CN105506075A - SNP (Single Nucleotide Polymorphism) marker related to resistance to alternaria kikuchiana and application - Google Patents
SNP (Single Nucleotide Polymorphism) marker related to resistance to alternaria kikuchiana and application Download PDFInfo
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- CN105506075A CN105506075A CN201510910376.0A CN201510910376A CN105506075A CN 105506075 A CN105506075 A CN 105506075A CN 201510910376 A CN201510910376 A CN 201510910376A CN 105506075 A CN105506075 A CN 105506075A
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Abstract
The invention discloses an SNP (Single Nucleotide Polymorphism) marker which is used for identifying alternaria brassicae of a sand pear variety, and belongs to the technical field of molecular genetic breeding of fruit trees. A method for identifying the alternaria brassicae of the sand pear variety comprises the following steps: (1) extracting a genomic DNA (Deoxyribonucleic Acid) of a to-be-detected pear; (2) carrying out a PCR (Polymerase Chain Reaction) amplification reaction by taking the genomic DNA of the to-be-detected pear as a template and utilizing a primer pair; (3) detecting a PCR amplification product, wherein through sequence alignment, basic group in an S83.0_275630 position is the SNP marker of a high-susceptible variety or a high-resistance variety for identifying the alternaria brassicae of the sand pear variety, and the primer pair comprises a forward primer S83-F, SEQ ID NO.1 and a reverse primer S83-R, SEQ ID NO.2. According to the SNP molecular marker, which is screened by using the method and is used for identifying the resistance to the alternaria brassicae of the sand pear variety, disclosed by the invention, a theoretical foundation is laid for the research on molecular breeding of resistance to the alternaria brassicae and a mechanism of resistance to the alternaria brassicae of sand pears.
Description
Technical field
The invention belongs to fruit tree molecular genetic breeding technical field, provide a kind of SSR molecular marker method identifying the anti-black spot of Sand Pear, can be used for Sand Pear anti-black spot Forepart identification and the anti-black spot breeding of Chinese pear, to improve Chinese pear breeding for disease resistance efficiency, be applicable to be engaged in pears scientific research, the R&D institution of breeding and production and company.
Background technology
Pear black spot cause of disease is Kikuchi chain lattice born of the same parents (AlternariakikuchianaTanaka) that Deuteromycotina chain lattice born of the same parents belong to.Germ with conidium and mycelium killed branch, sick leaf, sick fruit and fall within ground invalid body on survive the winter.Second Year borrows wind and rain to propagate after producing conidium spring, from pore, hole skin with directly invade host tissue and cause First aggression.The rear germ of First aggression morbidity can cause in field infects again.General late April starts morbidity, and tender leaf is very easily injured.6 ~ July is in case of rainy, more easily popular.Main harm fruit, blade and young sprout, often cause early leaf fall and tender top drying extremely, cause dehiscent fruit and early stage shedding, seriously undermine tree vigo(u)r.Mainly Agro-chemicals control is adopted in current production.Use Agro-chemicals control not only wasting manpower and material resources, contaminate environment, also makes fruit pesticide residue increase.
Along with the develop rapidly of Protocols in Molecular Biology, molecular breeding technology accelerates the process of breeding for disease resistance greatly, significantly shorten the seed selection cycle of disease-resistant new lines.The prerequisite of molecular breeding obtains and the closely-related molecule marker of objective trait or functional gene.Utilize molecular marker assisted selection objective trait, can efficiency of selection be improved, shorten breeding cycle.Although black spot is one of important disease, but relatively weak for the Position Research of anti-black spot gene.Current report mainly concentrates on the inoculated identification of Pear black spot, not yet has SNP marker for the report of pears breeding character assisted Selection.
Summary of the invention
The present invention aims to provide one and resists sex-linked SNP marker and application with Pear black spot.
In order to realize above-mentioned object, the present invention adopts following technical measures:
A SNP marker relevant to Pear black spot resistance, its be positioned at pears genome S83.0_275630 place base be T then for anti-black spot, for C is then sense black spot.
The primer pair of SNP marker relevant to Pear black spot resistance described in 1 is required, forward primer S83-F, SEQIDNO.1, reverse primer S83-R, SEQIDNO.2 for test right.
For the identification of a method for the SNP marker of Sand Pear black spot, comprise the steps:
1) pears genomic dna to be measured is extracted;
2) with the genomic dna of pears to be measured for template, utilize primer pair to carry out pcr amplification reaction;
3) pcr amplification product is detected; Be the height sense kind of qualification Sand Pear black spot or the SNP marker of high resistance kind through sequence alignment in S83.0_275630 place base;
Wherein step 2) in PCR amplification system cumulative volume be 20 μ l, comprise 10ng/ μ l template DNA 2 μ l, 2 × EsTaqMasterMix10 μ l, each 2 μ l and H of the positive anti-primer of 10mM
2o4 μ l;
Described primer pair is forward primer S83-F, SEQIDNO.1, reverse primer S83-R, SEQIDNO.2;
Pcr amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, annealing temperature 60 DEG C of lasting 45s, 72 DEG C extend 60s, 35 circulations; Last 72 DEG C extend 10min;
Above-mentioned base is the genotype of T is then high sense kind, and this place's base is the genotype of C is high resistance kind.
Further, the application of above-mentioned SNP marker in pears molecular mark.
Above-mentioned application, utilize pears genomic dna, primer pair is adopted to be forward primer S83-F, SEQIDNO.1, reverse primer S83-R, SEQIDNO.2 carries out pcr amplification program, and as then felt kind for high through sequence alignment in the genotype that S83.0_275630 place base is T, this place's base is the genotype of C is high resistance kind.
Beneficial effect:
The present invention utilizes the early stage assisted Selection that can be used for pears anti-black spot cross-breeding offspring based on Super-BSA Policy Filtering to the SNP marker chain with Pear black spot, compared with observing with traditional field natural occurrence, environmental influence can not be subject to, get rid of interference from human factor, result more accurately and reliably.Compared with inoculated identification black spot resistance, eliminate the program that the separation of pathogenic bacteria, cultivation and inoculated identification etc. are numerous and diverse, it is more convenient to operate, and accelerates the breeding process of pears high resistance black spot new variety.
Accompanying drawing explanation
The black spot resistance trait that Fig. 1 pears F1 generation is different is separated
Fig. 2 parents and anti-sense mix the sequence alignment of pond in target SNP site.
Embodiment
Embodiment 1:
Resist the exploitation of sex-linked SNP marker with Pear black spot, obtain in the following manner:
1, screen the anti-sense material of pears, build Pear black spot genetics of resistance Research Group
Choose and feel black spot kind ' rouge and powder ' through the investigation of many years of field natural occurrence and pears high resistance black spot kind ' Jin Jing ' that screens of artificial infection idenfication and height and hybridize as parent, obtain 341 strain F1 generation individual plants.
2, Pear black spot resistance inoculated identification
Leaf age young sprout tender leaf that is consistent, that just launch completely is selected to inoculate.Each individual plant inoculates 2, does 3 repetitions.Through screening, bacterial strain is defined as that sporulation quantity is large, virulence is strong, distribution H bacterial strain comparatively widely, and spore concentration is 5 × 10
5/ ml, spray inoculation.25 DEG C of moisturizings, 5 days " Invest, Then Investigate " incidences (Fig. 2).Classification is carried out according to disease index.
3, based on group's Mixed method (BulkedsegregatingAnalysis) Policy Filtering Pear black spot resistance linked marker
Select high resistance and high sense individual plant 60 parts according to incidence survey result, sampling CTAB method extracts the genomic dna of each individual plant respectively, balanced mix, sets up disease-resistant gene respectively and mixes pond and susceptible gene mixes pond.Adopt SLAF-seq(Specific-LocusAmplifiedFragmentSequencing) technology to F1 heredity segregating population (the extreme proterties of 2 parents and 60+60 mixes pond) check order, GATK software tool pack is used to detect the SNP site that parent and filial generation mix pond and filter, then BSA is adopted to carry out association analysis, final acquisition 1 SNP site and scaffold83.0_275630.According to design of primers principle, adopt Primer software design primer, its upstream forward primer 5'-ATGAAATAATGGCACGCTCC-3'(S83-F, SEQIDNO.1) and reverse downstream primer 5'-GATGCTGGCTTATGTGCTGA-3'(S83-R, SEQIDNO.2).Amplified production size is 502bp.PCR amplification system cumulative volume is 20 μ l, comprises 10ng genomic templates DNA2 μ l, 2 × EsTaqMasterMix10 μ l, each 2 μ l and H of the positive anti-primer of 10mM
2o4 μ l.Amplification program is 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, annealing temperature 60 DEG C of lasting 45s, 72 DEG C extend 60s, 35 circulations; Last 72 DEG C extend 10min.Utilize this primer equal Successful amplification in ' Jin Jing ' ' rouge and powder ', anti-pond and sense pond, PCR primer carries out sequencing analysis.Find ' rouge and powder ' and feel pond in S83.0_275630 place base to be T through sequence alignment, ' Jin Jing ' and anti-pond are then C(Fig. 2 in this place's base).
Claims (5)
1. a SNP marker relevant to Pear black spot resistance, is characterized in that, its be positioned at pears genome S83.0_275630 place base be T then for anti-black spot, for C is then sense black spot.
2. require the primer pair of SNP marker relevant to Pear black spot resistance described in 1 for test right, it is characterized in that:
Forward primer S83-F, SEQIDNO.1,
Reverse primer S83-R, SEQIDNO.2.
3. for the identification of a method for the SNP marker of Sand Pear black spot, it is characterized in that, comprise the steps:
1) pears genomic dna to be measured is extracted;
2) with the genomic dna of pears to be measured for template, utilize primer pair to carry out pcr amplification reaction;
3) pcr amplification product is detected; Be the height sense kind of qualification Sand Pear black spot or the SNP marker of high resistance kind through sequence alignment in S83.0_275630 place base;
Wherein step 2) in PCR amplification system cumulative volume be 20 μ l, comprise 10ng/ μ l template DNA 2 μ l, 2 × EsTaqMasterMix10 μ l, each 2 μ l and H of the positive anti-primer of 10mM
2o4 μ l;
Described primer pair is forward primer S83-F, SEQIDNO.1, reverse primer S83-R, SEQIDNO.2;
Pcr amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, annealing temperature 60 DEG C of lasting 45s, 72 DEG C extend 60s, 35 circulations; Last 72 DEG C extend 10min;
Wherein said base is the genotype of T is then high sense kind, and this place's base is the genotype of C is high resistance kind.
4. the application of method in pears molecular mark as claimed in claim 3.
5. apply as claimed in claim 4, it is characterized in that, utilize pears genomic dna, primer pair is adopted to be forward primer S83-F, SEQIDNO.1, reverse primer S83-R, SEQIDNO.2 carry out pcr amplification program, as then felt kind for high through sequence alignment in the genotype that S83.0_275630 place base is T, this place's base is the genotype of C is high resistance kind.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925680A (en) * | 2016-05-06 | 2016-09-07 | 中国农业科学院蔬菜花卉研究所 | Method for developing marker through tetraploid potato high-throughput sequencing and application of method |
| CN113355445A (en) * | 2021-06-22 | 2021-09-07 | 山东大丰园农业有限公司 | Pear variety specific molecular marker and screening method and application thereof |
| CN116121420A (en) * | 2022-12-30 | 2023-05-16 | 四川农业大学 | SNP (Single nucleotide polymorphism) marker for detecting black spot resistance of walnut and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640671A (en) * | 2012-05-02 | 2012-08-22 | 湖北省农业科学院果树茶叶研究所 | Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm |
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- 2015-12-10 CN CN201510910376.0A patent/CN105506075B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640671A (en) * | 2012-05-02 | 2012-08-22 | 湖北省农业科学院果树茶叶研究所 | Method for identifying Alternaria altemata (Fr.) Keissler resistance of pear germplasm |
Non-Patent Citations (2)
| Title |
|---|
| JUN WU等: "The genome of the pear (Pyrus bretschneideri Rehd.)", 《GENOME RES.》 * |
| 周 贺等: "砂梨果皮转录组SNP位点发掘及其功能注释分析", 《青岛农业大学学报(自然科学版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925680A (en) * | 2016-05-06 | 2016-09-07 | 中国农业科学院蔬菜花卉研究所 | Method for developing marker through tetraploid potato high-throughput sequencing and application of method |
| CN113355445A (en) * | 2021-06-22 | 2021-09-07 | 山东大丰园农业有限公司 | Pear variety specific molecular marker and screening method and application thereof |
| CN116121420A (en) * | 2022-12-30 | 2023-05-16 | 四川农业大学 | SNP (Single nucleotide polymorphism) marker for detecting black spot resistance of walnut and application thereof |
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