CN114854896B - Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application - Google Patents
Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application Download PDFInfo
- Publication number
- CN114854896B CN114854896B CN202210583626.4A CN202210583626A CN114854896B CN 114854896 B CN114854896 B CN 114854896B CN 202210583626 A CN202210583626 A CN 202210583626A CN 114854896 B CN114854896 B CN 114854896B
- Authority
- CN
- China
- Prior art keywords
- rape
- bnmes
- methylselenocysteine
- molecular marker
- content
- 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
- XDSSPSLGNGIIHP-VKHMYHEASA-N Se-methyl-L-selenocysteine Chemical compound C[Se]C[C@H]([NH3+])C([O-])=O XDSSPSLGNGIIHP-VKHMYHEASA-N 0.000 title claims abstract description 46
- 239000003147 molecular marker Substances 0.000 title abstract description 21
- 238000012216 screening Methods 0.000 claims abstract description 11
- 238000009395 breeding Methods 0.000 claims description 19
- 230000001488 breeding effect Effects 0.000 claims description 19
- 240000007124 Brassica oleracea Species 0.000 claims description 6
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 6
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims description 6
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 6
- 210000000349 chromosome Anatomy 0.000 abstract description 12
- 239000003550 marker Substances 0.000 abstract description 6
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract 1
- 240000002791 Brassica napus Species 0.000 description 21
- 235000011293 Brassica napus Nutrition 0.000 description 20
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 18
- 229910052711 selenium Inorganic materials 0.000 description 18
- 239000011669 selenium Substances 0.000 description 18
- 229940091258 selenium supplement Drugs 0.000 description 18
- 239000000463 material Substances 0.000 description 15
- 108700028369 Alleles Proteins 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 238000012098 association analyses Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 6
- 235000013311 vegetables Nutrition 0.000 description 6
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 4
- 235000006008 Brassica napus var napus Nutrition 0.000 description 4
- 240000000385 Brassica napus var. napus Species 0.000 description 4
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000007844 allele-specific PCR Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 229960001471 sodium selenite Drugs 0.000 description 3
- 235000015921 sodium selenite Nutrition 0.000 description 3
- 239000011781 sodium selenite Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- DWCZIOOZPIDHAB-UHFFFAOYSA-L methyl green Chemical compound [Cl-].[Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)[N+](C)(C)C)=C1C=CC(=[N+](C)C)C=C1 DWCZIOOZPIDHAB-UHFFFAOYSA-L 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 125000003748 selenium group Chemical group *[Se]* 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000037208 balanced nutrition Effects 0.000 description 1
- 235000019046 balanced nutrition Nutrition 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Botany (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to a molecular marker BnMes-2C1 closely linked with a rape methylselenocysteine content trait QTL and application thereof. The invention obtains a SNP marker which is obviously related to the content of the methylselenocysteine of rape, is positioned at 43772485 th base position on the C02 chromosome of the Darmor-bzh v genome, and can explain 7.1% of phenotype variation. The KASP detection primer designed according to the molecular marker can be used for auxiliary selection, so that the screening efficiency of the menu strain containing higher methyl selenocysteine can be improved.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to a molecular marker BnMes-2C1 closely linked with a rape methylselenocysteine content trait QTL and application thereof.
Background
Modern researches have shown that selenium with different forms has great difference in physiological functions, biological safety and the like. Wherein the bioavailability of the inorganic selenium is low and the risk of toxic and side effects is high; compared with inorganic selenium, the methylselenocysteine has higher human body absorption efficiency, physiological activity and biological safety, thus becoming a healthy selenium supplementing form recommended by nutritionists.
The plant is a main way for selenium element to enter the human food chain from the environment, and is also an important carrier for converting inorganic selenium in the environment into organic selenium forms such as methylselenocysteine and the like. Cabbage type rape is a type of cultivated rape mainly pushed in China, has excellent selenium enrichment capability generally, and vegetable seedlings and vegetable moss of the cabbage type rape have become vegetable products popular with consumers in recent years due to the advantages of good taste, balanced nutrition, easy processing and the like. Researches show that the selenium form composition and content of different rape varieties have large difference, and the organic selenium transformation related characters have rich genetic variation. It can be seen that rape is a good carrier for the transformation of plant-derived organic selenium. Deep excavation of genetic control sites for efficient synthesis of organic selenium in specific germplasm of rape for breeding improvement is helpful for relieving the increasing healthy selenium supplementing requirement in China.
The traditional breeding means is difficult to meet the current crop breeding demands due to long breeding years and low selection efficiency. With the rapid development of molecular biology and sequencing technology, the genotype selection is adopted to accelerate the breeding process, so that the method is widely applied to the breeding of finished product seeds. The molecular marker closely related to the high-efficiency synthesis character of the methylselenocysteine in rape is detected by using the molecular marker to assist selection, so that the difficulty in phenotype identification of selenium content in different forms can be overcome, the precise introduction or polymerization of characters can be guided, and the breeding efficiency can be greatly improved. At present, no report exists on the related research on the transformation efficiency of plant methylselenocysteine, and no report exists on the identification of related genetic control sites in rape.
The invention uses rape core germplasm resource group as material, utilizes whole genome association analysis to identify methyl selenocysteine with breeding application potential in rape to efficiently synthesize QTL locus, and develops molecular markers based on QTL locus information, thereby being beneficial to improving the breeding efficiency of high methyl selenocysteine rape varieties.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting 43772485 th base on a C02 chromosome of a Darmor-bzh v genome of brassica napus in screening and breeding of the methylselenocysteine content of the brassica napus, and screening and breeding of the synthetic capability of the methylselenocysteine of the brassica napus can be realized by detecting the 43772485 th base on the C02 chromosome of the Darmor-bzh v genome of the brassica napus.
In order to achieve the above object, the present invention adopts the following technical measures:
obtaining the high-efficiency synthetic trait QTL locus qBnMes-2C1 with rape methylselenocysteine:
(1) 327 cabbage type rape inbred lines from various countries in the world are collected as rape association groups, individual leaves of each strain of the association groups are collected, total DNA is extracted by a CTAB method, and genotype analysis is carried out on each sample by using a rape 50K Illumina SNP chip developed by Wuhan double green source core science and technology research institute Co.
(2) Marker heterozygosity (heterozygous rate), deletion rate (MISSING RATE), minimum allele frequency (minor allele frequency) of the population material at each locus was calculated using Illumina BeadStudio genotyping software (http:// www.illumina.com /). Filtering SNP markers by taking the unique matching of the deletion rate of less than or equal to 0.2, the heterozygosity rate of less than or equal to 0.2, the minimum allele frequency of more than 0.05 and the SNP markers in the brassica napus Darmor genome (Chalhoub et al., 2014) as screening criteria, and finally obtaining 21,243 high-quality SNP markers for whole genome association analysis.
(3) And importing genotype data of the obtained association analysis population into STRUCTURE v.2.3.4 for population STRUCTURE analysis, and dividing 327 parts of brassica napus germplasm resources into 3 subgroups. The genetic relationship between 327 parts of brassica napus germplasm resources is calculated by SPAGeDi software (HARDY AND VEKEMANS, 2002).
(4) Planting 327 parts of materials in a hydroponic greenhouse by utilizing a rape hydroponic culture system; after the culture is carried out until the three leaf stage, 10 mu M sodium selenite is added into the culture medium for continuous culture for 5 days, and vegetable seedling samples are collected for selenium morphological determination. Setting 3 times of biological repetition, taking 5 strains of materials from each sample, uniformly crushing, and measuring the content of methylselenocysteine by a liquid chromatography-morphological pretreatment device-atomic fluorescence combined instrument (LC-AFS, GH/T1135-2017).
(5) In combination with genotype data, population structure and canola seedling selenium content data, association analysis was performed using TASSEL 5.0.0 software (braddury et al, 2007) to detect on the C02 chromosome SNP markers C02-43772485 that are significantly associated with canola methylselenocysteine content, up to a interpretable 7.1% phenotypic variation, the SNP variation site (variation from C to T) being located at base 43772485 of the C02 chromosome of the brassica napus Darmor-bzh v (Rousseau-Gueutin et al, 2020), the methylselenocysteine tightly linked to the SNP site being designated as qBnMes-2C1.
The application of the reagent for detecting 43772485 th base on the C02 chromosome of the Darmor-bzh v genome of brassica napus in screening and breeding of rape methyl selenocysteine enrichment capability belongs to the protection scope of the invention.
The application of the reagent for detecting the rape sequence containing 43772485 th base on the C02 chromosome of the brassica napus Darmor-bzh v genome in screening and breeding of the rape methylselenocysteine enrichment capability also belongs to the protection scope of the invention.
In the above application, it is preferable that the rape sequence is shown in SEQ ID NO. 2.
The application of the primer designed aiming at the 43772485 th base on the C02 chromosome of the brassica napus Darmor-bzh v genome in screening and breeding of the enrichment capability of the methyl selenocysteine of the brassica napus also belongs to the protection scope of the invention.
In the above application, the applicant developed KASP marker BnMes-2C1 of its antisense strand based on the above SNP site, and designed primers based on this marker were:
qBnMes-2C1 low selenium enrichment allele-specific primer BnMes-2C1-F1: CCCTAAAGTTTGAATCACAGCTACTA A
QBnMes-2C1 high selenium enrichment allele-specific primer BnMes-2C1-F2: CCCTAAAGTTTGAATCACAGCTACTG A
Reverse primer BnMes-2C1-R: GCGTGATACGTAGCCGATTTC.
The primers need to be added with universal joints marked by KASP before use according to the rule of KASP marking development.
Compared with the prior art, the invention has the following advantages:
(1) The invention obtains the main effect QTL locus qBnMes-2C1 obviously related to the efficient synthesis of the methylselenocysteine of rape for the first time, can explain 7.1% of phenotype variation at most, and can be effectively applied to the genetic improvement of the efficient synthesis character of the methylselenocysteine of rape.
(2) The first research discovers that the molecular marker BnMes-2C1 which is obviously related to the efficient synthesis of the rape methylselenocysteine provides a reliable molecular marker source for the pre-selection of the efficient synthesis property of the rape methylselenocysteine.
(3) The molecular marker BnMes-2C1 can be used for rapidly selecting excellent allelic variation of qBnMe s-2C1 in rape varieties or lines in the growth period of rape seedlings, so that the workload of breeding and screening can be greatly reduced, the breeding period is shortened, and the breeding process of efficient synthesis of methyl selenocysteine of rape is accelerated.
Detailed Description
The technical scheme of the invention is conventional in the art unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available. In the present invention, unless otherwise specified, the brassica napus genome is referred to by Darmor-bzh v (Rousseau-Gueutin et al 2020). The cabbage type rape reference of the invention ：Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Ge netic Basis of Ecotype Divergence.
Example 1:
Obtaining a rape methyl selenocysteine content trait QTL locus qBnMes-2C 1:
(1) 327 cabbage type rape inbred lines from various countries in the world are collected as rape association groups, individual leaves of each strain of the association groups are collected, total DNA is extracted by a CTAB method, and genotype analysis is carried out on each sample by using a rape 50K Illumina SNP chip developed by Wuhan double green source core science and technology research institute Co.
(2) Marker heterozygosity (heterozygous rate), deletion rate (MISSING RATE), minimum allele frequency (minor allele frequency) of the population material at each locus was calculated using Illumina BeadStudio genotyping software (http:// www.illumina.com /). Filtering SNP markers by taking the unique matching of the deletion rate of less than or equal to 0.2, the heterozygosity rate of less than or equal to 0.2, the minimum allele frequency of more than 0.05 and the SNP markers in the brassica napus Darmor genome (Chalhoub et al., 2014) as screening criteria, and finally obtaining 21,243 high-quality SNP markers for whole genome association analysis.
(3) And importing genotype data of the obtained association analysis population into STRUCTURE v.2.3.4 for population STRUCTURE analysis, and dividing 327 parts of brassica napus germplasm resources into 3 subgroups. The genetic relationship between 327 parts of brassica napus germplasm resources is calculated by SPAGeDi software (HARDY AND VEKEMANS, 2002).
(4) Planting 327 parts of materials in a hydroponic greenhouse by utilizing a rape hydroponic culture system; after the culture is carried out until the three leaf stage, 10 mu M sodium selenite is added into the culture medium for continuous culture for 5 days, and vegetable seedling samples are collected for selenium morphological determination. Setting 3 times of biological repetition, taking 5 strains of materials from each sample, uniformly crushing, and measuring the content of methylselenocysteine by a liquid chromatography-morphological pretreatment device-atomic fluorescence combined instrument (LC-AFS, GH/T1135-2017).
(5) In combination with genotype data, population structure and canola seedling methylselenocysteine content data, association analysis was performed using TASSEL 5.0.0 software (Bradbury et al, 2007) to detect on the C02 chromosome SNP markers C02-43772485 that are significantly associated with canola methylselenocysteine content, up to a maximum of 7.1% of the phenotypic variation, the SNP variation site (variation from C to T) being located at base 43772485 of the C02 chromosome of the brassica napus Darmor-bzh v (Rousseau-Gueutin et al, 2020), the SNP site closely linked methylselenocysteine efficient synthesis major QTL site being designated qBnMes-2C1.
Example 2:
Obtaining a molecular marker primer closely linked with a methylselenocysteine content trait QTL locus qBnMes-2C 1: (1) Extracting 100bp sequences of upstream and downstream of 43772485 th basic groups of a brassica napus C02 chromosome, developing a KASP molecular marker BnMes-2C1 for an antisense strand according to the design principle of KASP (Kompetitive Allele-SPECIFIC PCR, namely competitive allele-specific PCR) molecular marker, wherein the marker comprises two competitive forward primers Bn Mes-2C1-F1 and BnMes-2C1-F2, complementary sequence basic groups T and C corresponding to SNP mutation sites respectively, and a reverse universal primer BnMes-2C1-R, and the primer sequences are as follows:
BnMes-2C1-F1：ccctaaagtttgaatcacagctacta
BnMes-2C1-F2：ccctaaagtttgaatcacagctactg
BnMes-2C1-R：gcgtgatacgtagccgatttc
The primers need to be added with universal joints marked by KASP before use according to the rule of KASP marking development.
Wherein the sequence of the splice added before BnMes-2C1-F1 is GAAGGTGACCAAGTTCATGCT and the sequence of the splice added before BnMes-2C1-F2 is GAAGGTCGGAGTCAACGGATT.
The amplified sequence in brassica napus Bingo is genotype A (i.e., genotype TT), and the sequence is shown below as ：gcgtgatacgtagccgatttctaaaatatttaacatgtttagctcaccaatctttgtcttataagcgggattttctagtagctgtgattcaaactttaggg(SEQ ID NO.1.
The amplified sequence in brassica napus Westar is genotype B (i.e., genotype CC), and the sequence is shown below as ：gcgtgatacgtagccgatttctaaaatatttaacatgtttagctcaccaatctttgtcttataagcgggattttccagtagctgtgattcaaactttaggg(SEQ ID NO.2.
(2) Genotyping is carried out on the markers in rape-associated groups by adopting a competitive allele-specific PCR technology, a five-primer amplified hindered mutation system (PAMS) is used as a kit for amplification, and 10-100ng of 10uL of a reaction system ：2×PARMS master mix 5μL,Allele X primer(10μM)0.15μL,Allele Y primer(10μM)0.15μL,Common R primer(10μM)0.4μL, rape genomic DNA is designed according to the specification of a PAMS pro SNP GENTYPING PCR mix kit. The amplification procedure was: 94 ℃ for 15min;94 ℃ for 20s,65-57 ℃ (Touch-down) for 1min, and 10 times of circulation; cycling for 30 times at 94 ℃ for 20s and 57 ℃ for 1 min; and collecting 1 fluorescence signal and outputting genotype result. And performing association analysis by using Tassel software to determine that BnMes-2C1 is obviously associated with the main QTL locus qBnMes-2C1 of the rape methylselenocysteine efficient synthesis property.
By using the method, the BnMes-2C1 mark is obviously associated with the main effect QTL site qBnMes-2C1 of the rape methyl selenocysteine high-efficiency synthesis property.
Example 3:
The application of the primer designed based on 43772485 th base of rape C02 chromosome in screening and breeding of rape methylselenocysteine with high-efficiency synthetic characters comprises the following steps:
(1) Selecting 25 parts of materials with higher methyl selenocysteine content and lower methyl selenocysteine content which are homozygous through multi-generation selfing in 327 parts of materials; and (3) planting the materials in a hydroponic greenhouse, culturing until the three leaf stage, adding 10 mu M sodium selenite into a culture medium, continuously culturing for 5 days, and collecting vegetable seedling samples for selenium morphological determination. Setting 3 times of biological repetition, taking 5 strains of materials from each sample, uniformly crushing, and measuring the content of methylselenocysteine by a liquid chromatography-morphological pretreatment device-atomic fluorescence combined instrument (LC-AFS, GH/T1135-2017).
(2) Examination of the distribution of the two genotypes of molecular marker BnMes-2C1 in the above-described materials with higher and lower methylselenocysteine content shows that the genotype of molecular marker BnMes-2C1 is A in 7 parts, B in 18 parts, and A in 18 parts and B in 7 parts in 25 parts of the materials with lower methylselenocysteine content (Table 1).
(3) The T test result shows that the genotypes A and B detected by the molecular marker BnMes-2C1 have very significant difference (P < 0.01) in the methylselenocysteine content of the rape seedlings.
The above results are sufficient to indicate that the prepared molecular marker BnMes-2C1 is highly correlated with the methyl selenocysteine content of rape seedlings, so that the molecular marker can be used for auxiliary selection of the high methyl selenocysteine acidic molecular marker of rape.
Table 1: genotype of molecular marker BnMes-SC7 in rape seedling methylselenocysteine content extreme material
Sequence listing
<110> Institute of oil crop and oil crop at national academy of agricultural sciences
<120> Molecular marker BnMes-2C1 closely linked with rape methylselenocysteine content trait QTL and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
gcgtgatacg tagccgattt ctaaaatatt taacatgttt agctcaccaa tctttgtctt 60
ataagcggga ttttctagta gctgtgattc aaactttagg g 101
<210> 2
<211> 101
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
gcgtgatacg tagccgattt ctaaaatatt taacatgttt agctcaccaa tctttgtctt 60
ataagcggga ttttccagta gctgtgattc aaactttagg g 101
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
ccctaaagtt tgaatcacag ctacta 26
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
ccctaaagtt tgaatcacag ctactg 26
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
gcgtgatacg tagccgattt c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
gaaggtgacc aagttcatgc t 21
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
gaaggtcgga gtcaacggat t 21
Claims (1)
1. Application of primer in cabbage type rape methylselenocysteine content screening breeding, wherein the primer is BnMes-2C1-F1：CCCTAAAGTTTGAATCACAGCTACTA,BnMes-2C1-F2：CCCTAAAGTTTGAATCACAGCTACTG,BnMes-2C1-R：GCGTGATACGTAGCCGATTTC.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210583626.4A CN114854896B (en) | 2022-05-25 | 2022-05-25 | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210583626.4A CN114854896B (en) | 2022-05-25 | 2022-05-25 | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114854896A CN114854896A (en) | 2022-08-05 |
| CN114854896B true CN114854896B (en) | 2024-07-05 |
Family
ID=82641833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210583626.4A Active CN114854896B (en) | 2022-05-25 | 2022-05-25 | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114854896B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112568075A (en) * | 2020-12-10 | 2021-03-30 | 施恩（恩施）生物医药开发有限公司 | Selenium-rich cultivation method for edible rape bolts and application of selenium-rich cultivation method |
| CN113652499A (en) * | 2021-09-16 | 2021-11-16 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with rape selenium high-efficiency character major QTL locus qSe.C07 and application |
-
2022
- 2022-05-25 CN CN202210583626.4A patent/CN114854896B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112568075A (en) * | 2020-12-10 | 2021-03-30 | 施恩（恩施）生物医药开发有限公司 | Selenium-rich cultivation method for edible rape bolts and application of selenium-rich cultivation method |
| CN113652499A (en) * | 2021-09-16 | 2021-11-16 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with rape selenium high-efficiency character major QTL locus qSe.C07 and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114854896A (en) | 2022-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113652499B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application | |
| CN113736903B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C03 and application | |
| CN106755482A (en) | A kind of SSR molecular marker III for identifying Gala apple Progeny plants and its application | |
| CN109929945B (en) | Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| CN111979345A (en) | Development and application of KASP (Kaempferi protein) marker related to wheat biomass and yield under salt stress condition | |
| CN114752702B (en) | Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof | |
| CN109182557A (en) | A kind of SNP marker and its application for identifying Pelteobagrus vachelli low dissolved oxygen tolerance and coefficient of condition | |
| CN116926229A (en) | Molecular marker closely linked with high-vitamin E main effect QTL locus qVE.C02 of rape seed and application | |
| CN117737279A (en) | Cadmium low-accumulation hybrid rice molecular marker, rice mutant OsNramp5 gene, and identification method, application and primer thereof | |
| CN110004242B (en) | Molecular marker BrSF0239 primer of main QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| CN116790797A (en) | KASP primer group related to wheat grain weight and application thereof | |
| CN113584219B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C04 and application | |
| CN106701967A (en) | Molecular marker for regulating and controlling major QTL (Quantitative Trait Loci) of included angle of corn leaves and application method of mMolecular marker | |
| CN108118100B (en) | SNP molecular marker for identifying and controlling formation of low-temperature yellowing character of Chinese cabbage leaves and application thereof | |
| CN114854896B (en) | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application | |
| CN110468229A (en) | Rice wide spectrum high resistance to hoja blanca gene Xa45's (t) isolates molecular labeling Hxjy-1 | |
| CN114990251B (en) | Molecular marker closely linked with rape methylselenocysteine content trait QTL and application thereof | |
| CN113564161B (en) | Molecular marker closely linked with bacterial wilt resistance of cultivated peanut and application thereof | |
| CN114525360A (en) | SNP marker closely linked with green fruit color Gv1 gene of eggplant and application | |
| CN110643728B (en) | Method for improving breeding efficiency of poplar crossbreeding | |
| CN105483281A (en) | SNP molecular marker for identifying Shanghai colorful waxy corn NO.1 and identifying method thereof | |
| CN114990250A (en) | Molecular marker BnMES-5A1 closely linked with rape methylselenocysteine content trait QTL and application thereof | |
| CN117683938B (en) | KASP molecular marker closely linked with tomato fruit width and application thereof | |
| CN113736906B (en) | SNP locus combination for detecting verticillium wilt resistance of tomatoes and application thereof | |
| CN114774578A (en) | Molecular marker BnMG-1A1 closely linked with rape magnesium content trait QTL 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 |