CN116606950B - Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape - Google Patents
Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape Download PDFInfo
- Publication number
- CN116606950B CN116606950B CN202310482113.9A CN202310482113A CN116606950B CN 116606950 B CN116606950 B CN 116606950B CN 202310482113 A CN202310482113 A CN 202310482113A CN 116606950 B CN116606950 B CN 116606950B
- Authority
- CN
- China
- Prior art keywords
- rape
- qsw
- parms
- thousand
- seed weight
- 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
- 239000003550 marker Substances 0.000 title abstract description 9
- 239000003147 molecular marker Substances 0.000 title abstract description 7
- 210000000349 chromosome Anatomy 0.000 claims abstract description 32
- 238000009395 breeding Methods 0.000 claims abstract description 15
- 230000001488 breeding effect Effects 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 description 15
- 238000001514 detection method Methods 0.000 description 12
- 239000000463 material Substances 0.000 description 8
- 235000006008 Brassica napus var napus Nutrition 0.000 description 7
- 240000000385 Brassica napus var. napus Species 0.000 description 7
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 7
- 108700028369 Alleles Proteins 0.000 description 6
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 6
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 240000002791 Brassica napus Species 0.000 description 5
- 238000012098 association analyses Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 235000011293 Brassica napus Nutrition 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 244000208060 Lawsonia inermis Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 102000054766 genetic haplotypes Human genes 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002689 soil Substances 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 field of biological breeding, in particular to application of a rape thousand seed weight associated PARMS molecular marker or marker combination, and applicant screens two thousand seed weight associated sitesqSW.A9‑4AndqSW.C2‑2the peak SNP markers Bn-A09-p6574470 and Bn-scaff_15712_2-p365609 of the rape A09 chromosome are located at base 38,530,546 of the 6,430,156 and C2 chromosomes respectively, which on average can explain the phenotypic variances of 10.5% and 11.5% with an average effect of 0.19 and 0.38 g. The PARMS markers developed by the two SNP are used for detecting the rape related population, the operation is simple and convenient, the typing is clear, the difference value of thousand grain weight of the two homozygous genotypes is 0.22 g and 0.40 g respectively, and the combination of the two homozygous genotypes is up to 0.51 g, so that the rape related population has good selection effect.
Description
Technical Field
The invention belongs to the technical field of biological breeding, and particularly relates to application of a PARMS molecular marker or a marker combination associated with thousand seed weight of rape.
Background
Under the same planting density condition, the single yield of rape depends on the single plant yield, and the single plant yield is directly determined by three constituent factors of the number of the whole plant fruits, the number of grains per corner and the grain weight. Although there is a negative correlation to some extent between the three components of rape yield, the coefficients are often not large, and thus yield can be increased by increasing the yield components (e.g. grain weight). The characteristic data of the yield of the winter rape registered varieties in the countries of 2005-2013 show that the yield of the rape is increased by 14.9% from 2490.99 kg/hectare in 2005 to 2861.25 kg/hectare in 2013; the thousand grain weight is increased from 3.46 g to 3.85 g, the amplification is 11.2%, the number of grains per corner is from 20.08 grains to 21.13 grains, and the amplification is 5.2%; whereas the whole plant silique showed a significant decrease in the number (from an average angle of 387.45 to 280.52 per plant) by 27.6% (Hu et al, 2017). Therefore, from the practical aspect of breeding, the increase of rape unit yield can be realized by improving the constitution factors such as grain weight. At present, the thousand seed weight of rape varieties is generally lower than 4g, and the maximum value of the thousand seed weight in rape germplasm resources can exceed 8 g (Chen Wei and the like, 2011), which indicates that the rape seed weight still has a large lifting space.
With the rapid development of modern biotechnology, the molecular marker technology is also updated and iterated continuously, and the initial RFLP, RA PD/AFLP and SSR are gradually developed into the current SNP markers. The current SNP detection methods are mainly divided into two main categories: one general class is the traditional classical detection methods based on gel electrophoresis, represented by single-strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DDGE), cleaved Amplified Polymorphic Sequences (CAPS), allele-specific PCR (AS-PCR), etc.; another major class is high throughput, high degree of automation detection methods represented by direct sequencing, DNA chips, denaturing High Performance Liquid Chromatography (DHPLC), mass spectrometry detection techniques, high resolution dissolution profiles (HRM), and the like. The two methods have advantages and disadvantages, and detection methods combining the two methods, such as KASP (Lister et., 2013) and PARMS (Lu et al, 2020) are available at present, so that the method is simple to operate, low in cost and capable of realizing high-flux detection.
Grain weight is a complex quantitative trait typically controlled by multiple genes, and phenotypes are continuously distributed and susceptible to environmental conditions. The inheritance of grain weight is mainly additive, dominant and superior, so that its heterosis is weak, and the grain weight of hybrid F1 is generally between parents (Li Na, 2015). The combination of quantitative genetics and molecular marker technology can break down complex quantitative traits into individual quantitative trait loci (quantitative trait loci, QTL) and then study multiple genes that control quantitative traits as quality traits. QTL localization is based on genetic segregating populations, and quantitative trait phenotype data of segregating populations are analyzed by means of QTL mapping software by means of molecular markers and genetic maps, so that the position and effect of quantitative trait genes on chromosomes are determined. Currently, with the methods of linkage mapping (linkage ma mapping) and association mapping (association mapping), there are a limited number of QTLs that work in different populations and environments, but it is difficult to meet practical breeding requirements, when rape seeds have been mapped to more than one hundred thousand grain weight QTLs (quejada et al, undell et al, 2006;Radoev et al, 2008, shi et al, 2009, wang Feng et al, 2010;Basunanda et al, 2010, fan et al, 2010, zhang et al, 2011, zhao Weiguo et al, 2017).
The invention utilizes thousand seed weight data of ten environments of rape natural population to carry out whole genome association analysis, aims at finding a new stable site with improvement effect on thousand seed weight of rape, develops a practical molecular marker with high flux and low cost for auxiliary selection according to the new stable site, and lays a foundation for subsequent whole genome selection breeding.
Disclosure of Invention
The invention aims at providing SNP molecular markers Bn-A09-p6574470 of a locus qSW.9-4 which is obviously associated with grain weight on a rape A09 chromosome, wherein the SNP is 6,430,156 th base of a rape DarmorV4.1 reference genome.
Another object of the present invention is to provide a combination of molecular markers Bn-A09-p6574470 and Bn-scaff_15712_2-p365609, which are related to thousand seed weight of rape, and are located at 6,430,156 th base and 38,530,546 th base of chromosome C02 of the A09 reference genome of DarmorV4.1 of rape, respectively.
It is a further object of the present invention to provide a PARMS-tagged primer directed against the above molecular tag or combination of molecular tags.
The final object of the invention is to provide the application of PARMS marked primer in rape thousand seed weight selection breeding. In order to achieve the above object, the present invention adopts the following technical measures:
acquisition of SNP loci related to thousand seed weight of rape:
(1) Total DNA of 331 lines of the canola associated population was extracted and genotyping was performed on each sample using a canola 60K SNP chip (Li et al 2020).
(2) The deletion rate (transmission rate), heterozygosity rate (heterozygous rate), secondary allele frequency (m inor allele frequency) of the population material at each site was calculated using Illumina BeadStudio genotyping software (http:// www.illumina.com /).
(3) And (3) planting 331 strains of the related population in 10 environments respectively, picking 10 single plants with uniform growth vigor in each cell in the mature period, harvesting, threshing the single plants respectively, sun-drying seeds for thousand-grain weight investigation, and calculating the average value of each strain.
(4) Full genome association analysis was performed using the high density SNP genotype data of the associated population, thousand grain weight phenotype data of 10 environments, population structure and kindred relationship using TASSEL 5.0 software (braddury et al, 2007). Finally, the sites qSW.A9-4 and qSW.C2-2 with obvious correlation with the grain weight are obtained, the peak SNP markers Bn-A09-p6574470 and Bn-scaff_15712_2-p365609 are respectively positioned at the 38,530,546 base of the A9 chromosome 6,430,156 and the 38,530,546 chromosome of the reference genome A9 of rape DarmorV4.1, and can be repeatedly detected in two environments and two models.
(5) Extracting the sequence of 100bp on the upstream and downstream of 6,430,156 base of the rape A09 chromosome, and obtaining a primer sequence according to the PARMS primer design principle: qSW.A9-4F: TTTTGTGTCAATTCTCTTACAACCC; qSW.A9-4Ra:GAAGGT GACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA；qSW.A9-4R g：GAAGGTCGGAGTCAACGGATTCCCGTATAACCTTGCAAAAAAGTG。
extracting the sequence of 100bp on the upstream and downstream of 38,530,546 th base of rape C02 chromosome, and obtaining a primer sequence according to the PARMS primer design principle: qSW.C2-2F: AAGGGTGTGTGACTTTCAAAATG; qSW.C2-2Ra:GAAGGTGACC AAGTTCATGCTCGGGTACCCGAACGCCCAGA；qSW.C2-2Rg：GAAGG TCGGAGTCAACGGATTCGGGTACCCGAACGCCCAGG。
the protection content of the invention comprises:
the application of a system for detecting 6,430,156 th base of a chromosome A09 of a rape DarmorV4.1 reference genome in rape thousand seed weight selection breeding is disclosed, wherein the system is a reagent, a kit or an instrument.
Rape thousand seed weight selection breeding's device, its characterized in that: the device comprises a data receiving module and a data processing module, wherein the data receiving module is configured to receive the genotype of 6,430,156 bases of a chromosome A09 of a rape DarmorV4.1 reference genome to be tested; the data processing module is used for converting the genotype from the receiving module into a judging result of the rape to be detected.
A computer readable storage medium for thousand seed weight selection breeding of canola, characterized in that: the computer readable storage medium causes a computer to execute the steps of: detecting the genotype of 6,430,156 th basic group of the DarmorV4.1 reference genome A09 chromosome of the rape to be detected, and converting the genotype into a judging result for judging the rape to be detected.
In the application, the reagent is a primer for detecting 6,430,156 base of the A09 chromosome of the DarmorV4.1 reference genome of rape.
In the application, the primer is as follows: qSW.A9-4F: TTTTGTGTCAATTCTCTTACAACC C; qSW.A9-4Ra: GAAGGTGACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA; qSW.A9-4Rg:GAAGGTCG GAGTCAACGGATTCCCGTATAACCTTGCAAAAAAGTG。
in the above application, the kit is a PARMS detection kit.
In the above application, when the genotype of the rape to be detected is AA, plants with higher thousand grain weight are judged, and when the genotype of the rape to be detected is GG, plants with lower thousand grain weight are judged.
The protection scope of the invention also comprises:
the application of a system for detecting 6,430,156 th base and 38,530,546 th base of a chromosome A09 and a chromosome C02 of a reference genome A09 of rape in thousand seed weight selection breeding of rape is disclosed, wherein the system is a reagent, a kit or an instrument.
Rape thousand seed weight selection breeding's device, its characterized in that: the device comprises a data receiving module and a data processing module, wherein the data receiving module is configured to receive genotypes of 6,430,156 th basic group and 38,530,546 th basic group of C02 chromosome of a reference genome A09 of rape DarmorV4.1 to be detected; the data processing module is used for converting the genotype from the receiving module into a judging result of the rape to be detected.
A computer readable storage medium for thousand seed weight selection breeding of canola, characterized in that: the computer readable storage medium causes a computer to execute the steps of: detecting genotypes of 6,430,156 th basic group and 38,530,546 th basic group of the A09 chromosome and the C02 chromosome of the DarmorV4.1 reference genome of the rape to be detected, and converting the genotypes into a judging result for judging the rape to be detected.
In the application, the reagent is a primer for detecting 6,430,156 th base of the A09 chromosome and 38,530,546 th base of the C02 chromosome of the rape DarmorV4.1 reference genome.
In the application, the primer is qSW.A9-4F: TTTTGTGTCAATTCTCTTACAACCC, qSW.A9-4Ra:GAAGGTGACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA and q SW.A9-4Rg: GAAGGTCGGAGTCAACGGATTCCCGTATAACCTTGCAAAAAAGTG; qSW.C2-2F: AAGGGTGTGTGACTTTCAAAATG, qSW.C2-2Ra:GAAGGTGACCAAGTT CATGCTCGGGTACCCGAACGCCCAGA and qSW.C2-2Rg:GAAGGTCGGAGTCAACGGA TTCGGGTACCCGAACGCCCAGG。
in the above application, the kit is a PARMS detection kit.
In the application, when the genotype of the 6,430,156 th base of the rape A09 chromosome to be detected is AA and the genotype of the 38,530,546 th base of the C02 chromosome is GG, judging that the plants with high thousand grain weight are plants; and when the genotype of 6,430,156 th basic group of the rape A09 chromosome to be detected is GG and the genotype of 38,530,546 th basic group of the C02 chromosome is AA, judging that the rape is a plant with lower thousand grain weight.
Compared with the prior art, the invention has the following advantages:
(1) The invention obtains the locus qSW.A9-4 which is obviously related to the thousand seed weight of the rape, can be repeatedly detected in a plurality of environments, can explain 10.5% of phenotype variance on average, has an average effect of 0.19 g, and can be effectively applied to the genetic improvement of the thousand seed weight of the rape.
(2) The invention obtains the locus qSW.C2-2 which is obviously related to the thousand seed weight of the rape, can be repeatedly detected in a plurality of environments, can explain 11.5% of phenotype variance on average, has an average effect of 0.38 g, and can be effectively applied to the genetic improvement of the thousand seed weight of the rape.
(3) The invention obtains the PARMS mark obviously associated with the thousand seed weight of the rape, and the detection method has simple operation, low cost and high flux, can select the genome haplotype region obviously associated with the thousand seed weight, and improves the selection efficiency and accuracy.
Drawings
FIG. 1 is a graph showing the frequency distribution of thousand seed weight phenotypes of 331 parts of material from a canola associated population in 10 environments.
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. The Version number of the brassica napus genome used in the present invention is Darmor-bzh (Version 4.1)https://www.genoscope.cns.fr/brassicanapus/。
Example 1:
acquisition of SNP markers remarkably related to thousand seed weight of rape
(1) Leaves of 331 core lines of rape-associated groups were collected, total DNA was extracted by CTAB method, and genotype analysis was performed on each sample using a rape 60K SNP chip (Li et al 2020).
(2) The marker heterozygosity (heterozygous rate), loss rate (transmission rate), minimum allele frequency (minor a llele frequency) was calculated for each locus using Illumina BeadStudio genotyping software (http:// www.illumina.com /). Filtering SNP markers by taking 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 unique match of the SNP markers in the brassica napus genome as screening standards, and finally obtaining 24,508 high-quality SNP markers for whole genome association analysis. Group structure analysis was performed on 331 core lines using structure2.3 software (Pritchard et al, 2000), and the relationships between 331 core lines were calculated using SPAGeDi software (Hardy and Vekemans, 2002).
(3) 331 lines of the related population were planted in 2012 (W12), 2013 (Z13, W13, and N13), 2014 (Z14, W14, and N14), 2015 (W15), 2016 (W16), and 2017 (W17) at the academy of agricultural science in henna (Z), the institute of oil crops in china (Yang Luoji) and the base of red soil in the province in the west of the river (N), respectively, for a total of 10 environments. And (3) selecting 10 single plants with uniform growth vigor in each cell in the mature period, harvesting, threshing the single plants respectively, sun-drying the seeds for investigation of thousand seed weight, and calculating the average value of each plant line. By analyzing the thousand-grain weight data in each environment of the association population, the result shows that the thousand-grain weight data in all 10 environments are normally distributed and have large variation amplitude (figure 1), and the method can be used for subsequent whole genome association analysis.
(4) Full genome association analysis was performed using four models (GLM-PCA, GLM-Q, MIM-PCA+K, MLM-Q+K) of TASSEL 5.0 software (Bradbury et al 2007) in combination with genotype data and thousand grain weight phenotype data of the associated population. Sites qsw.a9-4 and qsw.c2-2 associated with thousand kernel weight were obtained on the a09 chromosome by integrating the significance-related SNP markers detected in different environments and models, which could be repeatedly detected in both environments and models, respectively, with peak SNP markers Bn-a09-P6574470 and Bn-scaff_15712_2-P365609 at base 6,430,156 of the a09 chromosome and base 38,530,546 of the C02 chromosome, with highest significance levels p=8.75e-08 and 2.14E-07, mean interpretable phenotypic variances of 10.5% and 11.5%, and mean effects of 0.19 and 0.38 grams.
Information of Table I, grain weight associated sites qSW.A9-4 and qSW.C2-2
Example 2: development of PARMS markers markedly associated with thousand kernel weight
The relevant markers obtained in example 1 were derived from SNP chips, only probe sequence information for molecular hybridization. The rape SNP chip can detect tens of thousands of sites at a time, but the operation is complicated and special equipment is needed. In addition, the detection of a large number of breeding intermediate materials by using rape SNP chips is expensive, and therefore, it is necessary to convert them into a detection method based on PCR amplification, such as PARMS (Penta-primer Amplifica tion Refractory Mutation System, five-primer amplification-blocked mutation system) markers, which is simple to operate and low in cost. The labeling system comprises a pair of fluorescent universal primers (FAM and HEX are used as report fluorescence), a pair of SNP allele specific primers and a reverse common primer, so that SNP allele detection can be rapidly and simply performed.
(1) For the peak SNP marker Bn-A09-p6574470 associated with qSW.A9-4, 100bp sequences of the 64,30,156 th base upstream and downstream of the rape A09 chromosome are extracted. The primer sequences obtained according to the PARMS primer design principle are as follows: qSW.A9-4F: TTTTGTGTCAATTCTCTTACAACCC, qSW.A9-4Ra:GAAGGTGACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA and qSW.A9-4Rg: GAAGGTCGGAGTCAACGGATT CCCGTATAACCTTGCAAAAAAGTG;
for the peak SNP marker Bn-scaff_15712_2-p365609 associated with qSW.C2-2, 100bp sequences upstream and downstream of the 38,530,546 th base of the rape A09 chromosome were extracted. The primer sequences obtained according to the PARMS primer design principle are as follows: qSW.C2-2F: AAGGGTGTGTGACTTTCAAAATG, qSW.C2-2Ra: GAAGGTGACCAAGTTCATGCTCGGGTACCCGAACGCCCAGA and qSW.C2-2Rg:GAAGGTCGGAGTCAACGGA TTCGGGTACCCGAACGCCCAGG。
(2) And (3) taking genome DNA of rape related population as a template, carrying out fluorescent quantitative PCR amplification by using the primer, scanning FAM, HEX and ROX signals by using TecanF200, outputting the results, and finally converting the results into genotypes.
Example 3: application of PARMS mark in rape thousand seed weight screening
Of 331 relevant populations (Li et al 2020), 232 parts of AA genotype materials were detected with the PARMS marker qsw.a9-4 provided in example 2 and 59 parts of GG genotype (table two). The thousand grain weight difference between the two genotypes reached a significant level in six environments, with the thousand grain weight difference between the AA and GG genotypes ranging from 0.15 to 0.34, on average 0.21 grams.
Comparison of thousand-grain weights of two genotypes of Table II and PARMS marked qSW.A9-2 in ten environments of related populations
| Genotype of the type | N13 | N14 | W12 | W13 | W14 | W15 | W16 | W17 | Z13 | Z14 |
| AA(n＝232) | 4.81 | 4.12 | 4.11 | 3.77 | 3.88 | 4.10 | 3.55 | 3.71 | 3.73 | 4.08 |
| GG(n＝59) | 4.48 | 3.95 | 3.81 | 3.62 | 3.76 | 3.99 | 3.39 | 3.67 | 3.56 | 4.01 |
| AA-GG | 0.34** | 0.18* | 0.30** | 0.15* | 0.11 | 0.11 | 0.16* | 0.05 | 0.16* | 0.07 |
* And represents significance levels p=0.05 and 0.01, respectively.
Of 331 relevant populations, 267 parts of the material with genotype AA and 53 parts with genotype GG were detected using PARMS marker qSW.C2-2 provided in example 2 (Table three). The thousand grain weight difference between the two genotypes reached a significant level in nine environments, with the thousand grain weight difference between the two genotypes GG and AA ranging from 0.23 to 0.52, with an average of 0.40 grams.
Comparison of thousand grain weights of two genotypes of table three and PARMS marked qSW.A9-3 in ten environments of related populations
| Genotype of the type | N13 | N14 | W12 | W13 | W14 | W15 | W16 | W17 | Z13 | Z14 |
| AA(n＝267) | 4.31 | 4.12 | 4.13 | 3.76 | 3.89 | 4.12 | 3.63 | 3.74 | 3.74 | 4.08 |
| GG(n＝53) | 4.66 | 4.62 | 4.48 | 4.25 | 4.23 | 4.44 | 4.16 | 3.98 | 3.87 | 4.56 |
| GG-AA | 0.35** | 0.50*** | 0.36** | 0.49*** | 0.34** | 0.32** | 0.52*** | 0.23* | 0.14 | 0.48*** |
* Sum represents significance levels p= 0.05,0.01 and 0.001, respectively.
Of 331 relevant populations, the primers of example 2 providing PARMS markers qSW.A9-4 and qSW.C2-2 were used in combination, 34 of the materials of genotype AA in qSW.A9-4 and genotype GG in qSW.C2-2, 43 of the materials of genotype GG in qSW.A9-4 and genotype AA in qSW.C2-2, and the thousand-grain weight differences of the two genotypes reached significance levels in all 10 environments. Among them, the difference in 7 environments reached an extremely significant level, ranging from 0.44 to 0.61, with an average of 0.51 g.
Comparison of thousand-grain weights of two genotypes of Table four, PARMS markers qSW.A9-2 and qSW.A9-3 in ten environments of the relevant population
* Sum represents significance levels p= 0.05,0.01 and 0.001, respectively.
The above results are sufficient to demonstrate that the PARMS markers qSW.A9-4 and qSW.C2-2 prepared by us are highly correlated with thousand seed weight of canola, and have good selection effect both alone and in combination.
Claims (2)
1. Application of primer for detecting 6,430,156 th base of chromosome A09 of rape DarmorV4.1 reference genome in rape thousand seed weight selection breeding, wherein the primer is as follows:qSW.A9-4F：TTTTGTGTCAATTCTCTTACAACCC、qSW.A9- 4ra: GAAGGTGACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA and is provided withqSW.A9-4Rg：GAAGGTCGGAGTCAACGGATTCCCGTATAACCTTGCAAAAAAGTG。
2. Application of primer for detecting 6,430,156 th base and 38,530,546 th base of rape DarmorV4.1 reference genome A09 chromosome and C02 chromosome in rape thousand seed weight selection breeding, wherein the primer is as follows:qSW.A9-4F：TTTTGTGTCAATTCTCTTACAACCC、qSW.A9-4ra: GAAGGTGACCAAGTTCATGCTCCCGTATAACCTTGCAAAAAAGTA and is provided withqSW.A9-4Rg：GAAGGTCGGAGTCAACGGATTCCCGTATAACCTTGCAAAAAAGTG；qSW.C2-2F：AAGGGTGTGTGACTTTCAAAATG、qSW.C2-2Ra: GAAGGTGACCAAGTTCATGCTCGGGTACCCGAACGCCCAGA and is provided withqSW.C2-2Rg：GAAGGTCGGAGTCAACGGATTCGGGTACCCGAACGCCCAGG。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310482113.9A CN116606950B (en) | 2023-04-29 | 2023-04-29 | Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310482113.9A CN116606950B (en) | 2023-04-29 | 2023-04-29 | Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116606950A CN116606950A (en) | 2023-08-18 |
| CN116606950B true CN116606950B (en) | 2023-11-24 |
Family
ID=87677305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310482113.9A Active CN116606950B (en) | 2023-04-29 | 2023-04-29 | Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116606950B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157829A (en) * | 2019-04-16 | 2019-08-23 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular marker SNP A9-5 of rape mass of 1000 kernel and application |
| CN110184373A (en) * | 2019-04-16 | 2019-08-30 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular labeling of rape mass of 1000 kernel and application |
| CN114182041A (en) * | 2021-12-23 | 2022-03-15 | 中国农业科学院油料作物研究所 | PARMS molecular marker associated with number of ovules per ovary of rape and application |
| CN114231654A (en) * | 2021-12-23 | 2022-03-25 | 中国农业科学院油料作物研究所 | PARMS molecular marker associated with thousand grain weight of rape and application thereof |
| WO2023058064A1 (en) * | 2021-10-07 | 2023-04-13 | National Institute Of Plant Genome Research | Pan-genome genotyping array and uses thereof |
-
2023
- 2023-04-29 CN CN202310482113.9A patent/CN116606950B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157829A (en) * | 2019-04-16 | 2019-08-23 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular marker SNP A9-5 of rape mass of 1000 kernel and application |
| CN110184373A (en) * | 2019-04-16 | 2019-08-30 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular labeling of rape mass of 1000 kernel and application |
| WO2023058064A1 (en) * | 2021-10-07 | 2023-04-13 | National Institute Of Plant Genome Research | Pan-genome genotyping array and uses thereof |
| CN114182041A (en) * | 2021-12-23 | 2022-03-15 | 中国农业科学院油料作物研究所 | PARMS molecular marker associated with number of ovules per ovary of rape and application |
| CN114231654A (en) * | 2021-12-23 | 2022-03-25 | 中国农业科学院油料作物研究所 | PARMS molecular marker associated with thousand grain weight of rape and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Identification of Putative Candidate Genes for Water Stress Tolerance in Canola(Brassica napus);Zhang J.等;《Front.Plant Sci》;第6卷;摘要，第2 页，补充表2 * |
| 甘蓝型油菜千粒重全基因组关联分析;肖小军;《中国油料作物学报》;第45卷(第3期);第510-517页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116606950A (en) | 2023-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114231654B (en) | PARMS molecular marker associated with thousand grain weight of rape and application thereof | |
| CN109825621B (en) | Wheat spikelet number QTL (quantitative trait locus) linked SNP (single nucleotide polymorphism) molecular marker and application thereof | |
| CN101970669B (en) | Gray leaf spot tolerant maize and methods of production | |
| CN110157829B (en) | Molecular marker SNPA9-5 associated with thousand seed weight of rape and application | |
| CN110295251B (en) | SNP molecular marker linked with wheat effective tillering number QTL and application thereof | |
| CN110184373B (en) | Molecular marker associated with thousand seed weight of rape and application thereof | |
| CN111893209B (en) | Indel site detection marker related to thousand grain weight of wheat and application thereof | |
| CN114182041B (en) | PARMS molecular marker related to number of ovules of rape per ovary and application | |
| CN109762926B (en) | Molecular marker primer related to rape pod number and application | |
| CN115029465A (en) | KASP and dCAPS markers cosegregating with rape seed secondary dormancy major QTL and application thereof | |
| CN105603083B (en) | Method for carrying out corn-assisted efficient breeding by using two molecular markers including SSR (simple sequence repeat) and SNP (Single nucleotide polymorphism) | |
| CN116926229A (en) | Molecular marker closely linked with high-vitamin E main effect QTL locus qVE.C02 of rape seed and application | |
| CN116790797A (en) | KASP primer group related to wheat grain weight and application thereof | |
| CN116606950B (en) | Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.9-4 of rape | |
| CN109022432B (en) | Method for identifying wheat tillering angle character and special primer group thereof | |
| CN116555474B (en) | Application of rape thousand seed weight-related PARMS molecular markers or marker combinations | |
| CN116574833B (en) | Application of PARMS molecular marker or marker combination of rape thousand seed weight associated site qSW.A1-2 | |
| CN116555476B (en) | Application of PARMS molecular marker or marker combination of thousand seed weight associated locus qSW.C3-3 of rape | |
| CN107287210B (en) | Rice appearance quality gene qAQ7 and molecular marking method and application thereof | |
| CN106399495B (en) | SNP marker closely linked with soybean short stalk character and application thereof | |
| CN112080580B (en) | SNP marker for identifying broccoli variety Zhe Qing 60 | |
| CN114703316A (en) | Development and application of KASP marker of tomato ps-2 gene | |
| Yamada et al. | Genetic analysis of forage grasses based on heterologous RFLP markers detected by rice cDNAs | |
| CN112760399B (en) | Major QTL site for controlling grain length of wheat grains, KASP primer closely linked with major QTL site and application of KASP primer | |
| CN116640878B (en) | Molecular marker of wheat floret number QTL per spike developed based on novel mode of multi-single-combined-standard-evidence 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 |