CN101824472A - Cabbage type rape high oleic acid molecular marker, preparation method and application thereof - Google Patents
Cabbage type rape high oleic acid molecular marker, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of rape molecular breeding, in particular relates to a preparation method for a cabbage type rape high oleic acid codominant SCAR molecular marker and application as marker assisted selection in breeding high oleic acid cabbage type rape new products. The difference of nucleotide sequences of AA254 and AA177 is identified by cloning and checking sequences on DNA segments of fad2 gene of genomes of high oleic acid cabbage type rape strain AA254 and low oleic acid cabbage type rape strain AA177, primers YQ-Fad2a-1 and YQ-Fad2a-2 are developed by utilizing the difference of the nucleotide sequences provided by the invention, and then the codominant SCAR molecular marker which can distinguish cabbage type rape high oleic acid and low oleic acid is obtained. The invention provides a new marker for the rape molecular breeding. The invention also discloses the preparation method and the application of the molecular marker.
Description
Technical field
The invention belongs to the rape molecular breeding technical field, be specifically related to a kind of preparation and application of cabbage type rape high oleic acid molecular marker.Described molecule marker can be used as high oleic acid swede type rape breeding marker assisted selection, for the high oleic acid new rape variety of cultivating inheritance stability provides new molecule marker.
Background technology
Lipid acid is formed the quality directly determining rapeseed oil and to people's nutritive value in the Semen Brassicae campestris.Oleic acid is a kind of 18 carbon monounsaturated fatty acids, and high oleic acid content rapeseed oil is at edible oil and industrial very high using value arranged.The increase of monounsaturated fatty acids and the minimizing of polyunsaturated fatty acid can increase edible oil oxidation stability, reduce oxidation products.When deep fried foodstuff, high oleic acid edible oil high temperature does not play cigarette, can shorten the frying time, reduce oil and absorb excessive (Miller etc., Genetic control of high oleic acid content in sunflower oil.CropSciences, 1987,27:923-926.); In diet, high oleic acid can reduce the content of the low density lipoprotein cholesterol in the blood, thereby help stoping arterial sclerosis (Chang etc., Effects of the ratio of polyunsaturated and monounsaturated fatty acid onrat plasma and liver lipid concentration.Lipids, 1998,33:481-487).Because oleic 18 carbon chain lengths are similar to diesel component, high oleic acid rapeseed oil can be used for production high-quality biofuel, be important recyclability energy raw material (Piazza etc., Rapeseed oil for oleochemical uses.Eur J Lipid Sci Techno l, 2001,103:405-454.).According to another report, oleic acid also has the effect of cancer cells such as the mammary cancer of killing.Therefore significantly improve the important content that oleic content has become the rapeseed quality breeding.
Oleic acid content low (oleic acid content 8-14%) in traditional high erucic acid (content of erucic acid 40-55%) cabbage type rape variety, thereby nutritive value is lower.By reducing the breeding of content of erucic acid, oleic acid is brought up to 52-60% in the low erucic acid kind (erucic acid＜1%), and nutritive value improves greatly.Yet, further improve oleic acid content owing to be subjected to the restriction of genetic resources, make progress slower.Simultaneously, the low erucic acid kind that high oleic acid proterties is imported high yield is mainly utilized traditional Phenotypic Selection method, has shortcomings such as breeding cycle is long, efficient is low.Modern biotechnology provides strong tool for crop breeding, the molecular marker assisted selection technology is exactly a wherein important technology, compare with traditional Phenotypic Selection, molecular marker assisted selection can be carried out in any vegetative period, be not subjected to environmental influence, can get rid of that non-allelic genes interact and the interference that causes, have quick, economical, advantage such as efficient height, accuracy are strong.In the cabbage type rape high oleic acid breeding process, molecular marking technique is combined with back cross breeding, can carry out directly and fast carrying out foreground selection to the genotype of proterties, to get rid of the influence of environment and extraneous factor by molecule marker.Simultaneously can utilize molecule marker that background is selected, thereby accelerate the genetic background resume speed, shortening the breeding cycle and alleviate chain burden.
Aspect the location of high oleic acid purpose proterties, domestic and international research person utilizes the high-oleic acid material of different sources, has obtained certain progress.(Javidfar etc. such as Javidfar, Identification of molecular markers associated with oleic and linolenic acid in springoilseed rape (Brassica napus) .Plant Breeding, 2006,125:65-71) use high oleic acid, low linolenic strain TO99-5318-20 (oleic acid content＞79%, linolenic acid content＜2%) with high oleic acid, high linolenic DH is that (oleic acid content is about 68% to DH12075, linolenic acid content＞7%) hybridization, obtain 8 RAPD marks related with oleic acid and linolenic acid content, wherein the contribution rate of 2830 pairs of oleic acid of RAPD mark UBC and linolenic acid content variation is respectively 43% and 13%, and is converted to the SCAR mark.(Hu X etc. such as Hu, Mapping of the loci controllingoleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassicanapus L.) .Theor Appl Genet, 2006,113:497-507) from the DH mapping population, navigated to main imitate and little effect QTL relevant with linolenic acid content with swede type rape oleic acid.Wherein the high oleic main effect QTL of control is positioned on No. 5 karyomit(e) of genome, and contribution rate is 76.3%; Little effect QTL is positioned at that contribution rate is 9.4% on No. 1 karyomit(e).According to these QTL, design has obtained the SNP mark relevant with fad2, but the difference that only has one 3 ' end when the SNP of its acquisition is marked at design of primers, effect in swede type rape linolenic acid assisted selection is subjected to influences such as multiple factor such as dna profiling concentration, the synthetic quality of primer, primer concentration, PCR reaction system and parameter, makes its application in actual production exist and is very limited.The relevant application that these are marked in the cabbage type rape high oleic acid assisted selection does not appear in the newspapers as yet, does not utilize molecular marker assisted selection to carrying out the report that background is selected in the cabbage type rape high oleic acid breeding at present yet.
Summary of the invention
The objective of the invention is to develop a kind ofly being applicable to that seed selection possesses the codominance SCAR molecule marker of the swede type rape of high oleic acid proterties, for the cabbage type rape high oleic acid breeding provides a kind of simple, quick and effective supplementary breeding method.
The present invention realizes by following scheme:
The applicant is applicable to that by the test acquisition is a kind of seed selection possesses the swede type rape codominance SCAR molecule marker of high oleic acid proterties, and its nucleotide sequence is shown in sequence table SEQ IDNO:3 and SEQ ID NO:4.
Preparation is applicable to the method for dominant SCAR molecule mark of cabbage type rape high oleic acid proterties, according to following steps:
Utilize primer that the applicant designs to HY-Fad2 increase high oleic acid swede type rape strain A-grade in the first class 254 and low oleic acid swede type rape strain A-grade in the first class 177 genomic dnas, obtain two DNA cloning fragments, then described DNA cloning fragment is cloned, order-checking, wherein obtain the nucleotide sequence shown in SEQ ID NO:1 from increasing the A-grade in the first class 254, obtain nucleotide sequence shown in SEQ ID NO:2 from amplification the A-grade in the first class 177, the insertion sudden change of one 4 bp is arranged at the 567-568 bp place of SEQ ID NO:1, inserting base is AGCC, causes sequence specific amplification region polymorphism; This sudden change causes the frameshit of gene reading frame, and causes 667-669bp terminator codon TGA to occur; Design primer to YQ-Fad2a-1 and YQ-Fad2a-2 according to the nucleotide sequence difference shown in sequence table SEQ ID NO:1 and the SEQ ID NO:2, carry out pcr amplification, obtain distinguishing cabbage type rape high oleic acid and low oleic codominance SCAR molecule marker, its nucleotide sequence is shown in SEQ IDNO:3 and SEQ ID NO:4.
In aforesaid method, the right nucleotide sequence of the primer is as follows:
(name in sequence table is respectively SEQ ID NO:5 to primer to (1) HY-Fad2; SEQ ID NO:6):
Forward primer 5 '-ATGGGTGCAGGTGGAAGAAT-3 ',
Reverse primer 5 '-TCATAACTTATTGTTGTACCAG-3 '.
(name in sequence table is respectively SEQ ID NO:7 to primer to (2) YQ-Fad2a-1; SEQ ID NO:8):
Forward primer 5 '-CAGTTCACTCTCGGCAGC-3 ',
Reverse primer 5 '-GCAACTCCTTGGACAGCA-3 '.
(name in sequence table is respectively SEQ ID NO:9 to primer to (3) YQ-Fad2a-2; SEQ ID NO:10):
Forward primer 5 '-CAGTTCACTCTCGGCTGG-3 ',
Reverse primer 5 '-GATCAAAACTAAGAACCCG-3 '.
Wherein, primer is used for amplification nucleotide sequence shown in sequence table SEQ ID NO:1 and SEQ ID NO:2 to HY-Fad2.Primer is respectively applied for amplification nucleotide sequence shown in sequence table SEQ ID NO:3 and SEQ ID NO:4 to YQ-Fad2a-1, YQ-Fad2a-2.Positively effect of the present invention:
The present invention successfully obtains the codominance SCAR molecule marker of cabbage type rape high oleic acid, the assisted Selection of using this mark to possess the swede type rape of high oleic acid proterties can overcome the shortcoming that relies on phenotype to select in the traditional breeding method, reduce the breeding work amount, shortening the breeding cycle, accelerated the process of cabbage type rape high oleic acid breeding.
More detailed technical scheme is referring to " embodiment ".
Description of drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence of the dna fragmentation that amplification obtains from swede type rape A-grade in the first class 254 genomes, and sequence length is 1159bp;
Sequence table SEQ ID NO:2 is the nucleotide sequence of the dna fragmentation that amplification obtains from swede type rape A-grade in the first class 177 genomes, and sequence length is 1155bp;
Sequence table SEQ ID NO:3 is the nucleotide sequence of the swede type rape codominance SCAR molecule marker of the high oleic acid proterties for preparing of the present invention, and sequence length is 201bp;
Sequence table SEQ ID NO:4 is the nucleotide sequence of the swede type rape codominance SCAR molecule marker of the high oleic acid proterties for preparing of the present invention, and sequence length is 258bp;
Sequence table SEQ ID NO:5 is that primer is to HY-Fad2 forward primer sequence;
Sequence table SEQ ID NO:6 is that primer is to HY-Fad2 reverse primer sequence;
Sequence table SEQ ID NO:7 is that primer is to YQ-Fad2a-1 forward primer sequence;
Sequence table SEQ ID NO:8 is that primer is to YQ-Fad2a-1 reverse primer sequence;
Sequence table SEQ ID NO:9 is that primer is to YQ-Fad2a-2 forward primer sequence;
Sequence table SEQ ID NO:10 is that primer is to YQ-Fad2a-2 reverse primer sequence.
Fig. 1: utilize primer to HY-Fad2 at swede type rape strain A-grade in the first class 177 and A-grade in the first class 254 and F
1Genomic dna in amplification.Among the figure:
P
1Represent A-grade in the first class 177; P
2Represent A-grade in the first class 254; F
1Represent 254 * A-grade in the first class of A-grade in the first class 177; M representation DNA marker (clip size is followed successively by 1500,1200,1000,900,800,700,600,500,400,300,200 and 100bp).
Fig. 2: utilize primer to HY-Fad2 at swede type rape strain A-grade in the first class 177 and A-grade in the first class 254 and F
1Genome in the sequence dna fragment comparison of amplification, among the figure: NO 1, NO 2 represent the sequence of SEQ ID NO:1 and SEQ ID NO:2 in the sequence table respectively, trilateral is represented the insertion sudden change of the 4bp of 567-568bp place of sequence, and the primer location of design (being that primer is to YQ-Fad2a-1, YQ-Fad2a-2) is referring to the underscore place.
Fig. 3: utilize primer to HY-Fad2 at swede type rape strain A-grade in the first class 177 and A-grade in the first class 254 and F
1Genome in the sequence SEQ IDNO:1 that obtains of amplification and SEQ ID NO:2 aminoacid sequence comparison after translating.Among the figure: NO 1, NO 2 represent the sequence of SEQ ID NO:1 and SEQ ID NO:2 in the sequence table respectively, NO 1-aa, NO 2-aa represent the sequence translation back aminoacid sequence of SEQ ID NO:1 and SEQ ID NO:2 in the sequence table respectively, and * * represents terminator codon.
Fig. 4: the primer that the present invention obtains to YQ-Fad2a-1, YQ-Fad2a-2 at swede type rape strain A-grade in the first class 177 and A-grade in the first class 254, F
1And utilization (254 * A-grade in the first class of A-grade in the first class 177) F
1The part individual plant detected result of the DH colony that obtains.Among the figure: P
1Represent A-grade in the first class 177; P
2Represent A-grade in the first class 254; F
1Represent 254 * A-grade in the first class of A-grade in the first class 177; 1-8 represents high oleic acid in the DH colony (oleic acid content＞75%) individual plant; 9-16 represents low oleic acid (oleic acid content＜65%) individual plant in the DH colony; M representation DNA marker (clip size is followed successively by 1500,1200,1000,900,800,700,600,500,400,300,200 and 100bp).
Fig. 5: be techniqueflow chart of the present invention.
Embodiment
1, set up double haploid (DH) colony:
In the present embodiment, do with high oleic acid strain A-grade in the first class 254 that maternal (oleic acid content is 76.91%, the seed of this material has been delivered Chinese typical culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, its preserving number is CCTCC-P200909), (oleic acid content is 61.95% in conventional oil acid content rape strain A-grade in the first class 177, the seed of this material is delivered Chinese typical culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009, and its preserving number is CCTCC-P200908) obtain F as paternal hybrid
1, utilize F
1(the microspores culture method is referring to document through microspores culture for the pollen of plant, lingering remnants of past customs group etc., some the cultivation factor research that improves swede type rape sporule embryoid seedling rate, Acta Agronomica Sinica, 1997,23 (2): 165-168) obtain 190 double haploids (being called for short DH) colony.
2, extract the swede type rape genomic dna
Extract genomic dna DH segregating population that obtains from above-mentioned steps and parents''s (being the described swede type rape of above-mentioned steps A-grade in the first class 177 and A-grade in the first class 254) the fresh and tender blade, concrete preparation method is with reference to (Li Jia etc. such as Li Jia, the method of the total DNA of a kind of effective extraction rape leaf, Hua Zhong Agriculture University's journal, 1994,13 (5): 521-523) reported method is carried out, agarose gel electrophoresis with 1% detects the DNA quality, and detect DNA concentration with ultraviolet spectrophotometer (model: Pharmacia Biotech, GeneQuant II).
3, the mensuration of oleic acid content:
Seed is pressed individual plant results back gas chromatograph (HP6890, Genmany), analyze fatty acid content, after grinding, parent and offspring's compound sample picked at random 30-50 grain full seed pour the 10ml test tube into, in test tube, add 1ml ether, sherwood oil (volume ratio is 1: 1) mixed solution, and then add isopyknic methyl alcohol (containing 5%KOH) and carry out esterification, leave standstill more than 40 minutes it is fully reacted.Add aquae destillata at last and be settled to the 10ml extraction, get top ether layer solution and get sample introduction mensuration.If carry out half material analysis, the add-on of ether, sherwood oil (volume ratio is 1: 1) mixed solution is 100ul.
Lipid acid composition gas chromatography determination, chromatographic condition is as follows
Chromatographic instrument: Hewlett Packard (HP6890, Genmany), flame ionization ditector, hand sampling, sample size 0.4ul (half is analyzed sample size is 0.8ul), splitting ratio 1: 45; Chromatographic column HP-inowax19091N-133,30m * 0.25mm * 0.25um capillary column; Inspection side device and Sample Room temperature are respectively 250 ℃ and 280 ℃; Carrier gas: N
2, 30ml/min, tail blows 40min/min; Air velocity: 300ml/min; H
2Flow velocity: 30min/min; Furnace temperature: persistently overheating, 180 ℃ kept 2 minutes, rose to 220 ℃ with 10 ℃/min afterwards and kept and keep 7min.
The lipid acid composition determines that by the retention time and the standard substance contrast of position, peak content is then represented with area percentage.When being analyzed and put in order, the data of measuring only consider 7 kinds of main lipid acid, promptly brown eleostearic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linolic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:1), erucic acid (C22:1).
The oleic acid content frequency distribution the results are shown in Table 1 in the Brassica napus DH segregating population of measuring.Swede type rape oleic acid content data are carried out suitability (x
2) test, test method is edited referring to Agricultural University Of Nanjing, field test and statistical method, Chinese agriculture press, 1985, second edition.Consequently, (254 * A-grade in the first class of A-grade in the first class 177) F
1Set up DH colony through microspores culture and obtain 190 individual plants altogether, oleic acid content is lower than totally 97 of 70% individual plants, and the oleic acid content of 93 individual plants is more than or equal to 70%, and the ratio of high oleic acid and low oleic acid individual plant quantity meets 1: 1 segregation ratio.These data declaration swede type rape oleic acid contents are controlled by single genetic locus.Experiment sees Table 1.
The phenotype of the oleic acid content of Brassica napus DH segregating population is separated among table 1 the present invention
| Segregating population | Total strain number | Oleic acid 〉=70.0% | Oleic acid＜70.0% | Desired ratio | ????x 2 | The P value |
| (254 * A-grade in the first class of A-grade in the first class 177) F 1The DH colony that cultivates | ??190 | ????93 | ????97 | ????1∶1 | ??0.047 | ??0.75-1.0 |
Annotate: df=1; x
20.05=3.84, x
2 0.01=6.63.
4, utilize primer to HY-Fad2 amplification analysis parents and F
1Genomic dna
The primer that utilizes our unit to develop increases to HY-Fad2 (primer sees Table 2 to sequence) and analyzes swede type rape strain A-grade in the first class 177 and A-grade in the first class 254 and F
1Genomic dna.
The PCR reaction system is as follows: 1 * PCR buffer, 1.35mM MgCl
2, 0.08mM dNTPs, 1.0U Taq archaeal dna polymerase (all available from MBI Fermentas, Lithuania company), 100ng DNA, each 0.45 μ M of forward and reverse primer, ddH
2O is supplemented to final volume 20 μ l.Thermal circulation parameters is: 94 ℃ of 3min; 94 ℃ of 30sec, 54 ℃ of 45sec, 72 ℃ of 60sec, 29 circulations; 72 ℃ of 10min, 1 circulation; 4 ℃ of preservations, reaction are to finish on PTC-ALD1244 PCR instrument.Amplified production 1.0% agarose gel electrophoresis on the horizontal strip electrophoresis groove detects, and use 1 * TAE damping fluid (0.04M Tris-acetate, 0.001M EDTA, pH8.0), voltage 8V/cm, electrophoresis 35min.Electrophoresis finishes, gel imaging system (UVP) preservation of taking pictures.
Among the present invention primer to HY-Fad2 at parents and F
1Genomic dna in amplified production be single bright band about 1200bp, 1.0% agarose gel electrophoresis detection lug segment length indifference (experimental result is seen Fig. 1).
The primer of table 2 the present invention design is to nucleotide sequence
5, the dna fragmentation that recovery, clone, sequencing primer increase in parents to HY-Fad2
The dna fragmentation that the primer that obtains in the recovery above-mentioned steps increases in swede type rape strain A-grade in the first class 177 and A-grade in the first class 254 to HY-Fad2.Schedule of operation reclaims the method that test kit (available from Shanghai JaRa bio-engineering corporation) specification sheets provides by GenClean pillar DNA glue: the dna fragmentation that digs out amplification with blade from 1.0% agarose gel, put into the centrifuge tube of 1.5ml, add 300 μ lBinding Solution B by every 100mg sepharose, place 55 ℃ of water-baths to heat 10min, once every the 2min mixing; The sol solution that melts is transferred among the GenClean Column that is enclosed within the collection tube, and room temperature is placed 2min, 3, the centrifugal 30sec of 000rpm; Outwell the waste liquid in the collection tube, add 500 μ l WashSolution, 8, the centrifugal 30sec of 000rpm room temperature, this step repeats once; Outwell the waste liquid in the collection tube, GenClean Column is put into same collection tube, 10, the centrifugal 1min of 000rpm puts into GenClean Column the centrifuge tube of a new 1.5ml, add 30 μ l Elution Buffer in pillar film central authorities, room temperature is placed 2min; 10, the centrifugal 1min of 000rpm, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
The target dna fragment 2 μ l that get above-mentioned recovery make template, carry out pcr amplification with primer HY-Fad2 according to above-mentioned steps, and the agarose gel 1.0% detects the dna fragmentation of amplification.If the result who increases in the dna fragmentation length of amplification and the above-mentioned steps is inequality, need amplification again to reclaim; If that increases in the dna fragmentation length of amplification and the above-mentioned steps comes to the same thing, illustrate that reclaiming the dna fragmentation that obtains promptly is the target dna fragment, can be used for next step T-A clone.
The target dna fragment that reclaims is connected (this carrier is available from TaKaRa company, and precious biotechnology (Dalian) company limited is acted on behalf of) on the pMDT-18 carrier.The method that schedule of operation is introduced by the specification sheets of this test kit: earlier of short duration before use centrifugal it is collected in of reagent managed the bottom; Carry out ligation in the centrifuge tube of 0.5ml, the ligation system is DNA 2.0 μ l, pMDT-18 carrier 0.5 μ l and Solution I 2.5 μ l.Come resorption mixing several times with transfer pipet, put the ligation of spending the night of 4 ℃ of refrigerators; Prepare LB liquid nutrient medium and LB solid medium (containing the 100mg/ml penbritin, 5-bromo-4-chloro-3-indoles-α-D-galactoside of the sec.-propyl of 24mg/ml-sulfo-B-D-galactoside and 20mg/ml); From-70 ℃ of refrigerators, take out competent cell be placed on treat on ice it slowly thaw (about 5min); Centrifugal collection ligation liquid is got 2 μ l reaction solutions and is joined a 1.5ml centrifuge tube of having sterilized (being placed on precooling on ice); Flick with finger at the bottom of the pipe that competent cell is housed with mixing, get 50 μ l competent cells and add the 1.5ml centrifuge tube that 2 μ l ligation liquid are housed, flick mixing, be placed on 20min on ice with pointing; Heat shock 90sec (not shaking) in 42 ℃ of water-baths places 5min then on ice; Add behind the LB liquid nutrient medium of 500 μ l at 37 ℃ of shaking culture 1h (150rmp/min); Conversion fluid 200 μ l after the absorption shaking culture are coated on the aseptic LB solid medium, place 16-20h at 37 ℃; Carry out indigo plant, hickie screening, select 24 positive colonies and be numbered, and at aseptic liquid LB substratum (penbritin that contains 50ug/ml) shaking culture 16-20h; The bacterium liquid 2 μ l that get after the shaking culture make pcr template, with M13 do primer (forward primer: 5 '-CAGGGTTTTCCCAGTCACGA-3 '; Reverse primer: 5 '-CGGATAACAATTTCACACAGGA-3 ') amplification, PCR reaction is as described in the above-mentioned step.Amplification detects on 1.0% sepharose.If the dna fragmentation of gained than the big 200bp of target dna fragment about, illustrate to transform successfully, select 8 parts transform successful bacterium liquid respectively draw 100 μ l send China greatly Gene science limited-liability company carry out sequencing.Remaining the muddy bacterium liquid of 400 μ l adds 400 μ l, 50% aseptic glycerine and preserves in-70 ℃ of numberings in the aseptic centrifuge tube of 2ml.
Among the present invention, primer respectively repeats order-checking, wherein sequence results unanimity for 8 times to the dna fragmentation that HY-Fad2 increases in swede type rape strain A-grade in the first class 177 and A-grade in the first class 254.Primer is respectively 1155bp, 1159bp to the amplification of DNA fragments length of HY-Fad2 in A-grade in the first class 177, A-grade in the first class 254, and its nucleotide sequence is shown in sequence table SEQ ID NO:2 and SEQ ID NO:1.
6, in the preparation swede type rape with the SCAR mark of the high oleic acid linkage of characters
The primer that above-mentioned steps is obtained carries out sequence alignment to the fragment that HY-Fad2 increases with EBI Tools ClustalW (http://www.ebi.ac.uk/Tools/clustalw/index.html) software in A-grade in the first class 177 and A-grade in the first class 254.Comparison result shows, the dna fragmentation that amplifies in A-grade in the first class 254 is in the insertion sudden change that a 4bp arranged at the 567-568bp place, and inserting base is AGCC (seeing accompanying drawing 2), causes sequence specific amplification region polymorphism.
Nucleotide sequence (shown in sequence table SEQ ID NO:2 and NO:1) according to primer HY-Fad2 amplification of DNA fragments in swede type rape strain A-grade in the first class 177 and A-grade in the first class 254, utilize Primer Premier 5.0 softwares (http://www.PremierBiosoft.com) of report, forward primer of design between the 553-570bp in sequence SEQ ID NO:1.Requiring primer length during design is 17～22 bp, 50～62 ℃ of renaturation temperature, and GC content is 40～60%.Screening obtains the twin target primer, and (sequence sees Table in 2 primer to YQ-Fad2a-1, the position is seen shown in the underscore of NO 1 among Fig. 2), with its called after YQ-Fad2a-1, be expected at the fragment that amplifies 201 bp in the genomic dna of swede type rape A-grade in the first class 254, and in the genomic dna of A-grade in the first class 177 no any amplified production; According to identical method, forward primer of design between the 553-570bp in sequence SEQ ID NO:2.(sequence sees Table in 2 primer to YQ-Fad2a-2 to obtain the twin target primer by above-mentioned requirements screening, the position is seen shown in the underscore of NO_2 among Fig. 2), be expected at the fragment that amplifies 258bp in the genomic dna of parent A-grade in the first class 177, and in the genomic dna of parent A-grade in the first class 254 no any amplified production.
The present invention utilizes the primer of above-mentioned steps design to carry out PCR, and the PCR reaction system is same as above, 58 ℃ of renaturation temperature.Amplification detects on 1.2% agarose gel.Wherein primer to YQ-Fad2a-1 at swede type rape A-grade in the first class 254 and F
1Genomic dna in amplify the target fragment of 201bp, and in the genomic dna of swede type rape A-grade in the first class 177 no any amplified production (seeing accompanying drawing 4), so this amplified fragments can be used as the dominant SCAR molecule mark of cabbage type rape high oleic acid proterties.Primer to YQ-Fad2a-2 at swede type rape strain A-grade in the first class 177 and F
1Genomic dna in amplify the target fragment of 258bp, and in the genomic dna of swede type rape A-grade in the first class 254 no any amplified production (seeing accompanying drawing 4), this amplified fragments and primer can be used as the codominance SCAR molecule marker of cabbage type rape high oleic acid proterties to YQ-Fad2a-1 amplified fragments combination.
7, the checking of the codominance SCAR marker combination of cabbage type rape high oleic acid proterties
Utilize the DH segregating population (seeing Table 3) among cabbage type rape high oleic acid SCAR labeled analysis the present invention that above-mentioned steps obtains,
The oleic acid content of Brassica napus DH segregating population and marker detection result among table 3 the present invention
In the table: P
1Represent A-grade in the first class 177; P
2Represent A-grade in the first class 254.
Wherein 97 individual plants the same with amplified fragments in the parent A-grade in the first class 177 (promptly utilize primer to YQ-Fad2a-1 amplification do not have the band line, amplification has the band line to primer to YQ-Fad2a-2, be designated as " B banding pattern "), other 93 individual plants are the same with amplified fragments in the parent A-grade in the first class 254 (promptly to utilize primer to YQ-Fad2a-2 amplification nothing band line, amplification has the band line to primer to YQ-Fad2a-1, be designated as " A banding pattern "), by data in the analytical table as can be known, every primer is to YQ-Fad2a-1, YQ-FAD2a-2 detects, detected result is " an A banding pattern ", its oleic acid content is all more than 70.0%, and detected result is " a B banding pattern ", its oleic acid content is all less than 70.0%, and this explanation is isolating altogether with the cabbage type rape high oleic acid proterties at this mark of DH segregating population.Therefore, utilize primer can effectively identify the individuality that carries high preproinsulin gene in early days, can change into qualitative character to a quantitative character and select, improve accuracy and the validity selected YQ-Fad2a-1, YQ-Fad2a-2.
Sequence table
＜110〉Hua Zhong Agriculture University
＜120〉preparation method of cabbage type rape high oleic acid molecular marker and application
<130>
<141>2009-12-26
<160>4
<170>PatentIn?version?3.1
<210>1
<211>1159
<212>DNA
＜213〉swede type rape (Brassica napus)
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<223>
<220>
<221>primer_bind
<222>(1)..(20)
<223>
<400>1
atgggtgcag?gtggaagaat?gcaagtgtct?cctccctcca?aaaagtctga?aaccgacaac?????60
atcaagcgcg?taccctgcga?gacaccgccc?ttcactgtcg?gagaactcaa?gaaagcaatc????120
ccaccgcact?gtttcaaacg?ctcgatccct?cgctctttct?cctacctcat?ctgggacatc????180
atcatagcct?cctgcttcta?ctacgtcgcc?accacttact?tccctctcct?ccctcaccct????240
ctctcctact?tcgcctggcc?tctctactgg?gcctgccagg?gctgcgtcct?aaccggcgtc????300
tgggtcatag?cccacgagtg?cggccaccac?gccttcagcg?actaccagtg?gctggacgac????360
accgtcggcc?tcatcttcca?ctccttcctc?ctcgtccctt?acttctcctg?gaagtacagt????420
catcgacgcc?accattccaa?cactggctcc?ctcgagagag?acgaagtgtt?tgtccccaag???480
aagaagtcag?acatcaagtg?gtacggcaag?tacctcaaca?accctttggg?acgcaccgtg???540
atgttaacgg?ttcagttcac?tctcggcagc?ctggcctttg?tacttagcct?tcaacgtctc???600
ggggagacct?tacgacggcg?gcttcgcttg?ccatttccac?cccaacgctc?ccatctacaa???660
cgaccgtgag?cgtctccaga?tatacatctc?cgacgctggc?atcctcgccg?tctgctacgg???720
tctctaccgc?tacgctgctg?tccaaggagt?tgcctcgatg?gtctgcttct?acggagttcc???780
tcttctgatt?gtcaacgggt?tcttagtttt?gatcacttac?ttgcagcaca?cgcatccttc???840
cctgcctcac?tatgactcgt?ctgagtggga?ttggttgagg?ggagctttgg?ccaccgttga???900
cagagactac?ggaatcttga?acaaggtctt?ccacaatatc?acggacacgc?acgtggcgca???960
tcacctgttc?tcgaccatgc?cgcattatca?tgcgatggaa?gctacgaagg?cgataaagcc??1020
gatactggga?gagtattatc?agttcgatgg?gacgccggtg?gttaaggcga?tgtggaagga??1080
ggcgaaggag?tgtatctatg?tggaaccgga?caggcaaggt?gagaagaaag?gtgtgttctg??1140
gtacaacaat?aagttatga???????????????????????????????????????????????1159
<210>2
<211>1155
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>gene
<222>(1)..(1155)
<223>
<220>
<221>primer_bind
<222>(1134)..(1155)
<223>
<220>
<221>primer_bind
<222>(792)..(810)
<223>
<220>
<221>primer_bind
<222>(553)..(570)
<223>
<220>
<221>primer_bind
<222>(1)..(20)
<223>
<400>2
atgggtgcag?gtggaagaat?gcaagtgtct?cctccctcca?aaaagtctga?aaccgacaac?????60
atcaagcgcg?taccctgcga?gacaccgccc?ttcactgtcg?gagaactcaa?gaaagcaatc????120
ccaccgcact?gtttcaaacg?ctcgatccct?cgctctttct?cctacctcat?ctgggacatc????180
atcatagcct?cctgcttcta?ctacgtcgcc?accacttact?tccctctcct?ccctcaccct????240
ctctcctact?tcgcctggcc?tctctactgg?gcctgccagg?gctgcgtcct?aaccggcgtc????300
tgggtcatag?cccacgagtg?cggccaccac?gccttcagcg?actaccagtg?gctggacgac????360
accgtcggcc?tcatcttcca?ctccttcctc?ctcgtccctt?acttctcctg?gaagtacagt????420
catcgacgcc?accattccaa?cactggctcc?ctcgagagag?acgaagtgtt?tgtccccaag???480
aagaagtcag?acatcaagtg?gtacggcaag?tacctcaaca?accctttggg?acgcaccgtg???540
atgttaacgg?ttcagttcac?tctcggctgg?cctttgtact?tagccttcaa?cgtctcgggg???600
agaccttacg?acggcggctt?cgcttgccat?ttccacccca?acgctcccat?ctacaacgac???660
cgtgagcgtc?tccagatata?catctccgac?gctggcatcc?tcgccgtctg?ctacggtctc???720
taccgctacg?ctgctgtcca?aggagttgcc?tcgatggtct?gcttctacgg?agttcctctt???780
ctgattgtca?acgggttctt?agttttgatc?acttacttgc?agcacacgca?tccttccctg???840
cctcactatg?actcgtctga?gtgggattgg?ttgaggggag?ctttggccac?cgttgacaga???900
gactacggaa?tcttgaacaa?ggtcttccac?aatatcacgg?acacgcacgt?ggcgcatcac???960
ctgttctcga?ccatgccgca?ttatcatgcg?atggaagcta?cgaaggcgat?aaagccgata??1020
ctgggagagt?attatcagtt?cgatgggacg?ccggtggtta?aggcgatgtg?gagggaggcg??1080
aaggagtgta?tctatgtgga?accggacagg?caaggtgaga?agaaaggtgt?gttctggtac??1140
aacaataagt?tatga???????????????????????????????????????????????????1155
<210>3
<211>201
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>gene
<222>(1)..(201)
<223>
<220>
<221>primer_bind
<222>(1)..(18)
<223>
<220>
<221>primer_bind
<222>(184)..(201)
<223>
<400>3
cagttcactc?tcggcagcct?ggcctttgta?cttagccttc?aacgtctcgg?ggagacctta?????60
cgacggcggc?ttcgcttgcc?atttccaccc?caacgctccc?atctacaacg?accgtgagcg????120
tctccagata?tacatctccg?acgctggcat?cctcgccgtc?tgctacggtc?tctaccgcta????180
cgctgctgtc?caaggagttg?c??????????????????????????????????????????????201
<210>4
<211>258
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>gene
<222>(1)..(258)
<223>
<220>
<221>primer_bind
<222>(240)..(258)
<223>
<220>
<221>primer_bind
<222>(1)..(20)
<223>
<400>4
cagttcactc?tcggctggcc?tttgtactta?gccttcaacg?tctcggggag?accttacgac?????60
ggcggcttcg?cttgccattt?ccaccccaac?gctcccatct?acaacgaccg?tgagcgtctc????120
cagatataca?tctccgacgc?tggcatcctc?gccgtctgct?acggtctcta?ccgctacgct????180
gctgtccaag?gagttgcctc?gatggtctgc?ttctacggag?ttcctcttct?gattgtcaac????240
gggttcttag?ttttgatc??????????????????????????????????????????????????258
<210>5
<211>20
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(20)
<223>
<400>5
atgggtgcag?gtggaagaat????????????????????????????????????????????????20
<210>6
<211>22
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(22)
<223>
<400>6
tcataactta?ttgttgtacc?ag?????????????????????????????????????????????22
<210>7
<211>18
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(18)
<223>
<400>7
cagttcactc?tcggcagc??????????????????????????????????????????????????18
<210>8
<211>18
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(18)
<223>
<400>8
gcaactcctt?ggacagca????????????????????????????18
<210>9
<211>18
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(18)
<223>
<400>9
cagttcactc?tcggctgg????????????????????????????18
<210>10
<211>19
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>primer_bind
<222>(1)..(19)
<223>
<400>10
gatcaaaact?aagaacccg????????????????????????????19
Claims (5)
1. codominance SCAR molecule marker that is applicable to the seed selection of high oleic acid swede type rape, its nucleotide sequence is shown in sequence table SEQ ID NO:3 and SEQ ID NO:4.
2. be used to increase the primer of high oleic acid swede type rape codominance SCAR molecule marker to HY-Fad2a-1 and primer to HY-Fad2a-2, its nucleotide sequence is as follows:
Primer is to (1) YQ-Fad2a-1:
Forward primer 5 '-CAGTTCACTCTCGGCAGC-3 ',
Reverse primer 5 '-GCAACTCCTTGGACAGCA-3 ';
Primer is to (2) YQ-Fad2a-2:
Forward primer 5 '-CAGTTCACTCTCGGCTGG-3 ',
Reverse primer 5 ' GATCAAAACTAAGAACCCG-3 '.
3. the preparation method of one kind high oleic acid swede type rape codominance SCAR molecule marker, according to following steps:
Utilize primer HY-Fad2 increase high oleic acid swede type rape strain A-grade in the first class 254 and low oleic acid swede type rape strain A-grade in the first class 177 genomic dnas, obtain two DNA cloning fragments, then described DNA cloning fragment is cloned, order-checking, wherein amplification obtains nucleotide sequence shown in SEQ ID NO:1 from high oleic acid swede type rape strain A-grade in the first class 254, and amplification obtains the nucleotide sequence shown in SEQ ID NO:2 from low oleic acid swede type rape strain A-grade in the first class 177; The insertion sudden change of a 4bp is arranged at the 567-568bp place of the nucleotide sequence shown in the described SEQ IDNO:1, and the insertion base is AGCC, causes sequence specific amplification region polymorphism; According to the nucleotide sequence difference shown in SEQ ID NO:1 and SEQ ID NO:2 design primer to YQ-Fad2a-1 and primer to YQ-Fad2a-2, carry out pcr amplification, what obtain shown in SEQ ID NO:3 and SEQ ID NO:4 nucleotide sequence can distinguish cabbage type rape high oleic acid and low oleic codominance SCAR molecule marker.
4. the application of the described molecule marker of claim 1 in the high oleic acid swede type rape of assisted Selection.
5. the described special primer of claim 2 application in the high oleic acid swede type rape of seed selection to HY-Fad2a-1 and primer HY-Fad2a-2.
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| CN104278025A (en) * | 2013-07-10 | 2015-01-14 | 华中农业大学 | SCAR molecular markers of Brassica napus whole genomes and preparation method and application of SCAR molecular markers |
| EP2761031A4 (en) * | 2011-09-27 | 2015-09-23 | Dow Agrosciences Llc | Ho/ll canola with resistance to clubroot disease |
| CN105567856A (en) * | 2016-03-02 | 2016-05-11 | 四川农业大学 | Genotype detection method of fad2 gene of cabbage type rape |
| CN105613258A (en) * | 2016-01-26 | 2016-06-01 | 华中农业大学 | Cultivating method for high-oleic acid rapeseed variety |
| CN105671161A (en) * | 2016-03-02 | 2016-06-15 | 四川农业大学 | Method for determining high oleic acid in brassica napus on basis of BnaLCR78 gene |
| CN108239674A (en) * | 2017-11-19 | 2018-07-03 | 华中农业大学 | Cabbage type rape high oleic acid QTL and the molecular labeling with its close linkage |
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| CN100434534C (en) * | 2006-01-19 | 2008-11-19 | 华中农业大学 | Molecular marker for artificial yellow seed rape (brassica napus L.) and its application |
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| EP3447151A1 (en) * | 2011-09-27 | 2019-02-27 | Dow AgroSciences LLC | Ho/ll canola with resistance to clubroot disease |
| CN104278025B (en) * | 2013-07-10 | 2017-06-20 | 华中农业大学 | Cabbage type rape full-length genome SCAR molecular labelings and preparation method and application |
| CN104278025A (en) * | 2013-07-10 | 2015-01-14 | 华中农业大学 | SCAR molecular markers of Brassica napus whole genomes and preparation method and application of SCAR molecular markers |
| CN105613258A (en) * | 2016-01-26 | 2016-06-01 | 华中农业大学 | Cultivating method for high-oleic acid rapeseed variety |
| CN105671161A (en) * | 2016-03-02 | 2016-06-15 | 四川农业大学 | Method for determining high oleic acid in brassica napus on basis of BnaLCR78 gene |
| CN105567856B (en) * | 2016-03-02 | 2018-11-13 | 四川农业大学 | A kind of genotype detection method of the fad2 genes of cabbage type rape |
| CN105567856A (en) * | 2016-03-02 | 2016-05-11 | 四川农业大学 | Genotype detection method of fad2 gene of cabbage type rape |
| CN105671161B (en) * | 2016-03-02 | 2019-06-14 | 四川农业大学 | A method of containing high oleic acid according to BnaLCR78 genetic testing cabbage type rape |
| CN108239674A (en) * | 2017-11-19 | 2018-07-03 | 华中农业大学 | Cabbage type rape high oleic acid QTL and the molecular labeling with its close linkage |
| CN108239674B (en) * | 2017-11-19 | 2021-04-13 | 华中农业大学 | Cabbage type rape high oleic acid QTL and molecular marker closely linked with same |
| CN108504761A (en) * | 2018-04-19 | 2018-09-07 | 西南医科大学 | The detection kit and detection method of eclipta |
| CN110326532A (en) * | 2019-07-23 | 2019-10-15 | 华中农业大学 | A kind of selection of high oleic acid Rapeseed GMS system and its application |
| CN110699481A (en) * | 2019-11-20 | 2020-01-17 | 华中农业大学 | Gene closely related to glucosinolate content of rape leaves, molecular marker and application thereof |
| CN110699481B (en) * | 2019-11-20 | 2021-06-04 | 华中农业大学 | Gene closely related to glucosinolate content of rape leaves, molecular marker and application thereof |
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| CN101824472B (en) | 2012-02-08 |
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