CN107130019A - The molecular labeling primer of the thin spike of rice pest resistant gene of Local Rice Race and its application - Google Patents
The molecular labeling primer of the thin spike of rice pest resistant gene of Local Rice Race and its application Download PDFInfo
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- CN107130019A CN107130019A CN201710278281.0A CN201710278281A CN107130019A CN 107130019 A CN107130019 A CN 107130019A CN 201710278281 A CN201710278281 A CN 201710278281A CN 107130019 A CN107130019 A CN 107130019A
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Abstract
The invention belongs to crop molecular genetic breeding field, molecular labeling primer and its application of the thin spike of rice pest resistant gene of Local Rice Race are disclosed.Rice Thin spike of rice pest resistant gene Pb bd1 of the present invention molecular labeling, described molecular labeling BS23 sense primer is BS23 F：SEQ ID NO.1, anti-sense primer is BS23 R：SEQ ID NO.2, amplified production size is 114bp；Described molecular labeling RM7654 sense primer is RM7654 F：SEQ ID NO.3, anti-sense primer is RM7654 R：SEQ ID NO.4, amplified production size is 155bp.The Pyricularia Oryzae resistant gene Pb bd1 molecular labeling provided by the present invention can detect Rice Thin rice and its hybridization, be returned and whether the offspring that reestablishes diplomatic relations contains the gene, its resistance level to rice blast can be predicted, the efficiency of selection of Conferring Resistance To Neck Blast In Rice material is greatly improved, accelerates breeding for disease resistance process.
Description
Technical field
The invention belongs to crops molecular genetic breeding field, it is related to the thin spike of rice pest resistant gene of Local Rice Race
Molecular labeling primer and its application.
Background technology
Paddy rice is one of most important cereal crops of China, to ensureing that China's grain security and growth of the national economic have weight
Want meaning.Rice blast is the disease of most serious in China's Rice Production, with (Ling Zhong the features such as propagating wide fast, generation, harm weight
Specially etc., 1989, northern China rice region physiological races of rice blast fungus research Scientia Agricultura Sinicas, 22 (3):7-13).Further send out
Dig and using China's blast resistant gene resource, cultivate and plantation disease-resistant variety is the generation of control rice blast and reduces rice yield
Lose most economical effective approach.
So far, scientists from all over the world identify more than 100 blast resistant gene from paddy rice, wherein there is 24 disease-resistant gene quilts
Successful clone.These disease-resistant genes can introduce or be aggregated to modern varieties, select the high anti-, kind of wide spectrum.But tradition is educated
Kind of method is time-consuming, laborious, phenotypic evaluation is difficult, breeding efficiency is low, often exists because disease-resistant gene is mostly dominant, between gene upper
Position property interaction, disease-resistant gene polymerization is increasingly difficult.This problem can effectively be solved by molecular mark.
China Taihu Lake basin rice makees with a long history, it is considered to be one of japonica rice area of origin, and containing has abundant Rice Resources,
Li Peifu etc. (genetic analysis of 2007,4 Taihu Lake basin japonica rice local varieties blast resistances, heredity, 10 phases in 2007) report
Road China thin rice of Taihu Lake basin japonica rice local varieties to the Resistant expression wide spectrums of seedling pest and it is high anti-the characteristics of.In our research
In, it also shows high resistance to fringe pest.Neck blast resistance gene is identified and cloned from the distinctive germ plasm resource of China, can
To obtain the gene with independent intellectual property right, to improving China's rice anti-rice blast breeding level, the especially anti-rice of japonica rice
Seasonal febrile diseases breeding is of great importance.
The content of the invention
The purpose of the present invention is：The thin spike of rice pest resistant gene Pb-bd1 of rice varieties molecule labelling method is provided.Pass through
Detection and the molecular labeling of disease-resistant gene Pb-bd1 close linkages, it may be determined that whether there is disease-resistant gene Pb-bd1, and predict paddy rice
The rice blast resistance of plant, accelerates the seed selection progress of anti-rice blast rice new varieties.
The purpose of the present invention can be achieved through the following technical solutions：
Rice Thin spike of rice pest resistant gene Pb-bd1 molecular labeling, described molecular labeling is in BS23, RM7654
Any one；Described molecular labeling BS23 sense primer is BS23-F：SEQ ID NO.1, anti-sense primer is BS23-R：
SEQ ID NO.2, amplified production size is 114bp；Described molecular labeling RM7654 sense primer is RM7654-F：SEQ
ID NO.3, anti-sense primer is RM7654-R：SEQ ID NO.4, amplified production size is 155bp.
Rice Thin spike of rice pest resistant gene Pb-bd1 of the present invention molecular labeling primer, described molecular labeling
BS23 sense primer is BS23-F：SEQ ID NO.1, anti-sense primer is BS23-R：SEQ ID NO.2, amplified production size
For 114bp；Described molecular labeling RM7654 sense primer is RM7654-F：SEQ ID NO.3, anti-sense primer is
RM7654-R：SEQ ID NO.4, amplified production size is 155bp.
Application of the molecular labeling of the present invention in Pyricularia Oryzae resistant gene Pb-bd1 is differentiated.
Application of the molecular labeling primer of the present invention in Pyricularia Oryzae resistant gene Pb-bd1 is differentiated.
Application of the molecular labeling primer of the present invention in anti-rice blast rice is differentiated.
A kind of method of quick discriminating Pyricularia Oryzae resistant gene Pb-bd1 presence or absence, is comprised the following steps：
(1) water intaking rice sample, extracts paddy rice sample gene group DNA；
(2) using any pair or two pairs in molecular labeling primer of the present invention, to described paddy rice sample base
Because a group DNA enters performing PCR amplification, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if expanded
Increase the molecular labeling fragment for correspondence size, indicate the presence of Pb-bd1 genes.
The reaction system of described PCR amplifications is：10 × contain Mg2+μ l, the 4pmol/ μ l of buffer solution 1.0 the molecule mark
Remember μ l, the 5U/ μ l of 1 μ l, 2.5mM dNTPs of primer pair 0.2 μ l, the 10ng/ μ l of Taq enzyme 0.1 paddy rice sample gene group template
The μ l of DNA 1, add water to 10 μ l；Response procedures are：After 94 DEG C of DNA pre-degenerations 5 minutes；94 DEG C are denatured 40 seconds, and 60 DEG C are moved back 40 seconds,
72 DEG C of extension 40 second minutes of exhibition, circulate 30 times；Last 72 DEG C extend 10 minutes.
Beneficial effect
The molecule labelling method of Rice Thin spike of rice pest resistant gene provided by the present invention, with advantages below：
(1) thin rice is Taihu Lake basin japonica rice local varieties, the feature with high anti-fringe pest, its main effect disease-resistant gene Pb-bd1
For a new neck blast resistance gene, screening obtains the molecular labeling BS23 and RM7654 of close linkage therewith, is molecular labeling
Assisted selection and clone's Pb-bd1 genes are laid a good foundation.Utilize any pair of the present invention and Rice Thin rice neck blast resistance base
Because of the molecular labeling of Pb-bd1 close linkages, carry out neck blast resistance gene Pb-bd1 discriminating, efficiency of selection all reach 91% with
On, using the efficiency of selection of two pairs of molecular labeling primers up to 99.32%.
(2) accurate by the gene loci of Molecular mapping of the present invention, identification is convenient.Because these marks and fringe pest resist
Property gene Pb-bd1 recombination fraction is low (≤8.96%), by detecting these molecule marks with disease-resistant gene Pb-bd1 close linkages
Note, it may be determined that blast resistant gene Pb-bd1 presence or absence, predicts the rice blast resistance of rice plant, so that quick disease-resistant
Breeding process.
(3) assistant breeding selection target is clearly, cost-effective.In traditional disease resistant and breeding method, to the fringe of breeding material
Pest resistance carries out phenotypic evaluation, and larger by inoculation ambient influnence, the result reliability of phenotypic evaluation is low.Therefore breeding for disease resistance is not only
It is time-consuming, and difficulty is big, and cost is high.It by detecting neck blast resistance gene Pb-bd1, can be sampled in seedling stage, extract DNA, utilized
Above-mentioned mark can identify the individual plant of blast resisting, eliminate other plant, not only save production cost, control breeding population rule
Mould, and greatly improve the efficiency of selection of blast resisting individual.
Brief description of the drawings
Fig. 1 Pyricularia Oryzae resistant gene Pb-bd1 close linkage SSR markers BS23 8% non-denaturing polyacrylamide coagulates
Gel electrophoresis figure.Wherein：M：Molecular weight Marker；B：Thin rice；S：Suyunuo；F：Heterozygous；1-5：Susceptible RIL；6-
10：Disease-resistant RIL.
Fig. 2 Pyricularia Oryzae resistant gene Pb-bd1 close linkage SSR markers RM7654 8% non-denaturing polyacrylamide
Gel electrophoresis figure.
Wherein：M：Molecular weight Marker；B：Thin rice；S：Suyunuo；F：Heterozygous；1-5：Susceptible RIL；6-10：
Disease-resistant RIL.
Embodiment
Embodiment 1
(1) materials and methods：
Obtained 1. Li Peifu etc. is hybridized using the thin rice of anti-rice blast rice local varieties (♀) with susceptible variety Suyunuo (♂)
F1, selfing acquisition F2Segregating population, for genetic analysis, specifies thin rice to " resistance of northern 1 " fungus strain is main by single major gene resistance
Control.The present invention on this basis, F is built using single seed descent2:8Recombinant inbred lines, i.e. two parents obtain F1,
Selfing obtains F2, F2Individual plant selfing produces strain, and in the continuous generation of selfing 6, being finally built into includes the F of 212 strains2:8Recombinate selfing
It is colony.A main effect site related to anti-fringe pest is navigated to using the colony, this site is located at Sub_clause 11 chromosomal marker
Between BS23 and RM7654, and close linkage is marked with the two, genetic distance is 2.7cM, and its offer is not less than 51.56%
Resistance.
2. Spawn incubation and inoculation identification method, with reference to (rice in China science, 2007,21 such as Li Peifu：579~584)
And (the Tianjin Agr Sci, 2007,13 such as Liu:55-58).
3.DNA extracting methods：The DNA of each individual plant of segregating population is extracted with CTAB methods.
4. the determination of compact linkage molecule mark:
(1) mark polymorphism screening：DNA using the thin rice of parent and Suyunuo is template, with what is announced on Gramene websites
Paddy rice SSR marker (http://www.gramene.org), carry out polymorphism analysis through PCR reactions.
(2) the anti-sense pond screening of polymorphism mark:In recombinant inbred lines, 5 inoculated identifications of random selection are anti-
Family's based material (disease-resistant RIL) of sick phenotype and 5 inoculated identifications for susceptible phenotype family's based material (susceptible restructuring from
Hand over and be), mixed respectively after extracting DNA, form disease-resistant pond and susceptible pond, carrying out screening to polymorphism mark analyzes itself and anti-sense
Between relation, if the disease-resistant pond electrophoresis result of a certain mark is consistent with disease-resistant parent, susceptible pond electrophoresis result and Susceptible parent
Unanimously, then illustrate that the mark may be with disease-resistant gene close linkage.
(3) checking of compact linkage molecule mark：Will likely be with the molecular labeling of disease-resistant gene close linkage respectively in structure
Verified, if linkage relationship is implicitly present in, then entered in all family's based materials into 5 family's based materials in anti-sense pond
Row checking, according to chain polymorphism mark and corresponding anti-sense phenotype, evaluation of markers and intergenic recombination frequency are calculated
Mark the efficiency of selection of antagonism.
5.PCR reaction systems：Volume is 10 μ l, wherein 10 × buffer solution (contains Mg2+) 1.0 μ l, molecular labeling primer pair
(4pmol/ μ l) 1 μ l, 2.5mM dNTPs 0.2 μ l, the μ l of Taq enzyme (5U/ μ l) 0.1, the μ l of template DNA (10ng/ μ l) 1, are added water to
10μl.Response procedures are:After 94 DEG C of DNA pre-degenerations 5 minutes, (94 DEG C are denatured 40 seconds, and 55 DEG C are annealed 40 seconds, 72 DEG C of extension exhibitions 40
Minute) circulate 30 times, last 72 DEG C extend 10 minutes.In the enterprising performing PCR amplification of biometre amplification instruments, amplified production is 8%
Non-denaturing polyacrylamide gel is (containing 7.6 grams of acrylamides and the double acryloyls of 0.4 gram of methene in 100ml polyacrylamide solutions
Amine) on be separated by electrophoresis, then on ultraviolet transilluminator take a picture, record result.
(2) result and analysis：
Result of study discovery, SSR marker BS23, RM7654 and disease-resistant gene Pb-bd1 close linkages (Fig. 1, Fig. 2), in parent
Amplified band size in this is as shown in table 2.In 212 family's based materials of RIL, efficiency of selection computational methods are as follows
Occur 19 crossover-type gametes between SSR marker BS23 and disease-resistant gene Pb-bd1, recombination fraction is only 8.96%, should
The efficiency of selection of antagonism is marked up to 91.04%；
Occur 16 crossover-type gametes between SSR marker RM7654 and disease-resistant gene Pb-bd1, recombination fraction is only 7.55%,
The efficiency of selection of the mark antagonism is up to 92.45%；
The efficiency of selection of SSR marker BS23 and RM7654 double labelling screening is 1-8.96%*7.55%=99.32%；
Blast resistant gene Pb-bd1 presence or absence, single mark selection can efficiently be identified by above-mentioned 2 molecular labelings
Efficiency reaches 91%, and the efficiency of selection of double labelling combination is up to 99.32%.Water is effectively improved available for molecular marker assisted selection
The breeding process of rice disease-resistant variety, controls breeding population scale.
Table 2.
<110>Agricultural University Of Nanjing
<120>The molecular labeling primer of the thin spike of rice pest resistant gene of Local Rice Race and its application
<160> 4
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Rice Thin rice blast resistant gene Pb-bd1 molecular labeling BS23 sense primers BS23-F
<400> 1
cgcgactaat ctagactct 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Rice Thin rice blast resistant gene Pb-bd1 molecular labeling BS23 anti-sense primers BS23-R
<400> 2
tggacactta tgcaattagc 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Rice Thin rice blast resistant gene Pb-bd1 molecular labeling RM7654 sense primers RM7654-F
<400> 3
ctcatggttg tgtcgtggtc 20
<210> 4
<211> 219
<212> DNA
<213>Artificial sequence
<220>
<223>Rice Thin rice blast resistant gene Pb-bd1 molecular labeling RM7654 anti-sense primers RM7654-R
<400> 4
gtgcagtgcc agtggtacg 19
Sequence table
2
Claims (7)
1. Rice Thin spike of rice pest resistant gene Pb-bd1 molecular labeling, it is characterised in that described molecular labeling be selected from BS23,
Any one in RM7654；Described molecular labeling BS23 sense primer is BS23-F：SEQ ID NO.1, anti-sense primer
For BS23-R：SEQ ID NO.2, amplified production size is 114bp；Described molecular labeling RM7654 sense primer is
RM7654-F：SEQ ID NO.3, anti-sense primer is RM7654-R：SEQ ID NO.4, amplified production size is 155bp.
2. the molecular labeling primer of the Rice Thin spike of rice pest resistant gene Pb-bd1 described in claim 1, it is characterised in that described
Molecular labeling BS23 sense primer be BS23-F：SEQ ID NO.1, anti-sense primer is BS23-R：SEQ ID NO.2, expand
Increasing primer size is 114bp；Described molecular labeling RM7654 sense primer is RM7654-F：SEQ ID NO.3, downstream is drawn
Thing is RM7654-R：SEQ ID NO.4, amplified production size is 155bp.
3. application of the molecular labeling in Pyricularia Oryzae resistant gene Pb-bd1 is differentiated described in claim 1.
4. application of the molecular labeling primer in Pyricularia Oryzae resistant gene Pb-bd1 is differentiated described in claim 2.
5. application of the molecular labeling primer in anti-rice blast rice is differentiated described in claim 2.
6. a kind of method of quick discriminating Pyricularia Oryzae resistant gene Pb-bd1 presence or absence, it is characterised in that include following step
Suddenly：
(1) water intaking rice sample, extracts paddy rice sample gene group DNA；
(2) using any pair or two pairs in the molecular labeling primer described in claim 2, to described paddy rice sample gene
Group DNA enters performing PCR amplification, and pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplification
Go out the molecular labeling fragment of correspondence size, indicate the presence of Pb-bd1 genes.
7. method according to claim 5, it is characterised in that the reaction system of described PCR amplifications is：10 × contain Mg2+'s
Taq enzyme 0.1 of μ l, the 4pmol/ μ l of buffer solution 1.0 molecular labeling primer to μ l, the 5U/ μ l of 1 μ l, 2.5mM dNTPs 0.2
μ l, the 10ng/ μ l μ l of paddy rice sample gene group template DNA 1, add water to 10 μ l；Response procedures are：94 DEG C of DNA, 5 points of pre-degenerations
Zhong Hou；94 DEG C are denatured 40 seconds, and 60 DEG C are moved back 40 seconds, 72 DEG C of extension 40 second minutes of exhibition, are circulated 30 times；Last 72 DEG C extend 10 minutes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154783A (en) * | 2020-01-16 | 2020-05-15 | 四川农业大学 | Application of maize ZmAKIN β gamma 1 gene in breeding lead stress resistant maize |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102162011A (en) * | 2011-05-09 | 2011-08-24 | 南京农业大学 | Molecule marking method of rice blast-resisting gene |
| CN105713983A (en) * | 2016-04-21 | 2016-06-29 | 南京农业大学 | Molecular marker closely interlocked with neck blast resistance gene of paddy rice Jiangnan lateness and application thereof |
-
2017
- 2017-04-25 CN CN201710278281.0A patent/CN107130019B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102162011A (en) * | 2011-05-09 | 2011-08-24 | 南京农业大学 | Molecule marking method of rice blast-resisting gene |
| CN105713983A (en) * | 2016-04-21 | 2016-06-29 | 南京农业大学 | Molecular marker closely interlocked with neck blast resistance gene of paddy rice Jiangnan lateness and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| HIROKI TAKAGI等: "QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations", 《THE PLANT JOURNAL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154783A (en) * | 2020-01-16 | 2020-05-15 | 四川农业大学 | Application of maize ZmAKIN β gamma 1 gene in breeding lead stress resistant maize |
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