistance gene for auxiliary selection, so that the breeding efficiency is improved.
At present, a plurality of genes for resisting the brown planthopper are cloned, wherein Bph9 is a gene for resisting the brown planthopper of rice from a resistant parent Pokkali and shows good resistance to ecotypes I, II and III of the brown planthopper. However, all Bph9 used in breeding are linked markers at present, and functional markers in genes are not reported. In addition, rice has a plurality of alleles Bph1, Bph2, Bph7, Bph10, Bph18, Bph21 and the like at the gene locus, and the genes are difficult to distinguish by the linked markers. These all affect the selection efficiency and accuracy of molecular marker assisted breeding, and limit the application of the Bph9 gene in the breeding process of brown planthopper resistance of rice.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects and the defects that the prior art has difficulty in accurately distinguishing the brown planthopper resistant gene of rice by adopting a linked marker, affects the selection efficiency and the accuracy of molecular marker assisted breeding, limits the application of the Bph9 gene in the brown planthopper resistant breeding process of rice and the like, the functional molecular marker, the identification method and the application of the brown planthopper resistant gene Bph9 of rice are provided. The functional molecular marker can effectively distinguish the Bph9 from other alleles Bph1, Bph2, Bph7, Bph10, Bph18, Bph21 and the like, is the PCR-type intra-gene functional molecular marker which is designed only aiming at the Bph9 gene at present, and can be used for carrying out rice breeding practice simply, quickly and in a high-throughput manner.
The invention adopts the following technical scheme to achieve the purpose of the invention.
Firstly, the invention provides a functional molecular marker of a brown planthopper resistant gene Bph9 of rice, wherein the molecular marker is Bph9-M, and the nucleotide sequence of the Bph9-M is shown in a sequence table SIPOS sequence listing 1.0. The nucleotide sequence of Bph9-M comprises 1950 nucleotides, specifically:
GTATTTAAGGAGTCAGTAAATTTGGATCAACAAGATCTTGAATTGATCAAAGAAGCGAAACCGATTCTAAAGAAGTGCAATGGACTTCCCCTTGCAATTGTCACCATAGGTGGTTTCTTGGCAAGCCGCCCCAAAACTACTTTGGAGTGGAGAAAATTGAATGAGCATATTAGTGCAGAGTTGGAGACAAACCCAGGGCTTGAGGCCATCAGAGCTGTCCTTAATATAAGCTACGACGGATTACCTTATCACCTCAAGTCTTGCTTCTTGTATCTGTCCATCTTTCCTGAAGATGGCAAGATTAGCAGAAAACGTTTGGTGCGTCGATGGTGTGCAGAGGGTTACTCAAGGGAGCTATGGGACAAATCTGCAGAGGAAATAGCAAACAACTACTTCTTTGAACTCATAGACAGAAGCATGATCCTACCAACTCAAAATTCAACTTACAGCAGTAGAGGGGCTGATTCTTGCCAGATCCATGATATCATGCGTGAGATAGCCATCTTGAAGTCAAAGGAGGAAAACCTTGTTCTTAGACTCGAAGGGGGTCCCAGGCTATACAATCATGACACAGTTCGGCATATTTCCATTACAAACATCAGCGAGGACTGGGAGACAGATGTCGATGAATTGAAGACAACAGTAGATATGTCCCGAATAAGATCATTAACAGTATTTGGGATGTGGAGACCTTTTTTTATTTCTGACAAGATGCAGTTACTACAAGTGCTAGACTTGGAAGACACAAAAGGTGTATATGATCATCATATTAAGCAAATTGGGAAGCTCCTTCACCTTAGATACCTTTCTCTAAGAGGATGTGGGAACATTACTTACCTGCCTGATTCCTTAGGTAACCTAAGGCAACTGGAGACACTAGATGTCAGAGGTACGTGCATACTCAGGTTGCAAAAGACCATCATTAATCTTCGCAAGCTAAAGTATCTCCGTGCTGTCCCAGAGTTATCTGACCCGTATGAAGACATAGCAGAGAAACTACCAGAGCTCATTAGGAACAGGCTATGCATTTCTGCGACTGCGTTGCTGGCGCTTTGCGTGTTATGCTCACCAAGTGATCAAGGGATTAGTACCCGTGACCTCTGCACCTTGTGTTGCTGCAGTATTCTCCCTGCCATTGCCATGCGCCTCGACGGGAATGGTGTAGTAGCACCGAGAGGGCTGAGGAGACTGACAGCCCTGCACACGCTAGGTGTGGTGGACATTTCATGGCAGCCATCAATTTTACAAGATATCAAGAGGCTCATCCAGCTGCGCAAACTGGGAGTGAGCGGTGTCAACAAGAAAAACAGCAAAAAGTTTTTATCTGCCCTTGTCGCTCTCAGCCGCCTGGAATCATTGTCACTGATCTCGAAGGGGAAGCCAGGTCTCTGGGGCTGTCTGGATGCTGATGAAAAGTTTTCGCCACCTAAGAATCTCAAGACTCTGAAGCTTCAAGGCAACCTGGTTGAGTTGCCAAAATGGATCGGGCAGCTCAACAATCTCGTGAAGCTGAAGCTATCAGAAACCGGGCTCAAGGATCATGATGCTGCTATACAAGTCCTTGGTAAGCTACGAAACCTGACCATCCTATGCCTGCTGGGCAAGTCATTTCACTCGCTTGAGGGTGGTGAACTCAATTTCTCGGAGGGATCTTTCAAAAGCCTGGTGGTTCTCGAGCTTGACTTCAGTGGGAGCAAATGCGTCAAGTTTCAACAAGGAGCATTCCACAATCTTGAGCTACTGGAGCTTCATTGTGAGCTACTGGAGCTTTCTGGTCATATTGAAGAAGTCGAAACTAAGTTCTCTGGGCTAGAATTTCTCCCAAGAATCAAGGAAGTCCGGCTCCAGGGTTATTTTTACGGATTTTATGACACACGAAAATTGATGGAGGACTTGCTGGCACAGCTTTCCGAGAACCCAAAGAAACCAATCCTGAAGCCTAGCGGGTGA
further, the primers Bph9-M are: a forward primer: TCATTAGGAACAGGCTATGCA, respectively; reverse primer: TTCTTGTTGACACCGCTCAC are provided.
Secondly, the invention provides an identification method of a functional molecular marker Bph9-M of a brown planthopper resistant gene of rice, which comprises the following steps:
(1) RIGW searches a locus gene sequence in indica rice, MSU searches a locus gene sequence in japonica rice, and obtains a gene sequence Bph9(ID number KU216221) and an allele sequence Bph1(ID number KX681949), Bph7(ID number KU221258), Bph10(ID number KX681950) and Bph21(ID number KX 681951);
(2) after a plurality of sequences are compared together, selecting an Indel sequence existing in the 3 rd exon, and carrying out molecular marker design by using online Primer design software Primer1, wherein the sequence is named as Bph 9-M; a forward primer: TCATTAGGAACAGGCTATGCA, respectively; reverse primer: TTCTTGTTGACACCGCTCAC is added.
Further, the indica rice comprises Zhenshan 97(OsZS _12T0336600) and Minghui 63(OsMH _12T 0305200); the japonica rice comprises Nipponbare (LOC _ Os12g 37290).
Finally, the invention also provides application of the gene function molecular marker Bph9-M in brown planthopper resistant breeding of rice. The breeding application comprises the following steps: (1) extracting rice genome DNA; (2) genotype identification, wherein the genotype identification takes rice genome DNA as a template to carry out PCR amplification detection; (3) and (4) carrying out phenotype identification on brown planthopper resistance.
Further, the rice genome DNA is extracted in the step (1) by a simple CTAB method.
Furthermore, the simple CTAB method comprises the following specific steps: shearing about 0.5g of leaves, putting the leaves into a 2ml centrifuge tube, putting a steel bead into the tube, covering a tube cover, adding 600 mu l of CTAB extracting solution into the centrifuge tube, and fixing the centrifuge tube on a proofing machine to vibrate at the speed of 23-26 r/S for about 60S; water bath at 65 deg.C for 30min, shaking and mixing for 2 times; opening the centrifuge tube, adding 600 μ l chloroform, shaking thoroughly, standing for 5min, and centrifuging at 12000rpm for 8 min; sucking 400 μ l of supernatant into a 1.5mL centrifuge tube prepared in advance, adding 1mL of anhydrous ethanol pre-cooled in a refrigerator at-20 deg.C, shaking, standing in the refrigerator at-20 deg.C for 20min, and centrifuging (12000rpm, 10 min); pouring the upper layer of absolute ethyl alcohol into a waste water tank, adding 200 mu l of ddH after air drying2O, storing at-20 ℃ for later use.
Further, in the PCR amplification detection of step (2), the PCR reaction system is (in 20 μ l): 2ul of 10xPCR reaction buffer, 1ul of each of 5pM primers F and R, 0.5ul of 10mM dNTP, 2ul of DNA sample, 0.5ul of Taq DNA polymerase, and ddH2O is complemented to 20 ul. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. The products of the PCR reaction were separated by 2% agarose electrophoresis, stained with ethidium bromide and imaged under a gel imaging system.
Further, the brown planthopper resistance phenotype identification in the step (3) adopts a standard seedling stage group screening method.
Furthermore, the standard seedling stage group screening method comprises the following specific steps: the test rice varieties are germinated and respectively sown in plastic pots, 8 varieties are planted in each variety, 3 times of repetition is set, when rice seedlings grow to 2 leaves and 1 heart, the whole plastic pot is covered by a gauze, 12 heads of each plant of rice of 2-3-year-old nymphs of brown planthoppers are inoculated, and resistance phenotype observation is carried out.
Has the advantages that:
(1) the functional molecular marker Bph9-M of the rice brown planthopper resistant gene Bph9 disclosed by the invention is a molecular marker located in the rice brown planthopper resistant gene Bph9, and the molecular marker Bph9-M cannot be subjected to exchange separation with the gene, so that different alleles on the gene site can be well distinguished, and the influence of environmental conditions is avoided. Overcomes the defects and the defects that the brown planthopper resistant gene of the rice is difficult to be accurately distinguished by adopting a linkage marker in the prior art, the selection efficiency and the accuracy of molecular marker assisted breeding are influenced, the application of the Bph9 gene in the brown planthopper resistant breeding process of the rice is limited, and the like.
(2) The identification method of the functional molecular marker Bph9-M of the brown planthopper resistance gene Bph9 of the rice, disclosed by the invention, is simple in identification operation method and low in cost, lays a solid foundation for the molecular marker-assisted selection work of the Bph9 gene in brown planthopper resistance breeding of the rice, and can be used for carrying out rice breeding practice simply, quickly and in a high-throughput manner.
Drawings
FIG. 1 shows the position of the functional molecular marker Bph9-M of Bph9 in the marker sequence.
Wherein: the positions of the primers before and after are underlined, and the dotted line indicates the deleted nucleotide sequence;
OsZS _12T0336600 is Zhenshan 97 from a RIGW website;
OsMH _12T0305200 is minghui 63 from the RIGW website;
nip _ Os12g37280 is nipponbare from MSU website;
bph9 is the gene sequence in NCBI Genbank (ID number KU 216221);
bph1 is the median gene sequence of NCBI Genbank (ID number KX 681949);
bph7 is the median gene sequence of NCBI Genbank (ID number KU 221258);
bph10 is the median gene sequence of NCBI Genbank (ID number KX 681950);
bph21 is the NCBI Genbank allele sequence (ID number KX 681951).
FIG. 2 shows the results of the detection of a plurality of different rice varieties by the marker Bph 9-M.
Wherein 1-26 are in sequence: lopa nationality 9, IR64 (containing Bph1), IR36 (containing Bph2), IR50 (containing Bph2), Lopa nationality 18 (containing Bph18), Liaojing 454, Longjing 9, Nanjing 46, Qingguang, Fujin, Tetep, Kasalath, KDM105, Xiangguoyang, Zhenhui 084, R644, middle group 14, T7954, Wushan Miao, Huazhan, Minghui 63, 93-11, Chenghui 727, Zhongzao 39, Neixiang 10B and Zhenshan 97B.
FIG. 3 shows F of Lopa 9/Jiangxiang B2The detection result of Bph9-M in the population.
Wherein: 1 is Lopa 9, 2 is Jiangxiang B, and the rest c-y is F of Lopa 9/Jiangxiang B2Different strains in the population.
Detailed Description
The present invention will be further described with reference to specific examples, but the present invention is not limited to the following examples. The experimental procedures in the following examples were carried out in a conventional manner unless otherwise specified, and materials, reagents and the like used in the following examples were commercially available unless otherwise specified.
The first embodiment is as follows: identification method of molecular marker Bph9-M of brown planthopper resistant gene Bph9 of rice
The identification method of the molecular marker Bph9-M of the brown planthopper resistant gene Bph9 of rice comprises the following steps:
the RIGW searches the gene sequence of the locus in the indica rice, wherein the indica rice is Shanzhen 97(OsZS _12T0336600) and/or Minghui 63(OsMH _12T 0305200); MSU searches the gene sequence of the site in japonica rice, wherein the japonica rice is Nipponbare (LOC _ Os12g 37280); gene sequences Bph9(ID number KU216221) and allele sequences Bph1(ID number KX681949), Bph7(ID number KU221258), Bph10(ID number KX681950) and Bph21(ID number KX681951) are obtained from NCBI Genbank.
After comparing the sequences together, the Indel sequence present in exon 3 was selected. The molecular marker design is carried out by using online Primer design software Primer1, and is named as Bph9-M, and a forward Primer: TCATTAGGAACAGGCTATGCA; reverse primer: TTCTTGTTGACACCGCTCAC are provided.
The position of a molecular marker Bph9-M sequence of the brown planthopper resistant rice gene Bph9 in the gene is shown in figure 1. Wherein:
underlined indicates the positions of the front and rear primers, Primer F is the position of the forward Primer, Primer R is the position of the reverse Primer, and the dotted line indicates the deleted nucleotide sequence;
OsZS _12T0336600 is Zhenshan 97 from a RIGW website;
OsMH _12T0305200 is minghui 63 from the RIGW website;
nip _ Os12g37280 is nipponica from MSU website;
bph9 is the gene sequence in NCBI Genbank (ID number KU 216221);
bph1 is the median gene sequence of NCBI Genbank (ID number KX 681949);
bph7 is the allele sequence of NCBI Genbank (ID number KU 221258);
bph10 is the allele sequence of NCBI Genbank (ID number KX 681950);
bph21 is the NCBI Genbank allele sequence (ID number KX 681951).
As can be seen from FIG. 1, the Bph9 with resistance function can amplify a 298bp band, while the other materials not carrying Bph9 can amplify 313bp or 319bp bands.
Example II detection of multiple different Rice varieties by marker Bph9-M
1. Test material
2. Extraction of DNA
The experiment adopts a CTAB simple method to extract the genome DNA of the rice sample, and the specific steps are as follows:
(1) shearing about 0.5g of leaves, putting the leaves into a 2ml centrifuge tube, putting a steel bead into the tube, covering a tube cover, adding 600 mu l of CTAB extracting solution into the centrifuge tube, and fixing the centrifuge tube on a proofing machine to vibrate at the speed of 23-26 r/S for about 60S.
(2) Water bath at 65 deg.c for 30min, and shaking and mixing for 2 times.
(3) The centrifuge tube was opened, 600. mu.l of chloroform was added thereto, the mixture was shaken well and then allowed to stand for 5min, and the mixture was centrifuged at 12000rpm for 8 min.
(4) 400. mu.l of the supernatant was aspirated into a 1.5mL centrifuge tube prepared in advance, and 1mL of absolute ethanol pre-cooled in a refrigerator at-20 ℃ was added thereto, and after shaking, the mixture was left to stand in the refrigerator at-20 ℃ for 20min and centrifuged (12000rpm, 10 min).
(5) Pouring the upper layer of absolute ethyl alcohol into a waste water tank, air-drying, and adding 200 mu l of ddH2O, storing at-20 ℃ for later use.
3. Genotyping
2. mu.l of DNA was used as a template for PCR amplification detection. The PCR reaction was calculated in 20. mu.l: 2ul of 10xPCR reaction buffer, 1ul of each of 5pM primers F and R, 0.5ul of 10mM dNTP, 2ul of DNA sample, 0.5ul of Taq DNA polymerase, and ddH2O is complemented to 20 ul. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃. The products of the PCR reaction were separated by 2% agarose electrophoresis, stained with ethidium bromide and imaged under a gel imaging system. The detection result is shown in figure 2.
As can be seen from fig. 2: the expected 298bp band can be amplified from the material Loa gamma 9 containing Bph9, 313bp or 319bp band can be amplified from other materials not carrying Bph9, the electrophoresis band is specific and clear, the gene marker can well distinguish whether the rice material carries the anti-nilaparvata lugens gene Bph9, and the gene marker can be effectively used for marker-assisted breeding of the anti-nilaparvata lugens of rice.
Third embodiment, Lopa 9/Jiangxiang B and F thereof2Detection of Bph9-M in a population
1. Test material
The Lopa 9, gan Xiang B, and F combined with the Lopa 9/gan Xiang B2The isolated population (c-y) was used for validation of the marker Bph 9-M.
2. Extraction of DNA
The experiment adopts a CTAB simple method to extract the genome DNA of the rice sample, and the specific steps are as follows:
(1) shearing about 0.5g of leaves, putting the leaves into a 2ml centrifuge tube, putting a steel bead into the tube, covering a tube cover, adding 600 mu l of CTAB extracting solution into the centrifuge tube, and fixing the centrifuge tube on a sample making machine to vibrate at the speed of 23-26 r/S for about 60S.
(2) Water bath at 65 deg.C for 30min, shaking and mixing for 2 times.
(3) The centrifuge tube was opened, 600. mu.l of chloroform was added thereto, the mixture was shaken well and then allowed to stand for 5min, and the mixture was centrifuged at 12000rpm for 8 min.
(4) Sucking 400 μ l of the supernatant into a prepared 1.5mL centrifuge tube, adding 1mL of anhydrous ethanol pre-cooled in a-20 ℃ refrigerator, shaking, standing in the-20 ℃ refrigerator for 20min, and centrifuging (12000rpm, 10 min).
(5) Pouring the upper layer of absolute ethyl alcohol into a waste water tank, air-drying, and adding 200 mu l of ddH2O, storing at-20 ℃ for later use.
3. Genotyping
Taking 2. mu.l of DNA as a template to carry out PCR amplification detection. The PCR reaction was calculated in 20. mu.l: 2ul of 10xPCR reaction buffer, 1ul of each of 5pM primers F and R, 0.5ul of 10mM dNTP, 2ul of DNA sample, 0.5ul of Taq DNA polymerase, and ddH2The content of O is complemented to 20 ul. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃; cooling at 4 deg.C for 10 min. Products of the PCR reaction were separated by 6% polyacrylamide gel electrophoresis, stained with silver nitrate and sodium hydroxide, and imaged under a gel imaging system. And (5) judging and recording the band type of the progeny individual plant by referring to the amplified bands of the parents. The detection results are shown in figure 3.
As can be seen from FIG. 3, the genotype marker Bph9-M clearly distinguishes the genotype in the population. Different strains of F2 segregation population of Hoodia gamma 9/Ganxiang B, wherein the strains (e, F, i, q, s, t, w, x and y) contain electrophoretic bands consistent with the Hoodia gamma 9, and carry homozygous Bph9 genes; the strain (c, d, j, n, o, p, r) contains a marker consistent with that of the Ganxiang B and does not carry the gene Bph 9; strains (g, h, k, l, m, u, v) carry the bands of both parents, carrying the heterozygous Bph9 gene.
4. Anti-brown planthopper phenotype identification
And randomly taking different strains with Bph9 and no Bph9 in the F2 population, and carrying out brown planthopper resistance identification.
The standard seedling-stage group screening method is adopted, the tested paddy rice varieties are respectively sown in plastic pots after germination acceleration, 8 varieties are planted in each variety, and 3 repetitions are set. When the rice seedlings grow to 2 leaves and 1 heart, covering the whole plastic pot by using a gauze, inoculating 12 heads of each rice plant of 2-3 instar nymphs of brown planthopper, and carrying out resistance phenotype observation, wherein the observation result is as follows: different strains of the progeny of the rice Loa 9 show resistance to brown planthopper due to the strains with the Bph9, and the rice plants grow normally; and different rice Ganxiang B progeny plants of different rice plants do not have the Bph9 plant line and show that the rice plants cannot resist brown planthopper, and the rice plants are completely withered and die.
The result shows that the brown planthopper resistance of the strain with the Bph9 gene is obviously stronger than that of the strain without the Bph9 gene.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the above-described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the present invention, without departing from the spirit and scope of the invention.
Nucleotide sequence table of brown planthopper resistant gene Bph9-M of rice
Sequence listing
<110> institute of Paddy Rice research of academy of agricultural sciences in Jiangxi province
<120> functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice
<140> 2019102349683
<141> 2019-03-27
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
tcattaggaa caggctatgc a 21
<210> 2
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
<210> 3
<211> 1950
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
gtatttaagg agtcagtaaa tttggatcaa caagatcttg aattgatcaa agaagcgaaa 60
ccgattctaa agaagtgcaa tggacttccc cttgcaattg tcaccatagg tggtttcttg 120
gcaagccgcc ccaaaactac tttggagtgg agaaaattga atgagcatat tagtgcagag 180
ttggagacaa acccagggct tgaggccatc agagctgtcc ttaatataag ctacgacgga 240
ttaccttatc acctcaagtc ttgcttcttg tatctgtcca tctttcctga agatggcaag 300
attagcagaa aacgtttggt gcgtcgatgg tgtgcagagg gttactcaag ggagctatgg 360
gacaaatctg cagaggaaat agcaaacaac tacttctttg aactcataga cagaagcatg 420
atcctaccaa ctcaaaattc aacttacagc agtagagggg ctgattcttg ccagatccat 480
gatatcatgc gtgagatagc catcttgaag tcaaaggagg aaaaccttgt tcttagactc 540
gaagggggtc ccaggctata caatcatgac acagttcggc atatttccat tacaaacatc 600
agcgaggact gggagacaga tgtcgatgaa ttgaagacaa cagtagatat gtcccgaata 660
agatcattaa cagtatttgg gatgtggaga ccttttttta tttctgacaa gatgcagtta 720
ctacaagtgc tagacttgga agacacaaaa ggtgtatatg atcatcatat taagcaaatt 780
gggaagctcc ttcaccttag atacctttct ctaagaggat gtgggaacat tacttacctg 840
cctgattcct taggtaacct aaggcaactg gagacactag atgtcagagg tacgtgcata 900
ctcaggttgc aaaagaccat cattaatctt cgcaagctaa agtatctccg tgctgtccca 960
gagttatctg acccgtatga agacatagca gagaaactac cagagctcat taggaacagg 1020
ctatgcattt ctgcgactgc gttgctggcg ctttgcgtgt tatgctcacc aagtgatcaa 1080
gggattagta cccgtgacct ctgcaccttg tgttgctgca gtattctccc tgccattgcc 1140
atgcgcctcg acgggaatgg tgtagtagca ccgagagggc tgaggagact gacagccctg 1200
cacacgctag gtgtggtgga catttcatgg cagccatcaa ttttacaaga tatcaagagg 1260
ctcatccagc tgcgcaaact gggagtgagc ggtgtcaaca agaaaaacag caaaaagttt 1320
ttatctgccc ttgtcgctct cagccgcctg gaatcattgt cactgatctc gaaggggaag 1380
ccaggtctct ggggctgtct ggatgctgat gaaaagtttt cgccacctaa gaatctcaag 1440
actctgaagc ttcaaggcaa cctggttgag ttgccaaaat ggatcgggca gctcaacaat 1500
ctcgtgaagc tgaagctatc agaaaccggg ctcaaggatc atgatgctgc tatacaagtc 1560
cttggtaagc tacgaaacct gaccatccta tgcctgctgg gcaagtcatt tcactcgctt 1620
gagggtggtg aactcaattt ctcggaggga tctttcaaaa gcctggtggt tctcgagctt 1680
gacttcagtg ggagcaaatg cgtcaagttt caacaaggag cattccacaa tcttgagcta 1740
ctggagcttc attgtgagct actggagctt tctggtcata ttgaagaagt cgaaactaag 1800
ttctctgggc tagaatttct cccaagaatc aaggaagtcc ggctccaggg ttatttttac 1860
ggattttatg acacacgaaa attgatggag gacttgctgg cacagctttc cgagaaccca 1920
aagaaaccaa tcctgaagcc tagcgggtga 1950
Claims (8)
1. Brown planthopper resistant gene of riceBph9The functional molecular marker primer is characterized in that the nucleotide sequence of the primer is as follows:
a forward primer: TCATTAGGAACAGGCTATGCA;
reverse primer: TTCTTGTTGACACCGCTCAC is added.
2. A method for identifying a functional molecular marker Bph9-M of a brown planthopper resistant gene of rice is characterized by comprising the following steps:
(1) RIGW searches the locus gene sequence in indica rice, MSU searches the locus gene sequence in japonica rice to obtain the gene sequenceBph9And allelic sequences thereofBph1、Bph7、Bph10、Bph21；
(2) After comparing the sequences together, selecting an Indel sequence existing in the 3 rd exon, and designing a molecular marker Primer by using an online Primer design software Primer1, wherein the Primer is defined in claim 1;
(3) the nucleotide sequence of the rice brown planthopper-resistant gene functional molecular marker Bph9-M is shown in SEQ ID No.3, the functional molecular marker primer in claim 1 is used for PCR amplification, a rice sample with Bph9-M with a resistance function can amplify a band with the size of 298bp, and other rice samples without Bph9-M can amplify bands with 313bp or 319 bp.
3. The method for identifying the functional molecular marker Bph9-M for the brown planthopper resistance gene in rice according to claim 2, wherein the method comprises the following steps: the indica rice comprises Zhenshan 97 and/or Minghui 63; the japonica rice comprises Nipponbare.
4. The application of the functional molecular marker primer in the brown planthopper resistant rice breeding of claim 1 is characterized by comprising the following steps:
(1) extracting rice genome DNA;
(2) identifying the genotype; the genotype identification is carried out by taking rice genome DNA as a template and utilizing the functional molecular marker primer of claim 1 to carry out PCR amplification detection, a rice sample with Bph9-M with a resistance function can amplify a strip with the size of 298bp, and other rice samples without Bph9-M can amplify strips with 313bp or 319 bp;
(3) and (4) carrying out phenotype identification on brown planthopper resistance.
5. The use of claim 4, wherein the step (1) of extracting genomic DNA from rice is carried out by a simple CTAB method.
6. The use of claim 4, wherein the PCR amplification assay of step (2) is performed under the following reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.
7. The use as claimed in claim 4, wherein the phenotypic identification of brown planthopper resistance in step (3) is by standard seedling stage group screening.
8. The use of claim 7, wherein the standard seedling group screening method comprises: the test rice varieties are germinated and respectively sown in plastic pots, 8 varieties are planted in each variety, 3 times of repetition is set, when rice seedlings grow to 2 leaves and 1 heart, the whole plastic pot is covered by a gauze, 12 heads of each plant of rice of 2-3-year-old nymphs of brown planthoppers are inoculated, and resistance phenotype observation is carried out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910234968.3A CN109762929B (en) | 2019-03-27 | 2019-03-27 | Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910234968.3A CN109762929B (en) | 2019-03-27 | 2019-03-27 | Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109762929A CN109762929A (en) | 2019-05-17 |
| CN109762929B true CN109762929B (en) | 2022-07-15 |
Family
ID=66459837
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910234968.3A Expired - Fee Related CN109762929B (en) | 2019-03-27 | 2019-03-27 | Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109762929B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951748B (en) * | 2019-12-16 | 2021-10-22 | 武汉大学 | Rice brown planthopper resistant gene Bph37, protein, vector, host cell, molecular marker, method and application |
| CN112592997B (en) * | 2020-12-17 | 2023-08-22 | 华智生物技术有限公司 | Auxiliary breeding molecular marker of brown planthopper resistant gene Bph9 and application thereof |
| CN112813188A (en) * | 2021-03-01 | 2021-05-18 | 广西壮族自治区农业科学院 | PARMS marker based on brown planthopper resistance gene Bph26 and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509791A (en) * | 2013-07-31 | 2014-01-15 | 江西省农业科学院水稻研究所 | Gene marker of major gene Bph14 for resisting brown planthopper in rice and application thereof |
| WO2014036946A1 (en) * | 2012-09-05 | 2014-03-13 | Wuhan University | Rice brown planthopper resistance gene bph9 and molecular markers, and uses thereof |
| WO2016165243A1 (en) * | 2015-04-13 | 2016-10-20 | 武汉大学 | Rice gene bph6 resistant to brown planthopper and closely linked molecular marker thereof |
-
2019
- 2019-03-27 CN CN201910234968.3A patent/CN109762929B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014036946A1 (en) * | 2012-09-05 | 2014-03-13 | Wuhan University | Rice brown planthopper resistance gene bph9 and molecular markers, and uses thereof |
| CN103667309A (en) * | 2012-09-05 | 2014-03-26 | 武汉大学 | Brown planthopper resistant rice gene Bph9 as well as molecular marker and application thereof |
| CN103509791A (en) * | 2013-07-31 | 2014-01-15 | 江西省农业科学院水稻研究所 | Gene marker of major gene Bph14 for resisting brown planthopper in rice and application thereof |
| WO2016165243A1 (en) * | 2015-04-13 | 2016-10-20 | 武汉大学 | Rice gene bph6 resistant to brown planthopper and closely linked molecular marker thereof |
Non-Patent Citations (4)
| Title |
|---|
| Identification of spontaneous mutation for broad-spectrum brown planthopper resistance in a large, long-term fast neutron mutagenized rice population;Wintai Kamolsukyeunyong等;《RICE》;20190319;第12卷(第1期);16 * |
| 分子标记辅助选择在水稻抗病虫基因聚合上的应用;裴庆利等;《中国水稻科学》;20110228;第25卷(第02期);119-129 * |
| 水稻抗褐飞虱基因Bph9共显性功能标记开发及应用;陈明亮等;《分子植物育种》;20210304;第1-10页 * |
| 水稻新品种抗性基因检测、籼粳属性鉴定及杂种优势预测;赵小燕;《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑(月刊)》;20160215(第02期);D047-30 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109762929A (en) | 2019-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109762929B (en) | Functional molecular marker, identification method and application of brown planthopper resistant gene Bph9 of rice | |
| CN110684858B (en) | Molecular marker of rice slender grain type gene and application thereof | |
| Jia et al. | Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR | |
| CN110512025B (en) | Molecular marker closely linked with wheat powdery mildew resistance gene PmJM23 and application thereof | |
| CN109055598B (en) | Rice brown planthopper resistant gene BPH6 codominant molecular marker and application thereof | |
| US12048280B2 (en) | Green bean plants with improved disease resistance | |
| CN106957897B (en) | Molecular marking method of cucumber young leaf yellowing gene | |
| CN107988409B (en) | Specific CAPS marker for identifying wild type or mutant of rice salt-tolerant gene OsRR22, primer and application of primer | |
| CN114480709B (en) | Molecular marker for detecting wheat leaf rust resistance gene Lr47, detection method and application thereof | |
| CN117887895B (en) | KASP molecular marker for pumpkin fruit length character and application thereof | |
| CN107201395B (en) | Molecular marker of major gene Bph30 for resisting brown planthopper of rice and application thereof | |
| CN112195268B (en) | Molecular marker, primer, application and variety breeding method closely linked with origin green peach aphid resistance character of cultivar | |
| Tamura et al. | Mapping of the Grh1 locus for Green Rice Leahopper Resistance in Rice Using RFLP Markers | |
| Řepková et al. | New CAPS marker for selection of a barley powdery mildew resistance gene in the Mla locus | |
| WO2009058335A1 (en) | Major qtls conferring resistance of corn to fijivirus | |
| CN114015701B (en) | Molecular marker for detecting barley grain shrinkage character and application thereof | |
| CN112266976B (en) | CAPS molecular marker, primers, detection method, detection kit and application based on tomato gray leaf spot resistance gene Sm | |
| Markussen et al. | Positioning of sex-correlated markers for Populus in a AFLP-and SSR-marker based genetic map of Populus tremula x tremuloides | |
| CN108315463B (en) | Primer group for detecting corn 1768 gene and application thereof | |
| CN107058545B (en) | SNP molecular marker of corn embryogenic callus induction related gene GRMZM2G020814 and application thereof | |
| CN109468400B (en) | Rice blast resistance gene Pi36 codominant molecular marker and application thereof | |
| CN106834485B (en) | Molecular marker of rice blast resistance gene Pi9 and application thereof | |
| CN113699273B (en) | SNP locus combination for detecting resistance of tomato root-knot nematode and application thereof | |
| CN114292951B (en) | InDel molecular marker linked with brown planthopper resistant major gene bph41 (t) of rice, and primers and application thereof | |
| CN111826461B (en) | Development and application of molecular marker of sorghum miscanthus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220715 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |