CN115029465B - KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof - Google Patents
KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof Download PDFInfo
- Publication number
- CN115029465B CN115029465B CN202210098380.1A CN202210098380A CN115029465B CN 115029465 B CN115029465 B CN 115029465B CN 202210098380 A CN202210098380 A CN 202210098380A CN 115029465 B CN115029465 B CN 115029465B
- Authority
- CN
- China
- Prior art keywords
- rape
- dormancy
- secondary dormancy
- kasp
- dcaps
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a KASP and dCAPS marker co-separated from a rape seed secondary dormancy major QTL and application thereof, belonging to the technical field of rape molecular breeding. Designing a primer composition of a KASP marker and a primer composition of a dCAPS marker based on a sequence of a flanking SNP marker A09_10470644 coseparated with a rape seed secondary dormancy main effect QTL qSSD.A09 as a basic sequence, and carrying out PCR amplification by taking genome DNA of a rape plant as a template to effectively convert the SNP marker into the KASP marker and the dCAPS marker. The detection system and the method provided by the invention can effectively predict the secondary dormancy characteristic of the rape seeds, assist in screening excellent varieties or strains with weak secondary dormancy characteristic, effectively improve the breeding accuracy and accelerate the improvement of the secondary dormancy characteristic of the rape seeds.
Description
Technical Field
The invention belongs to the technical field of rape molecular breeding, relates to an SNP locus, a molecular marker developed based on the SNP locus and application of the molecular marker, and particularly relates to KASP and dCAPS markers which are co-separated from a rape seed secondary dormancy main effect QTL qSSD.A09 and application of the KASP and dCAPS markers in auxiliary selection of rape seed secondary dormancy characteristics.
Background
Rape is one of the most important oil crops in the world, provides more than 15 percent of edible vegetable oil in the world, and is also important biofuel and feed. Mechanical behavior in rape production area during mechanical combined harvestAbout 6000 to 9000 grains/m are formed 2 The seeds are scattered in the field, and the manual harvest can cause more seeds to be scattered. The scattered seeds induce secondary dormancy due to adverse conditions such as drought and the like, can survive for several months or even ten years underground, cause the underground seed bank to exist for a long time, and once the external environment is proper, the secondary dormancy seeds germinate to form self-growing seedlings. On one hand, the rape self-growing seedlings can become weeds in the field, and the field management investment (manpower, herbicide and the like) is increased; on the other hand, rape is a common cross-pollinated crop, and rape self-growing seedlings cause variety mixing through pollen drift in the flowering period, so that the quality of the rape is reduced. More seriously, the self-growing seedlings of the transgenic rapes can cause gene drift and cause biological safety problems through cross pollination. Cultivating and applying a new species of weak secondary dormancy is one of the most economical and effective ways to solve the problem.
Single Nucleotide Polymorphism (SNP) refers to a DNA sequence polymorphism caused by a change such as a transition, a transversion, an insertion, or a deletion at a specific nucleotide position in DNA in a genome. The SNP markers have the advantages of large quantity, wide distribution, uniform distribution in the whole genome and the like, and become the most widely applied molecular markers.
KASP is a novel genotyping technology with low cost and high throughput characteristics by competitive allele specific PCR (KASP) developed by LGC company (Laboratory of the Government Chemist) (http:// www.lgcgenomics.com), carries out accurate double allele genotyping on SNP and InDel markers by specific matching of terminal bases of primers, and is widely applied to molecular marker-assisted selection of crops such as rice, wheat, soybean and the like. dCAPS (sequenced purified amplified polymorphic sequences, dCAPS) molecular marking technology utilizes enzyme cutting sites formed by mutation to carry out enzyme cutting detection on the amplified products of the mutation sites so as to judge the mutation condition. The method is simple to operate and low in cost, and can complete genotyping by combining with conventional laboratory equipment such as Polymerase Chain Reaction (PCR) and agarose gel electrophoresis.
The former people locate 4 Quantitative Trait Loci (QTL) related to secondary dormancy by linkage analysis, which are respectively located on chromosomes A05, C03, C05 and C08, but do not develop molecular markers which are tightly linked with the QTL and can be used for marker-assisted selective breeding. Therefore, the research utilizes the developed KASP and dCAPS markers co-separated from the main effect QTL qSSD.A09 related to the secondary dormancy of the rape seeds to accurately improve the secondary dormancy characteristics of the rape seeds, and has important significance in the production practice of the rape seeds.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a KASP and dCAPS marker which is co-separated from a rape seed secondary dormancy main effect QTL qSSD.A 09; the second technical problem to be solved by the invention is to provide a primer composition labeled with KASP and a primer composition labeled with dCAPS; the third technical problem to be solved by the invention is the application of KASP and dCAPS markers or primer compositions thereof in genetic analysis and fine positioning of rape seed secondary dormancy genes and the application in auxiliary selection or genetic improvement of rape seed secondary dormancy characteristics.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the method utilizes a PEG chemical induction method to identify the secondary dormancy rate of a backcross recombination inbred line group of 178 lines from 2017 to 2021, locates the QTL related to the secondary dormancy based on a complete interval mapping method built in IcMapping v4.2, screens out the main effect QTL qSSD.A09 of the secondary dormancy of rape seeds, and can explain 7.8-12.2% of phenotypic variation.
A flanking SNP marker A09_10470644 co-separated from a main effect QTL qSSD.A09 of rape seed secondary dormancy, wherein the SNP marker and the QTL qSSD.A09 related to the rape seed secondary dormancy are co-located in an interval of 4.9cM of a chromosome of rape A09, are located in 10.47-13.48Mb of a No. 09 chromosome of a double 11 reference genome A, and have polymorphism of A/C.
Further, the invention also provides a method for developing KASP markers and dCAPS markers based on SNP sites, which takes the sequence of A09_10470644 as a basic sequence, designs primer compositions of the KASP markers and primer compositions of the dCAPS markers, and takes genome DNA of rape plants as a template for PCR amplification to effectively convert the SNP markers into the KASP markers and the dCAPS markers; the nucleotide sequence of the primer composition marked by KASP is shown in SEQ ID NO. 1-3; the nucleotide sequence of the dCAPS labeled primer composition is shown in SEQ ID NO. 4-5.
A Primer composition of KASP marker co-separated from rape seed secondary dormancy major QTL qSSD.A09 comprises two specific primers PrimeR A and PrimeR C for amplifying the KASP marker and a universal Primer _ Common, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-3.
The method comprises the following specific steps:
the application is that the 5' end of the nucleotide sequence of a specific primer SEQ ID NO.1-2 is respectively added with a fluorescent modification group FAM and a fluorescent modification group HEX.
A primer composition of a dCAPS marker co-separated from a rape seed secondary dormancy main effect QTLqSSD.A09, wherein a forward primer of the dCAPS marker is shown as SEQ ID NO.4, and a reverse primer of the dCAPS marker is shown as SEQ ID NO.5, and comprises the following components:
the primer composition of the KASP marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 or the KASP marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 is applied to genetic analysis and fine positioning of rape seed secondary dormancy genes.
The primer composition of the KASP marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 or the KASP marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 is applied to molecular marker-assisted breeding of the rape seed secondary dormancy characteristics.
The verification of the KASP marking efficiency by utilizing 178 group strains shows that the KASP marking can effectively predict the secondary dormancy characteristic of rape seeds, and a new variety with weak secondary dormancy characteristic can be selected in an auxiliary mode in production.
The primer composition of the dCAPS marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 or the dCAPS marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 is applied to genetic analysis and fine positioning of rape seed secondary dormancy genes.
The primer composition of the dCAPS marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 or the dCAPS marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09 is applied to molecular marker-assisted breeding of the rape seed secondary dormancy characteristics.
The efficiency of the dCAPS marker is verified by utilizing 47 randomly selected group strains, which shows that the dCAPS marker can effectively predict the secondary dormancy characteristic of rape seeds, and can assist in screening of excellent varieties or strains with weak secondary dormancy characteristics.
A method for screening a weak dormancy genotype rape plant comprises the following steps: taking the genome DNA of a plant sample to be detected as a template, and carrying out PCR amplification on the template by using the KASP marked primer composition which is co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09, and carrying out genotype typing on the KASP amplification result; in the primer composition, different fluorescent modification groups are respectively added to the 5' ends of the primer sequences shown in SEQ ID NO.1-2, and the plants capable of reading the fluorescent groups marked by SEQ ID NO.2 are identified as the rape plants with weak dormancy genotypes.
The PCR amplification system is as follows: template DNA 20ng/ul 2.5ul,2x KASP Master mix 2.5ul, KASP Assay mix 0.07ul.
The PCR amplification procedure was: 15min at 94 ℃; the annealing temperature is reduced by 0.6 ℃ for 10 cycles in 10min at 94 ℃ and 61-55 ℃; 26 cycles of 94 ℃ for 20s and 55 ℃ for 1 min.
A method of screening a canola plant for a weakly dormant genotype comprising the steps of: and (2) taking the genome DNA of a plant sample to be detected as a template, carrying out PCR amplification on the template by using the primer composition of dCAPS marker co-separated from the rape seed secondary dormancy main effect QTL qSSD.A09, carrying out enzyme digestion and electrophoresis detection on a PCR product by using restriction enzyme Taq I, screening the rape plant with the weak dormancy genotype according to an electrophoresis detection result, wherein the rape plant with the strong dormancy genotype is obtained if the fragment is 89bp after enzyme digestion, and the rape plant with the weak dormancy genotype is obtained if the fragment is 59bp and 30bp after enzyme digestion.
The PCR amplification system is as follows: mu.L of template DNA, 10. Mu.L of 2 XKOD FX buffer, 4. Mu.L of 2mM dNTPs, 0.4. Mu.L of KOD FX, 10. Mu. Mol/L of each of the forward and reverse primers, 0.1. Mu.L of each of 3.4. Mu.L of ddH 2 O。
The PCR amplification procedure was: 5min at 94 ℃; 10s at 98 ℃, 30s at 57 ℃, 2min at 68 ℃ and 32 cycles; 10min at 68 ℃; keeping the temperature at 4 ℃.
The enzyme cutting system is as follows: 3 μ L of PCR product, 1 μ L of restriction enzyme Taq I10U/mL, 0.5 μ L of 10x Buffer,0.5 μ L of ddH 2 And O, placing the enzyme digestion system at the constant temperature of 37 ℃ for 2h to obtain a product after enzyme digestion.
Further, a kit for identifying the secondary dormancy genotype of rape seeds comprising said KASP-tagged primer composition or said dCAPS-tagged primer composition is also within the scope of the present application.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a main effect QTLqSSD.A09 coseparation KASP and dCAPS markers positioned on a rape A09 chromosome and related to secondary dormancy of seeds, which can be used for detecting the secondary dormancy characteristics of the rape seeds, and can efficiently and accurately carry out auxiliary selection on the secondary dormancy characteristics of the rape seeds by combining the markers, thereby overcoming the defects of long period and low selection efficiency of the traditional breeding, improving the breeding working efficiency, accelerating the genetic improvement process of the secondary dormancy characteristics of the rape seeds, and providing markers and excellent genetic materials for solving the problem of self-growing seedlings of the rape.
Drawings
FIG. 1 is a QTL scanning result chart of the secondary dormancy rates of rape in BILs group in 5 environments in 2017-2021;
FIG. 2 is a diagram of KASP tag development and verification; AA: a strongly dormant parent genotype; CC: a weakly dormant parent genotype;
FIG. 3 is a graph showing the results of typing of dCAPS markers; in the figure, H: a weakly dormant parent; v: a strongly dormant parent; B1-B200: backcrossing and recombining partial strains of the inbred line group; marker:20bp DNA Ladder (Code No.3420A; taKara); AA: a strongly dormant parent genotype; CC: a weakly dormant parent genotype.
Detailed Description
The invention is further described with reference to specific examples.
Example 1
1. Materials and field tests
Secondary dormancy super-strong variety Huaiyou-SSD-V1 (V for short, secondary dormancy rate) with obvious secondary dormancy character difference>90 percent) and a secondary dormancy ultra-weak variety Huaiyou-WSD-H2 (H for short, the secondary dormancy rate is less than 5 percent) as parents for hybridization to obtain a hybrid F 1 By continuous multi-generation backcross with weak secondary dormancy parent, a backcross recombinant inbred line population (BILs) containing 178 lines is constructed. Planting the group and the parents in a biological science and technology garden test base of Huaiyin academy of academic, huai, in 2017-2021 for 5 years, wherein all tests are designed in a random block mode, and a seedling transplanting mode is adopted, wherein 2 lines are planted in each cell, and 15 plants are planted in each line. And field management is carried out according to a conventional production mode, in order to ensure the purity, rape flowering phase is subjected to bagging on sampled plants, after the seeds are completely mature, seeds of 10 plants are collected from each family, and the plants are naturally dried and stored.
2. Phenotypic evaluation
And (3) identifying the secondary dormancy rate of the population from 2017 to 2021 by adopting an optimized PEG chemical induction method (national invention patent: ZL201210108031.X, 2013).
3. Genetic linkage map construction and positioning
178 materials were genotyped using a rape 90K illuminium SNP chip. All SNP markers were screened as follows: (1) there is polymorphism between parents; (2) the deletion ratio of the offspring genotype is not more than 50%; (3) Minor Allele Frequency (MAF) greater than 5%; (4) the heterozygosity rate is not more than 20%; (5) The allele proportion of the donor parent is less than 25 percent, and 12,959 high-quality SNP markers are finally obtained. The probe sequences of these markers were aligned to the reference genome (version ZS11, http:// cbi. Hzau. Edu. Cn/cgi-bin/rape/download _ ext) using blast software to obtain the locations of 12,339 SNP markers on 19 chromosomes of Brassica napus. Genetic map construction was performed using the JoinMap v4.0 software, and the inter-marker recombination rates were converted to genetic distances by Kosambi mapping functions. Finally, a full-length 2031.98cM is constructed, a high-density genetic linkage map containing 5738 SNP markers covers 19 chromosomes of rape, and the average map distance is 0.67eM. The linkage group length is distributed in the range of 51.76-203.71cM, the number of markers in each linkage group is 42-1343, and the average number of markers is 302. Based on the high-density genetic linkage map, an Ichimapping v4.2 software built-In Complete Interval Mapping (ICIM) is utilized to locate the rape seed secondary dormancy related QTL, a threshold LOD is set to be more than or equal to 2.5, and finally, a main effective locus (shown in figure 1) which is stable in multi-environment is located on the chromosome A09 and named as qSSD.A09. This QTL was identified in 5 environments in 2017-2021, accounting for 7.8-12.2% phenotypic variation. The QTL is positioned in the 4.9cM interval of rape A09 chromosome, and the corresponding physical positions of the reference genome of the medium and double 11 rape are as follows: 10.47-13.48Mb.
4. KASP tag development and validation
Aiming at the flanking SNP marker A09_10470644 of the rape seed secondary dormancy main effect QTL QSSD.A09 coseparation, the polymorphism of the marker is A/C. KASP molecular marker Primer sequences were designed using Primer premier 5.0 software, including 2 specific primers (Primer A and Primer C) and 1 universal Primer (Primer _ Common). Wherein, the 5' ends of 2 specific primers are respectively added with different fluorescent modification groups, and the primer sequences and the fluorescent groups are shown in table 1. 178 strains of BILs group are tested, genome DNA of a plant sample to be tested is extracted by a heaven root kit (DP 305) to be used as a template, and the template is amplified by an LGC Hydrocycler-16PCR instrument by using the 3 primer mixtures and the KASP kit (KBS-1016, LGC), wherein the amplification system is as follows: template DNA (20 ng/ul) 2.5ul,2x KASP Master mix 2.5ul, KASP Assay mix 0.07ul, amplification program: 15min at 94 ℃; the annealing temperature is reduced by 0.6 ℃ for 10 cycles in 10min at 94 ℃ and 61-55 ℃; 26 cycles of 94 ℃ for 20s and 55 ℃ for 1 min. After the reaction is finished, a Pherastar scanner is used for reading fluorescence data of the KASP product, and the result of fluorescence scanning is automatically converted into a graph. The BMG PHERstar instrument was used to detect the fluorescence signal and to look for typing.
TABLE 1 KASP primer sequences
As shown in FIG. 2, the KASP marker can accurately distinguish different genotype individuals, wherein the average secondary dormancy rate of 26 individuals with strong dormant genotype (AA) is 68.55%; the average secondary dormancy rate of 149 individuals with the weak dormancy genotype (CC) is 37.19%, and the difference of the secondary dormancy rate among different genotype individuals is extremely obvious (p is less than 0.001).
5. dCAPS marker development and validation
While the KASP marker has high flux, the KASP marker also has the characteristics of high cost, high hardware requirements required by fluorescence detection and the like, the dCAPS marker is simple to operate and low in cost, and typing can be completed by using conventional laboratory equipment such as a PCR instrument, agarose gel electrophoresis and the like.
Based on the above, the application aims at the flanking SNP marker A09_10470644 of the co-separation of the main effect QTL qSSD.A09 of the secondary dormancy of the rape seeds and utilizes a dCAPS Finder 2.0 online tool (A2 _ 10470644)http://helix.wustl.edu/dcaps/ dcaps.html) dCAPS primer development and endonuclease selection were performed while the corresponding downstream primers were designed using Primerpremier 5.0 software (Table 2). The primers were synthesized by Biotechnology engineering (Shanghai) Inc.
Performing PCR amplification by using the genomic DNA of the parent and the material to be detected as a template, wherein the PCR amplification system is 20 mu L: mu.L of template DNA, 10. Mu.L of 2 XKOD FX buffer, 4. Mu.L of 2mM dNTPs, 0.4. Mu.L of KOD FX, 0.1. Mu.L (10. Mu. Mol/L) of each of the forward and reverse primers, and 3.4. Mu.L of ddH 2 And O. The amplification procedure was: 5mi at 94 DEG Cn; 10s at 98 ℃, 30s at 57 ℃, 2min at 68 ℃ and 32 cycles; 10min at 68 ℃; keeping the temperature at 4 ℃. After the PCR product is detected to be qualified by 1% agarose gel electrophoresis, the PCR product is cut by enzyme respectively, the enzyme cutting system is 5 mu L: 3 mu L PCR product, 1 mu L restriction enzyme (Taq I, 10U/mL), 0.5 mu L10x Buffer,0.5 mu L ddH 2 And O, placing the enzyme digestion system at the constant temperature of 37 ℃ for 2 hours to obtain a product after enzyme digestion. The enzyme digestion products were detected by 6% polyacrylamide gel electrophoresis.
As shown in fig. 3, 47 BILs offspring were randomly picked for signature verification. Wherein, the number of the strong secondary dormancy strains is 26, and the average secondary dormancy rate is 68.55%; 21 weak secondary dormancy strains with the average secondary dormancy rate of 27.63%; the difference of the secondary dormancy rates among individuals with different genotypes is extremely obvious (p is less than 0.001).
TABLE 2 dCAPS tagged primers
Sequence listing
<110> Huaiyin college of learning professions
<120> KASP and dCAPS markers coseparated with rape seed secondary dormancy major QTL and application thereof
<130> 1
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> DNA
<213> PrimerA(artificial)
<400> 1
gaaggtgacc aagttcatgc tgtataacgt ccttcccttc ctact 45
<210> 2
<211> 45
<212> DNA
<213> PrimerC(artificial)
<400> 2
gaaggtcgga gtcaacggat tgtataacgt ccttcccttc ctacg 45
<210> 3
<211> 24
<212> DNA
<213> Primer_Common(artificial)
<400> 3
atgaaaaatg agtctttgtc caac 24
<210> 4
<211> 25
<212> DNA
<213> dCAPS-F1(artificial)
<400> 4
acgtataacg tccttccctt ccttc 25
<210> 5
<211> 24
<212> DNA
<213> dCAPS-R1(artificial)
<400> 5
agatcataaa attgatgaaa aatg 24
Claims (9)
1. A primer composition of KASP markers co-separated with a rape seed secondary dormancy major QTL is characterized by comprising two specific primers and a universal primer for amplifying the KASP markers, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-3.
2. A dCAPS marked primer composition which is co-separated with a rape seed secondary dormancy main effect QTL, wherein a dCAPS marked forward primer is shown as SEQ ID NO.4, and a dCAPS marked reverse primer is shown as SEQ ID NO. 5.
3. Use of a KASP-tagged primer composition co-segregating with a major QTL of secondary dormancy of oilseed rape seed as claimed in claim 1 for genetic analysis and fine-positioning of a gene for secondary dormancy of oilseed rape seed.
4. Use of a KASP-tagged primer composition co-segregating with a major QTL for secondary dormancy of oilseed rape as claimed in claim 1 for molecular marker assisted breeding of secondary dormancy traits of oilseed rape.
5. Use of the primer composition of dCAPS marker co-isolated with a rape seed secondary dormancy major QTL as claimed in claim 2 for genetic analysis and fine mapping of rape seed secondary dormancy genes.
6. Use of a dCAPS-tagged primer composition co-segregating with a rape seed secondary dormancy major QTL as defined in claim 2 for molecular marker assisted breeding of rape seed secondary dormancy characteristics.
7. A method for screening a rape plant with a weak dormancy genotype is characterized by comprising the following steps: carrying out PCR amplification on a template by using the genome DNA of a plant sample to be detected as the template and the KASP marked primer composition which is co-separated with the rape seed secondary dormancy major QTL and is disclosed in claim 1, and carrying out genotyping by using the amplification result; in the primer composition, different fluorescent modification groups are respectively added at the 5' end of the primer sequence shown in SEQ ID NO.1-2, and the plant capable of reading the fluorescent group marked by SEQ ID NO.2 is identified as the rape plant with weak dormancy genotype.
8. A method of screening a canola plant for a weak dormant genotype comprising the steps of: taking genome DNA of a plant sample to be detected as a template, carrying out PCR amplification on the template by using a dCAPS marked primer composition which is co-separated from the rape seed secondary dormancy main effect QTL and is described in claim 2, carrying out enzyme digestion and electrophoresis detection on a PCR product by using restriction enzyme TaqI, screening the rape plant with the weak dormancy genotype according to an electrophoresis detection result, wherein the rape plant with the strong dormancy genotype is obtained if the fragment after enzyme digestion is 89bp, and the rape plant with the weak dormancy genotype is obtained if the fragment after enzyme digestion is 59bp and 30 bp.
9. A kit for identifying a secondary dormancy genotype of a rape seed comprising the KASP-tagged primer composition of claim 1 or the dCAPS-tagged primer composition of claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210098380.1A CN115029465B (en) | 2022-01-27 | 2022-01-27 | KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210098380.1A CN115029465B (en) | 2022-01-27 | 2022-01-27 | KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115029465A CN115029465A (en) | 2022-09-09 |
| CN115029465B true CN115029465B (en) | 2023-03-24 |
Family
ID=83120032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210098380.1A Active CN115029465B (en) | 2022-01-27 | 2022-01-27 | KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115029465B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568502A (en) * | 2022-12-12 | 2024-02-20 | 江西农业大学 | Primer pair for screening rape aluminum toxin sensitive varieties and application and method thereof |
| CN117106802B (en) * | 2023-09-15 | 2024-04-30 | 西部(重庆)科学城种质创制大科学中心 | Cabbage type rape high-dehiscence-angle resistance gene and identification and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102612893B (en) * | 2012-04-13 | 2013-03-13 | 淮阴师范学院 | Chemical detection method for secondary dormancy characteristics of grown rape seeds |
| CN108300801B (en) * | 2018-04-25 | 2021-11-16 | 西南大学 | Molecular marker closely related to rape grain weight and silique length and application |
| CN110760608B (en) * | 2019-12-02 | 2020-07-14 | 浙江省农业科学院 | Rape BnFAD2 gene high oleic acid allelic mutation and development and application of SNP marker primer thereof |
-
2022
- 2022-01-27 CN CN202210098380.1A patent/CN115029465B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN115029465A (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115029465B (en) | KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL and application thereof | |
| CN110295251B (en) | SNP molecular marker linked with wheat effective tillering number QTL and application thereof | |
| CN111893209B (en) | Indel site detection marker related to thousand grain weight of wheat and application thereof | |
| CN114134247B (en) | Molecular marker closely linked with millet plant height character, primer sequence and application thereof | |
| CN112195264B (en) | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of tomato hybrid and application | |
| CN114657277B (en) | KASP molecular marker related to wheat grain length and application thereof | |
| CN106755413B (en) | Rice nitrogen absorption and utilization site qNUE6 and molecular marking method thereof | |
| CN110331222B (en) | Molecular marker related to cotton fertility restoration and application thereof | |
| CN115927718B (en) | KASP molecular marker for identifying grain weight and grain width of wheat and application | |
| CN116121445A (en) | KASP molecular marker for detecting grain weight and grain width of wheat in multiple environments and application | |
| CN115852021A (en) | SNP molecular marker for identifying wheat grain weight and grain length and application thereof | |
| CN113832251B (en) | SNP locus combination for detecting tomato mosaic virus resistance and application thereof | |
| CN113736866B (en) | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof | |
| CN114908188A (en) | Application of KASP molecular marker related to wheat grain weight and grain length and primer composition thereof | |
| CN111118192B (en) | KASP molecular marker of wheat ear base small ear fruition main effect QTL and application thereof | |
| CN110923355A (en) | Linkage KASP molecular marker for rice high temperature resistance character and application thereof | |
| CN111485032A (en) | Method for identifying cucumber female line and SNP primer combination used by same | |
| CN117286287B (en) | KASP (KASP) marker of soybean shade-tolerance gene GmYUC2 and application thereof | |
| CN116590453B (en) | SNP molecular marker related to dwarf trait of lotus plant and application thereof | |
| CN110724755B (en) | CAPS marker primer group linked with watermelon internode length and application thereof | |
| CN114908183B (en) | Method for identifying resistance of cucumber scab, SNP (Single nucleotide polymorphism) special primer set and application | |
| CN117265175B (en) | Molecular marker closely linked with main effect QTL of wheat sterile spike number and application thereof | |
| CN113736908B (en) | SNP locus combination for detecting tomato leaf mold resistance and application thereof | |
| CN116004889A (en) | KASP molecular marker of rice fertility gene and application thereof | |
| CN117887885A (en) | Soybean oil content-related major single nucleotide polymorphism site and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |