CN102162011B - Molecule marking method of rice blast-resisting gene - Google Patents
Molecule marking method of rice blast-resisting gene Download PDFInfo
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- CN102162011B CN102162011B CN 201110118819 CN201110118819A CN102162011B CN 102162011 B CN102162011 B CN 102162011B CN 201110118819 CN201110118819 CN 201110118819 CN 201110118819 A CN201110118819 A CN 201110118819A CN 102162011 B CN102162011 B CN 102162011B
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Abstract
The invention discloses a molecule marking method of a rice blast-resisting gene, belonging to the field of crop molecule heredity and breeding. The method comprises the following steps: (1) taking a rice sample and extracting a genome DNA (Deoxyribose Nucleic Acid) of the rice sample; and (2) carrying out PCR (Polymerase Chain Reaction) amplification on the genome DNA of the rice sample by utilizing any one pair of molecule-marked primers in RM6091 and RM26632, carrying out electrophoresis detection on a PCR amplification product, and if a molecule-marked DNA segment with corresponding size is amplified, showing that a Pi-bdl(t) gene exists. Through rice blast-resisting gene Pi-bdl(t) molecule marking in the invention, whether thin rice as well as crossbred descendants, backcross descendants and multiple cross descendants thereof contain the gene can be detected, the resistance level of the gene on rice blast can be forecasted, the selecting efficiency of rice blast resistant materials can be greatly increased, and the breeding process for disease resistance can be accelerated.
Description
Technical field
The invention belongs to the farm crop molecular genetic breeding and learn the field, relate to the molecule marking method of rice blast resistant gene.
Background technology
Paddy rice is one of most important food crop of China, and is significant to ensureing China's grain security and growth of the national economic.Rice blast is the most serious disease on China's Rice Production, have propagate fast, occur wide, harm heavily wait characteristics (Ling Zhongzhuan etc., 1989, physiological races of rice blast fungus research in northern China rice district is rushed state's agricultural sciences, 22 (3): 7-13).Further excavate and utilize China's blast resistant gene resource, cultivating and plant disease-resistant variety is that control rice blast occurs and the most economical effective approach of minimizing Rice Yield Loss Caused.
So far, the various countries scientist has identified more than 70 blast resistant gene from paddy rice, wherein has 17 disease-resistant genes successfully to be cloned.Modern varieties be introduced or be aggregated to these disease-resistant genes can, selects the kind of high resistance, wide spectrum.But traditional breeding way is time-consuming, effort, phenotypic evaluation difficulty, breeding efficiency are low, because that disease-resistant gene mostly is is dominant, often have epistatic interaction between gene, the disease-resistant gene polymerization is more difficult.Can effectively address this problem by molecular mark.
China Taihu Lake basin rice does with a long history, be considered to one of japonica rice area of origin, contain abundant Rice Resources is arranged, Li Peifu etc. (1999, the genetic research of two Taihu Lake basin japonica rice local variety blast resistings. the rice in China science, 13 (1): 11-14) the thin rice of report China's Taihu Lake basin japonica rice local variety is to the resistance performance wide spectrum of Pyricularia oryzae and the characteristics of high resistance.Identify from the distinctive germ plasm resource of China and clone's blast resistant gene, the gene that can obtain to have independent intellectual property right is of great importance to the blast resisting breeding that improves China rice anti-rice blast breeding level, especially japonica rice.
Summary of the invention
The objective of the invention is: the molecule marking method that the thin rice blast resistant gene of rice varieties Pi-bd1 (t) is provided.By detecting and the closely linked molecule marker of disease-resistant gene Pi-bd1 (t), can define without disease-resistant gene Pi-bd1 (t), and the prediction rice plant rice blast resistance, accelerate the seed selection progress of anti-rice blast rice new variety.
Purpose of the present invention can be achieved through the following technical solutions:
The molecule marking method of Rice Thin rice blast resistant gene Pi-bd1 (t) comprises following operation steps:
(1) water intaking rice sample extracts paddy rice sample gene group DNA;
(2) utilize any a pair of in the table 1 or two pairs and the closely linked molecule marker primer of Rice Thin rice blast resistant gene Pi-bd1 (t), described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection at 8% non-denaturing polyacrylamide gel, if amplify the molecular marker DNA fragment of corresponding size, indicate the existence of Pi-bd1 (t) gene:
Table 1
Wherein, the reaction system of described pcr amplification is: 10 * damping fluid (contains Mg
2+) 1.0 μ l, the molecule marker primer pair 1 μ l described in the table 1 of 4pmol/ μ l, 2.5mM dNTPs 0.2 μ l, the Taq enzyme 0.1 μ l of 5U/ μ l, the paddy rice sample gene group template DNA 1 μ l of 10ng/ μ l adds water to 10 μ l; Response procedures is: DNA94 ℃ of denaturation is after 5 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended exhibition 1 minute, circulate 35 times; Last 72 ℃ were extended 10 minutes.
Beneficial effect
The molecule marking method of Rice Thin rice blast resistant gene provided by the present invention has the following advantages:
(1) thin rice is Taihu Lake basin japonica rice local variety, feature with wide spectrum high resistant to rice blast, its main effect disease-resistant gene Pi-bd1 (t) is a new disease-resistant gene, screening obtains with it closely linked molecule marker RM6091 and RM26632, for molecular marker assisted selection breeding and clone Pi-bd1 (t) gene are laid a good foundation.Utilize the arbitrarily a pair of and closely linked molecule marker of Rice Thin rice blast resistant gene Pi-bd1 (t) of the present invention, carry out the discriminating of blast resistant gene Pi-bd1 (t), efficiency of selection all reaches more than 98.8%, utilizes the efficiency of selection of two pairs of molecule marker primers to reach 99.98%.
(2) accurate by the gene locus of molecule marker of the present invention location, it is convenient to identify.Because the recombination fraction low (≤1.2%) of these marks and disease-resistant gene Pi-bd1 (t), by detecting these and the closely linked molecule marker of disease-resistant gene Pi-bd1 (t), whether can determine the existence of blast resistant gene Pi-bd1 (t), the rice blast resistance of prediction rice plant, thereby quick disease-resistant breeding process.
(3) the assistant breeding select target is clear and definite, saves cost.In traditional disease resistant and breeding method, the rice blast resistance of breeding material is carried out phenotypic evaluation, generally at seedling stage or heading stage, inoculated environmental influence larger, the result reliability of phenotypic evaluation is low.Therefore breeding for disease resistance is not only time-consuming, and difficulty is large, and cost is high.By detecting blast resistant gene Pi-bd1 (t), can take a sample in seedling stage, extract DNA, utilize above-mentioned mark just can identify the individual plant of blast resisting, eliminate other plant, not only save production cost, control the breeding population scale, and greatly improve the efficiency of selection of blast resisting individuality.
Description of drawings
Fig. 1. the 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-bd1 (t) close linkage SSR mark RM6091.
Wherein: M: molecular weight Marker; B: thin rice; S: Suyunuo; F: heterozygous; 1-6: susceptible RIL; 7-13:
Disease-resistant RIL.
Fig. 2. the 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-bd1 (t) close linkage SSR mark RM26632.
Wherein: M: molecular weight Marker; B: thin rice; S: Suyunuo; F: heterozygous; 1-5: susceptible RIL; 6-13: disease-resistant RIL.
Embodiment
(1) materials and methods:
Li Peifu etc. utilize the thin rice of anti-rice blast rice local variety (♀) and susceptible variety Suyunuo (
) hybridization acquisition F
1, selfing obtains F
2Segregating population, be used for genetic analysis, clear and definite thin rice is controlled by a pair of main effect dominant gene the resistance of " north 1 " fungus strain, further thin rice and 12 Japanese rice blast differential variety hybridization are carried out allelomorphism mensuration, the result shows, thin rice is inequipotential to the entrained Known resistance gene of the resistant gene of northern 1 fungus strain and 12 differential varieties, is Pi-bd1 (t) (heredity with this unnamed gene, 2007,29 (10): 1249～1255).The present invention adopts single seed descent to make up F on this basis
2: 6Recombinant inbred lines, namely two parents are hybridized acquisition F
1, selfing obtains F
2, F
2Choose at random 166 individual plants, individual plant selfing produces strain, continuously 5 generations of selfing, finally is built into the F that comprises 166 strains
2: 6Recombinant inbred lines.Utilize this colony further Pi-bd1 (t) to be positioned between Sub_clause 11 karyomit(e) RM6091 and the RM26632, and with these two mark close linkages, genetic distance is 1.2CM.
2. spawn culture and inoculation identification method are with reference to (rice in China science, 2007,21:579～584) such as Li Peifu.
3.DNA extracting method: the DNA that extracts each individual plant of segregating population with the SDS method.
4. the compact linkage molecule mark is definite:
(1) mark polymorphism screening: take the DNA of the thin rice of parent and Suyunuo as template, with the paddy rice SSR mark (http://www.gramene.org) of announcing on the Gramene website, react through PCR and to carry out polymorphism analysis.
(2) the anti-sense pond of polymorphism mark screening: in recombinant inbred lines, 10 inoculated identifications of random selection are family's based material (disease-resistant RIL) and 10 family's based materials (susceptible RIL) that inoculated identification is susceptible phenotype of disease-resistant phenotype, mix respectively after extracting DNA, form disease-resistant pond and susceptible pond, polymorphism mark is screened the relation of analyzing between itself and the anti-sense, if the disease-resistant pond electrophoresis result of a certain mark is consistent with disease-resistant parent, susceptible pond electrophoresis result is consistent with Susceptible parent, then illustrate this mark may with the disease-resistant gene close linkage.
(3) checking of compact linkage molecule mark: may in 10 family's based materials that consist of anti-sense pond, verify respectively with the closely linked molecule marker of disease-resistant gene, when if linkage relationship exists really, in all family's based materials, verify again, according to chain polymorphism mark and corresponding anti-sense phenotype, evaluation of markers and intergenic recombination frequency calculate the antagonistic efficiency of selection of mark.
5.PCR reaction system: volume is 10 μ l, and wherein 10 * damping fluid (contains Mg
2+) 1.0 μ l, molecule marker primer pair (4pmol/ μ l) 1 μ l, 2.5mM dNTPs 0.2 μ l, Taq enzyme (5U/ μ l) 0.1 μ l, template DNA (10ng/ μ l) 1 μ l adds water to 10 μ l.Response procedures is: DNA94 ℃ of denaturation be after 5 minutes, (72 ℃ are extended exhibition 1 minute for 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds) circulation 35 times, and last 72 ℃ were extended 10 minutes.In the enterprising performing PCR amplification of biometre amplification instrument, amplified production carries out electrophoretic separation at 8% non-denaturing polyacrylamide gel (containing 7.6 gram acrylamides and 0.4 gram methylene diacrylamide in the 100ml polyacrylamide solution), then take a picture the record result at ultraviolet transilluminator.
(2) results and analysis:
Result of study is found, SSR mark RM6091, RM26632 and disease-resistant gene Pi-bd1 (t) close linkage (Fig. 1, Fig. 2), and the amplified band size in the parent is as shown in table 2.In 166 family's based materials of RIL (in 332 gametes), the efficiency of selection method of calculation are as follows
Occur 2 crossover-type gametes between SSR mark RM6091 and the disease-resistant gene Pi-bd1 (t), recombination fraction only is 1.2%, and the antagonistic efficiency of selection of this mark reaches 98.8%;
Occur 2 crossover-type gametes between SSR mark RM26632 and the disease-resistant gene Pi-bd1 (t), recombination fraction only is 1.2%, and the antagonistic efficiency of selection of this mark reaches 98.8%;
The efficiency of selection of SSR mark RM6091 and the screening of RM26632 double-tagging is 1-1.2%*1.2%=99.98%;
Can identify efficiently that by above-mentioned 2 molecule markers whether blast resistant gene Pi-bd1 (t) exists, single Marker selection efficient all reaches 98.8%, and the efficiency of selection of double-tagging combination reaches 99.98%.The breeding process that can be used for molecular marker assisted selection Effective Raise resistant rice cultivars, control breeding population scale.
Table 2.
Claims (2)
1. the molecule marking method of " thin rice " rice blast resistant gene Pi-bd1 (t) is characterized in that comprising the steps:
(1) gets " thin rice " paddy rice sample, extract paddy rice sample gene group DNA;
(2) primer of utilization and the closely linked molecule marker RM6091 of rice varieties " thin rice " blast resistant gene Pi-bd1 (t) and/or RM26632, described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection at 8% non-denaturing polyacrylamide gel, if amplify the molecule marker fragment of corresponding size, indicate the existence of Pi-bd1 (t) gene;
Wherein, described molecule marker RM6091 upstream primer sequence is SEQ ID NO.1, and the downstream primer sequence is SEQ ID NO.2, and the molecule marker clip size of its corresponding size is 195bp; Described molecule marker RM26632 upstream primer sequence is SEQ ID NO.3, and the downstream primer sequence is SEQ ID NO.4, and the molecule marker clip size of its corresponding size is 205bp.
2. the molecule marking method of " thin rice " rice blast resistant gene Pi-bd1 according to claim 1 (t) is characterized in that the reaction system of described pcr amplification is: 10 * contain Mg
2+Damping fluid 1.0 μ l, the described molecule marker primer pair 1 μ l of 4 pmol/ μ l, 2.5 mM dNTPs, 0.2 μ l, the Taq enzyme 0.1 μ l of 5 U/ μ l, the paddy rice sample gene group template DNA 1 μ l of 10 ng/ μ l adds water to 10 μ l; Response procedures is: behind 94 ℃ of denaturation 5 min of DNA; 94 ℃ of sex change 30 s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 35 times; Last 72 ℃ are extended 10min.
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| CN102703443B (en) * | 2012-05-23 | 2014-09-24 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103205506B (en) * | 2013-05-08 | 2014-07-23 | 辽宁省农业科学院 | Method for detecting rice blast resistant gene pi5 by using coseparation mark pi5-2-4 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
| CN103215369B (en) * | 2013-05-08 | 2014-07-23 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103215368A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2 |
| CN103805612A (en) * | 2014-02-08 | 2014-05-21 | 南京农业大学 | Rice gene OsRem1 and application thereof |
| CN105713983B (en) * | 2016-04-21 | 2019-03-05 | 南京农业大学 | A kind of molecular labeling and its application with rice Jiangnan evening neck blast resistance gene close linkage |
| CN105779622B (en) * | 2016-04-21 | 2018-12-14 | 南京农业大学 | A kind of molecular labeling and its application with rice Race specificity blast resistant gene close linkage |
| CN106636335B (en) * | 2016-10-09 | 2020-05-08 | 南京农业大学 | Molecular marking method of rice panicle blast resistance gene |
| CN106555001B (en) * | 2016-11-10 | 2019-07-26 | 福建省农业科学院生物技术研究所 | A kind of molecular labeling of rice blast resistant gene and its application |
| CN106957916A (en) * | 2017-04-25 | 2017-07-18 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene Xa27 molecular labeling primer and its application |
| CN107130019B (en) * | 2017-04-25 | 2020-07-17 | 南京农业大学 | Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof |
| CN106939346A (en) * | 2017-04-25 | 2017-07-11 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene xa5 molecular labeling and its application |
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| CN1648253A (en) * | 2005-01-07 | 2005-08-03 | 南京农业大学 | Molecule label linked with rice anti-rice blast gene |
| CN101643790B (en) * | 2009-09-07 | 2012-03-07 | 中国水稻研究所 | Specific molecular marker of rice blast resistant gene Pi 25 for rice and special primer thereof |
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