CN102433331B - Low-linolenic-acid molecular marker for brassica napus, and preparation method and application of low-linolenic-acid molecular marker - Google Patents
Low-linolenic-acid molecular marker for brassica napus, and preparation method and application of low-linolenic-acid molecular marker Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular breeding of rape, and particularly relates to a preparation method for a codominant single nucleotide polymorphism (SNP) molecular marker which is closely linked with the content of linolenic acid of brassica napus, and application of the marker to the auxiliary selection of low-linolenic-acid brassica napus. The preparation method comprises the following steps of: amplifying genomic deoxyribonucleic acid (DNA) of a low-linolenic-acid brassica napus line AA254 and a high-linolenic-acid brassica napus line AA177 by using primers to obtain two DNA-amplified fragments respectively, cloning the four DNA-amplified fragments, sequencing, and comparing nucleotide sequences; simultaneously mismatching 2nd basic groups of 3' terminals of a forward primer and a reverse primer according to the difference of the sequences so as to design a primer pair, namely YQ-fad32-1 and YQ-fad32-2; and performing polymerase chain reaction (PCR) amplification on the primer pair, namely the YQ-fad32-1 and the YQ-fad32-2 to obtain the codominant SNP molecular marker which is linked with a low linolenic acid gene of the brassica napus through PCR amplification and analysis of a population detection effect. The invention provides a novel marker for the molecular breeding of the rape. Nucleotide sequences of the novel marker are shown as SEQ ID NO:7 and SEQ ID NO:8.
Description
Technical field
The present invention is that application number is dividing an application of 200910273437.1 application cases.
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
In Semen Brassicae campestris, lipid acid forms the quality directly determining rapeseed oil and to people's nutritive value.Linolenic acid is the highest lipid acid of oxidation ratio in highly unsaturated fatty acids, with oxidation very easily occurs after air, light, heat etc. contact, forms the stench oxide compound, makes the rotten and storage tolerance not of rape oil.More seriously linolenic oxidation products is harmful, can cause cardiovascular atherosis (Chang etc., Effects of the ratio of polyunsaturated and monounsaturated fatty acid on rat plasma and liver lipid concentration.Lipids, 1998,33:481-487).After the double-strand break of unsaturated fatty acids, than being easier to form free radical, peroxidatic reaction of lipid appears, thereby structure and function (the hole auspicious sign of meeting damaging cells and cytolemma, the nutritional-physiological of essential trace element and clinical meaning. Hefei: the .1982:3-60 of Anhui Science Press), directly affect cell division, grow, breed and heredity.In rapeseed oil, after the alpha-linolenic acid oxidation, also can make grease with stronger sharp flavor.Therefore, reducing linolenic acid content in rapeseed oil is long-term objective (the Rachael Scarth etc. of breeding man aspect the swede type rape quality breeding, Designer oil canola-a review of new food-grade Brassica oils with focus on high oleic, low linolenic types, 10th Int.Rapeseed Congress, 1999).
The low linolenic genetic resources also lacks very much both at home and abroad at present, mainly utilizes traditional Phenotypic Selection method when the low linolenic proterties being imported to the high linolenic kind of high yield.Because linolenic acid content in swede type rape is subject to controlled by multiple genes, there are the shortcomings such as breeding cycle is long, efficiency is low.Molecular Marker Assisted Selection Technology in modern biotechnology, compared plurality of advantages with traditional Phenotypic Selection.Molecular marker assisted selection can be carried out in any vegetative period, was not subject to environmental influence, can get rid of that non-allelic genes interact and the interference that causes, have quick, economical, the characteristics that efficiency 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 by molecule marker, to get rid of the impact of environment and extraneous factor.Simultaneously can utilize molecule marker to be selected background, thereby accelerate the genetic background resume speed, shortening the breeding cycle and alleviate Linkage drag.And one of prerequisite of carrying out these work is the efficient molecule marker of exploitation.
Aspect the location of low linolenic purpose proterties, investigator both domestic and external has obtained a lot of achievements in research.(the Somers etc. such as Somers, Identification of Molecular Markers Associated with Linoleic Acid Desaturation in Brassica napus, Theoretical andApplied Genetics, 1998,96.897-903) utilize the BSA method in the DH colony formed by 115 genotype, find 16 RAPD marks relevant with linolenic acid content, be positioned at 3 linkage groups, explain respectively 32%, 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 altogether 51% variation.(the Hu J etc. such as HU, Mapping of a gene determining linolenic acid content in rapeseed with DNA based markers.Theor Appl Genet 1995,90:258-262) also find a RAPD dominant marker relevant to linolenic acid content, this mark amplified fragments size is 650bp.They further, by this fragment clone, order-checking, are converted into dominant SCAR mark.(Hu J etc., SCAR and RAPD marker associated with 18-carbon fatty acids in rapeseed, Brassica napus.Plant Breeding, 1999,118:145-150).(the Cheung etc. such as Cheung, Molecular mapping of seed quality traits in (Brassica juncea L.) czern.and coss.Acta Horticulturae, 1998,459:139-147) find that two mono-of QTLs that control linolenic acid contents are positioned at one, fae1 site and are positioned at the fad3 site.(the Jourdren etc. such as Jourdren, Specific molecular marker of the genes controlling linolenic acid content in rapeseed.Theor Appl Genet, 1996b, 93:512-518) located two QTLss relevant with linolenic acid content and the fad3 gene is closely related.(the Lionneton etc. such as Lionneton, Development of an AFLP-based linkage map and localization of QTLs for seed fatty acid content in condiment mustard (Brassica juncea) Genome 2002,45:1203-1215) discovery is positioned at the phenotypic variation in the QTL site soluble 41.2% on LG2 for linolenic acid content.
(the F.Javidfar etc. such as F.Javidfar, Identification of molecular markers associated with oleic and linolenic acid in spring 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 is DH12075 hybridization.Obtain 8 RAPD marks associated 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 2003/0150020A1, Aug.7,2003 such as Somers, Daryl Somers, US 7081564, July 25, 2006) be attained at the SNP mark of low linolenic content, (the Hu etc. such as Hu, Mapping ofthe loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.) .Theor Appl Genet, 2006, 113:497-507) navigated to the main effect QTL relevant with the swede type rape linolenic acid content from the DH mapping population, a main effect QTL (fad3c) of controlling low linolenic content is positioned at karyomit(e) No. 14, another main effect QTL is positioned at karyomit(e) No. 4, come from the A genome.According to these QTL, design has obtained two and fad2, the SNP that fad3 is relevant (Single Nucleotide Polymorphisms) mark.Zhao (the .Mapping QTL controlling fatty acid composition in a doubled haploid rapeseed population segregating for oil content.Mol Breed such as Zhao J, 2008,21:115-125) also detect one about linolenic QTL on swede type rape N14, explained 28% genetic mutation rate.But due to (Daryl Somers, US 2003/0150020A1, 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 while being marked at design of primers, effect in swede type rape linolenic acid assisted selection is subject to the impacts such as many factors 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 object of the invention is to develop a kind of molecule marker that seed selection has the swede type rape of low linolenic proterties that is applicable to, 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 by following scheme:
The applicant obtains a kind of swede type rape codominance SNP molecule marker that seed selection possesses the low linolenic proterties that is applicable to by test, and its nucleotide sequence is as shown in sequence table SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.The emphasis that the present invention asks for protection is the protection as molecule marker to the nucleotide sequence shown in sequence table SEQ ID NO:7 and 8.
The codominance SNP molecule marker of a kind of low linolenic acid of cabbage type rape content prepared by the present invention, its nucleotide sequence as shown in sequence table SEQ ID NO:7 and SEQ ID NO:8, as shown in sequence table SEQ ID NO:7 and SEQ ID NO:8 sequence the two be used singly or in combination the molecule marker as low linolenic acid of cabbage type rape SNP.
Preparation is applicable to the method for dominant SNP molecule marker of low linolenic acid of cabbage type rape proterties, according to following steps:
Primer HY-fad31, the 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 that utilize the applicant to design, obtain respectively two DNA cloning fragments, then described 4 DNA cloning fragments are cloned, checked order, wherein from amplification A-grade in the first class 254, obtain the nucleotide sequence as shown in SEQ ID NO:1 and SEQ ID NO:3, from amplification A-grade in the first class 177, obtain the nucleotide sequence as shown in SEQ ID NO:2 and SEQ ID NO:4.
With sequence table SEQ ID NO:2, compare, 5 single base mutations of existence and 2 insertion mutations (seeing accompanying drawing 3) at sequence table SEQ ID NO:1, the applicant utilizes the single nucleotide polymorphism (SNP of the mutational site 28T → 28C existed in SEQ ID NO:1 and SEQ ID NO:2, Single Nucleotide Polymorphisms), adopt primer 3 ' end mispairing technical tactic (Drenkard etc., A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.Plant Physiol, 2000 124:1483-1492) primer pair YQ-fad31-1 and YQ-fad31-2 have been designed.With sequence table SEQ ID NO:4, compare, single base mutation (seeing accompanying drawing 4) at 1 1450G → 1450A of existence of sequence table SEQ ID NO:3, the applicant utilizes the single nucleotide polymorphism of the mutational site 1450G → 1450A existed in SEQ ID NO:3 and SEQ ID NO:4, adopts primer 3 ' end mispairing technical tactic to design primer pair YQ-fad32-1 and YQ-fad32-2.Primer pair YQ-fad31-1, YQ-fad31-2, YQ-fad32-1 and YQ-fad32-2 are carried out to pcr amplification, 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 as shown in sequence table SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.Wherein in female case of the application's case, request is as the protection of molecule marker for the nucleotide sequence shown in sequence table SEQ ID NO:5, SEQ ID NO:6, and the emphasis that the present invention asks for protection is the protection as molecule marker to the nucleotide sequence shown in sequence table SEQ ID NO:7 and 8.
In aforesaid method, the right nucleotide sequence of the primer is as follows:
Primer pair (1) is numbered HY-fad31:
Forward primer 5 '-TGGACATGGGAGTTTCTC-3 ',
Reverse primer 5 '-TTAGTTGATTTTGGATTTGTC-3 '.
Primer pair (2) is numbered HY-fad32:
Forward primer 5 '-ACTCCACCCTGAGAACTT-3 ',
Reverse primer 5 '-TTAGTTGATTTTGGATTTGTC-3 '.
Primer pair (3) is numbered YQ-fad31-1:
Forward primer 5 '-GGAGTTTCTCAGACATTCGT-3 ',
Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3 '.
Primer pair (4) is numbered YQ-fad31-2:
Forward primer 5 '-GGAGTTTCTCAGACATTCGC-3 ',
Reverse primer 5 '-GTGTCGTCCAAGCCTCTC-3
Primer pair (5) is numbered YQ-fad32-1:
Forward primer 5 '-CCTTGGTACAGAGGCAACA-3 ',
Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '.
Primer pair (6) is numbered YQ-fad32-2:
Forward primer 5 '-CCTTGGTACAGAGGCAATG-3 ',
Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '.
Wherein, primer pair HY-fad31 is for the nucleotide sequence shown in extension increasing sequence table SEQ ID NO:1 and SEQ ID NO:2; Primer pair HY-fad32 is for the nucleotide sequence shown in extension increasing sequence table SEQ ID NO:3 and SEQ ID NO:4; Primer pair YQ-fad31-1, YQ-fad31-2 are respectively used to the nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6 in the extension increasing sequence table; Primer pair YQ-fad32-1, YQ-fad32-2 are respectively used to the nucleotide sequence shown in SEQ ID NO:7 and SEQ ID NO:8 in the extension increasing sequence table.
Positively effect of the present invention:
The present invention holds the mispairing application of policies in exploitation and the detection of swede type rape SNP polymorphism mark primer 3 ' for the first time; And utilize for the first time forward primer and reverse primer mispairing simultaneously means to develop PCR-based amplification and agarose gel electrophoresis SNP mark in allopolyploid crop swede type rape.The present invention successfully obtains the codominance SNP molecule marker of low linolenic acid of cabbage type rape, use in this assisted Selection that is marked at the low linolenic swede type rape and can overcome the shortcoming that relies on phenotype to be selected in traditional breeding method, 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 ".
The accompanying drawing explanation
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 254 that increases of the present invention 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 pair 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.In 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 pair 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.In 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 the amplification of primer pair HY-fad31 in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes, in figure: NO_1, NO_2 represent respectively the sequence of SEQ ID NO:1 and SEQ ID NO:2 in sequence table, the single base mutation of the 28T of solid black trilateral " ▲ " expression sequence → 28C, the primer location (being the forward primer position of primer pair YQ-fad31-1, YQ-fad31-2) of design is referring to the underscore place, and hollow triangle " ▽ " means the design of primers base mismatch.
Fig. 4: utilize the sequence dna fragment comparison of the amplification of primer pair HY-fad32 in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes, in figure: NO_3, NO_4 represent respectively the sequence of SEQ ID NO:3 and SEQ ID NO:4 in sequence table, the single base mutation of the 1450G of solid black trilateral " ▲ " expression sequence → 1450A, the primer location (being the forward primer position of primer pair YQ-fad32-1, YQ-fad32-2) of design is referring to the underscore place, and hollow triangle " ▽ " means the design of primers base mismatch.
Fig. 5: utilize the sequence dna fragment comparison of primer pair HY-fad31, the HY-fad32 amplification in swede type rape strain A-grade in the first class 254 and A-grade in the first class's 177 genomes, in figure: NO_1, NO_2, NO_3, NO_4 represent respectively the sequence of SEQ ID NO:1 in sequence table, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, the single base difference between the difference copy of the swede type rape fad gene that solid black trilateral " ▲ " expression primer pair HY-fad31, HY-fad32 increase in same parent; Pri.1 represents the reverse primer of primer pair YQ-fad31-1, YQ-fad31-2, Pri.2 represents the reverse primer of primer pair YQ-fad32-1, YQ-fad32-2, the primer location of design is referring to the underscore place, and arrow represents the primer direction, and hollow triangle " ▽ " means the 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 D H colony produced.In 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 DH colony that (under in figure, numerical value is the individual plant linolenic acid content to different linolenic acid content individual plants, unit: %); Fragment length is described primer extension product length.
Fig. 7: be techniqueflow chart of the present invention.
Embodiment
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 Representative culture collection center preservation in the Wuhan University of Wuhan City, Hubei Province on November 20th, 2009 to take the swede type rape strain A-grade in the first class 254 of low linolenic content (linolenic acid content is 2.28%) of our unit's seed selection, its preserving number is CCTCC-P200909, the biomaterial of this preservation is open in its female case application, the application number of female case application is that 200910273437.1 its publication numbers are: CN101818196A, its open day is on 09 01st, 2010) for maternal, common flax acid content (linolenic acid content is 7.78%) the rape advanced lines breeding of take choosing is that (seed of this material was delivered the Chinese Typical Representative 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, the biomaterial of this preservation is open in its female case application, the application number of female case application is that 200910273437.1 its publication numbers are: CN101818196A, its open day is on 09 01st, 2010) hybridized for male parent, obtain F
1seed.
The F that above-mentioned steps is obtained
1seed is sowed under field conditions (factors), obtains F
1plant, utilize 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 the DH segregating population obtained 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) 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) method of report 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 linolenic acid content:
Seed is used gas chromatograph (HP6890 after pressing the individual plant results, Genmany), analyze fatty acid content, parent and offspring's compound sample are chosen at random after 30-50 grain full seed grinds and are poured the 10ml test tube into, add 1ml ether, sherwood oil (volume ratio 1: 1) mixed solution in test tube, and then add isopyknic methyl alcohol (containing 5%KOH) to carry out esterification, standingly 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 Fatty acid component 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 ℃ keep 2 minutes, rise to 220 ℃ with 10 ℃/min afterwards and keep and keep 7min.
Fatty acid component is definite by retention time and the standard substance contrast of position, peak, and content means with area percentage.Only consider 7 kinds of main fatty acids, i.e. 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) to the data analysis measured with while arranging.
4, utilize primer pair HY-fad31, HY-fad32 Amplification Analysis parents and F
1genomic dna
Primer pair HY-fad31, the HY-fad32 that utilizes the applicant to develop (the primer pair sequence is in Table 2) comes Amplification Analysis 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 as follows: 1 * PCR buffer, 1.35mM MgCl
2, 0.08mM dNTPs, 1.0U Taq archaeal dna polymerase (all purchased 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 C 3min; 94 ℃ of C 30sec, renaturation temperature 45 C C sec, 72 ℃ of C 60sec, 29 circulations; 72 ℃ of C 10min, 1 circulation; 4 ℃ of C preserve, and reaction is to complete on the PTC-ALD1244PCR instrument.Two materials all carry out twice repetition.Amplified production 1.0% agarose gel electrophoresis on Horizontal electrophoresis tank detects, and uses 1 * TAE damping fluid (pH 8.0 for 0.04M Tris-acetate, 0.001M EDTA), voltage 8V/cm, electrophoresis 35min.Electrophoresis is complete, gel imaging system (UVP) preservation of taking pictures.
In the present invention, primer pair HY-fad31 is at parents and F
1genomic dna in amplified production be the bright band of about 2150bp, this bright band comprise very bright purpose band and than purpose band slightly bigger non-target fragment, parents and F
1amplified production is at 1.0% agarose gel electrophoresis detection lug segment length indifference; Primer pair HY-fad32 is at parents and F
1genomic dna in amplified production be the single bright band of about 1950bp, 1.0% agarose gel electrophoresis detection lug segment length indifference (Fig. 1 and table 2 are shown in experiment).
Numbering and the nucleotide sequence thereof of the primer pair of table 2 the present invention design
Annotate: runic and underscore base in (1) * table (as
g) represent that this base carried out the mispairing processing when design of primers.
(2) according to the order of describing, annex as specification sheets, in the nucleotide sequence provided separately and/or the copy of amino acid computer-reader form (referring to sequence table), the described primer sequence of above-mentioned table 2 is processed into respectively sequence table SEQ ID NO:9-20, and it is consistent with the primer sequence shown in table 2.
5, the DNA fragmentation that recovery, clone, sequencing primer increase in parents to HY-fad31, HY-fad32
The DNA fragmentation that primer pair HY-fad31, the HY-fad32 obtained 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.Schedule of operation reclaims by GenClean pillar DNA glue the method that test kit (purchased from Shanghai JaRa bio-engineering corporation) specification sheets provides: with blade, from 1.0% agarose gel, dig out the target DNA fragment of amplification, put into the centrifuge tube of 1.5ml, add 300 μ l Binding Solution B by every 100mg sepharose, be placed in 55 ℃ of C water-baths and heat 10min, mix once every 2min; The sol solution of thawing is transferred in the GenClean Column be enclosed within collection tube, room temperature is placed 2min, the centrifugal 30sec of 3,000rpm; Outwell the waste liquid in collection tube, add 500 μ l Wash Solution, the centrifugal 30sec of 8,000rpm room temperature, this step repeats once; Outwell the waste liquid in collection tube, GenClean Column is put into to same collection tube, the centrifugal 1min of 10,000rpm; GenClean Column is put into to the centrifuge tube of a new 1.5ml, in pillar film central authorities, add 30 μ l Elution Buffer, room temperature is placed 2min; The centrifugal 1min of 10,000rpm, the liquid in centrifuge tube is the DNA fragmentation of recovery, can use immediately or be stored in-20 ℃ of C standby.
The target dna fragment 2 μ l that get above-mentioned recovery make template, with primer pair HY-fad31, HY-fad32, according to above-mentioned steps, carry out pcr amplification, and the agarose gel 1.0% detects the DNA fragmentation of amplification.If the DNA fragmentation length of amplification is not identical with the result increased in above-mentioned steps, the recovery of need to again increasing; If that increases in the DNA fragmentation length of amplification and above-mentioned steps comes to the same thing, illustrate that reclaiming the DNA fragmentation obtained is the target dna fragment, can be used for next step T-A clone.
The target dna fragment of recovery is connected to (this carrier is purchased from TaKaRa company, that is, 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: reagent is first of short duration before use centrifugally is collected in the pipe bottom by it; 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.With transfer pipet, come resorption to mix several times, put the ligation of being spent the night of 4 ℃ of refrigerators; Prepare LB liquid nutrient medium and LB solid medium (containing the 100mg/ml penbritin, the chloro-3-indoles-α of the bromo-4-of 5-of the sec.-propyl of 24mg/ml-sulfo-β-D-galactoside and 20mg/ml-D-galactoside); From-70 ℃ of refrigerators, take out competent cell be placed on treat on ice it slowly thaw (about 5min); Centrifugal collection ligation liquid, get the 1.5ml centrifuge tube (being placed on precooling on ice) that 2 μ l reaction solutions join sterilizing; Flick with finger at the bottom of the pipe that competent cell is housed to mix, get 50 μ l competent cells and add the 1.5ml centrifuge tube that 2 μ l ligation liquid are housed, with finger, flick and mix, be placed on 20min on ice; Heat shock 90sec (not shaking) in 42 ℃ of water-baths, then place 5min on ice; Add after 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 aseptic LB solid medium, at 37 ℃, place 16-20h; Carry out indigo plant, hickie screening, select 24 positive colonies and be numbered, and at aseptic liquid LB substratum (containing the penbritin of 50ug/ml) shaking culture 16-20h; The bacterium liquid 2 μ l that get after 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 above-mentioned step.Amplification detects on 1.0% sepharose.If the DNA fragmentation of gained, than the large 200bp of target dna fragment left and right, illustrates and transforms successfully, select 5 parts of successful bacterium liquid of conversion respectively to draw 100 μ l and send Hua Da Gene science limited-liability company to carry out sequencing.Remaining the muddy bacterium liquid of 400 μ l adds in the aseptic glycerine of 400 μ l 50% centrifuge tube aseptic at 2ml in-70 ℃ of numbering preservations.
In 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 as 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 as shown in sequence table SEQ ID NO:3 and SEQ ID NO:4.
6, prepare in swede type rape the SNP mark with the low linolenic linkage of characters
EBI Tools ClustalW for fragment (http://www.ebi.ac.uk/Tools/clustalw/index.html) software that primer HY-fad31, the HY-fad32 that above-mentioned steps is obtained increases in A-grade in the first class 254 and A-grade in the first class 177 carries out sequence alignment.Comparison result shows, with the SEQ ID NO:2 shown in sequence table, compare, 4 single base mutations of the existence of SEQ ID NO:1 and 2 insertion mutations, 5 single base mutations are respectively: 28T → 28C, 390A → 390T, 1692T → 1703A, 1723G → 1734T, 1865C → 1876T; Insertion mutation is respectively and inserts a Nucleotide T at the 713-714 place, inserts the nucleotide fragments of a 10bp at the 1665-1666bp place, and inserting base is ATGATTAGTA (seeing accompanying drawing 3).With sequence table SEQ, IDNO:4 compares, at the single base mutation that only has 1 1450G → 1450A in total length 1963bp (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 sequence table are carried out to Multiple Sequence Alignment demonstration as a result, height homology between the different copies of the swede type rape fad3 gene amplified in same parent, the difference part mainly concentrates on low GC concentration areas, at GC content, be applicable to, in the zone (40～60%) of design primer, only having part single base mutation (seeing accompanying drawing 5).
Nucleotide sequence (as shown in sequence table SEQ ID NO:1 and SEQ ID NO:2) according to primer HY-fad31 at 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, a primer of mutational site design in sequence SEQ ID NO:1, a 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 pair of primers, reach the effect of the specific copy of amplification in specific parent.Requiring primer length during design is 17～25bp, 50～62 ℃ of C of renaturation temperature, GC content is 40～60%, is effectively to distinguish difference and the eliminating of this site between parents simultaneously, and the mispairing that utilizes primer 3 ' end mispairing strategy that the 2-4 bit base of primer 3 ' end is carried out to a base is processed.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 in Table 2 in primer pair YQ-fad31-1, the forward primer position is seen in accompanying drawing 3 shown in the NO_1 underscore, the reverse primer position is seen in accompanying drawing 5 shown in the NO_1 underscore), during design, 3 ' of forward primer and reverse primer being held to the 2nd bit base is G by the C mispairing respectively, by the A mispairing, be T, by 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 without any amplified production, by the above-mentioned requirements profit, use the same method, in sequence SEQ ID NO:2, screening obtains the twin target primer, between the 9-28bp of forward primer in sequence SEQ ID NO:2, between the 787-804bp of reverse primer in sequence SEQ ID NO:2 (sequence in Table 2 in primer pair YQ-fad31-2, the forward primer position is seen in accompanying drawing 3 shown in the NO_2 underscore, the reverse primer position is seen in accompanying drawing 5 shown in the NO_2 underscore), 3 ' of forward primer and reverse primer holds the 2nd bit base to be G, to be 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 without any amplified production.
According to primer HY-fad32 at the nucleotide sequence (as shown in sequence table SEQ ID NO:3 and SEQ ID NO:4) of amplification of DNA fragments in A-grade in the first class 254 and A-grade in the first class 177 and exploitation primer pair YQ-fad31-1, the requirement of YQ-fad31-2 and method, obtain the twin target primer in sequence SEQ ID NO:3, between the 1432-1450bp of forward primer in sequence SEQ ID NO:3, between the 1840-1859bp of reverse primer in sequence SEQ ID NO:3 (sequence in Table 2 in primer pair YQ-fad32-1, the forward primer position is seen in accompanying drawing 4 shown in the NO_3 underscore, the reverse primer position is seen in accompanying drawing 5 shown in the NO_3 underscore), during design, 3 ' of forward primer and reverse primer being held to the 2nd bit base is C by the G mispairing respectively, by the C mispairing, be G, by 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 without any amplified production, obtain the twin target primer in sequence SEQ ID NO:4 simultaneously, between the 1432-1450bp of forward primer in sequence SEQ ID NO:4, between the 1840-1859bp of reverse primer in sequence SEQ ID NO:4 (sequence in Table 2 in primer pair YQ-fad32-1, the forward primer position is seen in accompanying drawing 4 shown in the NO_4 underscore, the reverse primer position is seen in accompanying drawing 5 shown in the NO_4 underscore), during design, 3 ' of forward primer and reverse primer being held to the 2nd bit base is T by the G mispairing respectively, by the C mispairing, be G, 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 without 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, and the renaturation temperature is in 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 without any amplified production (seeing accompanying drawing 6), so this amplified fragments can be used as the dominant 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 without 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 without any amplified production (seeing accompanying drawing 6), so this amplified fragments can be used as the dominant 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 without 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
DH segregating population in SNP labeled analysis the present invention of the low linolenic acid of cabbage type rape proterties of utilizing above-mentioned steps to obtain, 33 individual plants the same with amplified fragments in parent A-grade in the first class 254 (i.e. " A/A banding pattern " in the table) wherein, 39 individual plants the same with amplified fragments in parent A-grade in the first class 177 (i.e. " B/B banding pattern " in the table), 76 individual plant detected results are " A/B banding pattern ", and 42 individual plant detected results are " B/A banding pattern " (in Table 3).Known by data in analytical table, every YQ-fad31, YQ-fad32 detection is " A/A banding pattern ", its linolenic acid content is all below 3.50%, it meets low linolenic breeding standard (the Rachael Scarth etc. that Rachael Scarth proposes in the international rape conference of 10Jie, Designer oil canola-a review of new food-grade Brassica oils with focus on high oleic, low linolenic types, 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 is selected, improve 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 in table 3 the present invention
Claims (3)
1. the codominance SNP molecule marker of a low linolenic acid of cabbage type rape content, its nucleotide sequence is as shown in sequence table SEQ ID NO:7 and SEQ ID NO:8.
2. primer pair YQ-fad32-1 and the YQ-fad32-2 of the codominance SNP molecule marker of amplification low linolenic acid of cabbage type rape content, its nucleotide sequence is as follows:
Primer pair YQ-fad32-1:
Forward primer 5 '-CCTTGGTACAGAGGCAACA-3 ',
Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '; With
Primer pair YQ-fad32-2:
Forward primer 5 '-CCTTGGTACAGAGGCAATG-3 ',
Reverse primer 5 '-TTGCCACCAAACTTTCCAGT-3 '.
3. the application of primer pair claimed in claim 2 in the assisted Selection of low linolenic swede type rape.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007016521A2 (en) * | 2005-08-01 | 2007-02-08 | Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Foods Canada | Low fiber yellow canola seeds comprising high oleic, low linolenic oil |
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2009
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007016521A2 (en) * | 2005-08-01 | 2007-02-08 | Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Foods Canada | Low fiber yellow canola seeds comprising high oleic, low linolenic oil |
Non-Patent Citations (4)
| Title |
|---|
| C.Jourdren et al..Specific molecular marker of the genes controlling linolenic acid content in rapeseed.《Theor Appl Genet》.1996,第93卷512-518. |
| Construction of an RAPD linkage map and localization of QTLs for oleic acid level using recombinant inbreds in mustard(Brassica juncea);R.Sharma et al.;《Genome》;20021231;第45卷;467-472 * |
| R.Sharma et al..Construction of an RAPD linkage map and localization of QTLs for oleic acid level using recombinant inbreds in mustard(Brassica juncea).《Genome》.2002,第45卷467-472. |
| Specific molecular marker of the genes controlling linolenic acid content in rapeseed;C.Jourdren et al.;《Theor Appl Genet》;19961231;第93卷;512-518 * |
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