CN113652499B - Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application - Google Patents
Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application Download PDFInfo
- Publication number
- CN113652499B CN113652499B CN202111087529.8A CN202111087529A CN113652499B CN 113652499 B CN113652499 B CN 113652499B CN 202111087529 A CN202111087529 A CN 202111087529A CN 113652499 B CN113652499 B CN 113652499B
- Authority
- CN
- China
- Prior art keywords
- rape
- selenium
- qse
- sec07j
- molecular marker
- 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
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 239000011669 selenium Substances 0.000 title claims abstract description 81
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 81
- 239000003147 molecular marker Substances 0.000 title abstract description 30
- 230000000694 effects Effects 0.000 title description 17
- 240000002791 Brassica napus Species 0.000 claims abstract description 34
- 235000011293 Brassica napus Nutrition 0.000 claims abstract description 29
- 238000009395 breeding Methods 0.000 claims abstract description 22
- 230000001488 breeding effect Effects 0.000 claims abstract description 22
- 210000000349 chromosome Anatomy 0.000 claims abstract description 18
- 238000012216 screening Methods 0.000 claims abstract description 15
- 239000003550 marker Substances 0.000 abstract description 12
- 230000035772 mutation Effects 0.000 abstract description 4
- 230000000692 anti-sense effect Effects 0.000 abstract description 3
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 23
- 238000012360 testing method Methods 0.000 description 14
- 238000000034 method Methods 0.000 description 9
- 108700028369 Alleles Proteins 0.000 description 8
- 238000012098 association analyses Methods 0.000 description 8
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 5
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 5
- 238000007844 allele-specific PCR Methods 0.000 description 5
- 230000002860 competitive effect Effects 0.000 description 5
- 240000007124 Brassica oleracea Species 0.000 description 4
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 4
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000001391 atomic fluorescence spectroscopy Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 150000004678 hydrides Chemical class 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000002441 reversible effect 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
- 230000005540 biological transmission Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000002068 genetic effect 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
- 230000008569 process Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 208000000668 Chronic Pancreatitis Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000019926 Keshan disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010033649 Pancreatitis chronic Diseases 0.000 description 1
- 206010039921 Selenium deficiency Diseases 0.000 description 1
- 244000128884 Zier Kohl Species 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 230000037208 balanced nutrition Effects 0.000 description 1
- 235000019046 balanced nutrition Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 201000011510 cancer Diseases 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
- 230000007812 deficiency Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 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
-
- 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)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of molecular biology and genetic breeding, and in particular relates to a major QTL locus for efficient traits of selenium of rapeqSe.C07Closely linked molecular markers and applications thereof. The invention obtains the stable QTL locus for controlling the high-efficiency character of the rape selenium for the first timeqSe.C07And examined the SNP marker of 36436561 bases on the C07 chromosome of the published Darmor-bzh v10 genome of brassica napus closely linked to the site, a phenotypic variation of 12.2% was explained. According to the antisense strand of the mutation site, a KASP molecular marker SeC07J is designed, and the marker can be used for identifying the rape selenium high-efficiency QTL siteqSe.C07The molecular marker can be used in combination with other molecular markers to further improve the screening breeding efficiency of the selenium enrichment capability of the rape.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to a molecular marker closely linked with a rape selenium high-efficiency trait QTL locus qSe.C07 and application thereof.
Background
Selenium is an indispensable trace element in human life activities, has various important biological regulation functions, and plays an important role in enhancing organism immunity, improving male reproductive capacity, improving myocardial nutrition, preventing and resisting cancer, delaying aging and the like. The deficiency of selenium intake over a long period of time can lead to a variety of diseases such as keshan disease, osteoarthritis, multiple sclerosis, chronic pancreatitis, etc. The recommended daily selenium intake of adults by the national dietary nutrient reference intake standard (WST 578.3-2017) is 60-400 micrograms.
Selenium-containing plants are the main way for human body to ingest selenium. However, the selenium deficiency phenomenon in the soil environment of China is common, and the selenium content of the produced vegetable food is generally low. Rape has the advantages of high yield of the flowering cabbage, good taste, balanced nutrition, strong regional adaptability and the like, and has become a vegetable product popular with consumers in recent years. And the early-stage research shows that part of rape varieties have the characteristic of high selenium efficiency, namely the high selenium efficiency rape can be enriched with higher selenium content in the same tissue part under the same production environment, namely the selenium enrichment capability of the rape. The rape resources are rich, the genetic diversity is high, so that the potential of 'selenium high efficiency' of the rape is fully explored for breeding improvement, and the method is an effective means for relieving the selenium intake demands of residents 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 selenium high-efficiency character in rape is detected by using molecular marker auxiliary selection, so that the difficulty in identifying the selenium content phenotype can be overcome, the precise introduction or polymerization of the character can be guided, and the breeding efficiency can be greatly improved. At present, no report that selenium enrichment or selenium high-efficiency character associated sites are identified in rape exists, and related breeding work is still mainly based on traditional large-scale phenotype screening.
The invention aims to find a main effect QTL locus with an improvement effect on the high-efficiency character of the rape selenium through the whole genome association analysis of the content of the rape seed shaft selenium, and develop practical molecular markers based on the main effect QTL locus for marker-assisted selection of the high-efficiency character of the rape selenium.
Disclosure of Invention
The invention aims at providing a molecular marker closely linked with a main effect QTL locus qSe.C07 of selenium efficient property of rape, wherein the molecular marker is an SNP marker and is positioned at 36436561 th base on a C07 chromosome of a published Darmor-bzh v10 genome of brassica napus.
The invention further aims to provide an application of a molecular marker closely linked with a main effect QTL locus qSe.C07 of the selenium-rich efficient property of the rape, and screening and breeding of the selenium-rich capability of the rape can be realized by detecting the 36436561 th base on the C07 th chromosome of the Darmor-bzh v10 genome of the cabbage type rape.
The final aim of the invention is to provide an application of a molecular marker closely linked with the main effect QTL locus qSe.C07 of the selenium efficient property of the rape in combination with other molecular markers.
In order to achieve the above object, the present invention adopts the following technical measures:
obtaining a main effect QTL locus qSe.C07 of the selenium of the rape:
(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) The marker heterozygosity (heterozygous rate), loss rate (transmission 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 relative relationship between 327 parts of brassica napus germplasm resources was calculated using SPAGeDi software (Hardy and Vekemans, 2002).
(4) 327 parts of materials are respectively planted in Yang Luo test bases of the national academy of agricultural sciences in 2018 and in Yang Luo test bases of the national academy of agricultural sciences in 2019 and in Wuhan test bases of the national academy of agricultural sciences in 2020 and in 9, 3 repeats are respectively arranged in each point test, 15cm rape seed samples are collected in the period from the growth of rape to the bud period (about 40cm in height), and the selenium content of 327 parts of rape seed lines is measured. 5 strains of material were randomly selected for each sample and crushed uniformly, and the total selenium content was determined by hydride atomic fluorescence spectrometry (GB 5009.93-2017).
(5) The association analysis is carried out by using TASSEL 5.0 software (Bradbury et al, 2007) in combination with the selenium content data, genotype data and population structure of 4 test points in 3 years, the SNP marker Bn-scaff_15754_1-p1435843 which is obviously associated with the selenium content of the rape is detected on a C07 chromosome, the detection can be repeated under a plurality of environments, the highest possible interpretation of the phenotype variation is 12.2%, the obvious level is 9.61E-06, the SNP variation site (variation from A to G) is positioned at 36436561 bases of a C07 genome of the Darmor-bzh v10 (Rousseau-Gueutein et al, 2020), and the selenium high-efficiency property main effect QTL site closely linked with the SNP site is named as qSe.C07.
The application of the reagent for detecting 36436561 th base on C07 chromosome of the Darmor-bzhv 10 genome of brassica napus in screening and breeding of selenium enrichment capability of brassica napus belongs to the protection scope of the invention.
The application of the reagent for detecting the rape sequence containing 36436561 th base on the C07 chromosome of the Darmor-bzh v10 genome of the brassica napus in screening and breeding of selenium enrichment capability of the rapes 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 36436561 th base on the C07 chromosome of the Darmor-bzh v10 genome of the brassica napus in screening and breeding of selenium enrichment capability of the brassica napus also belongs to the protection scope of the invention.
In the above application, the applicant developed the KASP marker SeC07J of its antisense strand based on the above SNP site, and designed primers based on this marker were:
qSe.C07 low selenium enrichment allele specific primer SeC07J-F1: AAAACTAATGAGTGCAATAGAACACTTT
qSe.C07 high selenium enrichment capacity allele specific primer SeC07J-F2: AAAACTAATGAGTGCAATAGAACACTTC
Reverse primer SeC07J-R: TTGACTAACATGAAAAACACATCACAT.
The primers need to be added with universal joints marked by KASP before use according to the rule of KASP marking development.
In the application, the detection is combined with the detection of 11408528 th base on the C03 chromosome of the Darmor-bzh v10 genome of the brassica napus, so that the selenium enrichment capacity screening breeding efficiency of the brassica napus can be improved.
Compared with the prior art, the invention has the following advantages:
(1) The invention obtains the main effect QTL locus qSe.C07 obviously related to the high-efficiency character of the rape selenium for the first time, can explain the phenotype variation of 12.2% at the highest, can be repeatedly detected in a plurality of environments, and can be effectively applied to the genetic improvement of the high-efficiency character of the selenium of the rape.
(2) The first research discovers that the molecular marker SeC07J which is obviously related to the high-efficiency character of the rape selenium provides a reliable molecular marker source for the high-efficiency pre-selection of the rape selenium.
(3) The molecular marker SeC07J can be used for rapidly selecting excellent allelic variation of qSe.C07 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 efficient breeding process of rape selenium is accelerated.
(4) The combination of the molecular marker SeC07J and the molecular marker SeC03J can further improve the screening breeding efficiency of the selenium enrichment capability of the rape.
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 referenced to Darmor-bzh v10 (Rousseau-Gueutein et al 2020).
Example 1:
obtaining a main effect QTL locus qSe.C07 of the selenium high-efficiency property of the rape:
(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) The marker heterozygosity (heterozygous rate), loss rate (transmission 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 relative relationship between 327 parts of brassica napus germplasm resources was calculated using SPAGeDi software (Hardy and Vekemans, 2002).
(4) 327 parts of materials are respectively planted in Yang Luo test bases of the national academy of agricultural sciences in 2018 and in Yang Luo test bases of the national academy of agricultural sciences in 2019 and in Wuhan test bases of the national academy of agricultural sciences in 2020 and in 9, 3 repeats are respectively arranged in each point test, 15cm rape seed samples are collected in the period from the growth of rape to the bud period (about 40cm in height), and the selenium content of 327 parts of rape seed lines is measured. 5 strains of material were randomly selected for each sample and crushed uniformly, and the total selenium content was determined by hydride atomic fluorescence spectrometry (GB 5009.93-2017).
(5) The association analysis is carried out by using TASSEL 5.0 software (Bradbury et al, 2007) in combination with the selenium content data, genotype data and population structure of 4 test points in 3 years, the SNP marker Bn-scaff_15754_1-p1435843 which is obviously associated with the selenium content of the rape is detected on a C07 chromosome, the detection can be repeated under a plurality of environments, the highest possible interpretation of the phenotype variation is 12.2%, the obvious level is 9.61E-06, the SNP variation site (variation from A to G) is positioned at 36436561 bases of a C07 genome of the Darmor-bzh v10 (Rousseau-Gueutein et al, 2020), and the selenium high-efficiency property main effect QTL site closely linked with the SNP site is named as qSe.C07.
At the same time, SNP markers seq-new-rs42200 which are obviously related to the selenium content of the rape are detected on a C03 chromosome, the SNP markers can be repeatedly detected under a plurality of environments, the highest possible phenotypic variation of 10.6% can be explained, the obvious level is 6.24E-06, the SNP variation site (variation from C to T) is positioned at the 11408528 th base of the C03 chromosome of the Darmor-bzhv 10 (Rousseau-Gueutein et al 2020) genome of the cabbage type rape, and the selenium high-efficiency property main effect QTL site closely linked with the SNP site is named as qSe.C03.
Example 2:
obtaining a molecular marker primer closely linked with a selenium efficient main effect QTL locus qSe.C07:
(1) Extracting 100bp sequences of the upstream and downstream of 36436561 bases of the C07 chromosome of brassica napus, developing a KASP molecular marker SeC07J for the antisense strand according to the design principle of a KASP (Kompetitive Allele-Specific PCR, i.e. competitive allele-Specific PCR) molecular marker, wherein the marker comprises two competitive forward primers SeC07J-F1 and SeC07J-F2, complementary sequence bases T and C corresponding to SNP mutation sites respectively, and a reverse universal primer SeC07J-R, and the primer sequences are as follows:
SeC07J-F1：AAAACTAATGAGTGCAATAGAACACTTT
SeC07J-F2：AAAACTAATGAGTGCAATAGAACACTTC
SeC07J-R：TTGACTAACATGAAAAACACATCACAT
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 adaptor added before SeC07J-F1 is GAAGGTGACCAAGTTCATGCT, seC J-F2 and the sequence of the adaptor added before SeC07J-F2 is GAAGGTCGGAGTCAACGGATT.
The amplified sequence in brassica napus 74273 (CN 110476744 a) is genotype a (i.e., TT genotype), and the sequence is as follows: AAAACTAATGAGTGCAATAGAACACTTTTGGTGGAATAGTGACGTGACGAAAAACATGTGATGTGTTTTTCATGTTAGTCAA (SEQ ID NO. 1).
The sequence amplified in brassica napus 74296 (CN 110476744 a) is genotype B (i.e., genotype CC), and the sequence is shown below: AAAACTAATGAGTGCAATAGAACACTTCTGGTGGAATAGTGACGTGACGAAAAACATGTGATGTGTTTTTCATGTTAGTCAA (SEQ ID NO. 2).
(2) Extracting 100bp sequences of the 11408528 th base upstream and downstream of the brassica napus C03 chromosome, developing a KASP molecular marker SeC03J according to the design principle of a KASP (Kompetitive Allele-Specific PCR, i.e. competitive allele-Specific PCR) molecular marker, wherein the marker comprises two competitive forward primers SeC03J-F1 and SeC03J-F2, which correspond to SNP mutation C and T bases respectively, and a reverse universal primer SeC03J-R, and the primer sequences are as follows:
SeC03J-F1：CAGTGATGCTCAAACCAACTTCAC
SeC03J-F2：CAGTGATGCTCAAACCAACTTCAT
SeC03J-R：AAGTGTTAGGTTTGGATTCTGTATAGTG
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 adaptor added before SeC03J-F1 is GAAGGTCGGAGTCAACGGATT, seC J-F2 and the sequence of the adaptor added before SeC03J-F2 is GAAGGTGACCAAGTTCATGCT.
The amplified sequence in brassica napus 74273 is genotype a (i.e., genotype CC), and the sequence is shown below: CAGTGATGCTCAAACCAACTTCACATACACTATACAGAATCCAAACCTAACACTT (SEQ ID NO. 3).
The amplified sequence in brassica napus 74296 is genotype B (i.e., TT genotype) and is shown below: CAGTGATGCTCAAACCAACTTCATATACACTATACAGAATCCAAACCTAACACTT (SEQ ID NO. 4).
(3) Genotyping the markers in the rape-associated population by adopting a competitive allele-specific PCR technology, and using a five-primer amplified blocked mutation system (PAMS) as an amplification kit, designing a 10uL reaction system according to the instruction of a PAMS pro SNP Gentyping PCR mix kit: 2 XPARMS master mix 5. Mu.L, allle X primer (10. Mu.M) 0.15. Mu.L, allle Y primer (10. Mu.M) 0.15. Mu.L, common R primer (10. Mu.M) 0.4. Mu.L, rape genomic DNA 10-100ng. 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 SeC07J is obviously associated with the major QTL locus qSe.C07 of the selenium efficient property of the rape.
By using the method, the obvious association between the SeC03J mark and the major QTL locus qSe.C03 of the selenium efficient property of the rape is clarified.
Example 3:
the application of the primer designed based on 36436561 th base of rape C07 chromosome in screening and breeding rape selenium high-efficiency characteristics comprises the following steps:
(1) 32 parts of materials with high selenium content and low selenium content, which are homozygous through multi-generation selfing, and 32 parts of brassica napus 74273 and 74296 materials are selected, and planted in a male patrol test base of oil crop institute of China academy of agricultural sciences in 9 months in 2020, wherein the selenium content in the soil of the base is about 0.163mg/kg, 3 repeats are arranged for each material, the materials are sampled in the bud period, and the total selenium content of a sample is determined by utilizing a hydride atomic fluorescence spectrometry (GB 5009.93-2017).
(2) Examination of the distribution of the two genotypes of the molecular marker SeC07J in the above-mentioned materials having a high selenium content and a low selenium content revealed that the genotype of the molecular marker SeC07J was A in 7 parts, B in 25 parts, and A in 19 parts and B in 13 parts in 32 parts of the materials having a low selenium content (Table 1).
(3) The T test result shows that the genotypes A and B detected by the molecular marker SeC07J have extremely obvious difference (P < 0.01) in the selenium content of the rape bolt.
The above results are sufficient to indicate that the prepared molecular marker SeC07J is highly correlated with the selenium content of the brassica napus, so that the method can be used for molecular marker assisted selection of the high-efficiency character of the brassica napus selenium.
Example 4:
application of primers designed based on 36436561 th base of rape C07 chromosome and other site primers in screening and breeding of rape selenium high-efficiency characters:
(1) The material obtained in example 3 was tested using the molecular marker SeC03J and the results were as follows: the genotype of the molecular marker SeC03J was A in 4 parts and B in 28 parts in 32 parts of the material having a high selenium content, and A in 22 parts and B in 10 parts in 32 parts of the material having a low selenium content (Table 1). The T test result shows that the genotypes A and B detected by the molecular marker SeC03J have extremely obvious difference (P < 0.01) in the selenium content of the rape bolt.
(2) After the combination of SeC03J and SeC07J markers, 22 parts of the material having the B genotype for each of SeC03J and SeC07J, 17 parts of the material having the B genotype for SeC03J and the A genotype for SeC07J, 18 parts of the material having the A genotype for SeC03J and the B genotype for SeC07J, and 9 parts of the material having the A genotype for each of SeC03J and SeC07J were obtained in example 3. The T test result shows that the molecular markers SeC03J and SeC07J are B genotype materials, which are extremely higher than other three types of materials (P < 0.01) in the selenium content of the brassica napus.
The above results are sufficient to illustrate that the prepared molecular markers SeC07J and SeC03J can be used for improving the selenium enrichment capacity of the rape after being combined, so that the molecular markers can be combined for the auxiliary selection of the selenium efficient property of the rape.
Table 1: genotype of molecular markers SeC03J and SeC07J in selenium content extreme materials of brassica napus
Sequence listing
<110> institute of oil crop and oil crop at national academy of agricultural sciences
<120> molecular marker closely linked with main effect QTL locus qSe.C07 of rape selenium high-efficiency character and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aaaactaatg agtgcaatag aacacttttg gtggaatagt gacgtgacga aaaacatgtg 60
atgtgttttt catgttagtc aa 82
<210> 2
<211> 82
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aaaactaatg agtgcaatag aacacttctg gtggaatagt gacgtgacga aaaacatgtg 60
atgtgttttt catgttagtc aa 82
<210> 3
<211> 55
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cagtgatgct caaaccaact tcacatacac tatacagaat ccaaacctaa cactt 55
<210> 4
<211> 55
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cagtgatgct caaaccaact tcatatacac tatacagaat ccaaacctaa cactt 55
<210> 5
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
aaaactaatg agtgcaatag aacacttt 28
<210> 6
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aaaactaatg agtgcaatag aacacttc 28
<210> 7
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ttgactaaca tgaaaaacac atcacat 27
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
cagtgatgct caaaccaact tcac 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cagtgatgct caaaccaact tcat 24
<210> 10
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
aagtgttagg tttggattct gtatagtg 28
<210> 11
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gaaggtcgga gtcaacggat t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gaaggtgacc aagttcatgc t 21
Claims (4)
1. Application of primer designed aiming at 36436561 base on C07 chromosome of brassica napus Darmor-bzh v10 genome in screening and breeding of selenium enrichment capability of rape.
2. The use according to claim 1, wherein the primers are: seC07J-F1: AAAACTAATGAGTGCAATAGAACACTTT, seC07J-F2: AAAACTAATGAGTGCAATAGAACACTTC and SeC07J-R: TTGACTAACATGAAAAACACATCACAT.
3. Application of primer designed aiming at 36436561 th base on C07 chromosome of brassica napus Darmor-bzh v10 genome combined with primer designed on 11408528 th base on C03 chromosome in screening and breeding of selenium enrichment capability of rape.
4. The use according to claim 3, wherein the primers are: seC07J-F1: AAAACTAATGAGTGCAATAGAACACTTT, seC07J-F2: AAAACTAATGAGTGCAATAGAACACTTC and SeC07J-R: TTGACTAACATGAAAAACACATCACAT; seC03J-F1: CAGTGATGCTCAAACCAACTTCAC, seC03J-F2: CAGTGATGCTCAAACCAACTTCAT and SeC03J-R: AAGTGTTAGGTTTGGATTCTGTATAGTG.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111087529.8A CN113652499B (en) | 2021-09-16 | 2021-09-16 | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111087529.8A CN113652499B (en) | 2021-09-16 | 2021-09-16 | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113652499A CN113652499A (en) | 2021-11-16 |
| CN113652499B true CN113652499B (en) | 2023-06-09 |
Family
ID=78483772
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111087529.8A Active CN113652499B (en) | 2021-09-16 | 2021-09-16 | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113652499B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115812541B (en) * | 2021-09-17 | 2024-08-09 | 中国农业科学院油料作物研究所 | Production method and application of plant source ultrahigh organic selenium |
| CN114752702B (en) * | 2022-05-25 | 2023-08-11 | 中国农业科学院油料作物研究所 | Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof |
| CN114854896B (en) * | 2022-05-25 | 2024-07-05 | 中国农业科学院油料作物研究所 | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application |
| CN114990251B (en) * | 2022-05-25 | 2024-07-16 | 中国农业科学院油料作物研究所 | Molecular marker closely linked with rape methylselenocysteine content trait QTL and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6399855B1 (en) * | 1997-12-22 | 2002-06-04 | Pioneer Hi-Bred International, Inc. | QTL mapping in plant breeding populations |
| CN107002144A (en) * | 2014-12-18 | 2017-08-01 | 美国陶氏益农公司 | The identification of the SNP marker of marker assisted selection for these characters of fiber content and the related QTL of kernel seed coat colour the character finely positioning with checking and from yellow seed coat (YSC) Canola system YN01 429 and its pedigree |
| CN110476744A (en) * | 2019-09-18 | 2019-11-22 | 中国农业科学院油料作物研究所 | Floral leaf character is increasing the application on rape group child's hair twisted in a knot-childhood pericarp area |
| CN110527738A (en) * | 2019-08-28 | 2019-12-03 | 中国农业科学院油料作物研究所 | Main effect QTL site, SNP marker and its application of cabbage type rape seed oleic acid content |
| CN110592251A (en) * | 2019-06-17 | 2019-12-20 | 中国农业科学院油料作物研究所 | Development and application of major QTL (quantitative trait locus) and SNP (Single nucleotide polymorphism) molecular marker for flowering phase characters of brassica napus |
-
2021
- 2021-09-16 CN CN202111087529.8A patent/CN113652499B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6399855B1 (en) * | 1997-12-22 | 2002-06-04 | Pioneer Hi-Bred International, Inc. | QTL mapping in plant breeding populations |
| CN107002144A (en) * | 2014-12-18 | 2017-08-01 | 美国陶氏益农公司 | The identification of the SNP marker of marker assisted selection for these characters of fiber content and the related QTL of kernel seed coat colour the character finely positioning with checking and from yellow seed coat (YSC) Canola system YN01 429 and its pedigree |
| CN110592251A (en) * | 2019-06-17 | 2019-12-20 | 中国农业科学院油料作物研究所 | Development and application of major QTL (quantitative trait locus) and SNP (Single nucleotide polymorphism) molecular marker for flowering phase characters of brassica napus |
| CN110527738A (en) * | 2019-08-28 | 2019-12-03 | 中国农业科学院油料作物研究所 | Main effect QTL site, SNP marker and its application of cabbage type rape seed oleic acid content |
| CN110476744A (en) * | 2019-09-18 | 2019-11-22 | 中国农业科学院油料作物研究所 | Floral leaf character is increasing the application on rape group child's hair twisted in a knot-childhood pericarp area |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113652499A (en) | 2021-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113736903B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C03 and application | |
| CN113652499B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C07 and application | |
| CN107201404B (en) | Molecular biological identification method for sex of asparagus hermaphrodite plants and application thereof | |
| CN110157829B (en) | Molecular marker SNPA9-5 associated with thousand seed weight of rape and application | |
| CN110184373B (en) | Molecular marker associated with thousand seed weight of rape and application thereof | |
| CN109929945B (en) | Molecular marker BrSF2604 primer of main effect QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| CN113637789A (en) | Wheat stripe rust resistant gene YRTD121 linked KASP molecular marker, primer, kit and application | |
| CN112391488A (en) | SNP marker for identifying broccoli variety Zhe Qing 80 | |
| CN110305980B (en) | Breeding method and application of anti-clubroot high-oleic-acid rape | |
| CN113584219B (en) | Molecular marker closely linked with rape selenium efficient property main effect QTL locus qSe.C04 and application | |
| CN116926229A (en) | Molecular marker closely linked with high-vitamin E main effect QTL locus qVE.C02 of rape seed and application | |
| CN112029898B (en) | SNP marker for identifying broccoli variety Zhe Qing 100 | |
| CN114752702B (en) | Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof | |
| CN117737279A (en) | Cadmium low-accumulation hybrid rice molecular marker, rice mutant OsNramp5 gene, and identification method, application and primer thereof | |
| CN116790797A (en) | KASP primer group related to wheat grain weight and application thereof | |
| CN113564161B (en) | Molecular marker closely linked with bacterial wilt resistance of cultivated peanut and application thereof | |
| CN112080580B (en) | SNP marker for identifying broccoli variety Zhe Qing 60 | |
| CN101942512B (en) | Development and application of molecule marker for corn with considerable number of kernels and excellent allele function in low-nitrogen adverse environment | |
| CN114525360A (en) | SNP marker closely linked with green fruit color Gv1 gene of eggplant and application | |
| CN114854896B (en) | Molecular marker BnMes-2C1 closely linked with rape methyl selenocysteine content trait QTL and application | |
| CN114990251B (en) | Molecular marker closely linked with rape methylselenocysteine content trait QTL and application thereof | |
| CN114774578B (en) | Molecular marker BnMg-1A1 closely linked with rape magnesium content trait QTL and application thereof | |
| CN108265124B (en) | Molecular marker associated with rape root surface area and application thereof | |
| Malaviya et al. | Genetic diversity and lineage based on SSR markers of two genomic resources among trifolium collections held within the australian pastures Genebank | |
| CN115812541B (en) | Production method and application of plant source ultrahigh organic selenium |
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 |