CN112048567B - Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof - Google Patents
Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof Download PDFInfo
- Publication number
- CN112048567B CN112048567B CN202011052970.8A CN202011052970A CN112048567B CN 112048567 B CN112048567 B CN 112048567B CN 202011052970 A CN202011052970 A CN 202011052970A CN 112048567 B CN112048567 B CN 112048567B
- Authority
- CN
- China
- Prior art keywords
- hybrid
- pol
- cms
- primer
- purity
- 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 claims abstract description 74
- 230000035558 fertility Effects 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 18
- 240000002791 Brassica napus Species 0.000 claims description 53
- 235000011293 Brassica napus Nutrition 0.000 claims description 53
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 241000196324 Embryophyta Species 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 37
- 238000012408 PCR amplification Methods 0.000 claims description 36
- 239000000499 gel Substances 0.000 claims description 29
- 238000003384 imaging method Methods 0.000 claims description 29
- 239000011259 mixed solution Substances 0.000 claims description 26
- 238000001962 electrophoresis Methods 0.000 claims description 23
- 240000007124 Brassica oleracea Species 0.000 claims description 19
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 19
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 claims description 19
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 19
- 239000011543 agarose gel Substances 0.000 claims description 17
- 230000035784 germination Effects 0.000 claims description 17
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 15
- 239000002480 mineral oil Substances 0.000 claims description 15
- 235000010446 mineral oil Nutrition 0.000 claims description 15
- 238000000926 separation method Methods 0.000 claims description 14
- 210000001161 mammalian embryo Anatomy 0.000 claims description 12
- 108010006785 Taq Polymerase Proteins 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 210000002257 embryonic structure Anatomy 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 5
- 239000011541 reaction mixture Substances 0.000 claims description 4
- 230000006641 stabilisation Effects 0.000 claims description 4
- 238000011105 stabilization Methods 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 11
- 239000003921 oil Substances 0.000 description 35
- 108020004414 DNA Proteins 0.000 description 23
- 238000012360 testing method Methods 0.000 description 20
- 238000004519 manufacturing process Methods 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000012634 fragment Substances 0.000 description 12
- 238000012216 screening Methods 0.000 description 11
- 239000012530 fluid Substances 0.000 description 9
- 230000003321 amplification Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000003147 molecular marker Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 101000708283 Oryza sativa subsp. indica Protein Rf1, mitochondrial Proteins 0.000 description 5
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 5
- 101710130262 Probable Vpr-like protein Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 108010044467 Isoenzymes Proteins 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 208000000509 infertility Diseases 0.000 description 4
- 230000036512 infertility Effects 0.000 description 4
- 208000021267 infertility disease Diseases 0.000 description 4
- 238000012257 pre-denaturation Methods 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000009331 sowing Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 206010021929 Infertility male Diseases 0.000 description 2
- 208000007466 Male Infertility Diseases 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000031787 nutrient reservoir activity Effects 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108060006613 prolamin Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification 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
-
- 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 discloses a specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seeds thereof, which comprises a forward primer F and a reverse primer R with the following sequences: forward primer F:5'-AGTTAGCTCCTCTGTGGTTTGC-3'; reverse primer R:5'-CTACAGCTCGATTAGGGATCGT-3'. Has stronger universality and has more practical significance in the purity identification of the hybrid seeds or the parent seeds thereof. The application of the specific SSR marker primer in judging rape Pol-CMS seed fertility, rape Pol-CMS hybrid or parent seed purity identification thereof is also disclosed, the operation is simple, the identification result is accurate, the purity identification of new varieties and new combinations can be greatly simplified, and a new identification technology and method are provided for seed researchers, seed management and law enforcement departments.
Description
Technical Field
The invention belongs to the technical field of rape breeding, and particularly relates to a specific SSR marker primer for identifying the purity of a Pol-CMS hybrid of brassica napus or a parent seed of the Pol-CMS hybrid of brassica napus and application of the specific SSR marker primer.
Background
The Bolima cytoplasmic sterility (polima cytoplasmic male sterility, pol CMS) is the first cytoplasmic sterility type of cabbage type rape with production value, and is also the sterility source of the hybrid pollination control system with the widest application range of rape in China at present. The sterility of the sterile line of the system is controlled by the interaction of a sterile gene located in a mitochondrial genome and a nuclear genome, and a fertility restorer gene (Rfp) is located in the nuclear genome. The fertility of the sterile line is easily affected by the environmental temperature, and the micro-powder selfing and maturing phenomenon easily occurs in the large-area hybrid seed production process, so that the female parent false hybrid is produced. Because the female parent false hybrid is a sterile plant, the consistency of varieties is affected, and the exertion of the yield advantage of the varieties is more seriously affected, the strict purity and quality identification of seeds are required before the rape hybrid is applied and produced.
At present, three major categories of rape purity quality indoor identification mainly include isozyme electrophoresis, alcohol soluble protein electrophoresis and molecular marking technology. The isozyme electrophoresis and the prolamin electrophoresis have the advantages of simple technology, rapid identification, low required cost and the like, but the polymorphism of isozymes and storage proteins is relatively low, and the parent and hybrid seeds of the isozymes and the storage proteins are difficult to distinguish for certain hybrid combinations, so that the practical application of the technology is greatly limited. The molecular markers based on the genome DNA sequences, especially SSR markers developed in the early 90 s, are extremely abundant in number and high in polymorphism, and compared with other molecular markers, the molecular markers have relatively simple operation technology and low cost, and become the preferred molecular marker types in the aspects of crop variety purity and authenticity identification. However, as the genetic diversity of breeding resources becomes narrower, the workload of SSR marker screening is increased. Meanwhile, when the phenotype characters are basically consistent, at present, breeders start parent propagation and hybrid seed production when hybrid parents are bred, the phenomenon of separation of genetic information on a molecular level still exists, the phenomenon that the identification purity of hybrid SSR markers is inconsistent with the fertility investigation result of field planting identification easily occurs in actual production, and quality risks are brought to production seeds.
Meanwhile, the markers for SSR identification of the purity of the cabbage type rape hybrid are obtained by screening the father and mother of the hybrid from a whole genome database, and different identification markers need to be screened for different hybrid combinations, so that a large number of marker primer sequences need to be synthesized in advance for screening, the identification cost and the early investment are increased, and the popularization and the application of the technology are not facilitated. Meanwhile, SSR markers screened aiming at specific hybrid combinations of rape can only be used for detecting the genotype of a hybrid body of the specific hybrid combinations, and in actual production, hybrid seed production not only involves hybrid seed production, but also carries out hybrid parent seed propagation. At present, the common SSR markers are limited by combination specificity, so that the purity and quality of the hybrid parent seeds are difficult to detect. Therefore, the common SSR markers have the problems of poor universality and easy deviation between the identification result and field identification in rape hybrid purity identification, and cannot be used for detecting the purity of the parent seeds of the hybrid, so that the development of a more effective genotyping specific SSR marker has important significance for meeting the actual production requirements of hybrid production quality identification.
Disclosure of Invention
The invention aims to solve the technical problems and overcome the defects and shortcomings in the background technology, and provides a specific SSR marker primer for identifying the purity of rape Pol-CMS hybrid or parent seeds thereof and application thereof.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a specific SSR marker primer for identifying the purity of rape Pol-CMS hybrid or parent seeds thereof, wherein the specific SSR marker primer is RfpSSR19, and comprises a forward primer F and a reverse primer R with the following sequences:
forward primer F:5'-AGTTAGCTCCTCTGTGGTTTGC-3' (SEQ ID NO. 1);
reverse primer R:5'-CTACAGCTCGATTAGGGATCGT-3' (SEQ ID NO. 2).
The specific SSR marker primer is preferably located at NC_0243803.1:34437588 to 34437748 of the brassica napus genome at an amplification site.
Preferably, the length of the amplified DNA fragment of the specific SSR marker primer in the brassica napus Pol-CMS sterile line is 162bp, and the length of the amplified DNA fragment in the restorer line is 184bp.
Studies have shown that the gene controlling the restoration of the fertility of the Pol CMS of rape is the monogenic Rfp. In recent years, scholars have made a great deal of genetic mapping work on the gene and developed a series of molecular markers linked to fertility restorer genes, the Rfp genes were sequentially and respectively located at N 9 Or N 18 Linkage groups. Until 2014 Rfp was located in the N of Brassica napus 9 Linkage group, and restriction of Rfp gene to cabbage type rape genome A by molecular marker analysis 9 Upper 29.2 k. This region contains 7 ORFs, of which ORF2 is most likely involved in fertility restoration. Later, the scholars carry out genetic transformation on the ORF2 in the cabbage type rape sterile line, the result shows that the ORF2 has fertility restoration function, and the result of further sequence analysis shows that the coding region of the ORF2 is 1953bp long. Meanwhile, the genome sequencing information of the cabbage and the brassica napus is respectively published in databases in 2011 and 2014, and an available reference platform is provided for molecular marker development.
Aiming at the actual production requirements of hybrid seed production and parent quality identification of the current Pol CMS hybrid, the invention utilizes the sequence information of Rfp candidate gene region to carry out BLAST analysis on target sequences of cabbage type rape and cabbage genome, determines the position and sequence of PPR genes related to fertility restoration, develops SSR markers in the nearby regions, and is used for purity identification research of Pol CMS hybrid varieties so as to provide a more accurate and efficient detection method for rape hybrid purity identification work.
Based on a general inventive concept, the invention also provides an application of the specific SSR marker in judging rape Pol-CMS seed fertility, wherein the application method comprises the following steps:
s1, germinating and culturing a brassica napus Pol-CMS seed sample, adding a NaOH solution into a young embryo obtained by culture, placing the young embryo in an ultrasonic cleaner (WD-9415B type of Beijing Liuyi instrument factory) for ultrasonic crushing, and finally adding a Tris-HCl solution, and uniformly mixing to obtain a DNA template mixed solution for PCR amplification;
s2, adding a PCR reaction mixed solution containing the specific SSR marker primer RfpSSR19 into the DNA template mixed solution obtained in the step S1, and then adding mineral oil for covering to perform PCR amplification reaction;
s3, carrying out electrophoresis separation on the PCR amplification product obtained in the step S2 in agarose gel, and reading a gel imaging result; if the gel imaging result shows that only a single stripe with the length of 162bp appears, judging that the brassica napus Pol-CMS seed sample is a sterile single plant; if the gel imaging result shows that a band with the length of 184bp appears, judging that the brassica napus Pol-CMS seed sample is a normal fertile single plant.
In the above application, preferably, the NaOH solution is added in an amount of: adding 40-50 mu L of 0.25mol/L NaOH solution into each young embryo; the addition amount of the Tris-HCl solution is as follows: 120-150 mu L of 0.1mol/L Tris-HCl solution is added into each young embryo;
the addition amounts of the PCR reaction mixed solution and the mineral oil are as follows: adding 8-10 mu L of PCR reaction mixed solution into every 2-3 mu L of the mixed solution obtained in the step S1, and adding 10-20 mu L of mineral oil to cover; the PCR reaction mixture contained 1. Mu.L of 10 XBuffer (Mg-containing 2+ ) 0.2 mu L of 10mmol/L dNTP,0.5U of Taq DNA polymerase, and 35ng of each of the forward primer and the reverse primer of the specific SSR marker primer RfpSSR 19.
More preferably, the germination culture specifically comprises the following steps: placing the brassica napus Pol-CMS seed sample in a germination box of tiled absorbent paper, and performing germination culture at 25-28deg.C for 4-5 days until the length of hypocotyl is more than 2cm and cotyledons are completely green; the ultrasonic crushing parameter conditions are as follows: the crushing time is 5-10min, the water temperature is controlled at 70-80 ℃, and the power is controlled at 200-250W; the agarose gel adopted by the electrophoresis separation is 3.0-3.5% of the agarose gel by mass percent, and the electrophoresis voltage is 120-150V for stabilizing the pressure.
Based on a general inventive concept, the invention also provides an application of the specific SSR marker primer in the purity identification of rape Pol-CMS hybrid or parent seeds thereof, and the application method comprises the following steps:
(1) Sampling the Pol-CMS hybrid of the brassica napus or the parent seed sample and the female parent seed sample according to a 4-division method, and randomly selecting 300-400 seeds per part;
(2) Germinating and culturing the seeds selected in the step (1), and randomly taking 192-240 young embryos obtained by culture and placing the young embryos in a 96-well plate;
(3) Adding NaOH solution into each hole of a 96-hole plate, placing into an ultrasonic cleaner for ultrasonic crushing, and finally adding Tris-HCl solution, and uniformly mixing to obtain DNA template mixed solution for PCR amplification;
(4) Adding PCR reaction mixed liquor containing the specific SSR marker primer RfpSSR19 into each hole in the DNA template mixed liquor in the 96-hole plate obtained in the step (3), and then adding mineral oil for covering to carry out PCR amplification reaction;
(5) Performing electrophoresis separation on the PCR amplification product obtained in the step (4) in agarose gel, and reading a gel imaging result, wherein if the gel imaging result only shows a single band with the length of 162bp, the band shows that the brassica napus Pol-CMS seed sample is taken as a female parent; if the gel imaging result shows that only a single band with the length of 184bp appears, the brassica napus Pol-CMS seed sample is indicated as a male parent; if the gel imaging result shows that two bands with the lengths of 162bp and 184bp respectively appear, the brassica napus Pol-CMS seed sample is a hybrid; counting the final hybrid seed banding pattern number, female parent banding pattern number and male parent banding pattern number;
(6) Calculating SSR purity of the brassica napus Pol-CMS hybrid sample or purity of the parent sample according to the following formula:
cabbage type rape Pol-CMS hybrid sample SSR purity (%) = (number of detected plants-number of female parent bands-number of male parent bands) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid female parent sample SSR purity (%) = (number of detected plants-number of hybrid banding patterns-number of male parent banding patterns) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid male parent sample SSR purity (%) = (number of detected plants-number of hybrid bands-number of female parent bands) ×100/number of detected plants.
In the above application, preferably, in the step (2), the germination culture specifically includes the following steps: placing the seeds selected in the step (1) into a germination box of tiled water-absorbing paper, and performing germination culture for 4-5 days at 25-28 ℃ until the length of the hypocotyl is more than 2cm and the cotyledons are completely green;
in the step (3), the addition amount of the NaOH solution is as follows: adding 40-50 mu L of 0.25mol/L NaOH solution into each hole; the addition amount of the Tris-HCl solution is as follows: 120-150 mu L of 0.1mol/L Tris-HCl solution is added into each hole; the ultrasonic crushing parameter conditions are as follows: the crushing time is 5-10min, the water temperature is controlled at 70-80 ℃, and the power is controlled at 200-250W;
in the step (4), the addition amounts of the PCR reaction mixed solution and the mineral oil are as follows: 2-3 mu L of each hole in the mixed solution obtained in the step (3) is sucked and placed in another 96-well plate, 8-10 mu L of PCR reaction mixed solution is added into each hole, and 10-20 mu L of mineral oil is added for covering; the PCR reaction mixture contained 1. Mu.L of 10 XBuffer (Mg-containing 2+ ) 0.2 mu L of 10mmol/L dNTP,0.5U of Taq DNA polymerase, and 35ng of each of the forward primer and the reverse primer of the specific SSR marker primer RfpSSR 19;
in the step (5), the agarose gel adopted in the electrophoresis separation is 3.0-3.5% of agarose gel by mass percent, and the electrophoresis voltage is 120-150V for pressure stabilization.
More preferably, the cycle parameters of the PCR amplification reaction: pre-denaturation at 94℃for 2min, denaturation at 94℃for 20sec, annealing at 55℃for 20sec, elongation at 72℃for 30sec for a total of 35 cycles; extending at 72 deg.C for 5min, and storing at 4 deg.C.
The specific SSR marker primer is RfpSSR19, and is an SSR marker developed according to the flanking sequence of the homologous sequence locus of the brassica napus genome by comparing the candidate gene coding sequence of the disclosed Borima cytoplasmic male sterility restoring gene Rfp with the whole genome sequence of the brassica napus. Because the marker is positioned at the-3962 bp to-3801 bp section (namely the NC_024383.1:34437588 to 34437748 position of the brassica napus genome) at the upstream of the Rfp translation initiation site and is difficult to recombine with a restoring gene, the fertility of the single plant of the male sterile hybrid of the Borima cytoplasm can be judged theoretically by using the marker, and the result of purity identification by using the marker is more consistent with the fertility of the single plant identified by field planting. Meanwhile, the marker is unique in the amplification site of the brassica napus genome, and co-dominant inheritance is shown between the Borima cytoplasmic male sterile line and the restorer line, so that the influence of false negatives (no PCR amplification strip caused by genome DNA extraction) of the dominant marker on the result in detection can be overcome in practical application, and at the same time, the electrophoresis band of the PCR amplification product is at most 2, so that electrophoresis separation, detection and reading are facilitated, the accuracy of the result of the false positive influence caused by electrophoresis separation is avoided, 3% agarose gel electrophoresis can be directly utilized for detection, and the operation is simpler and more convenient than that of common markers such as AFLP and SSR. In addition, the marker is a co-dominant marker, namely, the bands from the father and the mother can be displayed in the hybrid, so that in purity identification, not only can the false hybrid (only one common band is shown) generated by the selfing of the mother be detected, but also the false hybrid (only one common band is shown) of the father mixed by mechanical hybridization can be detected, and the purity identification and quality monitoring of the seeds of hybrid seed production are facilitated.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention develops a novel specific SSR marker primer, can directly judge the fertility expression of the single plant by detecting the Pol-CMS seeds of the brassica napus, has stronger universality and has more practical significance in the purity identification of the hybrid seeds or the parent seeds thereof; the specific SSR marker primer can also effectively judge the type of male parent and female parent false hybrid in the hybrid sample.
2. The specific SSR marker primer is applied to judging the fertility of the Pol-CMS seeds of the brassica napus, and the purity identification of the Pol-CMS hybrid seeds of the brassica napus or the parent seeds thereof, has simple operation, accurate identification result and stronger universality, can greatly simplify the purity identification of new varieties and new combinations, and provides a novel identification technology and method for seed researchers, seed management and law enforcement departments.
3. The specific SSR marker primer developed by the invention can be used for purity quality identification of Pol CMS parent propagation seeds, particularly for the purity identification of male parent restorer lines, solves the technical problem that hybrid seeds heterozygous for restoring gene loci are difficult to pass fertility identification in planting identification, and has important practical production significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PCR amplification results of a specific SSR marker primer RfpSSR19 of the invention among 12 tested parents of a promoted hybrid variety;
FIG. 2 is an alignment of PCR amplified product sequences of the specific SSR marker primer RpfSSR19 of the present invention in 2 pol CMS sterile lines and 2 restorer lines;
FIG. 3 shows the PCR amplification result and fertility performance of the specific SSR marker primer RpfSSR19 of the invention on the individual plants of the pol CMS hybrid oil 737F2 isolate;
FIG. 4 shows the result of SSR-PCR amplification of genomic DNA of a sample of the test variety Feng oil 737 hybrid;
FIG. 5 shows the SSR-PCR amplification results of genomic DNA of a sample of a test variety Fengshi 958 hybrid;
FIG. 6 shows the result of SSR-PCR amplification of genomic DNA of a sample of a test variety Feng you 823 hybrid.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1:
the specific SSR marker primer RfpSSR19 comprises a forward primer F and a reverse primer R with the following sequences:
forward primer F:5'-AGTTAGCTCCTCTGTGGTTTGC-3' (SEQ ID NO. 1);
reverse primer R:5'-CTACAGCTCGATTAGGGATCGT-3' (SEQ ID NO. 2).
The amplification site of the specific SSR marker primer is positioned at NC_024803.1:34437588 to 34437748 of the brassica napus genome; the length of the amplified DNA fragment of the specific SSR marker primer in the brassica napus Pol-CMS sterile line is 162bp, and the length of the amplified DNA fragment in the restorer line is 184bp.
Example 2:
the application method of the specific SSR marker primer of the embodiment 1 in the identification of the Pol-CMS seed fertility of the brassica napus comprises the following steps:
s1, placing a brassica napus Pol-CMS seed sample in a germination box of tiled absorbent paper, and performing germination culture for 4-5 days at 25-28 ℃ until the length of an hypocotyl is more than 2cm and cotyledons are completely green; adding 50 mu L of 0.25mol/L NaOH solution into each young embryo obtained by culture, and then placing the young embryo into an ultrasonic lotion device for ultrasonic crushing for 10min, wherein the water temperature is controlled at 70-80 ℃ and the power is controlled at 200-250W; finally, 150 mu L of 0.1mol/L Tris-HCl solution is added, and the mixture obtained after uniform mixing is used for PCR amplification;
s2, adding 10 mu L of a solution containing 1 mu L of 10 XBuffer (Mg-containing solution) to the mixed solution obtained in the step S1 2+ ) The PCR reaction mixed solution of 35ng of the forward primer F and the reverse primer R of the specific SSR marking primer RfpSSR19, 0.2 mu L of 10mmol/L dNTP and 0.5U of Taq DNA polymerase is added, and then 10 mu L of mineral oil is added for covering, so as to carry out PCR amplification reaction;
PCR cycle parameters: pre-denaturation at 94℃for 2min, denaturation at 94℃for 20sec, annealing at 55℃for 20sec, elongation at 72℃for 30sec for a total of 35 cycles; extending at 72℃for 5min. After amplification, storing at 4 ℃;
s3, performing electrophoretic separation on the PCR amplification product obtained in the step S2 in agarose gel with the mass percentage of 3.0% by using 120V voltage stabilization, and finally observing and photographing in a gel imaging system to read gel imaging results; if the gel imaging result shows that only a single stripe with the length of 162bp appears, judging that the brassica napus Pol-CMS seed sample is a sterile single plant; if the gel imaging result shows that a band with the length of 184bp appears, judging that the brassica napus Pol-CMS seed sample is a normal fertile single plant.
Example 3:
the application method of the specific SSR marker primer of the embodiment 1 in the identification of the purity of the brassica napus Pol-CMS hybrid comprises the following steps:
(1) Sampling seed samples of the brassica napus Pol-CMS hybrid variety according to a 4-segment method, and randomly taking about 300-400 seeds per seed;
(2) Placing the seeds in a germination box of flat water-absorbing paper, germinating and culturing at 25-28deg.C for 4-5 days until the length of hypocotyl is more than 2cm and cotyledons are completely green, randomly taking 192-240 young embryo, and placing in 96-well plate;
(3) Adding 50 mu L of 0.25mol/L NaOH solution into each well of a 96-well plate, placing into an ultrasonic cleaner for ultrasonic crushing for 10min, controlling the water temperature at 70-80 ℃ and the power at 200-250W; finally, 150 mu L of 0.1mol/L Tris-HCl solution is added, and the DNA template mixed solution obtained after uniform mixing is used for PCR amplification;
(4) Will be put onThe DNA template mixture of the 96-well plate was pipetted into another 96-well plate at a volume of 2. Mu.L per well, and 10. Mu.L of a solution containing 1. Mu.L of 10 XBuffer (Mg-containing solution was added per well 2+ ) The PCR reaction mixed solution of 35ng of the forward primer F and the reverse primer R of the specific SSR marking primer RfpSSR19, 0.2 mu L of 10mmol/L dNTP and 0.5U of Taq DNA polymerase is added, and then 10 mu L of mineral oil is added for covering, so as to carry out PCR amplification reaction;
PCR cycle parameters: pre-denaturation at 94℃for 2min, denaturation at 94℃for 20sec, annealing at 55℃for 20sec, elongation at 72℃for 30sec for a total of 35 cycles; extending at 72℃for 5min. After amplification, storing at 4 ℃;
(5) Separating PCR amplification products in agarose gel with the mass percentage of 3.0% by using 120V stabilized voltage electrophoresis, finally observing and photographing in a gel imaging system to obtain a gel imaging result, and if the gel imaging result only shows a single strip with the length of 162bp, indicating that the brassica napus Pol-CMS seed sample is taken as a female parent; if the gel imaging result shows that only a single band with the length of 184bp appears, the brassica napus Pol-CMS seed sample is indicated as a male parent; counting the final female parent banding pattern number and male parent banding pattern number;
(6) Calculating SSR purity of the brassica napus Pol-CMS hybrid sample according to the following formula:
cabbage type rape Pol-CMS hybrid sample SSR purity (%) = (number of detected plants-number of female parent bands-number of male parent bands) ×100/number of detected plants.
Example 4:
the application method of the specific SSR marker primer of the embodiment 1 in the identification of the purity of the brassica napus Pol-CMS parent seed comprises the following steps:
(1) Sampling parent seed samples of the Pol-CMS of the brassica napus according to a 4-point method, and randomly taking about 300-400 seeds per seed;
(2) Placing the seeds in a germination box of flat water-absorbing paper, germinating and culturing at 25-28deg.C for 4-5 days until the length of hypocotyl is more than 2cm and cotyledons are completely green, randomly taking 192-240 young embryo, and placing in 96-well plate;
(3) Adding 50 mu L of 0.25mol/L NaOH solution into each well of a 96-well plate, placing into an ultrasonic cleaner for ultrasonic crushing for 10min, controlling the water temperature at 70-80 ℃ and the power at 200-250W; finally, 150 mu L of 0.1mol/L Tris-HCl solution is added, and the DNA template mixed solution obtained after uniform mixing is used for PCR amplification;
(4) mu.L of the DNA template mixture of the 96-well plate was pipetted into another 96-well plate, and 10. Mu.L of a solution containing 1. Mu.L of 10 XBuffer (Mg-containing solution was added to each well 2+ ) The PCR reaction mixed solution of 35ng of the forward primer F and the reverse primer R of the specific SSR marking primer RfpSSR19, 0.2 mu L of 10mmol/L dNTP and 0.5U of Taq DNA polymerase is added, and then 10 mu L of mineral oil is added for covering, so as to carry out PCR amplification reaction;
PCR cycle parameters: pre-denaturation at 94℃for 2min, denaturation at 94℃for 20sec, annealing at 55℃for 20sec, elongation at 72℃for 30sec for a total of 35 cycles; extending at 72℃for 5min. After amplification, storing at 4 ℃;
(5) Separating PCR amplification products in agarose gel with the mass percentage of 3.0% by using 120V stabilized voltage electrophoresis, finally observing and photographing in a gel imaging system to obtain a gel imaging result, and if the gel imaging result only shows a single strip with the length of 162bp, indicating that the brassica napus Pol-CMS seed sample is taken as a female parent; if the gel imaging result shows that only a single band with the length of 184bp appears, the brassica napus Pol-CMS seed sample is indicated as a male parent; if the gel imaging result shows that two bands with the lengths of 162bp and 184bp respectively appear, the brassica napus Pol-CMS seed sample is a hybrid; counting the final female parent banding pattern number and male parent banding pattern number;
(6) The SSR purity of the male and female samples of brassica napus Pol-CMS was calculated according to the following formula:
cabbage type rape Pol-CMS hybrid female parent sample SSR purity (%) = (number of detected plants-number of hybrid banding patterns-number of male parent banding patterns) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid male parent sample SSR purity (%) = (number of detected plants-number of hybrid bands-number of female parent bands) ×100/number of detected plants.
To demonstrate the application effect advantage of examples 1-3 of the present invention, the following experiments were performed:
1 materials and methods
1.1 test materials
Test material: 12 parts of cabbage type rape Pol CMS hybrid parent seed, 5 parts of sterile line and 7 parts of Pol-CMS restorer line, and the details are shown in table 1; 3 parts of seed of hybrid seed production sample of Pol-CMS hybrid seed, fender oil 737, fender oil 320, fender oil 958, fender oil 520, fender oil 823, qin You, qin You, and F of Fender oil 737 1 Single plant selfing F 2 And (5) group seeds.
Table 1: name and source of 12 tested rape parents
1.2 sample sampling for samples
Sowing 12 parts of Pol CMS hybrid parent materials at the end of 9 months in 2018 in a Hunan province crop institute test field (the last crop is rice), picking up 5 young leaves of single plants from each part of materials in a seedling stage (about 7 leaf stage), placing the young leaves in a 2mL centrifuge tube, and preserving at-20 ℃ for primer screening;
selecting hybrid seed samples of tested varieties of oil Fender 737, oil Fender 320, oil Fender 958, oil Fender 520, oil Fender 823, qin You, no. Qin You, no. Qin You and SC3 sample seeds of a female parent sterile line 20A and a male parent restorer line of oil Fender 520, taking about 1000 seeds at random each part, dividing the seeds into two parts on average, wherein one part is used for identifying the purity of a sample molecular marker, and the other part is used for identifying the purity of the sample field planting;
about 500 test varieties Feng you 737 hybrid F are selected 2 Group seeds are sowed in a test field of a crop research institute in Hunan province, 8 rows of cells are identified per sample to be tested, the row length is 2.0m, the row spacing is 0.5m, uniform sowing is performed, and no seedling thinning is performed in the whole growth period.
1.3 fertility restorer Gene-linked SSR marker development
ORF published by Liu et al, 2016 2 BLAST analysis was performed on the brassica napus genome and brassica napus genome (http:// brassica db. Org/brad /) to determine the physical location of high homology to the Rfp gene associated with fertility restoration (Liu Guosheng et al 2016); and based on the positionSSR sites are found in 5K,10K and 15K regions on two sides of the primer, and SSR primers are designed on line through http:// wshartins. And then, carrying out PCR amplification on genome DNA of different restorer lines and sterile lines by using designed SSR primers, and screening primers with differences as linkage markers of Rfp genes.
1.4 fertility segregating population coseparation verification
In the 7-9 leaf stage, sequentially selecting oil-fluid 737 hybridization F 2 192 single plants in the community are subjected to drop numbering, the genomic DNA of the single plants of the drop is extracted, PCR amplification is carried out by using the screened primers, the amplified products are subjected to electrophoresis separation by 3.0% agarose gel, finally, photographing and reading are observed in a gel imaging system, and the amplified band types of the single plants are recorded. The full bloom stage, the individual fertility performance of the 192 drop numbers tested was investigated.
1.5 molecular marker purity identification of hybrid and parental samples
After germination of the samples to be tested of the test variety and parent, the young embryo genomic DNA is extracted by the CTAB method according to Chen Ying (Chen Ying., 1993). And (3) carrying out PCR amplification on the young embryo genome DNA of the samples of the oil pack 737, the oil pack 320, the oil pack 958, the oil pack 520, the oil pack 823, qin You No. 10, qin You No. 7 and 20A, SC by utilizing the primers obtained by screening in the step 1.3, carrying out electrophoresis separation on the amplified products by using 3.0% agarose gel, finally observing photographing and reading in a gel imaging system, and calculating the purity of the samples according to a formula.
Purity calculation formula:
hybrid sample SSR purity (%) = (number of detected plants-number of female parent bands-number of male parent bands) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid female parent sample SSR purity (%) = (number of detected plants-number of hybrid banding patterns-number of male parent banding patterns) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid male parent sample SSR purity (%) = (number of detected plants-number of hybrid bands-number of female parent bands) ×100/number of detected plants.
1.6 identification of purity of hybrid samples and parents in field planting
Sowing samples to be detected of the tested varieties in a test field of a crop research institute of Hunan province, wherein 8 rows of cells are identified for each sample to be detected, the row length is 2.0m, the row spacing is 0.5m, the samples are uniformly sown, no seedlings are thinning in the whole growth period, and the purity of the samples of each identified cell is investigated according to fertility in the full bloom period.
The calculation formula is as follows:
the field hybrid identification purity (%) = (number of detected plants-number of sterile plants) ×100/number of detected plants;
the field female parent identification purity (%) = (number of detected plants-number of fertile plants) ×100/number of detected plants;
the field male parent identification purity (%) = (number of detected strains-number of sterile strains×3) ×100/number of detected plants.
2 results and analysis
2.1 Pol CMS fertility restorer gene linkage SSR marker development
1953bp sequence of ORF2 published by Liu et al in 2016 is used in brassica napus A 9 The chromosome obtains a highly homologous region (homology 99%), 32 SSR sites are obtained in the 15k regions on the upper and the lower stream of the region, and 32 pairs of SSR primers are designed and synthesized on line. PCR amplification is carried out on 10 tested parent material genome DNA by using the 32 pairs of primer pairs, 1 pair of SSR marker primers RfpSSR19 are obtained by screening, the amplification site is positioned at NC_024803.1:34437588 to 34437748 of the brassica napus genome, obvious polymorphism exists between 4 sterile lines and 6 restorer lines to be tested (see figure 1), and the markers are co-dominant markers of Pol CMS sterile lines and restorer lines.
Sequencing results of PCR amplified fragments of the sterile line and the restorer line show that the amplified fragment size in the sterile line is 162bp, and the amplified fragment size in the restorer line is 184bp; there is one 15bp and one 7bp insert in the restorer amplified fragment compared to the sterile line (see FIG. 2). Comparing the RfpSSR19 amplified product sequence with the brassica napus genome, the marker is found to be positioned in a promoter region (-3962 bp to-3801 bp) at the upstream of the translation start site of the Pol CMS fertility restorer gene Rfp, which shows that the marker RfpSSR19 obtained by the research is closely linked with the Rfp gene, and can be used for judging the fertility of Pol CMS seeds and identifying the purity of hybrid varieties and parent seeds thereof.
2.2 F 2 SeparationGroup individual plant field positioning test
In the F2 isolated population obtained after hybridization of Pol CMS and Feng oil 737, fertility examination is carried out in the flowering phase, simultaneously genotype analysis is carried out on 192 randomly selected individuals by adopting SSR marker RfpSSR19, and the result shows that the individual fertility of 184bp fragments in the amplified product is normal, the individual fertility of only one single 162bp fragment is amplified to be sterile, and the obtained PCR amplified banding pattern and the individual fertility expression are coseparated (see figure 3).
2.3 comparison of purity identification results of test hybrid samples
PCR amplification is carried out on sample seedling genome DNA of the test hybrid varieties of fluid oil 737, fluid oil 320, fluid oil 958, fluid oil 520, fluid oil 823, qin You No. 10 and Qin You by using the SSR primer RfpSSR19 obtained by screening, so as to obtain a clear and stable target amplified band (taking fluid oil 737, fluid oil 958 and fluid oil 823 hybrid samples as an example, the PCR amplification results are respectively shown in figures 4, 5 and 6, and female parent false hybrid is marked in the figures), and reliable SSR identification results are obtained by counting the band results.
Comparing the purity results of field planting identification with the results of marker identification (see table 2), it can be seen that the results of identification by using the obtained SSR marker primer RfpSSR19 are highly consistent with the results of field planting identification, and the difference of the identification results of 21 test samples is within 4%, which indicates that the purity identification result of the test hybrid variety sample studied by using the marker RpfSSR19 of the invention is reliable.
Table 2: purity results of field planting identification and SSR marker primer identification of hybrid seed sample of test variety
2.4 comparison of the results of the purity identification of the parent sample under test
SSR specific marker purity identification is carried out on female parent sterile line and male parent restorer line SC3 parent sample seedlings of the test Pol CMS hybrid oil 520 by using the SSR marker primer RfpSSR19 obtained by screening, and the result of field planting identification purity is compared with the result of marker identification (the result is shown in table 3), so that the identification result of the obtained SSR marker primer RfpSSR19 is highly consistent with the result of field planting identification, the difference of the identification results of 4 test samples is within 2%, and the fact that the purity identification result carried out on the test hybrid sample materials adopted by the research by using the marker RpfSSR19 is reliable is indicated. For the SSR identification purity value of the sample SC3-2 is different from 100% of field planting identification, the study carries out isolated selfing seed reserving on the group single plants identified in the field, and the fact that Pol CMS sterile plants appear in the next generation is found, which indicates that hybrid single plants with heterozygous restoring gene loci exist in the group with 100% of field planting identification purity in the previous generation. The fertility is still normal and fertile due to the existence of the restoring genes in the mixed single plant, and the distinguishing is difficult to carry out through planting identification, and the difficult problem can be overcome by utilizing the specific mark of the invention, and the identification result is more effective and accurate.
Table 3: purity results of field planting identification and SSR marker primer identification of parent sample to be tested
Discussion 3
The temperature sensitivity problem of sterile line exists in the rape Pol CMS hybrid seed production system, and the quality monitoring of seed purity, especially the accurate identification of female parent false hybrid in hybrid seeds, has been an indispensable link in rape hybrid seed production for a long time. At present, a molecular marker technology based on PCR amplification, in particular an SSR marker technology, has the advantages of high quantity, high polymorphism, stable result and very rich genetic information content, and becomes a preferred method for indoor purity identification of rape hybrid seeds. However, as breeders keep good preference among varieties and the backbone parents are repeatedly utilized in a large amount for a long time, the relatedness among the hybrid parents is more and more similar, and the primer screening workload is more and more great in the actual seed purity identification. The research is based on the actual requirement of Pol CMS hybrid purity identification, develops Rfp gene related molecular markers by utilizing mass information brought by rape molecular biology development, and performs inspection and application in production, thereby having stronger practical significance.
The invention utilizes the 1,953bp coding sequence of the published Pol CMS restoring gene to obtain the region where Rfp is located in the genome, and obtains 32 SSR sites in the region and the upstream and downstream 15K regions; wherein, 1 locus has polymorphism between the Pol CMS sterile line and the restorer line to be tested, compared with the sterile line, the PCR amplified product sequence of the restorer line has a 15bp fragment insertion and a 7bp fragment insertion, so the marker is a co-dominant marker; through F in oil Feng 737 2 The segregation population verifies that the specific SSR marker primer RfpSSR19 and fertility restoration traits are co-segregation inheritance, and can be used for judging fertility performance of a single plant theoretically; further utilizing the specific SSR marker primer RfpSSR19 to identify the purity of seed production samples of 7 different hybrid varieties such as oil pack 737, oil pack 320, oil pack 958, oil pack 520, oil pack 823, qin You, qin You and the like, wherein the identification result is consistent with the field fertility identification result, so that the accuracy of the specific SSR marker primer RfpSSR19 developed by the invention in the purity identification of the hybrid varieties is shown; meanwhile, the marker has strong universality, can greatly simplify the screening work of purity identification primers of new varieties and new combinations, and can provide a new technology and method for seed management and law enforcement departments.
Sequence listing
<110> Hunan province crop institute
<120> specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
agttagctcc tctgtggttt gc 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ctacagctcg attagggatc gt 22
Claims (7)
1. The application of a specific SSR marker primer in judging rape Pol-CMS seed fertility is characterized in that the specific SSR marker primer is RfpSSR19, and comprises a forward primer F and a reverse primer R with the following sequences:
forward primer F:5'-AGTTAGCTCCTCTGTGGTTTGC-3';
reverse primer R:5'-CTACAGCTCGATTAGGGATCGT-3'.
2. The use according to claim 1, characterized in that the method of application comprises the steps of:
s1, germinating and culturing a brassica napus Pol-CMS seed sample, adding a NaOH solution into young embryos obtained by culture, putting the young embryos into an ultrasonic cleaner for ultrasonic crushing, and finally adding a Tris-HCl solution, and uniformly mixing to obtain a DNA template mixed solution for PCR amplification;
s2, adding a PCR reaction mixed solution containing the specific SSR marker primer RfpSSR19 into the DNA template mixed solution obtained in the step S1, and then adding mineral oil for covering to perform PCR amplification reaction;
s3, carrying out electrophoresis separation on the PCR amplification product obtained in the step S2 in agarose gel, and reading a gel imaging result; if the gel imaging result shows that only a single stripe with the length of 161bp appears, judging that the brassica napus Pol-CMS seed sample is a sterile single plant; if the gel imaging result shows that a band with the length of 183bp appears, judging that the brassica napus Pol-CMS seed sample is a normal fertile single plant.
3. The use according to claim 2, wherein the NaOH solution is added in an amount of: adding 40-50 mu L of 0.25mol/L NaOH solution into each young embryo; the addition amount of the Tris-HCl solution is as follows: 120-150 mu L of 0.1mol/L Tris-HCl solution is added into each young embryo;
the addition amounts of the PCR reaction mixed solution and the mineral oil are as follows: adding 8-10 mu L of PCR reaction mixed solution into every 2-3 mu L of the mixed solution obtained in the step S1, and adding 10-20 mu L of mineral oil to cover; the PCR reaction mixture contains 1 mu L of 10 XBuffer, 0.2 mu L of 10mmol/L dNTP,0.5U of Taq DNA polymerase, and 35ng of each of the forward primer and the reverse primer of the specific SSR marker primer RfpSSR 19.
4. Use according to claim 2 or 3, characterized in that the germination culture comprises in particular the following steps: placing the brassica napus Pol-CMS seed sample in a germination box of tiled absorbent paper, and performing germination culture at 25-28deg.C for 4-5 days until the length of hypocotyl is more than 2cm and cotyledons are completely green; the ultrasonic crushing parameter conditions are as follows: the crushing time is 5-10min, the water temperature is controlled to be 70-80 ℃, and the power is controlled to be 200-250W; the agarose gel adopted by the electrophoresis separation is 3.0-3.5% of the agarose gel by mass percent, and the electrophoresis voltage is 120-150V for voltage stabilization.
5. The application of a specific SSR marker primer in the purity identification of rape Pol-CMS hybrid or parent seeds thereof is characterized in that the specific SSR marker primer is RfpSSR19, and comprises a forward primer F and a reverse primer R with the following sequences:
forward primer F:5'-AGTTAGCTCCTCTGTGGTTTGC-3';
reverse primer R:5'-CTACAGCTCGATTAGGGATCGT-3'.
6. The use according to claim 5, characterized in that the method of application comprises the steps of:
(1) Sampling the Pol-CMS hybrid of the brassica napus or the parent seed sample and the female parent seed sample according to a 4-division method, and randomly selecting 300-400 seeds per part;
(2) Germinating and culturing the seeds selected in the step (1), and randomly taking 192-240 young embryos obtained by culture and placing the young embryos in a 96-well plate;
(3) Adding NaOH solution into each hole of a 96-hole plate, placing into an ultrasonic cleaner for ultrasonic crushing, and finally adding Tris-HCl solution, and uniformly mixing to obtain DNA template mixed solution for PCR amplification;
(4) Adding PCR reaction mixed liquor containing the specific SSR marker primer RfpSSR19 into each hole in the DNA template mixed liquor in the 96-hole plate obtained in the step (3), and then adding mineral oil for covering to carry out PCR amplification reaction;
(5) Performing electrophoresis separation on the PCR amplification product obtained in the step (4) in agarose gel, and reading a gel imaging result, wherein if the gel imaging result only shows a single band with the length of 161bp, the band shows that the brassica napus Pol-CMS seed sample is taken as a female parent; if the gel imaging result shows that only a single band with the length of 183bp appears, the brassica napus Pol-CMS seed sample is indicated as a male parent; if the gel imaging result shows that two bands with the lengths of 161bp and 183bp respectively appear, the brassica napus Pol-CMS seed sample is a hybrid; counting the final hybrid seed banding pattern number, female parent banding pattern number and male parent banding pattern number;
(6) Calculating SSR purity of the brassica napus Pol-CMS hybrid sample or purity of the parent sample according to the following formula:
cabbage type rape Pol-CMS hybrid sample SSR purity% = (number of detected plants-number of female parent bands-number of male parent bands) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid female parent sample SSR purity% = (number of detected plants-number of hybrid banding patterns-number of male parent banding patterns) ×100/number of detected plants;
cabbage type rape Pol-CMS hybrid male parent sample SSR purity% = (number of detected plants-number of hybrid bands-number of female parent bands). Times.100/number of detected plants.
7. The use according to claim 6, wherein in step (2), the germination culture comprises in particular the steps of: placing the seeds selected in the step (1) into a germination box of tiled water-absorbing paper, and performing germination culture for 4-5 days at 25-28 ℃ until the length of the hypocotyl is more than 2cm and the cotyledons are completely green;
in the step (3), the addition amount of the NaOH solution is as follows: adding 40-50 mu L of 0.25mol/L NaOH solution into each hole; the addition amount of the Tris-HCl solution is as follows: 120-150 mu L of 0.1mol/L Tris-HCl solution is added into each hole; the ultrasonic crushing parameter conditions are as follows: the crushing time is 5-10min, the water temperature is controlled to be 70-80 ℃, and the power is controlled to be 200-250W;
in the step (4), the addition amounts of the PCR reaction mixed solution and the mineral oil are as follows: 2-3 mu L of each hole in the mixed solution obtained in the step (3) is sucked and placed in another 96-well plate, 8-10 mu L of PCR reaction mixed solution is added into each hole, and 10-20 mu L of mineral oil is added for covering; the PCR reaction mixture contains 1 mu L of 10 XBuffer, 0.2 mu L of 10mmol/L dNTP,0.5U of Taq DNA polymerase, and 35ng of each of a forward primer and a reverse primer of the specific SSR marker primer RfpSSR 19;
in the step (5), the agarose gel adopted in the electrophoresis separation is 3.0-3.5% of agarose gel by mass percent, and the electrophoresis voltage is 120-150V for voltage stabilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011052970.8A CN112048567B (en) | 2020-09-29 | 2020-09-29 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011052970.8A CN112048567B (en) | 2020-09-29 | 2020-09-29 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112048567A CN112048567A (en) | 2020-12-08 |
| CN112048567B true CN112048567B (en) | 2024-01-16 |
Family
ID=73605412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011052970.8A Active CN112048567B (en) | 2020-09-29 | 2020-09-29 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112048567B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667768A (en) * | 2021-07-14 | 2021-11-19 | 甘肃省农业科学院作物研究所 | SSR marker combination for identifying Longyou za 2 and fingerprint establishment and application |
| CN114438251B (en) * | 2022-02-24 | 2023-12-01 | 江苏沿海地区农业科学研究所 | Primer combination for identifying authenticity of rape and radish hybrid and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101430273A (en) * | 2008-12-01 | 2009-05-13 | 湖南省作物研究所 | Fast appraisal method for purity of Brassica napus L. hybrid seed |
| CN102220316A (en) * | 2011-04-20 | 2011-10-19 | 中国农业科学院油料作物研究所 | Preparation method and application of molecular marker of rape male sterile restoring gene |
| CN102321767A (en) * | 2011-10-18 | 2012-01-18 | 湖南省作物研究所 | Simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity detection method |
| CN107129991A (en) * | 2016-02-29 | 2017-09-05 | 华中农业大学 | Cabbage type rape pol cytoplasmic male sterility (pol cms) restoring gene Rfp and its application |
| CN108935068A (en) * | 2018-06-19 | 2018-12-07 | 湖南省作物研究所 | A method of improvement cabbage type rape pol CMS infertility line fertility temperature sensitivity |
| CN111286554A (en) * | 2020-03-18 | 2020-06-16 | 广东省农业科学院蔬菜研究所 | SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer |
-
2020
- 2020-09-29 CN CN202011052970.8A patent/CN112048567B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101430273A (en) * | 2008-12-01 | 2009-05-13 | 湖南省作物研究所 | Fast appraisal method for purity of Brassica napus L. hybrid seed |
| CN102220316A (en) * | 2011-04-20 | 2011-10-19 | 中国农业科学院油料作物研究所 | Preparation method and application of molecular marker of rape male sterile restoring gene |
| CN102321767A (en) * | 2011-10-18 | 2012-01-18 | 湖南省作物研究所 | Simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity detection method |
| CN107129991A (en) * | 2016-02-29 | 2017-09-05 | 华中农业大学 | Cabbage type rape pol cytoplasmic male sterility (pol cms) restoring gene Rfp and its application |
| CN108935068A (en) * | 2018-06-19 | 2018-12-07 | 湖南省作物研究所 | A method of improvement cabbage type rape pol CMS infertility line fertility temperature sensitivity |
| CN111286554A (en) * | 2020-03-18 | 2020-06-16 | 广东省农业科学院蔬菜研究所 | SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer |
Non-Patent Citations (8)
| Title |
|---|
| Development of SSR Markers Associated with Polima CMS Fertility Restorer in Brassica napus L. and Its Application;Tonghua Wang等;Molecular Plant Breeding;第12卷;1-7 * |
| Fine mapping and candidate gene analysis of the nuclear restorer gene Rfp for pol CMS in rapeseed (Brassica napus L.);Zhi Liu 等;Theor Appl Genet .;第125卷;773-779 * |
| XM_009119786;佚名;NCBI;1-2 * |
| 油菜Pol-CMS Rfp 候 选基因区域SSR 标记开发与应用;王同华等;分子植物育种;第20卷;1206-1213 * |
| 油菜细胞质雄性不育恢复基因分子标记的研究进展;尹明智;胡燕;张瑜;杨胜洁;杨露;;江西农业(第12期);105-107 * |
| 甘蓝型油菜pol CMS育性恢复基因的分子标记;刘平武;李;何庆彪;杨光圣;;中国油料作物学报(第01期);14-19 * |
| 甘蓝型油菜polCMS育性恢复位点的全基因组关联分析;魏大勇;谭传东;崔艺馨;吴道明;李加纳;梅家琴;钱伟;;中国农业科学;第50卷(第05期);802-819 * |
| 甘蓝型油菜细胞质雄性不育恢复基因的SSR标记及初步定位;罗莉斯;李大雄;张超;曾兵;;种子(第01期);77-79 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112048567A (en) | 2020-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101962640B (en) | Specific molecular markers of related genes of brassica napus grain weight and application thereof | |
| CN112981001B (en) | Molecular marker BnC04Y2255 for identifying dwarf cabbage type rape and application thereof | |
| KR101095220B1 (en) | DNA marker associated with resistance of cabbage clubroot disease and uses thereof | |
| CN112048567B (en) | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof | |
| WO2021017608A1 (en) | Indel marker for simultaneously identifying cotton cytoplasmic male sterility restoring genes rf1 and rf2 | |
| CN110747288A (en) | Rice large grain gene function marker and application | |
| CN111733274B (en) | SNP locus, primer set and method for identifying cauliflower Zhe nong piny flower 80-day variety | |
| CN102703438B (en) | Molecular marker of brassica napus L. grain weight character and preparation method and application | |
| CN110684858B (en) | Molecular marker of rice slender grain type gene and application thereof | |
| CN107988409A (en) | Differentiate Rice Salt gene OsRR22 wild types or specific C APS marks, primer and the primer purposes of mutant | |
| CN108660247B (en) | Primer SmemboI-2 for identifying purity of solanum torvum third eggplant based on SNP marker and application | |
| CN110904266A (en) | Identification of maize stalk lodging resistance QTL and development and application of molecular marker | |
| CN110468229B (en) | Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) | |
| CN110358861B (en) | Molecular marker R13I14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t) | |
| CN113528703A (en) | Development and application of KASP molecular marker of rice blast resistance gene Pid3-A4 | |
| CN110760613B (en) | Molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof | |
| CN110331222B (en) | Molecular marker related to cotton fertility restoration and application thereof | |
| CN115786567B (en) | Semi-dominant corn dwarf related molecular marker and application thereof | |
| CN110004242B (en) | Molecular marker BrSF0239 primer of main QTL sites in flowering phase and mature phase of brassica napus and application thereof | |
| CN108676903B (en) | Primer SmemboI-1 for identifying purity of solanum torvum third eggplant based on SNP marker and application | |
| CN105316345A (en) | Chuanyou 36 oilseed rape fertility restorergene, purity and homozygosity detecting method | |
| CN113736866B (en) | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof | |
| CN112795693B (en) | Molecular marker related to chlorophyll content of corn leaf and application thereof | |
| CN110358862B (en) | Molecular marker Hxjy-14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t) | |
| CN113862392A (en) | SSR molecular marker primer linked with Chinese cabbage yellow cotyledon gene Bryc 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 |