CN101418349A - Molecular marker method of rice variety brown planthopper resistance main gene Bph3 - Google Patents
Molecular marker method of rice variety brown planthopper resistance main gene Bph3 Download PDFInfo
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Abstract
The invention relates to a molecule tagging method for a brown paddy plant hopper resistant master gene Bph3 of rice cultivars, which belongs to the technical field of genetic thremmatology. The brown paddy plant hopper resistant master gene Bph3 of an insect resistant variety Rathu Heenati is obtained by genetic linkage analysis of the brown paddy plant hopper resistant level of gene types of various single plants of F2 and various genealogies of F 2:3 obtained after hybridization of the insect resistant rice variety Rathu Heenati (female) and an insect susceptible variety 02428(male). The gene is positioned between a molecular marker A4 and a molecular marker RM16533, and the selection efficiency of three Indel indexes namely RH784, RH786 and RH007 of the interval is approximately 97 percent. Detection is made whether the insect resistant variety Rathu Heenati and derivative varieties (systems) of the insect resistant variety Rathu Heenati contain the master gene through the molecular markeres of the brown paddy plant hopper resistant master gene; the brown paddy plant hopper resistance level of the master gene can be predicted; and the selection efficiency of brown paddy plant hopper resistant rice can be greatly improved.
Description
One, technical field
The invention provides the molecule marking method of rice varieties brown planthopper resistant key-gene Bph3, belong to the molecular genetics field, be exclusively used in the seed selection of paddy rice brown planthopper resistant kind and the utilization of germ plasm resource.
Two, background technology
Paddy rice is the important food crop of China.Brown paddy plant hopper is a kind of paddy rice monophagy insect, belongs to the Homoptera Delphacidae.Suck rice stem stalk phloem juice by lancet, be typical sucking pest.Cause rice strain bottom blackening, rotten smelly, paralysed when serious, be referred to as " lice burning ", cause the paddy rice underproduction or mistake to be received.At present, the control of brown paddy plant hopper is mainly depended on chemical pesticide, but this measure has not only increased production cost, and contaminate environment, therefore, economical and effective and the prophylactico-therapeutic measures that do not pollute is the application of pest-resistant cultivar the most.
So far, 13 brown planthopper resistant key-genes have been found and have identified.Wherein, 6 of dominant genes, i.e. Bph-1 (Athwal et al., 1971), Bph-3 (Lakshiminarayana and Khush, 1977), Bph-6 (Kabir and Khush, 1988), Bph-9 (Ikeda et al., 1985; Nemoto et al., 1989a), Bph-10 (t) (Multani et al., 1994; Ishii et al., 1994) and Bph-13 (t) (Liu Guoqing etc., 2001).7 of recessive genes, i.e. bph-2 (Athwal et al., 1971), bph-4 (Lakshiminarayana and Khush, 1977), bph-5 (Khush et al., 1985) and bph-7 (Kabir and Khush, 1988), bph-8 (Ikeda, 1985; Nemoto et al., 1989a), bph-11 (t) and bph-12 (t) (Hirabayashi and Ogawa, 1999).QTL (quantitive trait locus, the quantitative trait locus) Position Research of brown planthopper resistant has also been carried out in other researchs, has identified 26 brown planthopper resistant QTL.But the brown planthopper resistant gene that really is used for the paddy rice breeding for pest resistance is also few.
The International Rice Research Institute from 1973 seed selection successively a series of rice varieties that carry brown planthopper resistant key-genes such as Bph-1, bph-2 and Bph-3 respectively, controlled breaking out of brown paddy plant hopper effectively in the area of these kinds of plantation.Yet because the generation of brown paddy plant hopper true tumor type, the brown planthopper resistant kind is lost resistance gradually or is faced the danger of resistant lose (Pathak and Khush, 1979; Pathak and Saxena, 1980; Heinrichs, 1986; Saxena and Khan, 1989; Heinrichs, 1994; Gallagher et al., 1994).China also successively breeds a series of kinds (combination) that contain brown planthopper resistant gene Bph-1, has play a part positive to the control of brown paddy plant hopper.3328 parts of new rice varieties (being) that Lv Zhongxian etc. (2002) are national to 1986~2000 years and Zhejiang Province's breeding tackling key problem cooperative groups provides have carried out brown planthopper resistant evaluation and screening, found that from " the Seventh Five-Year Plan ", " eight or five " to " 95 " pest-resistant cultivar rate of reflecting out on a declining curvely, the breeding of paddy rice brown planthopper resistant is not subjected to enough attention.At present, domestic brown paddy plant hopper causes the strong Bangladesh's type biotype ratio of harmful power and rises based on biotype 2, has the pest-resistant cultivar of Bph-1 originally, loses resistance gradually, thereby presses for the new pest-resistant cultivar that carries a plurality of resistant genes of cultivation.
Because the complicacy that insect-resistance is identified utilizes the conventional breeding means often to be difficult to the different anti insect gene of polymerization effectively.And finding with the anti insect gene close linkage or be total on the basis of isolating molecule marker, by molecular marker assisted selection (Marker-assisted selection, MAS) technology then can on purpose be carried out the polymerization of anti insect gene or QTL, seed selection durable resistance kind delays the degeneration time limit of pest-resistant cultivar and prevents the generation of brown paddy plant hopper true tumor type.
Three, summary of the invention
Technical problem
The objective of the invention is: the molecule marking method that rice varieties Rathu Heenati brown planthopper resistant key-gene Bph3 is provided, by detecting and the chain molecule marker of these brown planthopper resistant key-genes, can predict the brown paddy plant hopper resistance of rice plant, accelerate the selection progress of brown planthopper resistant paddy rice.
Technical scheme
The molecule marking method of rice varieties brown planthopper resistant key-gene Bph3, rice varieties Rathu Heenati is controlled by a key-gene Bph3 the resistance of brown paddy plant hopper, it is characterized in that:
Use labeled primer A4,
Left end sequence A AGCAGCATAAACTGATTGA
Right-hand member sequence TCATCTTCTGAAAAAGCAAT
Perhaps use labeled primer RM16533,
Left end sequence TTTGCTTAGTCGGCAGATGTCC
Right-hand member sequence C ATAAGAACGTACCTCCACTGATTCC
Perhaps use labeled primer RH7841,
Left end sequence TGCCAAGTATGTATGCCTAT
Right-hand member sequence TTTTAGAGACCGTGTCCTTG
Perhaps use labeled primer RH786,
Left end sequence TTTGAAGTTCTTTCCATCTGA
Right-hand member sequence A AATGTGCTATCTGGGGTAAA
Perhaps use labeled primer RH007,
Left end sequence C TTGCGTTCCGTAGGAGAAG
Right-hand member sequence TGAGTGTAACCCGAAGTGGC
Amplifying rice brown planthopper resistant kind or breeding material DNA, if can amplify the amplified fragments of 193bp with primer A4, or can amplify the amplified fragments of 285bp with primer RM16533, or can amplify the amplified fragments of 213bp with primer RH7841, or can amplify the amplified fragments of 176bp with primer RH786, or can amplify the amplified fragments of 182bp with primer RH007, all indicate the existence of this rice varieties brown planthopper resistant key-gene Bph3, this key-gene is positioned on paddy rice the 4th the short arm of a chromosome.
The molecule marking method of beneficial effect rice varieties brown planthopper resistant provided by the present invention key-gene Bph3 has the following advantages:
(1) the key-gene Bph3 by the brown planthopper resistant among the present invention rice varieties Rathu Heenati that used SSR mark Fine Mapping in the world first.
(2) by the localized key-gene locality specific of molecule marker of the present invention, it is convenient to identify.By detecting these and the chain molecule marker of brown planthopper resistant gene, promptly can predict the brown paddy plant hopper resistance of rice plant, the genotype detection that is used for rice varieties or strain, judging whether this kind or strain have the brown paddy plant hopper resistance, and then rapid screening disease-resistant variety or strain are used for rice breeding.The key-gene site easy to detect fast, not affected by environment;
(3) the assistant breeding select target is clear and definite, saves cost.In traditional breeding way, at first to collect parent and Cultivar and carry out a series of hybridization, and will carry out individual plant to the brown paddy plant hopper resistance and select with anti insect gene.The paddy rice brown planthopper resistant is carried out the phenotypic evaluation complexity, simultaneously affected by environment, at first to obtain the worm source, raise brown paddy plant hopper, to obtain to inoculate the worm source in addition and rice seedling synchronous, unusual difficulty, the result reliability of phenotypic evaluation is low.Therefore breeding for pest resistance is not only time-consuming, and difficulty is big, the cost height.By detecting brown planthopper resistant key-gene site, can just identify the individual plant of high brown planthopper resistant in seedling stage, eliminate other plant, not only save production cost but also improve the efficiency of selection of brown planthopper resistant paddy rice greatly.
(4) purity that can be used for pest-resistant Hybrid simultaneously identify and seedling stage Rapid identification.
Four, description of drawings
The distribution of Fig. 1 rice varieties Rathu Heenati brown planthopper resistant gene on karyomit(e).
Five, embodiment
Studies show that the brown planthopper resistant gene resource mainly is present in (Ikeda and Vaughau, 1991) in Sri Lanka and India's long-grained nonglutinous rice and the wild seed rice.Athwal et al. (1971) report Mudgo, CO22 and MTU15 carry same brown planthopper resistant gene Bph-1, and ASD7 carries a recessive anti insect gene bph-2.Athwal andPathak (1972) report MGL2 contains anti insect gene Bph-1, and Ptb18 contains bph-2.Martinez andKhush (1974) report IR747B2-6 contains Bph-1, and R1154-243 and IR4-93 contain bph-2.Lakshiminarayana and Khush (1977) report Sri Lanka pest-resistant cultivar Rathu Heenati is by the dominant gene Bph-3 control with the Bph-1 independent separate; Kind Babawee is then by the recessive gene bph-4 control with the bph-2 independent separate.The rice varieties that Sidhu and Khush (1978) report carries Bph-3 or bph-4 all shows resistance to all brown paddy plant hopper biotypes.Thailand rice varieties Col.5Thailand and Col.11 Thailand, Burma rice varieties Chin Saba controls (Ikeda, 1985) by same with bph-2 and the inequipotential recessive anti insect gene bph-8 of bph-4.Identify and the genetic research of these anti insect genes is that the cultivation of pest-resistant cultivar provides the foundation, and wherein Bph-1, bph-2 and Bph-3 are applied in the breeding for pest resistance.But, because the complicacy that insect-resistance is identified utilizes the conventional breeding means often to be difficult to the different anti insect gene of polymerization effectively.And finding with the anti insect gene close linkage or be total on the basis of isolating molecule marker, by molecular marker assisted selection (Marker-assisted selection, MAS) technology then can on purpose be carried out the polymerization of anti insect gene and QTL, seed selection durable resistance kind delays the degeneration time limit of pest-resistant cultivar and prevents the generation of brown paddy plant hopper true tumor type.
By insect-proof rice kind Rathu Heenati brown planthopper resistant key-gene of the present invention and the evaluation in minor gene site and the discovery of molecule marker, special brown planthopper resistant key-gene site can be used to instruct the seed selection work of brown planthopper resistant rice varieties, with chain with it molecule marker pest-resistant cultivar is screened, make different pest-resistant key-genes site rapid polymerization in same plant, thereby improve breeding efficiency greatly.
(1) Rathu Heenati/02428 F
2Colony makes up and phenotypic evaluation
(1) with brown planthopper resistant kind Rathu Heenati (Ikeda and Kaneda 1981, Jpn J Breed, 31 (3): be maternal 279-285), sense brown paddy plant hopper japonica rice variety 02428 (Zou Jiangshi etc., Scientia Agricultura Sinica, 1989,22 (1): 6-14) be male parent, hybridization has made up the Rathu Heenati/02428 F that comprises 156 individual plants
2Segregating population, each F
2Individual plant obtains corresponding Rathu Heenati/02428 F by selfing
2:3Family is carried out pest-resistant evaluation.(2) adopt the inoculation in seedling stage that parent, F1, F2:3 are carried out insect-resistance and identify that brown paddy plant hopper is biotype 1 and biotype 2 mixed populations (Sun, LH etc. 2005).For guaranteeing parent, F
1And F
2:3Each family growth in the colony is consistent, and all distinguish presoaking and germinating prior to seeding for the examination material.Each family (kind) is got 25 planting seeds respectively in a diameter 8.5cm, high 9.0cm, fills with (aperture is arranged at the alms bowl bottom, is convenient to osmotic absorbent) in the round plastic alms bowl of nutrition soil, and spacing in the rows is 2.5cm.Each kind is planted 3 alms bowls at random.Per 28 polypotss place the plastic box of a 65cm * 44cm * 14cm interior (keeping about water layer 2cm in the case).Sow thinning after 7 days, eliminate sick and weak seedling, remain into every alms bowl 20 strains, treat seedling length to two leaves during one heart stage in the ratio of 10/seedling inoculation 2-3 brown paddy plant hopper in age nymph, nylon yarn guard on the back cover, as sense worm kind TN1 (Sun, LH etc. 2005) when all dead, with reference to (1971) such as Athwal, the method of (2001) such as IRRI (1988) and Huang carries out 0,1,3 to each individual plant, 5,7 or 9 grades evaluation of resistance (table 1) to each family of parent material and colony resistance rank by this family of weighted average calculation, and is inferred this individual plant genotype according to the resistance rank.
The anti-sense brown paddy plant hopper judgement criteria that this institute of table 1 uses
(2) Rathu Heenati/02428 F
2The molecular marker analysis of colony
(1) extracts parent, F with the SDS method
1And F
2The DNA of each strain system of colony.
(2) ssr analysis is with reference to the program of Chen et al. (1997).10 μ l reaction systems comprise: 10mM Tris-HCl pH 8.3,50mM KCl, 1.5mM MgCl2,50 μ M dNTPs, 0.2 μ M primer, 0.5U Taqpolymerase (TaKaRa, Dalian) and 20ng of dna profiling.Amplified reaction carries out on PTC-200 (MJResearch Inc.) PCR instrument: 94 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 35 circulations; 72 ℃ of 7min.Amplified production separates with 8% non-sex change PAGE glue, dyes colour developing by silver, and silver dyes program and formulates according to the method for Sanguinetti et al. (1994) and form.The lamp box that luminescent lamp is equipped with in the DNA band utilization of amplification is observed.The record result has polymorphic primer at F between the parent
2Analyze in the colony, obtain colony's genotype data;
(3), utilize MAPMARKER/EXP3.0 software colony's genotype data of each molecule marker to be made up the genetic linkage maps of paddy rice according to chain exchange rule.
(4) (Composite interval mapping CIM), is that step-length scans in full genome range with 2cM to utilize Windows QTL Cartographer V2.0 software (Wang et al., 2003) composite interval mapping method.5% overall conspicuous level is adopted in the detection of QTL, and the remarkable threshold of corresponding LOD statistic estimates that with permutation test (Permutation test) (Churchill and Doerge, 1994) method duplicate sampling is 1000 times altogether.The additive effect and the contribution rate in each site have been estimated simultaneously.Genome sequence according to the warm and fine rice variety 9311 of japonica rice variety Japan that provides in the instrument public database, utilize the PowerBlast analysis software, seek the insertion/deletion segment that exists in Xian, the round-grained rice kind genome sequence, utilize Primer Premier 5.0 Software development InDel marks.
(3) result and analysis:
(1) resistance is identified
Seedling stage, group's resistance identify to show Rathu Heenati, 02428 and F
1Pest-resistant rank be respectively 0.3,8.1 and 1.1, show Rathu Heenati brown planthopper resistant and the insect-resistance of 02428 sense brown paddy plant hopper and Rathu Heenati is controlled 156 F by dominant gene
2:3Family is continuous distribution to the pest-resistant rank frequency distribution of brown paddy plant hopper, and minimum is 0.1, is 9.00 to the maximum, and 3 tangible peak values occur 1,5 and 8 three position.According to the pest-resistant rank of brown paddy plant hopper with F
2:3Family is divided into, and pest-resistant, anti-sense separates and three kinds of phenotypes of sense worm, and corresponding F
2The genotype of individual plant then is respectively and is designated as three kinds of RR (it is pest-resistant to isozygoty), Rr (heterozygosis is pest-resistant) and rr (the sense worm of isozygotying).F
2Colony separates the ratio (χ that meets 1:2:1 to the anti-sense of brown paddy plant hopper
2=1.69, χ
2 0.05,2=5.99) (table 2).
Table 2 Rathu Heenati/02428 F
2156 individual plants of segregating population are to the anti-sense of brown paddy plant hopper segregation ratio
A)RR, it is pest-resistant to isozygoty; Rr, heterozygosis is pest-resistant; Rr, the sense worm of isozygotying
B)1RR:2Rr:1rr test of goodness of fit value χ
2Be 1.69 (χ
2 0.05,2=5.99);
C)This column is pest-resistant rank codomain; RS, Resistance Score (pest-resistant rank)
(2) linkage analysis
Linkage analysis is positioned at the brown planthopper resistant gene Bph3 among the Rathu Heenati between RM8213 and the RM5953, with two marks respectively at a distance of 3.6cM and 3.2cM (Fig. 1), this site is 83.9% to the contribution rate of brown paddy plant hopper resistance, is resistance key-gene site.Mark RM8213 band is 168bp, the RM8213 primer: left end sequence A GCCCAGTGATACAAAGATG and right-hand member sequence GCGAGGAGATACCAAGAAAG; Mark RM5953 band is 195bp, the RM5953 primer: left end sequence A AACTTTCTGTGATGGTATC and right-hand member sequence A TCCTTGTCTAGAATTGACA.
The result shows that resistance separates to separate with the genotype value of two SSR mark RM5953 and RM8213 and has significant correlation that further prove the close linkage of this brown planthopper resistant gene and these two marks, this key-gene is positioned on paddy rice the 4th the short arm of a chromosome.
(3) accurately locate
Being the site of further Fine Mapping key-gene Bph3, improving efficiency of selection, is female parent with brown planthopper resistant kind RathuHeenati, and sense brown paddy plant hopper japonica rice variety 02428 is a male parent, and hybridization has made up the F that comprises 5687 individual plants
2Segregating population, therefrom the individual plant of exchange takes place in selection markers RM8213 and RM5953, and the exchange individual plant of acquisition obtains corresponding F by selfing
2:3Family is carried out pest-resistant evaluation.
Genome sequence according to the warm and fine rice variety 9311 of japonica rice variety Japan that provides in the instrument public database, utilize the PowerBlast analysis software, seek the insertion/deletion segment that exists in Xian, the round-grained rice kind genome sequence, utilize Primer Premier 5.0 Software development InDel marks, made up the saturated linkage map between RM8213 and the RM5953, Bph3 has been positioned between two molecule marker A4 and the RM16533 (Fig. 1) in conjunction with the phenotype qualification result.From above-mentioned 5687 strain F
2In select 250 strains immediately, 3 Indel mark RH784, the RH786 in this interval and the efficiency of selection of RH007 are assessed, the result shows that the efficiency of selection of these three marks all reaches about 97% (table 3).Promptly use labeled primer A4,
Left end sequence A AGCAGCATAAACTGATTGA
Right-hand member sequence TCATCTTCTGAAAAAGCAAT
Perhaps use labeled primer RM16533,
Left end sequence TTTGCTTAGTCGGCAGATGTCC
Right-hand member sequence C ATAAGAACGTACCTCCACTGATTCC
Perhaps use labeled primer RH7841
Left end sequence TGCCAAGTATGTATGCCTAT
Right-hand member sequence TTTTAGAGACCGTGTCCTTG
Perhaps use labeled primer RH786
Left end sequence TTTGAAGTTCTTTCCATCTGA
Right-hand member sequence A AATGTGCTATCTGGGGTAAA
Perhaps use labeled primer RH007
Left end sequence C TTGCGTTCCGTAGGAGAAG
Right-hand member sequence TGAGTGTAACCCGAAGTGGC
Amplifying rice brown planthopper resistant kind or breeding material DNA, if can amplify the amplified fragments of 193bp with primer A4, or can amplify the amplified fragments of 285bp with primer RM16533, or can amplify the amplified fragments of 213bp with primer RH7841, or can amplify the amplified fragments of 176bp with primer RH786, or can amplify the amplified fragments of 182bp with primer RH007, all indicate the existence of this rice varieties brown planthopper resistant key-gene Bph3, this key-gene is positioned on paddy rice the 4th the short arm of a chromosome.
Table 3 F
2The pest-resistant index of colony is classified by RH784, RH786 and RH007 marker genotypes
A)The genotype of 1/1 expression 02428, the genotype of 2/2 expression Rathu Heenati, 1/2 expression hybrid gene type
Predict the rice plant resistance by above-mentioned molecular markers for identification key-gene site, expectation can improve the breeding process of China's paddy rice brown planthopper resistant kind rapidly.
Sequence table
<110〉Agricultural University Of Nanjing
<120〉molecule marking method of rice varieties brown planthopper resistant key-gene Bph3
<130〉specification sheets
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<170>PatentIn?version?3.1
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<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer A4 left end
<222>(1)..(20)
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<210>2
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer A4 right-hand member
<222>(1)..(20)
<223>
<400>2
<210>3
<211>22
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RM16533 left end
<222>(1)..(22)
<223>
<400>3
<210>4
<211>26
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RM16533 right-hand member
<222>(1)..(26)
<223>
<400>4
<210>5
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH7841 left end
<222>(1)..(20)
<223>
<400>5
<210>6
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH7841 right-hand member
<222>(1)..(20)
<223>
<400>6
<210>7
<211>21
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH786 left end
<222>(1)..(21)
<223>
<400>7
<210>8
<211>21
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH786 right-hand member
<222>(1)..(21)
<223>
<400>8
<210>9
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH007 left end
<222>(1)..(20)
<223>
<400>9
<210>10
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉labeled primer RH007 right-hand member
<222>(1)..(20)
<223>
<400>10
<210>11
<211>20
<212>DNA
<213〉synthetic
<220>
<221〉RM8213 primer left end
<222>(1)..(20)
<223>
<400>11
<210>12
<211>20
<212>DNA
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<221〉RM8213 primer right-hand member
<222>(1)..(20)
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<220>
<221〉RM5953 primer left end
<222>(1)..(20)
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<221〉RM5953 primer right-hand member
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Claims (1)
1, the molecule marking method of rice varieties brown planthopper resistant key-gene Bph3, rice varieties Rathu Heenati is controlled by a key-gene Bph3 the resistance of brown paddy plant hopper, it is characterized in that:
Use labeled primer A4
Left end sequence A AGCAGCATAAACTGATTGA
Right-hand member sequence TCATCTTCTGAAAAAGCAAT
Perhaps use labeled primer RM16533
Left end sequence TTTGCTTAGTCGGCAGATGTCC
Right-hand member sequence C ATAAGAACGTACCTCCACTGATTCC
Perhaps use labeled primer RH7841
Left end sequence TGCCAAGTATGTATGCCTAT
Right-hand member sequence TTTTAGAGACCGTGTCCTTG
Perhaps use labeled primer RH786
Left end sequence TTTGAAGTTCTTTCCATCTGA
Right-hand member sequence A AATGTGCTATCTGGGGTAAA
Perhaps use labeled primer RH007
Left end sequence C TTGCGTTCCGTAGGAGAAG
Right-hand member sequence TGAGTGTAACCCGAAGTGGC
Amplifying rice brown planthopper resistant kind or breeding material DNA, if can amplify the amplified fragments of 193bp with primer A4, or can amplify the amplified fragments of 285bp with primer RM16533, or can amplify the amplified fragments of 213bp with primer RH7841, or can amplify the amplified fragments of 176bp with primer RH786, or can amplify the amplified fragments of 182bp with primer RH007, all indicate the existence of rice varieties brown planthopper resistant key-gene Bph3, this key-gene is positioned on paddy rice the 4th the short arm of a chromosome.
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CN104263737A (en) * | 2014-08-30 | 2015-01-07 | 四川省农业科学院作物研究所 | Rice anti-Nilaparvata-lugens gene Bph28 and application thereof |
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