CN104388576A - Molecular marking method of indica rice variety subjected to anti-brown planthopper host gene Bph27 transfer - Google Patents
Molecular marking method of indica rice variety subjected to anti-brown planthopper host gene Bph27 transfer Download PDFInfo
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Abstract
The invention discloses a molecular marking method of an indica rice variety subjected to anti-brown planthopper host gene Bph27 transfer. The developed and designed gene Bph27 is closely interlocked with molecular marking primers B471 and B58, and differential fragments can be amplified from an anti-brown planthopper donor Balamawee and a susceptible indica rice variety; the anti-brown planthopper host gene Bph27 is genetically transferred through molecular marking of a gene Bph27 locus, the required fragments can be accurately and quickly guided, the selection efficiency of the gene Bph27 is greatly improved, and a new anti-brown planthopper indica rice variety 991RB is developed.
Description
Technical field
The invention belongs to molecular genetics field, relate to the molecule marking method of brown planthopper resistant key-gene Bph27 transformation rice variety.
Background technology
Brown paddy plant hopper (Nilaparvata lugens
) also known as brown back rice plant hopper, being a kind of world-renowned migratory pest, is one of primary pest of paddy rice, has a strong impact on output and the quality thereof of paddy rice.In recent years, annual hazard area reaches more than 200,000,000 mu, large generation year after year especially since 2005.2005, China's planthopper hazard area reached 3.86 hundred million mu, and within 2006,2007, occurring area reaches 500,000,000 mu times.So far, main insect protected measure uses chemical insecticide, and long-term a large amount of chemical insecticide that uses not only makes environment increasingly worsen, serious harm human health, also impel insect to produce resistance to chemical insecticide, thus injure species diversity, destroy the eubiosis.Therefore, changing chemical pesticide is main insect protected strategy, seeks new pest-resistant approach imperative.The application of pest-resistant cultivar is considered to the most cost-effective prophylactico-therapeutic measures usually.
After nineteen sixties, the brown paddy plant hopper that Ge Dao district, Asia breaks out in succession is caused harm and impels geneticist and breeding man set about screening and utilize brown planthopper resistant germ plasm resource, carries out excavation and the utilization of brown planthopper resistant new gene, seed selection BPH-resistant rice varieties.At present, 28 brown planthopper resistant key-genes have been identified.Current excavation some brown planthopper resistant genes of location, great majority all derive from wild-rice or the relatively poor local variety of some economical characters, and directly utilization is more difficult, limits the breeding utilization of brown planthopper resistant gene to a certain extent.So, first by the brown planthopper resistant gene transformation in wild-rice or local variety in excellent Cultivar, the brown planthopper resistant rice germplasm of some excellences should be created, for brown planthopper resistant breeding provides intermediate materials, the efficiency of brown planthopper resistant breeding could be improved.
Although also in succession cultivated some BPH-resistant rice varieties in the past few decades, as IRRI is proposed the pest-resistant cultivar IR26 with Bph1 gene, the IR36 containing bph2, has had the IR56 etc. of Bph3.But because brown paddy plant hopper has extremely strong adaptive faculty to pest-resistant cultivar, after pest-resistant cultivar is promoted, brown paddy plant hopper forms the population adapting to pest-resistant cultivar soon, causes varietal resistance to be lost.Brown paddy plant hopper population is that brown paddy plant hopper has extremely strong adaptive key point to pest-resistant cultivar in the individual difference of height, find during as the Population of Rice Brown Planthopper being distributed in Philippines's 5 different geographic regions is raised respectively in the kind with different anti insect gene, in each colony, all have some individualities to survive on pest-resistant cultivar and to lay eggs.This means when a pest-resistant cultivar be newly bred as is at field planting, most of brown paddy plant hopper is individual dead because not adapting to, but have minority individuality can normal existence and breeding of future generation, due to offspring and parent, to take food ability similar, the average viability of colony and reproductivity increase along with increase from generation to generation, final formed can on pest-resistant cultivar the Population of Rice Brown Planthopper of Fast-propagation.1973, after there is the pest-resistant cultivar IR26 release of Bph1 gene, in the countries and regions of all this kinds of plantation, Population of Rice Brown Planthopper quantity obviously declines, but less than time of 2 years, this varietal resistance is degenerated, and Population of Rice Brown Planthopper significantly rises again, and the kind at IR26 etc. with Bph1 resistant gene is broken out; 1976, IRRI was proposed again the kind of the tool bph2 resistant genes such as IR36, and another makes Population of Rice Brown Planthopper quantity decline, but in the kind of tool bph2 resistant gene, has broken out again brown paddy plant hopper harm after the several years.Thus in the urgent need to cultivating new pest-resistant cultivar.The complicacy of the variability of Biotypes of The Brown Planthopper Nilaparvata Lugens Stal and insect-resistance qualification causes conventional breeding means efficiency lower, and breeding for pest resistance is made slow progress.For this reason, utilize and be polymerized anti insect gene with the closely linked molecule marker of target gene and in conjunction with conventional breeding means, seed selection durable resistance kind will become the trend of brown planthopper resistant breeding.
Summary of the invention
The object of the invention is: the molecule marking method providing brown planthopper resistant key-gene Bph27 transformation rice variety, belongs to molecular genetics field.Bph27 compact linkage molecule labeled primer B471 and B58 of development and Design of the present invention, can amplify differential fragment at brown planthopper resistant donor parents Balamawee and sense worm rice variety.Achieved the hereditary transformation of brown planthopper resistant gene Bph27 by the molecule marker of Bph27 gene locus, can accurately and rapidly import required fragment, greatly improve the efficiency of selection of Bph27, promote Bph27 utilization.
Object of the present invention realizes by following technical scheme:
The molecule marking method of brown planthopper resistant key-gene Bph27 transformation rice variety, with molecule marker primer B471 or molecule marker primer B58, molecule marker is carried out to brown planthopper resistant site Bph27: the DNA using the brown planthopper resistant material of molecule marker primer B471 amplification BPH-resistant rice varieties Balamawee and transformation thereof, obtain Bph27 site-tag clip size and be about 130bp; Amplification sense worm rice variety DNA, obtains labeled fragment size and is about 120bp; Increase with molecule marker primer B58 the DNA of brown planthopper resistant material of BPH-resistant rice varieties Balamawee and transformation thereof, obtains Bph27 site-tag clip size and be about 88bp; Amplification sense worm rice variety DNA, obtains labeled fragment size and is about 118bp; Wherein molecule marker primer B471 left end primer sequence is as shown in SEQ ID NO.1, and right-hand member primer sequence is as shown in SEQ ID NO.2; Molecule marker primer B58 left end primer sequence is as shown in SEQ ID NO.3, and right-hand member primer sequence is as shown in SEQ ID NO.4.
For marking the molecule marker primer of brown planthopper resistant key-gene Bph27, be selected from molecule marker primer B471 or molecule marker primer B58 any one, molecule marker primer B471 left end primer sequence is as shown in SEQ ID NO.1, and right-hand member primer sequence is as shown in SEQ ID NO.2; Molecule marker primer B58 left end primer sequence is as shown in SEQ ID NO.3, and right-hand member primer sequence is as shown in SEQ ID NO.4.
The application of molecule marker primer of the present invention in the new lines of cultivating brown planthopper resistant.
Described application preferably includes following steps:
Hybridize with brown planthopper resistant local variety Balamawee and rice variety 9311, obtain hybridizing F
1, with hybrid F
1do maternal, make recurrent parent with 9311, obtain backcross population BC
1f
1, from BC
1f
1start just to detect with mark B471 and B58 in primer pair Bph27 site according to claim 2 in seedling stage, BC
1f
1strain containing Bph27 site, just makes female parent in flowering period and hybridizes with background parent 9311, use the same method from generation to generation, until BC at each
5f
1from generation to generation, selfing obtains BC
5f
2colony, by molecule marking method, obtain the individual plant that isozygotys of B471 and B58 site, obtain a stable long-grained nonglutinous rice new lines by this individual plant selfing, Brown Planthopper Resistance further by seedling stage and the time of infertility is identified, obtains and carries brown planthopper resistant gene Bph27 and the new lines stablizing brown planthopper resistant.
Described application also preferably includes: carry brown planthopper resistant gene Bph27 by obtaining and stablize the application method of conventional variety preparing hybrid kind and the seed selection conventional variety that the new lines of brown planthopper resistant and long-grained nonglutinous rice, japonica rice and breeding are utilizing.
Beneficial effect
(1) obtained by the present invention and derive from the closely linked molecule marker of rice variety Balamawee brown planthopper resistant site Bph27.And pass through exploitation and the screening of a large amount of mark, obtain molecule marker B471 and B58 between the rice varieties such as Balamawee and 9311 with polymorphism.
(2) can be carried out the molecule marker transformation of rice variety to Bph27 brown planthopper resistant site by the present invention, the fragment locality specific of selection, qualification is convenient, accurately and reliably.The method is fast easy to detect, not affected by environment.
(3) Bph27 compact linkage molecule labeled primer B471 and B58 of development and Design of the present invention, can amplify differential fragment at brown planthopper resistant donor parents Balamawee and sense worm rice variety.Achieved the hereditary transformation of brown planthopper resistant gene Bph27 by the molecule marker of Bph27 gene locus, can accurately and rapidly import required fragment, greatly improve the efficiency of selection of Bph27, promote Bph27 utilization.
(4) assistant breeding select target is clear and definite, cost-saving.In brown planthopper resistant traditional breeding way, first to plant larger secondary segregating population, need to drop into a large amount of manpower, material resources and financial resources.Secondly, the cycle of breeding is long, and the seed selection of a conventional variety needs 8-10.Again, the workload of Brown Planthopper Resistance qualification is large, poor reliability.And by the Marker-assisted selection in early stage fertility site, can in less segregating population, the detection marked by compact linkage molecule, seedling stage just can identify carry brown planthopper resistant gene individual plant, eliminate other plant, not only save production cost but also greatly improve efficiency of selection.By this method, brown planthopper resistant long-grained nonglutinous rice strain 991RB only used for 4 years just selected, and more conventional breeding method shifted to an earlier date for 4 years.
Accompanying drawing explanation
Fig. 1 utilizes Bph27 site compact linkage molecule mark B471 to carry out Single-plant selection.M is Marker; 1: pest-resistant parent Balamawee; 2, sense worm receptor parent 9311; 3-12, sense brown paddy plant hopper individual plant; 13-24: brown planthopper resistant individual plant.
Fig. 2 utilizes Bph27 site compact linkage molecule mark B58 to carry out Single-plant selection.M is Marker; 1: pest-resistant parent Balamawee; 2, sense worm receptor parent 9311; 3-12, sense brown paddy plant hopper individual plant; 13-24: brown planthopper resistant individual plant.
The Brown Planthopper Resistance qualification in seedling stage of Fig. 3 brown planthopper resistant long-grained nonglutinous rice strain 991RB.
The Brown Planthopper Resistance qualification in the time of infertility of Fig. 4 brown planthopper resistant long-grained nonglutinous rice strain 991RB.
Embodiment
Materials and methods:
(1) material and informative population
Brown planthopper resistant kind Balamawee and 02428 hybridizes the F obtained
1, selfing obtains the F that comprises 135 individual plants
2primary separation colony, and each F
2the corresponding F that individual plant is obtained by selfing
2:3family.Further expansion genetic group, utilizes a F including the Balamawee/02428 of 18308 individual plants
2segregating population carries out the Fine Mapping of Bph27.
(2) phenotypic evaluation
The rice terrace in the suburbs, Nanjing is picked up from worm source at first, and in platform, local No. 1 (TN1) has raised 10 generations more than.In this research, brown paddy plant hopper breeds on TN1, to provide qualification required worm source.Raise in the greenhouse of Agricultural University Of Nanjing.Note anti-natural enemy and ant.
Adopt seedling stage group's assay method carry out Balamawee, 02428, F
1individual plant and 135 F
2:3the insect-resistance of family measures.Consistent for guaranteeing the growth of each family, all examination materials that supplies distinguish presoaking and germinating prior to seeding.Each family is seeded in a diameter 8cm, high 8cm respectively, fills with in the nutrition pot of experiment soil (have an aperture bottom nutrition pot, be convenient to osmotic absorbent).Every 28 nutrition pots (comprising parent, pest-resistant contrast and each alms bowl of sense worm contrast) are placed in the plastic box of a 65cm × 44cm × 14cm and (keep water layer about 2cm in case).Each nutrition pot sows 35 grain germination seeds.Thinning in about 7 days, eliminates sick and weak seedling, retains the rice seedlings that 30 strains are uniform.Each family plants two repetitions.Until seedling grow to two leaves wholeheartedly time, in 8-10 head/seedling ratio access 2 ~ 3 age brown paddy plant hopper nymph.When feel worm parent TN1 whole withered time, with reference to the method for IRRI (1988) and Athwal et al. (1971), the grading of 0-9 level is carried out to each individual plant.By each family of weighted average calculation (kind) pest-resistant index.
(2) molecular marker analysis and linkage analysis
The method prepared with reference to (1983) such as Dellaporta of DNA sample.The DNA extracted is dissolved in (10mM Tris base, 0.1mM EDTA) in TE damping fluid, carries out DNA quality and Concentration Testing with MBA 2000UV/VIS spectrophotometer (Perkin Elemer Co.).Every part of DNA is all diluted to the concentration of 20ng/ μ l with dd H2O, as the template of pcr analysis.
Ssr analysis is with reference to the program of Chen et al (1997).10 μ l reaction systems comprise: 10mM Tris-HCl pH 8.3,50mMKCl, 1.5mM MgCl2,50 μMs of dNTPs, 0.2 μM of primer, 0.5U Taq polymerase (TaKaRa, Dalian) and 20ngDNA template.Amplified reaction carries out in PTC-200 (MJ Research Inc.) PCR instrument: 94 DEG C of 4min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1.5min, 35 circulations; 72 DEG C of 7min.The amplified production Native PAGE glue of 8% is separated, by the colour developing of silver dye.
Utilize the existing SSR marker be uniformly distributed on paddy rice 12 karyomit(e)s in this laboratory to carry out polymorphic detection to parent Balamawee and 02428, be then used in there is between parent polymorphic differences mark to F
2colony carries out Preliminary Linkage Analysis, and the linked marker that primary dcreening operation obtains utilizes F further
2target group carries out the analysis of molecular marker gene type, and utilizes MAPMAKER/3.0 analysis software to carry out the compartment analysis (Lander et al.1987) of Brown Planthopper Resistance and SSR marker to the data obtained.And Kosambi function is converted to genetic distance (cM), draw linkage map.According to the information of restructuring individual plant by the assignment of genes gene mapping between the fixing molecule marker in position, realize the chromosomal Primary Location of gene.
(3) molecular marker assisted selection of Bph27 utilizes
Hybridize with brown planthopper resistant local variety Balamawee and rice variety 9311, obtain hybridizing F1, do with Hybrids F1 maternal, make recurrent parent with 9311, obtain backcross population BC
1f
1, from BC
1f
1start just to detect with mark B471 and B58 in Bph27 site in seedling stage, BC
1f
1strain containing Bph27 site, just makes female parent in flowering period and hybridizes with background parent 9311, use the same method from generation to generation at each, until BC5F1 is from generation to generation, and selfing acquisition BC
5f
2colony, by molecule marking method, obtains the individual plant that isozygotys of B471 and B58 site, is derived obtain a stable long-grained nonglutinous rice new lines by this individual plant, further by the Brown Planthopper Resistance qualification in seedling stage and the time of infertility.
Results and analysis:
The hazard level of group's mensuration display in seedling stage Balamawee and 02428 is respectively 0 and 8.3, and this shows Balamawee pole brown planthopper resistant and 02428 feels brown paddy plant hopper.F
1the hazard level of plant is 0.7, to brown paddy plant hopper performance high resistance.This shows that the insect-resistance of Balamawee is controlled by dominant gene.
135 F
2:3family carries out brown planthopper resistant phenotypic evaluation, and hazard level frequency distribution is continuous distribution, and minimum is 0, is 9 to the maximum, and hazard level <3 is high resistance, and >7 is high sense, be more than or equal to 3 be less than or equal to 7 be in anti-.135 F
2:3family phenotype can be divided into pest-resistant, anti-sense to be separated and sense worm three types to the hazard level of brown paddy plant hopper according to it, and corresponding F
2the genotype of individual plant is then by corresponding F
2:3the phenotypic number supposition of family obtains, and is respectively and is designated as RR (isozygotying pest-resistant), Rr (heterozygosis is pest-resistant) and rr (sense worm of isozygotying) three kinds.135 F
2:3the anti-sense of family is separated into 32:67:36.Chi-square test shows, F
2the anti-sense of colony to brown paddy plant hopper is separated the ratio (χ meeting 1:2:1
2 0.05=0.249 < 5.99).This shows that the phenotype of the brown planthopper resistant of Balamawee is by a dominant Dominant gene.
Linkage analysis find be positioned at tetrasome long-armed on SSR marker B5742 and Brown Planthopper Resistance phenotype significant correlation.Select further to be positioned at around B5742 and there are other polymorphic SSR marker between parent and carry out linkage analysis, build linkage map.Brown planthopper resistant gene in Balamawee is positioned between B471 and B5742, respectively with it at a distance of 18.2cM and 4.7cM.
Between first positioning area, the InDel mark of some PCR-based has 13 according to the sequence self-developing of Nipponbare and 93-11 further.First utilize 1172 F
2individual plant has screened 46 and has exchanged individual plant between B471 and B5742SSR mark, is locked between j20690 and B13 by this gene further in conjunction with phenotypic data and recombination data.These two are utilized to mark 17136 F
2colony carries out pcr analysis, obtains 117 strain restructuring individual.Utilize this 117 strain to recombinate individual, be locked between B52 and B20 in conjunction with phenotypic data and this gene of recombination data, both sides have 4 and 7 strains to exchange respectively.According to the sequence of Nipponbare, this interval physical distance is about 63Kb.
In order to utilize molecular marker assisted selection, Bph27 is proceeded to rice variety 9311, first-selection is screened the polymorphism that the closely linked molecule marker of Bph27 carries out between Balamawee and 9311 independently developed 20, accurate linked marker B471 and the B58 being wherein positioned at Bph27 both sides has obvious polymorphism (Fig. 1, Fig. 2) between Balamawee and 9311.Determine subsequently to utilize B471 and B58 to carry out the molecular marker assisted selection application of Bph27 in long-grained nonglutinous rice.Utilize brown planthopper resistant local variety Balamawee and rice variety 9311 to hybridize simultaneously, obtain hybridizing F
1, with hybrid F
1do maternal, make recurrent parent with 9311, obtain backcross population BC
1f
1, from BC
1f
1start just to detect with mark B471 and B58 in Bph27 site in seedling stage, BC
1f
1strain containing Bph27 site, just makes female parent in flowering period and hybridizes with background parent 9311, use the same method from generation to generation, until BC at each
5f
1from generation to generation, selfing obtains BC
5f
2colony, pass through molecule marking method, obtain the individual plant isozygotied in B471 and B58 site, derived by this individual plant and obtain a stable long-grained nonglutinous rice new lines, Brown Planthopper Resistance further by seedling stage and the time of infertility is identified, show this strain no matter seedling stage or all there is the resistance (Fig. 3, Fig. 4) of higher brown paddy plant hopper, by tentative for this strain 991RB by name the time of infertility.
Claims (5)
1. the molecule marking method of brown planthopper resistant key-gene Bph27 transformation rice variety, it is characterized in that, with molecule marker primer B471 or molecule marker primer B58, molecule marker is carried out to brown planthopper resistant site Bph27: the DNA using the brown planthopper resistant material of molecule marker primer B471 amplification BPH-resistant rice varieties Balamawee and transformation thereof, obtain Bph27 site-tag clip size and be about 130bp; Amplification sense worm rice variety DNA, obtains labeled fragment size and is about 120bp; Increase with molecule marker primer B58 the DNA of brown planthopper resistant material of BPH-resistant rice varieties Balamawee and transformation thereof, obtains Bph27 site-tag clip size and be about 88bp; Amplification sense worm rice variety DNA, obtains labeled fragment size and is about 118bp; Wherein molecule marker primer B471 left end primer sequence is as shown in SEQ ID NO.1, and right-hand member primer sequence is as shown in SEQ ID NO.2; Molecule marker primer B58 left end primer sequence is as shown in SEQ ID NO.3, and right-hand member primer sequence is as shown in SEQ ID NO.4.
2. for marking the molecule marker primer of brown planthopper resistant key-gene Bph27, it is characterized in that being selected from molecule marker primer B471 or molecule marker primer B58 any one, molecule marker primer B471 left end primer sequence is as shown in SEQ ID NO.1, and right-hand member primer sequence is as shown in SEQ ID NO.2; Molecule marker primer B58 left end primer sequence is as shown in SEQ ID NO.3, and right-hand member primer sequence is as shown in SEQ ID NO.4.
3. the application of molecule marker primer according to claim 2 in the new lines of cultivating brown planthopper resistant.
4. application according to claim 3, is characterized in that comprising:
Hybridize with brown planthopper resistant local variety Balamawee and rice variety 9311, obtain hybridizing F
1, with hybrid F
1do maternal, make recurrent parent with 9311, obtain backcross population BC
1f
1, from BC
1f
1start just to detect with mark B471 and B58 in primer pair Bph27 site according to claim 2 in seedling stage, BC
1f
1strain containing Bph27 site, just makes female parent in flowering period and hybridizes with background parent 9311, use the same method from generation to generation, until BC at each
5f
1from generation to generation, selfing obtains BC
5f
2colony, by molecule marking method, obtain the individual plant that isozygotys of B471 and B58 site, obtain a stable long-grained nonglutinous rice new lines by this individual plant selfing, Brown Planthopper Resistance further by seedling stage and the time of infertility is identified, obtains and carries brown planthopper resistant gene Bph27 and the new lines stablizing brown planthopper resistant.
5. application according to claim 4, characterized by further comprising: carry brown planthopper resistant gene Bph27 by obtaining and stablize the application method of conventional variety preparing hybrid kind and the seed selection conventional variety that the new lines of brown planthopper resistant and long-grained nonglutinous rice, japonica rice and breeding are utilizing.
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