CN105624328A - High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof - Google Patents
High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-flux molecular marker for identifying tomato leaf mold resistance, and a marking method and application thereof. According to the molecular marker, a primer Cf-5-Al lele-F1, a primer Cf-5-Al lele-F2 and a primer Cf-5-R are designed according to the key specific sites of the target gene, and a primer tail end base is specifically matched with the target gene to carry out genotyping. A PCR (polymerase chain reaction)-based SNP Line platform is a high-flux molecular marker system, and thus, is capable of implementing the full-automatic operation procedure and lowering the human errors. The molecular marker has high analysis flux, can complete 0.5 million SNP genotyping every day, and is suitable for simultaneously detecting abundant samples. The high-detection-flux KASP molecular marker designed on the basis of tomato-leaf-mold-resistant gene Cf-5 is used for high-flux screening and identification of tomato-leaf-mold-resistant plants, can greatly save the time and labor cost, enhances the breeding efficiency of molecular marker assisted selection, has important meanings for culturing leaf-mold-resistant tomato leading varieties and effectively controlling hazards of tomato leaf mold, and is important breeding method innovation and technical innovation.
Description
Technical field
The invention belongs to agricultural biological technical field, it relates to a kind of high-throughput molecule marker for the identification of leaf muld of tomato resistance and marking method thereof and application.
Background technology
Tomato is one of important vegetable crop of China, occupies extremely important status in vegetable at protected field is produced. Leaf muld of tomato (CladosporiumfulvumCooke) is one of worldwide disease threatening tomato in greenhouse production, seriously affects the quality of tomato, causes the tomato underproduction, produces to tomato in greenhouse and brings massive losses. Leaf muld of tomato was reported in 1883 first in Britain, because the speed of spreading is fast, can break out and cause disaster in the short period of time, was just subject to the great attention of scientists very soon. Leaf muld of tomato, occurred successively in China mainland the thirties in 20th century after Taiwan occurs, and along with the expansion of protecting field area, its hazardness is more obvious.
In recent years, leaf muld of tomato has been carried out deeply studying widely and exploring by domestic and international scientist. Research shows, cultivating leaf mold disease-resistant variety is prevent and treat the most effective approach of leaf muld of tomato. Along with the progress of research, multiple leaf muld of tomato disease-resistant gene is identified and clones. Wherein, Cf-5 gene is one of important leaf muld of tomato disease-resistant gene. In the numerous disease-resistant tomato variety at home and abroad cultivated, Cf-5 plays irreplaceable effect. Therefore, develop the efficient molecule marker of Cf-5 particularly important. Wordregen etc. (1994) utilize molecule marker mapping to develop the SCAR molecule marker REX-1 with Mi and Cf-2/Cf-5 close linkage in " WSL6 " tomato. But, there is restructuring to a certain extent in Mi gene and Cf-5 gene, seriously affects the selection accuracy of molecule marker REX-1. Yu Shuancang etc. (2005) develop codominance CAPS molecule marker according to Cf-5 gene order, and this mark needs enzyme to cut, and cost height, experimental arrangement are loaded down with trivial details, detection efficiency is low.
In sum, at present, tomato breeding still lacks the molecule marker of accurate and efficient Cf-5 gene, seriously affects the breeding efficiency of Tomato Leaf Mould Resistant kind. Therefore, developing accurate, efficient and practical Cf-5 high-throughput molecule marker, to the anti-leaf muld of tomato improved seeds of cultivation, effectively the harm of control leaf muld of tomato, significant.
Summary of the invention
The present invention seeks to for prior art needing enzyme cut, cost height, experimental arrangement is loaded down with trivial details, and detection efficiency is low waits not enough and defect, it is provided that the high-throughput molecule marker of the qualification leaf muld of tomato resistant gene Cf-5 that a kind of cost is low, quickness and high efficiency, accuracy rate are high and marking method thereof and application.
Technical solution of the present invention is as follows:
High-throughput molecule marker for detecting resistant gene Cf-5 provided by the invention comprises primer Cf-5-Allele-F1, primer Cf-5-Allele-F2 and primer Cf-5-R. Primer is the primer comprising following sequence, and sequence is as follows:
Cf-5-Allele-F1:
5���CGAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3��
(underscore part is FAM fluorescence labels sequence)
Cf-5-Allele-F2:
5��-GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3��
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 ' CTTAGATTTCCAGTCGAGATCAAG-3 '
More concrete, design primer Cf-5-F1, primer Cf-5-F2, primer Cf-5-R, and add corresponding fluorescence labels sequence respectively at the 5 ' end of above-mentioned primer Cf-5-F1 and primer Cf-5-F2.
Wherein, primer Cf-5-F1, primer Cf-5-F2, primer Cf-5-R are the primer comprising following sequence:
Cf-5-F1:5 ' GCTGAAATCATGTTTCCTGATCTT-3��
Cf-5-F2:5 ' GCTGAAATCATGTTTCCTGATCTC-3��
Cf-5-R:5 ' CTTAGATTTCCAGTCGAGATCAAG-3 '
Preferably, fluorescence labels sequence is:
Cf-5-F1adaptor:5 ' GAAGGTGACCAAGTTCATGCT-3 ' (FAM fluorescence labels sequence)
Cf-5-F2adaptor:5 ' GAAGGTCGGAGTCAACGGATT-3 ' (HEX fluorescence labels sequence)
Preferably, molecule marker primer is:
Cf-5-Allele-F1:
5���CGAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3��
(underscore part is FAM fluorescence labels sequence)
Cf-5-Allele-F2:
5���CGAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3��
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 ' CTTAGATTTCCAGTCGAGATCAAG-3 '
Present invention also offers a kind of high-throughput molecule marking method identifying leaf muld of tomato resistance, key step is:
A uses CTAB method to extract tomato complete genome DNA;
B utilizes primer Cf-5_Allele-F1, primer Cf-5_Allele-F2 and primer Cf-5-R to carry out pcr amplification by template of tomato complete genome DNA;
Pcr amplification product is carried out fluorescent scanning by c;
D analyzes allelic gene typing result.
Preferably, in above-mentioned steps b, PCR reaction system is KASP gene type PCR reaction system: 5ngDNA, 0.07ulKASP72 �� assaymix, 2.5ulKASPV4.02 �� MasterMix, adds ddH2O to 5ul.
Preferably, in above-mentioned KASP gene type PCR reaction system, KASP72 �� assaymix is primer Cf-5_Allele-F1, primer Cf-5_Allele-F2 and primer Cf-5-R and the ddH of 100uM by concentration2O is mixed to get by the volume ratio of 12 12 30 46.
In KASP gene type PCR reaction system, KASPV4.02 �� MasterMix is by fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B, high-fidelity enzyme and dNTP composition. Above-mentioned fluorescent probe A is: the 5 ' end at 5 '-GAAGGTGACCAAGTTCATGCT-3 ' connects 1 FAM fluorophor; Fluorescent probe B is: the 5 ' end at 5 '-GAAGGTCGGAGTCAACGGATT-3 ' connects 1 HEX fluorophor; Quenching probes A is: the 3 ' end at 5 '-AGCATGAACTTGGTCACCTTC-3 ' connects quenching group BHQ; Quenching probes B is: the 3 ' end at 5 '-AATCCGTTGACTCCGACCTTC-3 ' connects quenching group BHQ.
Amplified reaction program is:
Stage 1:94 DEG C of denaturation 15min; Stage 2:94 DEG C of 20s, 65-55 DEG C of (each cycle down 1.0 DEG C) 1min, totally 10 circulations; Stage 3:94 DEG C of 20s, 55 DEG C of 1min, totally 26 circulations.
Preferably, to allelic gene typing and result analysis in steps d: gained amplified production is carried out fluorescent signal scanning, analyze tomato Cf-5 genotype according to scanning result. If described tomato amplified production fluorescent signal data to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then described tomato Cf-5 genotype to be measured is T T, is leaf muld of tomato disease-resistant plant; If presenting redness, then genotype is C C, is susceptible plant; If presenting green, then genotype is T C, is assorted conjunction disease-resistant plant.
Present invention also offers the application of the high-throughput molecule marker for the identification of leaf muld of tomato resistant gene Cf-5 in anti-leaf muld of tomato breed breeding.
Owing to have employed technique scheme, the invention has the beneficial effects as follows: the present invention is based on KASP (KompetitiveAllele-SpecificPCR, competitive ApoE gene) the SNPline genotype tests technology platform of technology, according to the crucial specific position design primer of goal gene, utilize the specially coupling of prime end base that target gene is carried out SNP somatotype. And the SNPLine platform of PCR-based is high-throughput molecule marker system, institute's operating process that can realize in this approach is full-automatic, reduces personal errors; Analysis throughput height, compatible 96,384,1536 porous plates, can complete 20 to 500000 SNP genotypings every day, are applicable to very much a large amount of sample and detect simultaneously. The high throughput testing KASP molecule marker designed with leaf muld of tomato disease-resistant gene Cf-5, when the high flux screening and the qualification that are applied to leaf muld of tomato disease-resistant plant, it is possible to greatly save time and cost of labor, it is to increase breeding efficiency.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is that the present invention utilizes tomato Cf-5 high-throughput molecular marker analysis 1536 hole sample panel SNP somatotype result schematic diagram;
In figure, A-T T genotype (disease-resistant) is blueness;
B-T C genotype (assorted conjunction is disease-resistant) is green;
C-C C genotype (susceptible) is redness;
D-pink colour represents that DNA poor quality or concentration are too low, and amplified production is not by clear and definite somatotype.
Embodiment
Below in conjunction with drawings and Examples, set forth the present invention further.
Embodiment 1: the high-throughput molecule marker design of qualification leaf muld of tomato disease-resistant gene Cf-5
(1) the tomato material with Cf-5 resistance getting different sources and each more than 20 parts of tomato material without Cf-5 resistance, extract its complete genome DNA by CTAB method:
1) by tomato leaf lyophilize, getting 0.6g sample and join in the 1.5mL centrifuge tube containing 1mL preheating CTAB, 65 DEG C of water-bath 30min after grinding, period puts upside down 3-5 time;
2) 400 �� L chloroforms are added: primary isoamyl alcohol mixes liquid (volume ratio 24 1), turns upside down mixed even, the centrifugal 15min of 12000rpm;
3) get supernatant, add equal-volume Virahol, put upside down mixed even, place 30min in-20 DEG C;
4) the centrifugal 10min of 12000rpm, abandons supernatant, and 70% alcohol washs 1 time;
5) dry, add 60 �� LddH2O dissolves, and-20 DEG C of storages are for subsequent use.
(2) the high-throughput molecular markers development of tomato Cf-5 gene
Retrieving tomato Cf-5 gene C DS sequence at ncbi database, design primer carries out pcr amplification by template of above-mentioned tomato sample DNA, carries out pcr amplification.
The primer sequence increased for above-mentioned specific fragment is:
1F:5 '-AGCAGATGAAATCCCTCGGTC-3 '
1R:5 '-CCTCGCTGCTTCTTTCTCCTT-3 '
PCR reaction system (20 �� L system) is: 2 �� TaqMix10 �� L, 1F (10 ��Ms) 0.5 �� L, 1R (10 ��Ms) 0.5 �� L, DNA profiling 20-100ng, ddH2O supplies.
Pcr amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 60s, 32 circulations; 72 DEG C extend 10min. Wherein, PCR instrument is the S1000ThermalCycler of BIO-RAD company. PCR primer agarose electrophoresis detects, and the object band that length is about 960bp is reclaimed in rubber tapping, send Beijing Invitrogen company to check order after TA connection, clone. By sequencing result compare of analysis, obtain 7 single base mutation sites and 1 insertion mutational site. It is respectively: between 2750T/C, 2770G/A, 2867A/C, 2928T/C, 2943G/A, 3159T/G, 3498A/G, 3254-3255, insert T.
In tomato Cf-5 gene C DS sequence there is T-C single base mutation site in 2750 Nucleotide places, is designed for the KASP molecule marker of high flux screening according to this SNP site:
Cf-5-F1:5 '-GCTGAAATCATGTTTCCTGATCTT-3��
Cf-5-F2:5 '-GCTGAAATCATGTTTCCTGATCTC-3��
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
At 5 ' end, Cf-5-F1 and Cf-5-F2 is added corresponding fluorescence labels sequence, and sequence is as follows:
Cf-5-F1adaptor:5 ' GAAGGTGACCAAGTTCATGCT-3 ' (FAM fluorescence labels sequence)
Cf-5-F2adaptor:5 ' GAAGGTCGGAGTCAACGGATT-3 ' (HEX fluorescence labels sequence)
Obtaining corresponding Cf-5 gene high-throughput molecule marker primer is the primer comprising following sequence:
Cf-5-Allele-F1:
5���CGAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3��
(underscore part is FAM fluorescence labels sequence)
Cf-5-Allele-F2:
5���CGAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3��
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 ' CTTAGATTTCCAGTCGAGATCAAG-3 '
Above-mentioned primer is synthesized by Shanghai Invitrogen biotech firm.
Embodiment 2: the foundation utilizing high-throughput Markers for Detection tomato Cf-5 genetic method
One, genomic dna is extracted
With reference to embodiment 1 genome DNA extracting method.
Two, pcr amplification
Taking different tomato dna group DNA as template, utilize molecule marker of the present invention to carry out pcr amplification, obtain pcr amplification product.
Gene type PCR reaction system:
96 orifice plates: 10ng genomic dna, 5ulKASPV4.02 �� MasterMix, 0.14ulKASP72 �� assaymix, adds ddH2O to 10ul.
384 orifice plates: 5ngDNA, 2.5ulKASPV4.02 �� MasterMix, 0.07ulKASP72 �� assaymix, adds ddH2O to 5ul.
1536 orifice plates: 5ngDNA, 2.5ulKASPV4.02 �� MasterMix, 0.07ulKASP72 �� assaymix, adds ddH2O to 5ul.
Wherein, KASPV4.02 �� MasterMix is LGC company product, and the KASPV4.02 �� MasterMix products catalogue for 96/384 orifice plate is numbered KBS-1016-002; KASPV4.02 �� MasterMix catalog number for 1536 orifice plates is KBS-1016-011.
In KASP gene type PCR reaction system, KASPV4.02 �� MasterMix is by fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B, high-fidelity enzyme and dNTP composition. Above-mentioned fluorescent probe A is: the 5 ' end at 5 '-GAAGGTGACCAAGTTCATGCT-3 ' connects 1 FAM fluorophor; Fluorescent probe B is: the 5 ' end at 5 '-GAAGGTCGGAGTCAACGGATT-3 ' connects 1 HEX fluorophor; Quenching probes A is: the 3 ' end at 5 '-AGCATGAACTTGGTCACCTTC-3 ' connects quenching group BHQ; Quenching probes B is: the 3 ' end at 5 '-AATCCGTTGACTCCGACCTTC-3 ' connects quenching group BHQ.
KASP72 �� assaymix is by the primer Cf-5-Allele-F1 that concentration is 100uM, primer Cf-5-Allele-F2, primer Cf-5-R and ddH2O is mixed to get by the volume ratio of 12 12 30 46.
KASP gene type pcr amplification reaction program is:
Stage 1:94 DEG C of denaturation 15min; In stage 2:94 DEG C of 20s, 65-55 DEG C of (each cycle down 1.0 DEG C) 1min, circulates 10 times altogether; In stage 3:94 DEG C of 20s, 55 DEG C of 1min, circulate 26 times altogether. Wherein PCR water-bath thermal cycling is the Hydrocycler16-32 high throughput thermally recycle system, is applicable to 96,384 and 1536 orifice plates.
The single reaction system that experiment is arranged simultaneously is not added the blank of template DNA, 2 blanks are only set in each PCR plate.
Three, the fluorescent scanning of pcr amplification product
Adopt two-way singly excite read plate instrument PHERAstar pcr amplification product is scanned, FAM excitation wavelength is 485nm, and emission wavelength is 520nm, HEX excitation wavelength is 528nm, emission wavelength is 560nm, and system ginseng is 575nm than fluorescence ROX excitation wavelength, and emission wavelength is 610nm.
Each pcr amplification product sample arranges at least 3 repetitions.
Four, allelic gene typing
Adopt KrakenTM software to be analyzed by reading plate instrument PHERAstar scan-data, determine the concrete genotype (referring to accompanying drawing 1) of tomato Cf-5 gene to be measured according to analytical results. Being aggregated in the sample genotype close to X-axis display blueness is the allelotype (referring to Figure 1A) connecting FAM fluorescence labels sequence, being aggregated in the sample genotype close to Y-axis display redness is the allelotype (referring to Fig. 1 C) connecting HEX fluorescence labels sequence, the sample genotype of middle display green is heterozygous (referring to Figure 1B), the sample of display pink colour may be too low due to DNA poor quality or concentration, and amplified production is not by clear and definite somatotype (referring to Fig. 1 D).
Further result is analyzed as follows: if described tomato amplified production fluorescent signal data to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then described tomato Cf-5 genotype to be measured is T T, is leaf muld of tomato disease-resistant plant; If presenting redness, then genotype is C C, is susceptible plant; If presenting green, then genotype is T C, is assorted conjunction disease-resistant plant.
Embodiment 3: the application of the high-throughput molecule marker of tomato Cf-5 gene in tomato variety seed selection
1) being detected by tomato breeding material with the high-throughput molecule marker of tomato Cf-5 gene, comprise F3 for material 200 parts, F4 for breeding material 200 parts, F5 for breeding material 200 parts and F6 for breeding material 200 parts, concrete grammar is with reference to embodiment 2.
2) the single strain of tomato Markers for Detection crossed carries out leaf muld of tomato bacterium artificial infection idenfication. Bacteria suspension (under 200 �� visual field) the sprinkling tomato blade face being 7-8 spore in 4-5 leaf phase concentration is inoculated, and observes, records tomato plant incidence after 2 weeks.
3) by high-throughput molecular marker analysis result and artificial inoculation's result system compare of analysis, it has been found that genotype and phenotype coincide rate up to 100% (see table 1).
Table 1 tomato Cf-5 gene high-throughput molecular marker gene type analysis result and the identical rate of phenotype investigation
Claims (10)
1. identify the high-throughput molecule marker of leaf muld of tomato resistance, it is characterised in that: described molecule marker is by design primer Cf-5-F1, primer Cf-5-F2 and primer Cf-5-R; Described primer Cf-5-F1 and described primer Cf-5-F2, after 5 ' end adds corresponding fluorescence labels sequence, realizes together with described primer Cf-5-R; Described primer Cf-5-F1, primer Cf-5-F2 and primer Cf-5-R are the primer comprising following sequence:
Cf-5-F1:5 '-GCTGAAATCATGTTTCCTGATCTT-3 ';
Cf-5-F2:5 '-GCTGAAATCATGTTTCCTGATCTC-3 ';
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '.
2. the high-throughput molecule marker of qualification leaf muld of tomato resistance as claimed in claim 1, it is characterised in that: described fluorescence labels sequence is:
Cf-5-F1adaptor:5 '-GAAGGTGACCAAGTTCATGCT-3 ', FAM fluorescence labels sequence;
Cf-5-F2adaptor:5 '-GAAGGTCGGAGTCAACGGATT-3 ', HEX fluorescence labels sequence.
3. the high-throughput molecule marker of qualification leaf muld of tomato resistance as claimed in claim 2, it is characterised in that: the primer of described molecule marker is:
Cf-5-Allele-F1:
5 '-GAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3 ';
Cf-5-Allele-F2:
5 '-GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3 ';
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '.
4. identify the high-throughput molecule marking method of leaf muld of tomato resistance, it is characterised in that:
A uses CTAB method to extract tomato complete genome DNA;
B utilizes the primer Cf-5-Allele-F1 described in claim 3, primer Cf-5-Allele-F2 and primer Cf-5-R to carry out pcr amplification by template of tomato complete genome DNA;
Pcr amplification product is carried out fluorescent scanning by c;
D analyzes allelic gene typing result.
5. the high-throughput molecule marking method of qualification leaf muld of tomato resistance as claimed in claim 4, it is characterized in that: in described step b, PCR reaction system is KASP gene type PCR reaction system: 5ngDNA, 0.07ulKASP72 �� assaymix, 2.5ulKASPV4.02 �� MasterMix, adds ddH2O to 5ul; Amplified reaction program is: stage 1:94 DEG C of denaturation 15min; Stage 2:94 DEG C of 20s, 65-55 DEG C of 1min, wherein each circulation decline 1.0 DEG C, totally 10 circulations; Stage 3:94 DEG C of 20s, 55 DEG C of 1min, totally 26 circulations.
6. the high-throughput molecule marking method of qualification leaf muld of tomato resistance as claimed in claim 5, it is characterized in that: in described KASP gene type PCR reaction system, KASP72 �� assaymix is diluted to after concentration is 100uM by primer Cf-5-Allele-F1 according to claim 3, primer Cf-5-Allele-F2 and primer Cf-5-R respectively, with ddH2O is mixed to get by the volume ratio of 12 12 30 46.
7. the high-throughput molecule marking method of qualification leaf muld of tomato resistance as claimed in claim 5, it is characterized in that: in described KASP gene type PCR reaction system, KASPV4.02 �� MasterMix includes fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B, high-fidelity enzyme and dNTP.
8. the high-throughput molecule marking method of qualification leaf muld of tomato resistance as claimed in claim 7, it is characterised in that: described fluorescent probe A sequence is 5 '-GAAGGTGACCAAGTTCATGCT-3 ', and 5 ' end connects 1 FAM fluorophor; Described fluorescent probe B sequence is 5 '-GAAGGTCGGAGTCAACGGATT-3 ', and 5 ' end connects 1 HEX fluorophor; Described quenching probes A sequence is 5 '-AGCATGAACTTGGTCACCTTC-3 ', and 3 ' end connects quenching group BHQ; Described quenching probes B sequence is 5 '-AATCCGTTGACTCCGACCTTC-3 ', and 3 ' end connects quenching group BHQ.
9. the high-throughput molecule marking method of qualification leaf muld of tomato resistance as claimed in claim 4, it is characterized in that: in described step c, fluorescent scanning adopts Kraken software to be analyzed by scan-data, the genotype of tomato Cf-5 is determined according to analytical results, if described tomato amplified production fluorescent signal data to be measured presents blueness through Kraken software analysis in gained somatotype dendrogram, then described tomato Cf-5 genotype to be measured is T T, is leaf muld of tomato disease-resistant plant; If presenting redness, then genotype is C C, is susceptible plant; If presenting green, then genotype is T C, is assorted conjunction disease-resistant plant.
10. identify the application of the high-throughput molecule marker of leaf muld of tomato resistance, it is characterised in that: the application of described molecule marker in anti-leaf muld of tomato breed breeding.
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| CN106048076A (en) * | 2016-08-19 | 2016-10-26 | 中源协和基因科技有限公司 | Kit for clopidogrel personalized medicine related gene SNP detection, and detection method thereof |
| CN106319049A (en) * | 2016-08-19 | 2017-01-11 | 中源协和基因科技有限公司 | Kit for detecting personalized medication related gene SNP of warfarin, and detection method thereof |
| CN106434943A (en) * | 2016-10-20 | 2017-02-22 | 中源协和基因科技有限公司 | Kit and detection method thereof for SNP detection of aspirin individualized medication related genes |
| CN106434940A (en) * | 2016-10-20 | 2017-02-22 | 中源协和基因科技有限公司 | Kit used for SNP detection of personalized medicine relaed genes of statin lipid-lowering medicine and detecting method thereof |
| CN108330201A (en) * | 2017-01-18 | 2018-07-27 | 中国种子集团有限公司 | Identify molecular labeling and its application of Tomato Mosaic Virus resistant gene |
| CN114703316A (en) * | 2022-04-21 | 2022-07-05 | 河南农业大学 | Development and application of KASP marker of tomato ps-2 gene |
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