CN101818196B - Molecular marker for low linolenic acid of cabbage type rape, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of rape molecular breeding, in particular relates to a preparation method of a codominant SNP (Single Nucleotide Polymorphism) molecular marker closely linked with the linolenic acid content of a cabbage type rape and application thereof as the marker in the auxiliary selection of the low linolenic acid cabbage type rape. The low linolenic acid cabbage type rape strain A254 and high linolenic acid cabbage type rape strain A177 genomes DNA are amplified by using primers to respectively obtain two DNA amplified fragments, then cloning, sequencing and nucleotide sequences comparison are carried out on the four DNA amplified fragments; according to the difference of the sequences, the second bases at 3'-end of a forward primer and a reverse primer are mispaired, primer pairs of YQ-fad31-1, YQ-fad31-2, YQ-fad32-1 and YQ-fad32-2 are designed, the PCR (Polymerase Chain Reaction) amplification is carried out on the primer pairs of YQ-fad31-1, YQ-fad31-2, YQ-fad32-1 and YQ-fad32-2, and the PCR amplification and group detection effects are analyzed to obtain the codominant SNP molecular marker linked with low linolenic acid genes of the cabbage type rape. The invention provides a new marker for the rape molecular breeding. In the invention, a mispairing strategy of the 3'-end of the primer is firstly applied to the development and the detection of the SNP polymorphic marker of the cabbage type rape, and a means of synchronously mispairing the forward primer and the reverse primer is firstly adopted to develop the SNP marker based on the PCR amplification and an agarose gel electrophoresis in the cabbage type rape of an allopolyploid crop. The invention synchronously discloses a method for preparing the molecular marker and the application thereof.

Description

Molecular marker for low linolenic acid of cabbage type rape and preparation method thereof and application

Technical field

The invention belongs to the rape molecular breeding technical field, be specifically related to a kind of preparation and application of molecular marker for low linolenic acid of cabbage type rape.Described molecule marker can be used as low linolenic swede type rape breeding marker assisted selection, for the low linolenic rape new variety of cultivating inheritance stability provide 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.Linolenic acid is the highest lipid acid of oxidation ratio in the highly unsaturated fatty acids, forms the stench oxide compound with oxidation very easily takes place after air, light, heat etc. contact, and makes the rotten and storage tolerance not of rape oil.Even more serious is that linolenic oxidation products is harmful; Can cause cardiovascular atherosis (Chang etc.; Effects of the ratio ofpolyunsaturated and monounsaturated fatty acid on rat plasma and liver lipid concentration.Lipids; 1998,33:481-487).Behind the double-strand break of unsaturated fatty acids; Than being easier to form radical; Peroxidatic reaction of lipid promptly appears; Thereby can damaging cells with the structure of cytolemma and function (hole auspicious sign, the nutritional-physiological of essential trace element and clinical meaning. Hefei: the .1982:3-60 of Anhui Science Press), directly influence cell division, grow, breed and hereditary.Also can make grease have stronger sharp flavor in the rapeseed oil after the alpha-linolenic acid oxidation.Therefore; Linolenic acid content is long-term objective (the Rachael Scarth etc. of breeding man aspect the swede type rape quality breeding in the reduction rapeseed oil; Designer oil canola-a review of new food-grade Brassica oils with focus on high oleic; Low linolenic types, 10thInt.Rapeseed Congress, 1999).

The low linolenic genetic resources also lacks very much both at home and abroad at present, when the low linolenic proterties being imported to the high linolenic kind of high yield, mainly utilizes traditional Phenotypic Selection method.Because linolenic acid content receives controlled by multiple genes in the swede type rape, has shortcomings such as breeding cycle is long, efficient is low.Molecular marker assisted selection technology in the modern biotechnology has been compared plurality of advantages with traditional Phenotypic Selection.Molecular marker assisted selection can be carried out in any vegetative period, did not receive environmental influence, can get rid of that non-allelic genes interact and the interference that causes, have quick, economical, the characteristics that efficient is high, accuracy is strong.In the low linolenic acid of cabbage type rape 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 molecule marker capable of using is selected background, thereby accelerates the genetic background resume speed, shortening the breeding cycle with alleviate chain burden.And one of prerequisite of carrying out these work is to develop molecule marker efficiently.

Aspect the location of low linolenic purpose proterties, domestic and international research person has obtained a lot of achievements in research.(Somers etc. such as Somers; Identification of Molecular Markers Associated with Linoleic Acid Desaturation in Brassica napus, Theoreticaland Applied Genetics, 1998; 96.897-903) utilize the BSA method in the DH colony that forms by 115 genotype; Find 16 RAPD marks relevant, be positioned at 3 linkage groups, explain 32% respectively with linolenic acid content; 14% and 5% phenotypic variation, the swede type rape fad3 assignment of genes gene mapping is on this linkage group of explaining 14% variation.Show additive effect between three QTLs, explain 51% variation altogether.(Hu J etc. such as HU; Mapping of a gene determining linolenic acid content in rapeseed with DNA based markers.Theor Appl Genet1995; 90:258-262) also find a RAPD dominant marker relevant with linolenic acid content, this mark amplified fragments size is 650bp.They are converted into the SCAR mark of dominance further with this fragment cloning, order-checking.(Hu J etc., SCAR and RAPD marker associated with18-carbon fatty acids in rapeseed, Brassica napus.Plant Breeding, 1999,118:145-150).(Cheung etc. such as Cheung; Molecular mapping of seed quality traits in (Brassica juncea L.) czern.and coss.Acta Horticulturae; 1998,459:139-147) one of the QTLs of two control of discovery linolenic acid contents is positioned at one in fael site and is positioned at the fad3 site.(Jourdren etc. such as Jourdren; Specific molecular marker of the genes controlling linolenic acid content in rapeseed.Theor Appl Genet; 1996b 93:512-518) has located two QTLss relevant with linolenic acid content and fad3 gene and has been closely related.(Lionneton etc. such as Lionneton; Development of an AFLP-based linkage map and localization of QTLs for seed fatty acid content incondiment mustard (Brassica juncea) Genome 2002,45:1203-1215) discovery is positioned at the phenotypic variation in the QTL site soluble 41.2% on the LG2 for linolenic acid content.

(F.Javidfar etc. such as F.Javidfar; Identification of molecular markers associated with oleic and linolenic acid inspring oilseed rape (Brassica napus) .Plant Breeding, 2006,125:65-71) with a high oleic acid; Low linolenic kind (C18:1＞79%; C18:3＜2%) T099-5318-20 and a high oleic acid, high linolenic (C18:1=68%, C18:3＞7%) DH are DH12075 hybridization.Obtain 8 RAPD marks related with oleic acid and linolenic acid content, wherein RAPD mark UBC2830 is respectively 43% and 13% to the contribution rate of oleic acid and linolenic acid content variation.RAPD mark UBC153550 is 19% to the contribution rate of linolenic acid content variation.Mark UBC2830 is transformed into the SCAR mark.(Daryl Somers, US 2003/0150020 Al, Aug.7,2003 such as Somers; Daryl Somers; US 7081564; July 25,2006) be attained at the SNP mark of low linolenic content, (Hu 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 the main effect QTL relevant with the swede type rape linolenic acid content, a main effect QTL (fad3c) of control low linolenic content is positioned at karyomit(e) No. 14; Another main effect QTL is positioned at karyomit(e) No. 4, comes from the A genome.According to these QTL, design has obtained two and fad2, SNP (Single NucleotidePolymorphisms) mark that fad3 is relevant.Zhao (.Mapping QTL controlling fatty acid composition in a doubled haploidrapeseed population segregating for oil content.Mol Breed such as Zhao J; 2008; 21:115-125) on swede type rape N14, also detect one, explained 28% genetic mutation rate about linolenic QTL.But because (Daryl Somers, US 2003/0150020 A1, Aug.7,2003 such as Somers; Daryl Somers; US 7081564; July 25; 2006) and the SNP that obtains of Hu etc. (2006) only have the difference of one 3 ' end when being marked at design of primers, the effect in swede type rape linolenic acid assisted selection receives influences such as multiple factor such as dna profiling concentration, the synthetic quality of primer, primer concentration, PCR reflection system and parameter, makes its application in actual production exist and is very limited.

Summary of the invention

The objective of the invention is to develop a kind ofly being applicable to that seed selection has the molecule marker of the swede type rape of low linolenic proterties, for the low linolenic acid of cabbage type rape breeding provides a kind of simple, quick and effective supplementary breeding method.

The present invention realizes through following scheme:

The applicant is applicable to that through the test acquisition is a kind of seed selection possesses the swede type rape codominance SNP molecule marker of low linolenic proterties, and its nucleotide sequence is shown in sequence table SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

Preparation is applicable to the dominance SNP molecular marker method of low linolenic acid of cabbage type rape proterties, according to following steps:

Utilize the applicant's designed primer HY-fad31, HY-fad32 amplification low linolenic swede type rape strain A-grade in the first class 254 and high linolenic swede type rape strain A-grade in the first class 177 genomic dnas; Obtain two DNA cloning fragments respectively; Then said 4 DNA cloning fragments are cloned, checked order; Wherein obtain the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:3, obtain the nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:4 from amplification the A-grade in the first class 177 from amplification the A-grade in the first class 254.

Compare with sequence table SEQ ID NO:2; 5 single base mutations of existence and 2 insertion sudden changes (seeing accompanying drawing 3) at sequence table SEQ ID NO:1; The applicant utilizes the SNP (SNP of the mutational site 28T → 28C that exists among SEQ ID NO:1 and the SEQ ID NO:2; Single Nucleotide Polymorphisms); Adopt primer 3 ' end mispairing technical tactic (Drenkard etc.; A simple procedure for theanalysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.Plant Physiol 2000124:1483-1492) has designed primer to YQ-fad31-1 and YQ-fad31-2.Compare with sequence table SEQ ID NO:4; Single base mutation (seeing accompanying drawing 4) at 1 1450G → 1450A of existence of sequence table SEQ IDNO:3; The applicant utilizes the SNP of the mutational site 1450G → 1450A that exists among SEQ ID NO:3 and the SEQ ID NO:4, adopts primer 3 ' end mispairing technical tactic to design primer to YQ-fad32-1 and YQ-fad32-2.Primer is carried out pcr amplification to YQ-fad31-1, YQ-fad31-2, YQ-fad32-1 and YQ-fad32-2; Obtain the codominance SNP molecule marker (being target molecule mark of the present invention) with low linolenic acid of cabbage type rape gene linkage, its nucleotide sequence is shown in sequence table SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

In aforesaid method, the right nucleotide sequence of the primer is as follows:

Primer is numbered HY-fad31 to (1):

Forward primer 5 '-TGGACATGGGAGTTTCTC-3 ',

Reverse primer 5 '-TTAGTTGATTTTGGATTTGTC-3 '.

Primer is numbered HY-fad32 to (2):

Forward primer 5 '-ACTCCACCCTGAGAACTT-3 ',

Reverse primer 5 '-TTAGTTGATTTTGGATTTGTC-3 '.

Primer is numbered YQ-fad31-1 to (3):

Forward primer 5 '-GGAGTTTCTCAGACATTCGT-3 ',

Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3 '.

Primer is numbered YQ-fad31-2 to (4):

Forward primer 5 '-GGAGTTTCTCAGACATTCGC-3 ',

Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3

Primer is numbered YQ-fad32-1 to (5):

Forward primer 5 '-CCTTGGTACAGAGGCAACA-3 ',

Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '.

Primer is numbered YQ-fad32-2 to (6):

Forward primer 5 '-CCTTGGTACAGAGGCAATG-3 ',

Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '.

Wherein, primer is used for the nucleotide sequence shown in extension increasing sequence table SEQ ID NO:1 and the SEQ ID NO:2 to HY-fad31; Primer is used for the nucleotide sequence shown in extension increasing sequence table SEQ ID NO:3 and the SEQ ID NO:4 to HY-fad31; Primer is respectively applied for the nucleotide sequence shown in the SEQ ID NO:5 and SEQ ID NO:6 in the extension increasing sequence table to YQ-fad31-1, YQ-fad31-2; Primer is respectively applied for the nucleotide sequence shown in the SEQ ID NO:7 and SEQ ID NO:8 in the extension increasing sequence table to YQ-fad32-1, YQ-fad32-2.

Positively effect of the present invention:

The present invention holds the mispairing application of policies in the exploitation and the detection of swede type rape SNP polymorphism mark primer 3 ' for the first time; And utilize forward primer and reverse primer mispairing simultaneously means in allopolyploid crop swede type rape, to develop PCR-based amplification and agarose gel electrophoresis SNP mark for the first time.The present invention successfully obtains the codominance SNP molecule marker of low linolenic acid of cabbage type rape; Use and to overcome the shortcoming that relies on phenotype to select in the traditional breeding method in this assisted Selection that is marked at the low linolenic swede type rape; Reduce the breeding work amount; Shortening the breeding cycle, accelerated the process of low linolenic acid of cabbage type rape breeding.

More detailed technical scheme is referring to " embodiment ".

Description of drawings

Sequence table SEQ ID NO:1-4 is 4 dna fragmentations (nucleotide sequence) that the genomic dna of the swede type rape strain A-grade in the first class that increases of the present invention 254 and high linolenic swede type rape strain A-grade in the first class 177 genomic dnas obtains;

Sequence table SEQ ID NO:5-8 is the nucleotide sequence of the swede type rape linolenic acid content molecule marker for preparing of the present invention.

Fig. 1: utilize primer to HY-fad31 at swede type rape strain A-grade in the first class 254 and A-grade in the first class 177 and F ₁Genomic dna in amplification.Among the figure: P ₁Represent A-grade in the first class 254; P ₂Represent A-grade in the first class 177; 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 3000,2600,2050,1650,1000,700,500 and 278bp).

Fig. 2: utilize primer to HY-fad32 at swede type rape strain A-grade in the first class 254 and A-grade in the first class 177 and F ₁Genomic dna in amplification.Among the figure: P ₁Represent A-grade in the first class 254; P ₂Represent A-grade in the first class 177; 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 3000,2600,2050,1650,1000,700,500 and 278bp).

Fig. 3: utilize the sequence dna fragment comparison of primer to the amplification of HY-fad31 in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes; 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; The single base mutation of the 28T → 28C of solid black trilateral " ▲ " expression sequence; Designed primer position (being the forward primer position of primer to YQ-fad31-1, YQ-fad31-2) is referring to the underscore place, and hollow triangle is represented the design of primers base mismatch.

Fig. 4: utilize the sequence dna fragment comparison of primer to the amplification of HY-fad32 in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes; Among the figure: NO_3, NO_4 represent the sequence of SEQ ID NO:3 and SEQ ID NO:4 in the sequence table respectively; The single base mutation of the 1450G → 1450A of solid black trilateral " ▲ " expression sequence; Designed primer position (being the forward primer position of primer to YQ-fad32-1, YQ-fad32-2) is referring to the underscore place, and hollow triangle

is represented the design of primers base mismatch.

Fig. 5: utilize the sequence dna fragment comparison of primer to HY-fad31, the amplification of HY-fad32 in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes; Among the figure: NO_1, NO_2, NO_3, NO_4 represent the sequence of SEQ ID NO:1 in the sequence table, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively, the single base difference between the difference copy of the swede type rape fad gene that solid black trilateral " ▲ " expression primer increases in same parent to HY-fad31, HY-fad32; Pri.1 represents the reverse primer of primer to YQ-fad31-1, YQ-fad31-2; Pri.2 represents the reverse primer of primer to YQ-fad32-1, YQ-fad32-2; The designed primer position is referring to the underscore place; Arrow is represented the primer direction, hollow triangle

expression design of primers base mismatch.

Fig. 6: primer YQ-fad31-1, YQ-fad31-2, YQ-fad32-1 and YQ-fad32-2 that the present invention obtains are at swede type rape strain A-grade in the first class 254, first 177, 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 produces.Among the figure: P ₁Represent A-grade in the first class 254; P ₂Represent A-grade in the first class 177; F ₁Represent 254 * A-grade in the first class of A-grade in the first class 177; 1-18 represents in the DH colony different linolenic acid content individual plants, and (numerical value is the individual plant linolenic acid content under among the figure, unit: %); Fragment length is said primer extension product length.

Fig. 7: be techniqueflow chart of the present invention.

Embodiment

Embodiment 1

1, preparation F ₁For cross-fertilize seed with set up double haploid (DH) colony:

In the present embodiment; (seed of this material was delivered the Chinese typical culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009 with the swede type rape strain A-grade in the first class 254 of the low linolenic content (linolenic acid content is 2.28%) of our unit's seed selection; Its preserving number is CCTCC-P200909) for maternal; With common linolenic acid content (linolenic acid content is 7.78%) rape advanced lines breeding choosing is that (seed of this material was delivered the Chinese typical culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009 in A-grade in the first class 177; Its preserving number is CCTCC-P200908) hybridize for male parent, obtain F ₁Seed.

The F that above-mentioned steps is obtained ₁Seed is sowed under field conditions (factors), obtains F ₁Plant utilizes F ₁The pollen of plant obtains 190 double haploids (being called for short DH) colony through microspores culture.

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 detects DNA concentration with ultraviolet spectrophotometer (model: Pharmacia Biotech, GeneQuant II).

3, the mensuration of linolenic acid content:

Seed is pressed individual plant results back with gas chromatograph (HP6890; Genmany); Analyze fatty acid content, pour the 10ml test tube into after parent and offspring's compound sample picked at random 30-50 grain full seed grind, in test tube, add 1ml ether, sherwood oil (volume ratio 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.Last adding distil water is settled to the 10ml extraction, gets top ether layer solution and gets 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.

The lipid acid composition is used gas chromatography determination, and chromatographic condition is following

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 confirms that by the RT 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), Triple Pressed Stearic Acid (C18:0), oleic acid (C18:1), linolic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:1), erucic acid (C22:1).

4, utilize primer that parents and F are analyzed in HY-fad31, HY-fad32 amplification ₁Genomic dna

The primer that utilizes the applicant to develop increases to HY-fad31, HY-fad32 (primer is seen table 2 to sequence) and analyzes cabbage type rape variety A-grade in the first class 254 and A-grade in the first class 177 and F ₁Genomic dna.

The PCR reaction system is following: 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 C 3min; 94 C 30sec, renaturation temperature 45 C sec, 72 C 60sec, 29 circulations; 72 C 10min, 1 circulation; 4 C preserve, and reaction is on PTC-ALD1244 PCR appearance, to accomplish.Two materials all carry out twice repetition.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-fad31 at parents and F ₁Genomic dna in amplified production be the bright band about 2150bp, this bright band comprises very bright purpose band and the non-target fragment bigger slightly than purpose band, parents and F ₁Amplified production is at 1.0% agarose gel electrophoresis detection lug segment length indifference; Primer to HY-fad32 at parents and F ₁Genomic dna in amplified production be the single bright band about 1950bp, 1.0% agarose gel electrophoresis detection lug segment length indifference (Fig. 1 and table 2 are seen in experiment).

Numbering that table 2 designed primer of the present invention is right and nucleotide sequence thereof

Annotate: on behalf of this base, runic and underscore base (like G) when design of primers, carried out the mispairing processing in (1) * table.

(2) according to the order of describing; In the copy of the end of this specification sheets and nucleotide sequence that provides and/or amino acid computer-reader form; The described primer sequence of above-mentioned table 2 is processed into sequence table SEQ ID NO:9-20 respectively, and it is consistent with the primer sequence shown in the table 2.

5, the dna fragmentation that recovery, clone, sequencing primer increase in parents to HY-fad31, HY-fad32

The dna fragmentation that the primer that obtains in the recovery above-mentioned steps increases in cabbage type rape variety A-grade in the first class 254 and A-grade in the first class 177 to HY-fad31, HY-fad32.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 target DNA fragment that digs out amplification with blade from 1.0% agarose gel; Put into the centrifuge tube of 1.5ml; Add 300 μ l Binding Solution B by every 100mg sepharose; Place 55 C water-baths to heat 10min, every separated 2min mixing once; 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 Wash Solution, 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; GenClean Column is put into 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, and it is subsequent use to use or be stored in-20 C immediately.

The target dna fragment 2 μ l that get above-mentioned recovery make template, with primer HY-fad31, HY-fad32 are carried out pcr amplification 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, explain 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) ltd 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; In the centrifuge tube of 0.5ml, carry out ligation, 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-β-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 at the bottom of the pipe that competent cell is housed with mixing with finger, 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 number, 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, the PCR reaction is of above-mentioned step.Amplification detects on 1.0% sepharose.If the dna fragmentation of gained than the big 200bp of target dna fragment about, explain to transform successfully, select 5 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 in the aseptic centrifuge tube of 2ml, preserves in-70 ℃ of numberings.

Among the present invention, each repeats order-checking for 5 the dna fragmentation that primer HY-fad31, HY-fad32 increase in A-grade in the first class 254 and A-grade in the first class 177, and its sequence results is consistent.Wherein the amplification of DNA fragments length of primer HY-fad31 in A-grade in the first class 254, A-grade in the first class 177 is respectively 2148bp, 2137bp, and its nucleotide sequence is shown in sequence table SEQ ID NO:1 and SEQ ID NO:2; The amplification of DNA fragments length of primer HY-fad32 in A-grade in the first class 254, A-grade in the first class 177 is 1963bp, and its nucleotide sequence is shown in sequence table SEQ ID NO:3 and SEQ ID NO:4.6, in the preparation swede type rape with the SNP mark of the low linolenic linkage of characters

The primer HY-fad31 that above-mentioned steps is obtained, the fragment that HY-fad32 increases in A-grade in the first class 254 and A-grade in the first class 177 are carried out sequence alignment with EBI Tools ClustalW (http://www.ebi.ac.uk/Tools/clustalw/index.html) software.Comparison result shows; Compare with the SEQ IDNO:2 shown in the sequence table; 4 single base mutations of the existence of SEQ ID NO:1 and 2 insertion sudden changes, 5 single base mutations are respectively: 28T → 28C, 390 A → 390T, 1692 T → 1703 A, 1723 G → 1734T, 1865 C → 1876T; Insert sudden change and be respectively Nucleotide T of insertion at the 713-714 place, insert the nucleotide fragments of a 10bp at the 1665-1666bp place, inserting base is ATGATTAGTA (seeing accompanying drawing 3).IDNO:4 compares with sequence table SEQ, at the single base mutation that in total length 1963bp, only has 1 1450G → 1450A (seeing accompanying drawing 4) of sequence table SEQ ID NO:3.Simultaneously; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 in the sequence table are carried out the multisequencing comparison result shows; Height homology between the different copies of the swede type rape fad3 gene that in same parent, amplifies; The difference part mainly concentrates on low GC concentration areas, is fit to only have part single base mutation (seeing accompanying drawing 5) in the zone (40～60%) of design primer at GC content.

Nucleotide sequence (shown in sequence table SEQ ID NO:1 and SEQID NO:2) according to primer HY-fad31 amplification of DNA fragments in A-grade in the first class 254 and A-grade in the first class 177; Utilize Primer Premier 5.0 softwares (http://www.PremierBiosoft.com) of report; Primer of mutational site design in sequence SEQID NO:1; Primer of pleomorphism site design between the difference copy of the different swede type rape fad3 genes of same parental source; Thereby get rid of the interference to pcr amplification of other fad3 copies in the swede type rape genome; Two primers of above-mentioned requirements are composed of a pair of primer, reach the effect of amplification specific copy in specific parent.Requiring primer length during design is 17～25bp; Renaturation temperature 50～62 C; GC content is 40～60%, is simultaneously effectively to distinguish difference and the eliminating of this site between parents, utilizes primer 3 ' end mispairing strategy the 2-4 bit base of primer 3 ' end to be carried out the mispairing processing of a base.Obtain the twin target primer by above-mentioned requirements; Between the 9-28bp of forward primer in sequence SEQ ID NO:1; Between the 788-805bp of reverse primer in sequence SEQ ID NO:1 (sequence is seen in the table 2 primer to YQ-fad31-1, and the forward primer position sees in the accompanying drawing 3 shown in the NO_1 underscore, and the reverse primer position is seen in the accompanying drawing 5 shown in the NO_1 underscore); 3 ' end the 2nd bit base with forward primer and reverse primer during design is G, is T by the A mispairing by the C mispairing respectively; With its called after YQ-fad31-1, be expected at the fragment that amplifies 797bp 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; Use the same method by the above-mentioned requirements profit; Screening obtains the twin target primer in sequence SEQ ID NO:2; Between the 9-28bp of forward primer in sequence SEQ ID NO:2; (sequence sees that primer is to YQ-fad31-2's in the table 2 between the 787-804bp of reverse primer in sequence SEQ ID NO:2; The forward primer position sees in the accompanying drawing 3 shown in the NO_2 underscore, and the reverse primer position is seen in the accompanying drawing 5 shown in the NO_2 underscore), 3 ' end the 2nd bit base of forward primer and reverse primer is G, is T by the A mispairing by the C mispairing respectively; Be expected at the fragment that amplifies 796bp 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.

According to the nucleotide sequence (shown in sequence table SEQ ID NO:3 and SEQID NO:4) of primer HY-fad32 amplification of DNA fragments in A-grade in the first class 254 and A-grade in the first class 177 and develop requirement and the method for primer to YQ-fad31-1, YQ-fad31-2; In sequence SEQ ID NO:3, obtain the twin target primer; Between the 1432-1450bp of forward primer in sequence SEQ ID NO:3; (sequence sees that primer is to YQ-fad32-1 in the table 2 between the 1840-1859bp of reverse primer in sequence SEQ ID NO:3; The forward primer position is seen in the accompanying drawing 4 shown in the NO_3 underscore; The reverse primer position is seen in the accompanying drawing 5 shown in the NO_3 underscore), during design 3 ' end the 2nd bit base of forward primer and reverse primer is C, is G by the C mispairing by the G mispairing respectively, with its called after YQ-fad32-1; Be expected at the fragment that amplifies 428bp 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; In sequence SEQ ID NO:4, obtain the twin target primer simultaneously; Between the 1432-1450bp of forward primer in sequence SEQ ID NO:4; (sequence sees that primer is to YQ-fad32-1 in the table 2 between the 1840-1859bp of reverse primer in sequence SEQ ID NO:4; The forward primer position sees in the accompanying drawing 4 shown in the NO_4 underscore, and the reverse primer position is seen in the accompanying drawing 5 shown in the NO_4 underscore), during design 3 ' end the 2nd bit base of forward primer and reverse primer is T, is G by the C mispairing by the G mispairing respectively; Be expected at the fragment that amplifies 428bp 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 above-mentioned steps designed primer to carry out PCR, and the PCR reaction system is with the above, and the renaturation temperature is seen table 2.Amplification detects on 1.2% agarose gel.Wherein primer YQ-fad31-1 is at swede type rape A-grade in the first class 254 and F ₁Genomic dna in amplify the target fragment of 797bp, and in the genomic dna of swede type rape A-grade in the first class 177 no any amplified production (seeing accompanying drawing 6), so this amplified fragments can be used as the dominance SNP molecule marker of low linolenic acid of cabbage type rape proterties.Primer YQ-fad31-2 is at swede type rape A-grade in the first class 177 and F ₁Genomic dna in amplify the target fragment of 796bp; And in the genomic dna of swede type rape A-grade in the first class 254 no any amplified production (seeing accompanying drawing 6), this amplified fragments and YQ-fad31-1 combination can be used as the codominance SNP molecule marker of low linolenic acid of cabbage type rape proterties.

Wherein primer YQ-fad32-1 is at swede type rape A-grade in the first class 254 and F ₁Genomic dna in amplify the target fragment of 428bp, and in the genomic dna of swede type rape A-grade in the first class 177 no any amplified production (seeing accompanying drawing 6), so this amplified fragments can be used as the dominance SNP molecule marker of low linolenic acid of cabbage type rape proterties.Primer YQ-fad32-2 is at swede type rape A-grade in the first class 177 and F ₁Genomic dna in amplify the target fragment of 428bp; And in the genomic dna of swede type rape A-grade in the first class 254 no any amplified production (seeing accompanying drawing 6), this amplified fragments and YQ-fad32-1 combination can be used as the codominance SNP molecule marker of low linolenic acid of cabbage type rape proterties.

7, the checking of the codominance SNP marker combination of low linolenic acid of cabbage type rape proterties

Utilize the DH segregating population among SNP labeled analysis the present invention of the low linolenic acid of cabbage type rape proterties that above-mentioned steps obtains; Amplified fragments the same (i.e. " A/A banding pattern " in the table) in 33 individual plants and the parent A-grade in the first class 254 wherein; Amplified fragments the same (i.e. " B/B banding pattern " in the table) in 39 individual plants and the parent A-grade in the first class 177; 76 individual plant detected results are " A/B banding pattern ", and 42 individual plant detected results are " B/A banding pattern " (seeing table 3).Can know through data in the analytical table; Every YQ-fad31, YQ-fad32 detection are " A/A banding pattern "; Its linolenic acid content is all below 3.50%; It meets low linolenic breeding standard (Rachael Scarth etc., Designer oil canola-a review of new food-grade Brassica oils with focuson high oleic, low linolenic types that Rachael Scarth proposes in the 10th international rape conference; 10th Int.Rapeseed Congress, 1999).Therefore; Utilize YQ-fad31, YQ-fad32 can effectively identify the individuality that all carries the low linolenic gene on two sites of swede type rape; Can change into qualitative character to a quantitative character selects; Improve the accuracy and the validity of seed selection low linolenic content swede type rape, thereby accelerate the seed selection of low linolenic content swede type rape.

The linolenic acid content of Brassica napus DH segregating population and marker detection result among table 3 the present invention

Sequence table

< 110>Hua Zhong Agriculture University

< 120>molecular marker for low linolenic acid of cabbage type rape and preparation method thereof and application

<130>

<141>2009-12-27

<160>20

<170>PatentIn?version?3.1

<210>1

<211>2148

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(2148)

<223>

<220>

<221>mutation

<222>(1665)..(1666)

<223>

<220>

<221>mutation

<222>(1865)..(1865)

<223>

<220>

<221>mutation

<222>(1723)..(1723)

<223>

<220>

<221>mutation

<222>(1692)..(1692)

<223>

<220>

<221>mutation

<222>(713)..(714)

<223>

<220>

<221>mutation

<222>(390)..(390)

<223>

<220>

<221>primer_bind

<222>(2128)..(2148)

<223>

<220>

<221>primer_bind

<222>(788)..(805)

<223>

<220>

<221>primer_bind

<222>(9)..(28)

<223>

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<220>

<221>mutation

<222>(28)..(28)

<223>

<400>1

tggacatggg?agtttctcag?acattcccct?tctgaatact?gcggttggtc?atattcttca 60

ttccttcatt?ctcgttccat?accatggttg?gtaagtcatt?tattttaact?tcttttttca 120

tgcaaattta?ttcttgtttt?cgtatttctt?acattttcct?tgtcattctt?ggtgcatgtt 180

agcaaacagt?aatctgataa?ctgaaaatat?attaattttt?catagtaaaa?taatgcatgt 240

gactaaaagc?atcaaaatct?ttagcatcga?agaaaaaaga?accaaacttt tatttaatgc 300

tatgggccta?tttatggtcc?aattagctat?tatcatatga?catgtccttg?aataaattaa 360

tgtataagtt?taatataata?tttatatatt?tttgttttaa?tggcttattt?tattgttaaa 420

tggatacatc?agcttgaaat?atctacgaac?atgcatcatt?ttcctagata?catttgtttg 480

ttgctcaaaa?aatgaataac?gtagttaaac?gagtgagatt?cttagcatct?gcctcgaaaa 540

cgatatgtta?ttgacaattc?caatttcatt?tttatgaaaa?taaaataata?gtttatttta 600

taattggggg?tggttgcagg?agaataagcc?atcggacaca?ccaccagaac?catggccatg 660

ttgaaaacga?cgagtcttgg?gttccggtaa?tccccctctc?atattttttt?ttttcttttt 720

ttgaaactct?ttcattttaa?ttttcttaga?attctatgta?tttattttaa?tcaatccttt 780

ttccagtgtg?aggcttggac?gaccacttgt?cagatttgtc?gtttagctgt?agtaaacaac 840

tgatttaaat?tgtttatggt?actgtagtta?actttaacaa?cgggccactt?atattcgagc 900

cattggcata?aaatgattct?tctcgaaatt?cgtttacttt?tcttagtatt?tttcagtttt 960

gtagtttacg?tagaactaat?aaaaagaaaa?aaacttataa?acacaccaca?tgcaatgaat 1020

aaattcgaat?atataaccat?actgttaaat?attaattaac?attttaatct?taattttgca 1080

ttccagttgc?cagaaaaatt?atacaagaat?ttgtcccaca?gtacacggat?gctcagatac 1140

actgtccctc?tccccatgct?cgcttaccct?ctctatctgg?taaatcctaa?ttcctcattt 1200

ttcttcctga?ttataattac?aattttgaat?ttttagattt?tgagtattaa?ctaaatataa 1260

attaaatttg?tttggggatg?actacagtgg?tacagaagtc?ctggtaaaga?agggtcacat 1320

tataacccat?acagtagttt?atttgcccca?agcgagagaa?agcttattgc?aacttcaact 1380

acttgctggt?cgatcatgtt?ggccactctt?gtttatctat?cattcctcgt?tggtccagtc 1440

acagttctaa?aagtctatgg?tgttccttac?attgtaagtt?tcatatattt?cattattata 1500

tcattgctaa?tataatttgt?ttttgacata?aagttttgga?aaaatttcag?atctttgtaa 1560

tgtggttgga?cgctgtcacg?tacttgcatc?atcatggtca?cgatgataag?ttgccttggt 1620

acagaggcaa?ggtaagtaga?tcaacattaa?tttataagaa?gcaacaatga?ttagtatttg 1680

attaatctaa?attattgatg?ttatgtgtac?aataatagga?atggagttat?ttatgtggag 1740

gattaacaac?tattgataga?gattacggga?tcttcaacaa?cattcatcac?gatattggaa 1800

ctcacgtgat?ccatcatctt?ttcccacaaa?tccctcacta?tcacttggtt?gatgccgtga 1860

gtgatctcgc?tctctttcta?gtttcatttg?attaaaatta?aagggtgatt?aattactaaa 1920

ttagtgatct?taattaatga?tatgcgacag?acgaaatcag?ctaaacatgt?gttgggaaga 1980

tactacagag?aaccaaagac?gtcaggagca?ataccgatcc?acttggtgga?aagtttggtg 2040

gcaagtatta?agaaagatca?ttacgtcagt?gacactggtg?atattgtctt?ctacgagaca 2100

gatccagatc?tctacgttta?tgcttctgac?aaatccaaaa?tcaactaa 2148

<210>2

<211>2137

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(2137)

<223>

<220>

<221>primer_bind

<222>(2117)..(2137)

<223>

<220>

<221>primer_bind

<222>(787)..(804)

<223>

<220>

<221>primer_bind

<222>(9)..(28)

<223>

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>2

tggacatggg?agtttctcag?acattcctct?tctgaatact?gcggttggtc?atattcttca 60

ttccttcatt?ctcgttccat?accatggttg?gtaagtcatt?tattttaact?tcttttttca 120

tgcaaattta?ttcttgtttt?cgtatttctt?acattttcct?tgtcattctt?ggtgcatgtt 180

agcaaacagt?aatctgataa?ctgaaaatat?attaattttt?catagtaaaa?taatgcatgt 240

gactaaaagc?atcaaaatct?ttagcatcga?agaaaaaaga?accaaacttt?tatttaatgc 300

tatgggccta?tttatggtcc?aattagctat?tatcatatga?catgtccttg?aataaattaa 360

tgtataagtt?taatataata?tttatatata?tttgttttaa?tggcttattt?tattgttaaa 420

tggatacatc?agcttgaaat?atctacgaac?atgcatcatt?ttcctagata?catttgtttg 480

ttgctcaaaa?aatgaataac?gtagttaaac?gagtgagatt?cttagcatct?gcctcgaaaa 540

cgatatgtta?ttgacaattc?caatttcatt?tttatgaaaa?taaaataata?gtttatttta 600

taattggggg?tggttgcagg?agaataagcc?atcggacaca?ccaccagaac?catggccatg 660

ttgaaaacga?cgagtcttgg?gttccggtaa?tccccctctc?atattttttt?tttctttttt 720

tgaaactctt?tcattttaat?tttcttagaa?ttctatgtat?ttattttaat?caatcctttt 780

tccagtgtga?ggcttggacg?accacttgtc?agatttgtcg?tttagctgta?gtaaacaact 840

gatttaaatt?gtttatggta?ctgtagttaa?ctttaacaac?gggccactta?tattcgagcc 900

attggcataa?aatgattctt?ctcgaaattc?gtttactttt?cttagtattt?ttcagttttg 960

tagtttacgt?agaactaata?aaaagaaaaa?aacttataaa?cacaccacat?gcaatgaata 1020

aattcgaata?tataaccata?ctgttaaata?ttaattaaca?ttttaatctt?aattttgcat 1080

tccagttgcc?agaaaaatta?tacaagaatt?tgtcccacag?tacacggatg?ctcagataca 1140

ctgtccctct?ccccatgctc?gcttaccctc?tctatctggt?aaatcctaat?tcctcatttt 1200

tcttcctgat?tataattaca?attttgaatt?tttagatttt?gagtattaac?taaatataaa 1260

ttaaatttgt?ttggggatga?ctacagtggt?acagaagtcc?tggtaaagaa?gggtcacatt 1320

ataacccata?cagtagttta?tttgccccaa?gcgagagaaa?gcttattgca?acttcaacta 1380

cttgctggtc?gatcatgttg?gccactcttg?tttatctatc?attcctcgtt?ggtccagtca 1440

cagttctaaa?agtctatggt?gttccttaca?ttgtaagttt?catatatttc?attattatat 1500

cattgctaat?ataatttgtt?tttgacataa?agttttggaa?aaatttcaga?tctttgtaat 1560

gtggttggac?gctgtcacgt?acttgcatca?tcatggtcac?gatgataagt?tgccttggta 1620

cagaggcaag?gtaagtagat?caacattaat?ttataagaag?caacacttga?ttaatctaaa 1680

ttattgatgt?tttgtgtaca?ataataggaa?tggagttatt?tacgtggagg?attaacaact 1740

attgatagag?attacgggat?cttcaacaac?attcatcacg?atattggaac?tcacgtgatc 1800

catcatcttt?tcccacaaat?ccctcactat?cacttggttg?atgccgtgag?tgatctcgct 1860

ctctctctag?tttcatttga?ttaaaattaa?agggtgatta?attactaaat?tagtgatctt 1920

aattaatgat?atgcgacaga?cgaaatcagc?taaacatgtg?ttgggaagat?actacagaga 1980

accaaagacg?tcaggagcaa?taccgatcca?cttggtggaa?agtttggtgg?caagtattaa 2040

gaaagatcat?tacgtcagtg?acactggtga?tattgtcttc?tacgagacag?atccagatct 2100

ctacgtttat?gcttctgaca?aatccaaaat?caactaa 2137

<210>3

<211>1963

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(1963)

<223>

<220>

<221>mutation

<222>(1450)..(1450)

<223>

<220>

<221>primer_bind

<222>(1943)..(1963)

<223>

<220>

<221>primer_bind

<222>(1840)..(1859)

<223>

<220>

<221>primer_bind

<222>(1432)..(1450)

<223>

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>3

actccaccct?gagaacttct?caataatcat?gctcttagtg?ctctaagaag?ggtccttaac 60

aaaatattaa?taataagata?tagtgtgggc?ccaaaaaaaa?acaaaaaacc?ggttacaaaa 120

gtcgcgaaag?aaggatcgat?tttggtcttt?tacttgtact?gtttgtggat?cccactggtg 180

gtggtccgcg?attggtttct?tttttaattt?aatttatttt?ttttaatcgg?agaaaaaaaa 240

ttaagaaacc?aaaaaacagt?tttaatcatg?gcctcatgtt?ggggttgagt?tttatattct 300

gataagaatc?ccatcttaaa?aaccccgtta?aacatgctct?taccatctgc?ttcgaaaatg 360

atatgttatt?gacaattcca?atttcatttt?tatgaaaata?aaataatagt?ttattttata 420

actgagggtg?gttgcaggag?aataagccat?cggacacacc?accagaacca?tggccatgtt 480

gaaaacgacg?agtcttgggt?tccggtaatc?tttccctctc?tcatattttt?ttttcttttt 540

ttttgaaatt?ctttcatttt?aattttctta?ggattctatg?tatttatttt?aatcaatcct 600

ttttccagtt?tgaggctagg?acgaccactt?gtcagatttg?tcgtttagct?gtagtaaaca 660

actgatttaa?attgtttata?gtactgtagt?taactttaac?aacggaccac?ttatattcga 720

gccattggca?taaaatgatt?cttctcgaaa?ttcgtttact?tttcttagta?tttttcaatt 780

ttggagttta?cgtagaacta?ataaaaagaa?aaacttataa?acacaccaca?tgcaatgaat 840

aaattcgaat?atataaccat?actgttaaat?attaatttac?attttaatct?taattttgca 900

ttccagttgc?cagaaaaatt?atacaagaat?ttgtcccaca?gtacacggat?gctcagatac 960

actgtccctc?tccccatgct?cgcttaccct?ctctatctgg?taaatcctaa?ttctaatttt 1020

cttctgatta?taattacaat?tttgaatttt?tagattttga?gtattaacta?aatataaatt 1080

aaatttgttt?ggggatgact?acagtggtac?agaagtcctg?gtaaagaagg?gtcacattat 1140

aacccataca?gtagtttatt?tgccccaagc?gagagaaagc?ttattgcaac?ttcaactact 1200

tgctggtcga?tcgtgttggc?cactcttgtt?tatctatcat?tcctcgttgg?tccagtcaca 1260

gttctaaaag?tctatggtgt?tccttacatt?gtaagtttca?tatatttctt?tattatatca 1320

ttgctaatat?aatttgtttt?tgacataaaa?gttttggaaa?aatttcagat?ctttgtaatg 1380

tggttggacg?ctgtcacgta?cttgcatcat?catggtcacg?atgataagct?gccttggtac 1440

agaggcaaga?taagtagatc?aacattattt?ataagaagca?ataatgatta?gtagttgaat 1500

aatctgaatt?tttgatgttt?ttgtacaata?ataggaatgg?agttatttac?gtggaggatt 1560

aacaactgtt?gatagagatt?acgggatctt?caacaacatt?catcacgata?ttggaactca 1620

cgtgatccat?catcttttcc?cacaaatccc?tcactatcac?ttggtcgatg?ccgtgagtga 1680

tctcgctctc?tctctagttt?catttgatta?tattaaaggg?tgattaatta?ctaaattagt 1740

gatcttaatt?aatgacatgc?gacagacgaa?agcagctaaa?catgtgttgg?gaagatacta 1800

cagagaacca?aagacgtcag?gagcaatacc?gatccactta?gtggaaagtt?tggtggcaag 1860

tattaagaaa?gatcattacg?tcagtgacac?tggtgatatt?gtcttctacg?agacagatcc 1920

agatctctac?gtttatgctt?ctgacaaatc?caaaatcaac?taa 1963

<210>4

<211>1963

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(1963)

<223>

<220>

<221>primer_bind

<222>(1943)..(1963)

<223>

<220>

<221>primer_bind

<222>(1840)..(1859)

<223>

<220>

<221>primer_bind

<222>(1432)..(1450)

<223>

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>4

actccaccct?gagaacttct?caataatcat?gctcttagtg?ctctaagaag?ggtccttaac 60

aaaatattaa?taataagata?tagtgtgggc?ccaaaaaaaa?acaaaaaacc?ggttacaaaa 120

gtcgcgaaag?aaggatcgat?tttggtcttt?tacttgtact?gtttgtggat?cccactggtg 180

gtggtccgcg?attggtttct?tttttaattt?aatttatttt?ttttaatcgg?agaaaaaaaa 240

ttaagaaacc?aaaaaacagt?tttaatcatg?gcctcatgtt?ggggttgagt?tttatattct 300

gataagaatc?ccatcttaaa?aaccccgtta?aacatgctct?taccatctgc?ttcgaaaatg 360

atatgttatt?gacaattcca?atttcatttt?tatgaaaata?aaataatagt?ttattttata 420

actgagggtg?gttgcaggag?aataagccat?cggacacacc?accagaacca?tggccatgtt 480

gaaaacgacg?agtcttgggt?tccggtaatc?tttccctctc?tcatattttt?ttttcttttt 540

ttttgaaatt?ctttcatttt?aattttctta?ggattctatg?tatttatttt?aatcaatcct 600

ttttccagtt?tgaggctagg?acgaccactt?gtcagatttg?tcgtttagct?gtagtaaaca 660

actgatttaa?attgtttata?gtactgtagt?taactttaac?aacggaccac?ttatattcga 720

gccattggca?taaaatgatt?cttctcgaaa?ttcgtttact?tttcttagta?tttttcaatt 780

ttggagttta?cgtagaacta?ataaaaagaa?aaacttataa?acacaccaca?tgcaatgaat 840

aaattcgaat?atataaccat?actgttaaat?attaatttac?attttaatct?taattttgca 900

ttccagttgc?cagaaaaatt?atacaagaat?ttgtcccaca?gtacacggat?gctcagatac 960

actgtccctc?tccccatgct?cgcttaccct?ctctatctgg?taaatcctaa?ttctaatttt 1020

cttctgatta?taattacaat?tttgaatttt?tagattttga?gtattaacta?aatataaatt 1080

aaatttgttt?ggggatgact?acagtggtac?agaagtcctg?gtaaagaagg?gtcacattat 1140

aacccataea?gtagtttatt?tgccccaagc?gagagaaagc?ttattgcaac?ttcaactact 1200

tgctggtcga?tcgtgttggc?cactcttgtt?tatctatcat?tcctcgttgg?tccagtcaca 1260

gttctaaaag?tctatggtgt?tccttacatt?gtaagtttca?tatatttctt?tattatatca 1320

ttgctaatat?aatttgtttt?tgacataaaa?gttttggaaa?aatttcagat?ctttgtaatg 1380

tggttggacg?ctgtcacgta?cttgcatcat?catggtcacg?atgataagct?gccttggtac 1440

agaggcaagg?taagtagatc?aacattattt?ataagaagca?ataatgatta?gtagttgaat 1500

aatctgaatt?tttgatgttt?ttgtacaata?ataggaatgg?agttatttac?gtggaggatt 1560

aacaactgtt?gatagagatt?acgggatctt?caacaacatt?catcacgata?ttggaactca 1620

cgtgatccat?catcttttcc?cacaaatccc?tcactatcac?ttggtcgatg?ccgtgagtga 1680

tctcgctctc?tctctagttt?catttgatta?tattaaaggg?tgattaatta?ctaaattagt 1740

gatcttaatt?aatgacatgc?gacagacgaa?agcagctaaa?catgtgttgg?gaagatacta 1800

cagagaacca?aagacgtcag?gagcaatacc?gatccactta?gtggaaagtt?tggtggcaag 1860

tattaagaaa?gatcattacg?tcagtgacac?tggtgatatt?gtcttctacg?agacagatcc 1920

agatctctac?gtttatgctt?ctgacaaatc?caaaatcaac?taa 1963

<210>5

<211>797

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(797)

<223>

<220>

<221>mutation

<222>(780)..(781)

<223>

<220>

<221>mutation

<222>(19)..(20)

<223>

<220>

<221>primer_bind

<222>(780)..(797)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>5

ggagtttctc?agacattcgc?cttctgaata?ctgcggttgg?tcatattctt?cattccttca 60

ttctcgttcc?ataccatggt?tggtaagtca?tttattttaa?cttctttttt?catgcaaatt 120

tattcttgtt?ttcgtatttc?ttacattttc?cttgtcattc?ttggtgcatg?ttagcaaaca 180

gtaatctgat?aactgaaaat?atattaattt?ttcatagtaa?aataatgcat?gtgactaaaa 240

gcatcaaaat?ctttagcatc?gaagaaaaaa?gaaccaaact?tttatttaat?gctatgggcc 300

tatttatggt?ccaattagct?attatcatat?gacatgtcct?tgaataaatt?aatgtataag 360

tttaatataa?tatttatata?tttttgtttt?aatggcttat?tttattgtta?aatggataca 420

tcagcttgaa?atatctacga?acatgcatca?ttttcctaga?tacatttgtt?tgttgctcaa 480

aaaatgaata?acgtagttaa?acgagtgaga?ttcttagcat?ctgcctcgaa?aacgatatgt 540

tattgacaat?tccaatttca?tttttatgaa?aataaaataa?tagtttattt?tataattggg 600

ggtggttgca?ggagaataag?ccatcggaca?caccaccaga?accatggcca?tgttgaaaac 660

gacgagtctt?gggttccggt?aatccccctc?tcatattttt?ttttttcttt?ttttgaaact 720

ctttcatttt?aattttctta?gaattctatg?tatttatttt?aatcaatcct?ttttccagtg 780

tgaggcttgg?acgacca 797

<210>6

<211>796

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(796)

<223>

<220>

<221>mutation

<222>(780)..(780)

<223>

<220>

<221>mutation

<222>(19)..(19)

<223>

<220>

<221>gene

<222>(779)..(796)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>6

ggagtttctc?agacattcgt?cttctgaata?ctgcggttgg?tcatattctt?cattccttca 60

ttctcgttcc?ataccatggt?tggtaagtca?tttattttaa?cttctttttt?catgcaaatt 120

tattcttgtt?ttcgtatttc?ttacattttc?cttgtcattc?ttggtgcatg?ttagcaaaca 180

gtaatctgat?aactgaaaat?atattaattt?ttcatagtaa?aataatgcat?gtgactaaaa 240

gcatcaaaat?ctttagcatc?gaagaaaaaa?gaaccaaact?tttatttaat?gctatgggcc 300

tatttatggt?ccaattagct?attatcatat?gacatgtcct?tgaataaatt?aatgtataag 360

tttaatataa?tatttatata?tatttgtttt?aatggcttat?tttattgtta?aatggataca 420

tcagcttgaa?atatctacga?acatgcatca?ttttcctaga?tacatttgtt?tgttgctcaa 480

aaaatgaata?acgtagttaa?acgagtgaga?ttcttagcat?ctgcctcgaa?aacgatatgt 540

tattgacaat?tccaatttca?tttttatgaa?aataaaataa?tagtttattt?tataattggg 600

ggtggttgca?ggagaataag?ccatcggaca?caccaccaga?accatggcca?tgttgaaaac 660

gacgagtctt?gggttccggt?aatccccctc?tcatattttt?tttttctttt?tttgaaactc 720

tttcatttta?attttcttag?aattctatgt?atttatttta?atcaatcctt?tttccagtgt 780

gaggcttgga?cgacca 796

<210>7

<211>428

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(428)

<223>

<220>

<221>mutation

<222>(18)..(19)

<223>

<220>

<221>primer_bind

<222>(409)..(428)

<223>

<220>

<221>primer_bind

<222>(1)..(19)

<223>

<400>7

ccttggtaca?gaggcaacat?aagtagatca?acattattta?taagaagcaa?taatgattag 60

tagttgaata?atctgaattt?ttgatgtttt?tgtacaataa?taggaatgga?gttatttacg 120

tggaggatta?acaactgttg?atagagatta?cgggatcttc?aacaacattc?atcacgatat 180

tggaactcac?gtgatccatc?atcttttccc?acaaatccct?cactatcact?tggtcgatgc 240

cgtgagtgat?ctcgctctct?ctctagtttc?atttgattat?attaaagggt?gattaattac 300

taaattagtg?atcttaatta?atgacatgcg?acagacgaaa?gcagctaaac?atgtgttggg 360

aagatactac?agagaaccaa?agacgtcagg?agcaataccg?atccacttac?tggaaagttt 420

ggtggcaa 428

<210>8

<211>428

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(428)

<223>

<220>

<221>mutation

<222>(18)..(18)

<223>

<220>

<221>gene

<222>(409)..(428)

<223>

<220>

<221>primer_bind

<222>(1)..(19)

<223>

<400>8

ccttggtaca?gaggcaatgt?aagtagatca?acattattta?taagaagcaa?taatgattag 60

tagttgaata?atctgaattt?ttgatgtttt?tgtacaataa?taggaatgga?gttatttacg 120

tggaggatta?acaactgttg?atagagatta?cgggatcttc?aacaacattc?atcacgatat 180

tggaactcac?gtgatccatc?atcttttccc?acaaatccct?cactatcact?tggtcgatgc 240

cgtgagtgat?ctcgctctct?ctctagtttc?atttgattat?attaaagggt?gattaattac 300

taaattagtg?atcttaatta?atgacatgcg?acagacgaaa?gcagctaaac?atgtgttggg 360

aagatactac?agagaaccaa?agacgtcagg?agcaataccg?atccacttac?tggaaagttt 420

ggtggcaa 428

<210>9

<211>18

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>9

tggacatggg?agtttctc 18

<210>10

<211>21

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(21)

<223>

<400>10

ttagttgatt?ttggatttgt?c 21

<210>11

<211>18

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>11

actccaccct?gagaactt 18

<210>12

<211>21

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(21)

<223>

<400>12

ttagttgatt?ttggatttgt?c 21

<210>13

<211>20

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>13

ggagtttctc?agacattcgt 20

<210>14

<211>18

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>14

gtgtcgtcca?agcctctc 18

<210>15

<211>20

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>15

ggagtttctc?agacattcgc 20

<210>16

<211>18

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(18)

<223>

<400>16

gtgtcgtcca?agcctctc 18

<210>17

<211> 19

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(19)

<223>

<400>17

ccttggtaca?gaggcaaca 19

<210>18

<211>20

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>18

ttgccaccaa?actttccagt 20

<210>19

<211>19

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(19)

<223>

<400>19

ccttggtaca?gaggcaatg 19

<210>20

<211>20

<212>DNA

< 213>swede type rape (Brassica napus)

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>20

ttgccaccaa?actttccagt 20

Claims (3)

1. the codominance SNP molecule marker of a low linolenic acid of cabbage type rape content, its nucleotide sequence is shown in sequence table SEQ IDNO:5 and SEQ ID NO:6.

2. the primer of the codominance SNP molecule marker of amplification low linolenic acid of cabbage type rape content is right, and its nucleotide sequence is as follows:

Primer is to YQ-fad31-1:

Forward primer 5 '-GGAGTTTCTCAGACATTCGT-3 ',

Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3 '; With

Primer is to YQ-fad31-2:

Forward primer 5 '-GGAGTTTCTCAGACATTCGC-3 ',

Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3 '.

3. the described primer of claim 2 is to the application in the assisted Selection of low linolenic swede type rape.

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