CN100434534C - Molecular marker for artificial yellow seed rape (brassica napus L.) and its application - Google Patents
Molecular marker for artificial yellow seed rape (brassica napus L.) and its application Download PDFInfo
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Abstract
The present invention belongs to the technical field of rape breeding, particularly to the technical field of the selective breeding of a cabbage type yellow-seed rape new species by the assistance of molecular marks. Appropriate parents are selected by the present invention for hybridization to obtain an F1 plant; a double haploid (DH) segregating group is obtained through the cultivation of microspore, and a segment with RAPD and AFLP marks, which is tightly locked with a cabbage type yellow-seed gene is obtained by procedures of design of a special primer and PCR, recovery, cloning, sequence measuring, etc. through building two gene pools of a rape yellow-seed group and a black-seed group. The DNA segment is named two molecular marks of SCS1130 and SCA1, and nucleotide sequences are respectively shown in SEQ ID NO. 1 and No. 2. The molecular marks are successfully applied to the cabbage type yellow-seed rape group and linkage genetic analysis. The present invention also discloses a method for preparing molecular markers and for assisting seed breeding. The present invention provides a new practical mark gene and a new using method for molecular breeding of rape.
Description
Technical field
The invention belongs to the rapeseed breeding technical field, be specifically related to a kind of synthetic yellow-seeded brassica napus molecule marker and utilize this molecule marker to carry out marker assisted selection, cultivate a kind of yellow seed rape new variety of inheritance stability.
Background technology
Studies show that in a large number under identical genetic background, the black seed of yellow seed rape has: oleaginousness and protein content height; Plant skin thin (being about half); Polyphenolic substance content such as Mierocrystalline cellulose, tannin and tannic acid are low; Oil is as clear as crystal, and impurity is few; Grouts are to the livestock and poultry good palatability; Ripening degree is more easily identified, is convenient to series of advantages such as in good time results.All there is natural yellow seed germplasm to exist at turnip type rape, mustard type rape and brassicacarinata, however the cabbage type rape yellow seed resource shortage, the yellow seed proterties instability of existing yellow-seeded brassica napus, selfing is many for still having black seed to occur, and color and luster is not bright-coloured.Many investigators utilize several different methods such as distant hybirdization, radioinduction and synthetic actively to create the cabbage type rape yellow seed germplasm.By orthoselection to the Brassica type variant, between kind and species hybridization, radioinduction, approaches and methods such as tissue culture can select good yellow-seeded brassica napus.Therefore quality traits such as yellow seed coat and floorboard with high oil content, low Mierocrystalline cellulose, low erucic acid, low-sulfur glycosides are combined, combine with heterosis utilization, biotechnology, carry out comprehensive proterties breeding, cultivate a kind of brand-new rape variety, become the raising seed oil content, improve rapeseed quality, increase seed output, improve an important channel of yield potential.
Yet the pool heredity of yellow-seeded brassica napus kind color of the leather is extremely unstable, and genetic mechanism is very complicated, and environment and extraneous factor influence are bigger.The breeding method that traditional phenotype is selected is subjected to environmental influence, has many shortcomings, and efficient is low.Molecular marker assisted selection is a kind of breeding method efficiently, compares with traditional Phenotypic Selection, and it can carry out in any vegetative period, be not subjected to environmental influence, can get rid of that non-allelic genes interact and the interference that causes, have quick, economical, advantage such as efficient height, accuracy are strong.In the yellow-seeded brassica napus breeding process, molecular marking technique is combined with back cross breeding, the genotype of yellow seed proterties is carried out directly and apace selecting, to get rid of the influence of environment and extraneous factor by molecule marker.Simultaneously background is selected, can be accelerated the genetic background resume speed like this, shortening the breeding cycle and the effect that alleviates chain burden.
In swede type rape, people such as Van Deyzne (Van Deynze etc., The identification of restriction fragment lengthpolymorphisms linked to seed colour gene in Brassicca napus., Genome, 1995,38::534-542) identified a RFLP mark chain with kernel seed coat colour.(Somers D J etc. such as Somers, .Identification of a major gene and RAPD marker foryellow seed coat colour in Brassica napus., Genome, 2001,44:1077-1082) adopt RAPD and BSA (Michelmore etc., Identification of markers linked to disease-resistance genes by bulked segregant analysis:a rapid methoddetect markers in specific genomic regions by using segregating populations., Proc Natl Acad Sci USA, 1991,88::9828-9832) bonded method, find a key-gene damp relevant with planting color of the leather, 8 RAPD marks damp chain have been obtained with planting color of the leather, one of them and chromogene (black seed) be divided into from, two with yellow seed gene close linkage.These work are that molecular marker assisted selection seed selection yellow seed rape kind is laid a good foundation.Molecular marker assisted selection (MAS) is used in swede type rape and is few at present, does not see the report that MAS is arranged in yellow-seeded brassica napus as yet.
Summary of the invention
The objective of the invention is to utilize dna molecular marker and, find and closely linked RAPD of rape yellow seed gene and AFLP mark, and they are changed into more stable dominant SCAR mark or codominance CAPS mark in conjunction with the BSA analytical procedure.Use that this SCAR and CAPS mark carry out fast yellow seed gene and select accurately, use RAPD simultaneously and the AFLP mark is selected its genetic background, to accelerate the seed selection of yellow seed rape new variety.
The present invention is achieved through the following technical solutions:
A kind of preparation and the closely linked rape molecular marker method of cabbage type rape yellow seed gene comprise the following steps:
1) acquisition of double haploid (DH) segregating population: with extensive 5148-2 is that (these product lie in and were deposited in Chinese typical culture collection center on January 18th, 2006 female parent, deposit number is CCTCC-P200601), (these product lie in and were deposited in Chinese typical culture collection center on January 18th, 2006 with yellow seed strain NO.2127-17, deposit number is CCTCC-P200602) be male parent, hybridization, the pollen of getting the F1 plant carries out microspores culture, and (the microspores culture method is referring to document, lingering remnants of past customs group etc., improve some cultivation factor research of swede type rape sporule embryoid seedling rate, Acta Agronomica Sinica, 1997,23 (2): 165-168), obtain double haploid (DH) segregating population.
2) gene pool of structure DH segregating population:
With group's analytical method (bulked seregation analysis, be called for short BSA, referring to document Michelmore RW etc., Identification ofmarkers linked todisease resistance gene by bulked segregant analysis:a rapid method to detect markers inspecific genomic regions using segregating population., Proc.Natl.Acad.Sci., USA, 88:9829-9832), make up two gene pools of yellow seed group and black seed group.
3) utilize above-mentioned steps 2) in the gene pool of the DH segregating population that makes up, screening and closely linked RAPD of cabbage type rape yellow seed gene and AFLP mark:
With discrepant primer between two parents pcr amplification is carried out in two DNA ponds that make up in above-mentioned 2, the discrepant primer of performance carries out pcr amplification and electrophoresis detection to the DNA sample of each individual plant between the pond again between two ponds.After being defined as the linked marker of this proterties, the DNA sample to each individual plant of DH colony carries out pcr amplification and electrophoresis detection then.
4) reclaim, that screen and the closely linked RAPD of yellow seed gene swede type rape and the fragment of AFLP mark among the clone, order-checking above-mentioned 3.
5) according to above-mentioned steps 4) in sequencing result design special primer (primer sequence sees that embodiment is described), be converted into the SCAR mark with what screen with closely linked RAPD of cabbage type rape yellow seed gene and AFLP mark.
6) with above-mentioned steps 5) in the SCAR mark that transforms of success develop into the CAPS mark.
When above-mentioned steps 5) in candidate SCAR primer can not amplify polymorphism, then carry out the CAPS restriction analysis.With restriction enzyme amplified production is carried out enzyme and cut, enzyme is cut the product electrophoretic separation and is analyzed with SCAR.
7) with above-mentioned steps 3), 5), 6) in the RAPD mark, the AFLP mark, SCAR mark and CAPS mark carry out population analysis and linkage inheritance analysis.
8) utilize above-mentioned steps 7) in the molecule marker and the method for assisted Selection yellow seed gene is imported in the recovery system of swede type rape pol cytoplasmic male sterility (pol cms), select yellow seed type rape and recover system.
More detailed technical scheme is by the following stated:
1, rape microspores culture method:
Initial bloom stage at rape is got F
1Flower bud on the plant about 3-4mm is put into sterilized small beaker (required device and liquid must be sterilized and be operated) under aseptic condition, add 70% alcohol disinfecting 1min, uses 0.1%HgCL
2Sterilization 10min uses aseptic water washing 3 times then, each 5min.Get the bud that disinfects and put into test tube, add the B of 1ml
5-13 (B
5Liquid nutrient medium, sucrose concentration are 13%) extracting solution, grind into homogenate with glass rod, pour in the funnel that 0.44 μ m nylon membrane is housed and filter, collect filtrate with the centrifuge tube of 10ml.Add B
5Extracting solution is to required scale, centrifugal 5min, rotating speed 1000rpm/min.Abandon supernatant liquor, add B
5-13 extracting solutions suspend again, centrifugal 5min, and rotating speed 500rpm/min repeats this step once.Abandon supernatant liquor, add NLN-16 substratum (containing the 13mg/L colchicine) and suspend again, divide to install in the culture dish that diameter is 6cm, density is 2 flower buds/ml, and every ware 2ml puts into 32 ℃, secretly cultivates two days.Every ware adds fresh NLN-13 substratum 2ml, puts into 25 ℃ and continues dark the cultivation 2-3 week.Treat the visible embryo of naked eyes, place on 25 ℃ of constant temperature shaking tables that in 1 week of shaking culture, rotating speed is 55rpm/min, the embryo that reaches cotyledon period changes solid B over to
5Succeeding transfer culture on-G (the containing 0.1mg/LGA3) substratum, Cheng Miaohou transplants in good time.
2, make up yellow seed group and two DNA ponds of black seed group: (Japanese Minolta company produces to utilize color colour-difference meter CR-300, cardinal principle is that instrument has 8 mm dia measuring heads, 6 highly sensitive 2 silicon photocells, adopt diffuse illumination and 0 degree viewing angle, the lonely lamp of pulse in the mixing section provides illumination for sample surfaces, there is the twin-beam feedback system of instrument to be used for measuring incident light and reflected light, and provide corresponding brightness value), seed to DH colony carries out the kernel seed coat colour measurement, and all samples observed value from 17 to 50 is continuous variation.We are after measuring a large amount of samples, with reference to the yellow seed sort research of forefathers result, and, all samples are divided into 6 grades by Huang to black in conjunction with the naked eyes range estimation, be respectively that 1 grade of (the most yellow) brightness value is greater than 40,2 grades of brightness values are 35-40,3 grades of brightness values are 30-35, and 4 grades of brightness values are 25-30, and 5 grades of brightness values are 20-25,6 grades of (the most black) brightness values are less than 20 (accompanying drawings 1), with same people by divide individual plant record selfed seed rank with quadrat method and standard.In the DH segregating population, measure and the color analysis result according to each DH system kind of color of the leather pool, in 1 grade, choose 10 parts of DH systems and in 6 grades, choose 10 parts of DH systems, respectively its DNA balanced mix is constituted yellow seed gene pool (Yellow-seeded bulk) and black seed gene pool (Black-seededbulk).
3, RAPD analyzes and aflp analysis
RAPD analyzes: (method is referring to Rohrer G A etc. available from Shanghai biotechnology company limited for the RAPD primer, The useof a randomly ampllified polymorphic DNA marker RAPD in an analysia of susceptibility to heamonchus andcoccidia infestations in goats, jounrnal of animal science, 69 2-3,1991).Through optimization Test, RAPD analyzes and determines following optimum PCR reaction system: 1 * PCR buffer, 1.35mmol/L MgCl
2, 0.08mmol/L dNTPs, and 1.0U Taq polymerase (above-mentioned four are MBI Fermentas, Lithuania), 0.45 μ mol/L 10-mer RAPD primer (Shanghai biotechnology company limited), the 50ng dna profiling adds ddH
2O is to final volume 20 μ l.(MJ Research carries out on USA) to react on PTC-225 amplification instrument.Thermal circulation parameters is: 94 3min; 94 30s, 40 45s, 72 60s, 38 circulations; 72 10min; 4 preserve.Amplified production is on horizontal strip electrophoresis groove (Beijing 61 instrument companies), use 1 * TAE damping fluid, voltage 3V/cm is about electrophoresis 2.5h, 1.2% sepharose (containing EB) electrophoretic separation is utilized Lambda DNA/HindIII+EcoRI marker estimation amplified production size.Electrophoresis finishes, in gel imaging system (UVP) preservation of taking pictures, and record polymorphism result.
Aflp analysis: the enzyme of (1) total DNA is cut and is connected by table 1 preparation reaction system: total DNA 200ng, (final volume is 20 μ l for MBI Fermentas, Lithuania) restriction enzyme for 2U EcoRI and 2U MseI.Earlier at 37 ℃ of water-bath 5hrs-8hrs, after change 65 ℃ of water-bath 45min over to, add 5 μ l behind the complete degestion and connect mixed solution, enzyme is cut connected mixed solution and place 22 ℃ of connections to spend the night.Connection finishes, and 70 ℃ of deactivation 10min use ddH again
2O is with 10 times of substrates as the pre-amplification of PCR of reaction solution dilution; (2) design of AFLP joint and primer sequence is all undertaken by (1995) report methods such as Vos, and is synthetic by Shanghai bio-engineering corporation.Joint preparation: (how many F R) adds the some amount ddH that sterilizes according to OD value with the left and right sides primer of EcoR I, Mse I joint earlier
2It is 100 μ mol/L that O unifies dissolved dilution, after (F R) is mixed in the same sterilization 0.5ml centrifuge tube, carries out renaturation with isopyknic left and right sides primer.The renaturation parameter is 65 10 minutes, 37 10 minutes, and 25 10 minutes, a circulation, (Perkin elmer Ltd. carries out on USA), is placed on 10 minutes on ice, and it is standby to place-20 to preserve at last at DNA-Thermal Cycler 480PCR instrument.
The EcoRI joint sequence:
Left side primer (EF): 5 '-CTC GTA GAC TGC GTA CC-3 '
Right primer (ER): 3 '-CTG ACG CAT GGT TAA-5 ';
Mse I joint sequence:
Left side primer (MF): 5 '-GAC GAT GAG TCC TGA G-3 '
Right primer (MR): 3 '-TA CTC AGG ACT CAT-5 '.
Table 1 DNA enzyme of the present invention is cut, the linked system design
(3) pre-amplification: in the anti-system of 20 μ l PCR, contain 0.125mmol/L dNTPs, 1U Taq polymerase, 1.35mmol/L MgCl
2, (four are MBI Fermentas to 1 * PCR buffer, and Lithuania), 50ng EA and 50ng MC (perhaps MG) pre-expansion primer, 3 μ l enzymes are cut and connected product (table 2).The PCR loop parameter is: 94 30s, and 56 30s, 72 1min, 20 circulations are finished on the PTC-225PCR instrument.Get 4 μ lPCR products and detect in 1.0% sepharose, (Smear, 50bp-1000bp), it is effective to show that pre-expansion increases if be the disperse shape.10-15 times of resultant product dilution is as the template of next step PCR selective amplification.
Corresponding pre-amplification primer sequence is:
EA is: 5 '-GAC TGC GTA CCA ATT CA-3 ';
MC is: 5 '-GAT GAG TCC TGA GTA AC-3 ';
MG is: 5 '-GAT GAG TCC TGA GTA AG-3 '
(4) selective amplification: in 15 μ l PCR reaction systems, contain 3 μ l pre-expansions dilution volume increase thing, 37.5ng the EA+2 primer (adds 2 selectivity bases in addition for EA pre-expansion primer, below similar) and 37.5ng MC/MG+2 primer, 0.15mmol/L dNTPs, 0.75U Taqpolymerase, 0.45mmol/L MgCl
2, (back is MBI Fermentas, Lithuania) (table 3) to 1 * PCR buffer.The PCR loop parameter is: first 94 ℃ of 30s that circulate, 65 ℃ of 30s, 72 ℃
Pre-amplification of table 2 and selective amplification reaction system
Table 3 AFLP selective amplification of the present invention design of primers
1min, after this each circulation renaturation temperature descends 0.7 ℃, amounts to 13 circulations; 94 ℃ of 30s then, 56 ℃ of 30s, 72 ℃ of 1min totally 23 circulations are finished on the PTC-225PCR instrument.Selective amplification primer sequence and numbering see Table 3.
(5) electrophoresis detection of amplified production
With washing composition electrophoresis glass is thoroughly cleaned, earlier with deionized water drip washing, the back is with dehydrated alcohol drip washing and dry.On long offset plate, evenly smear 2ml silication liquid (AMRFSCO) with slick medicated napkin.On short offset plate, evenly be coated with the anti-silication liquid of 1ml (95% ethanol, 0.5% glacial acetic acid, the anti-silication agent of 2 μ l), clean gently with 95% ethanol behind the 5min, remove unnecessary silication liquid and anti-silication liquid and dry 5min.Glass is assembled and separate, assemble electrophoresis system with edge strip (0.4mm).With syringe with 80ml denaturant gel liquid (6% acrylamide; 7mol/L urea; 0.5 * tbe buffer liquid; add 300 μ l, 10% ammonium persulphate and 30 μ l TEMED and rapid mixing before the encapsulating) slowly inject from glue folder bottom aperture; insert comb at last and add the clip protection, get final product electrophoresis behind the cohesion 2hrs.
The glue clamping plate of electrophoresis chamber bottom are taken off, clean the electrophoresis chamber glass outer side, with its vertical fixing on base, last groove and following groove add 0.5 * tbe buffer liquid 1000ml and 500ml respectively, wash the point sample hole immediately after comb extracted, connect power supply 120W electrophoresis preheating 30min, the used instrument of electrophoresis is Sequi-
Sequencing cell (Bio-Rad, USA).The selective amplification product adds isopyknic sample-loading buffer (98% deionized formamide, 10mmol/L EDTA, the blue or green FF of 0.005% dimethylbenzene, 0.005% tetrabromophenol sulfonphthalein), ice bath cooling immediately behind 95 ℃ of sex change 5min, point sample 2.2 μ l.85W left and right sides electrophoresis was ended electrophoresis (needing 2hrs approximately) when the blue or green FF of dimethylbenzene ran 2/3 offset plate.
Electrophoresis finishes, and carefully peels offset plate, and it is immersed in the 2L stationary liquid (10% glacial acetic acid), till shaking 30min gently or disappearing to indicator, uses the rinsed with deionized water offset plate then 2 times, each 5min.Wash completely, go to (0.1%AgNO in the 2L staining fluid
3, shake dyeing 30min in 0.056%HCHO) gently.Take out offset plate, rapid rinsing 10s is transferred to 2L precooling (10 ℃) developing solution (3%Na at once in distilled water
2CO
3, 0.056%HCHO, 2mg/L Na
2S
2O
35H
2O) in, shake to band high-visiblely gently, the back is taken out to put back to and is stopped in the stationary liquid (10% glacial acetic acid) developing, and uses the distilled water rinsing 5min of rinsing for the second time again, dries the preservation of taking pictures under the room temperature naturally.
4, target fragment reclaims: the target fragment of amplification reclaims with UNIQ-10 pillar DNA glue recovery test kit (Shanghai bio-engineering corporation) from 1% agarose gel.The method that schedule of operation provides by test kit: dig out the centrifuge tube that target fragment is put into 1.5ml with blade, add Bing Buffer, place 50-60 ℃ of water-bath to heat 10min, once every the 2min mixing; The glue that melts is transferred in the UNIQ-10 post that is enclosed within the collection tube, and room temperature is placed 2min, 8, the centrifugal 1min of 000rmp; Outwell the waste liquid in the collection tube, add 500 μ l Wash Solution, 8, the centrifugal 1min of 000rmp room temperature, inferior step repeats once; Outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, 12, the centrifugal 15sec of 000rmp; The UNIQ-10 post is put into the centrifuge tube of a new 1.5ml, add 30 μ l Elution Buffer or water (pH＞7.0) in pillar film central authorities, room temperature or 37 ℃ are placed 2min; 12, the centrifugal 1min of 000rmp, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
5, reclaim fragment cloning and order-checking: the target fragment 3 μ l that get above-mentioned recovery make template, carry out pcr amplification with corresponding primer, and the agarose gel 1% detects whether amplified fragments is required target fragment.If not then needing the recovery of increasing again; If required target fragment is then carried out next step TA-clone operations.The target fragment that reclaims is connected
-easy carrier (available from U.S. Promega company, the white agency of biotech firm in Yuanping City, Beijing).The method that schedule of operation provides by test kit: earlier of short duration before use centrifugal it is collected in of reagent managed the bottom in the test kit; In the centrifuge tube of 0.5ml, set up following ligation system:
Table 4 target fragment is connected
The reaction system of-easy carrier
Come resorption mixing several times with transfer pipet, be placed on the ligation of spending the night of 4 ℃ of refrigerators; Prepare SOC substratum (this medium component and consumption referring to document :) and LB substratum (containing ampli, IPTG and X-Gal); From-70 ℃ of refrigerators, take out competent cell be placed on treat on ice it slowly thaw (approximately 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 mixing at the bottom of the pipe that competent cell is housed 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 2min then on ice; Cultivate 1.5hrs (150rmp/min) at 37 ℃ of ends that shake after adding the SOC substratum of 950 μ l; Conversion fluid 100 μ l after the absorption shaking culture are coated on the aseptic LB substratum (containing ampli, IPTG and X-Gal), place 16-24hrs at 37 ℃; Blue, hickie screening are selected positive colony at aseptic liquid LB substratum (containing ampli) shaking culture 16-24hrs; On Bechtop, draw 400 μ l and become muddy bacterium liquid, wherein 200 μ l bacterium liquid add the aseptic glycerine of 200 μ l 50% and preserve in-70 ℃ of numberings in the aseptic centrifuge tube of 1.5ml, remain 200 μ l bacterium liquid centrifugal collecting precipitations, add 200 μ l sterilized water mixings, inhale 3 μ l bacterium liquid and make pcr template, use corresponding primer amplification, on 1% agarose gel, detect target fragment and whether transform success.If transform successfully, get corresponding numbers and give order-checking company and carry out sequencing.
6, the conversion of SCAR mark
The SCAR design of primers: according to the information of full sequence, on the Internet with the Primer3 primer-design software (
Http:// redb. Croplab.org/modules/redbtools/primer3.php) design different primers, requiring GC content is 40%-70%, the Tm value is 60 ℃-70 ℃, no secondary structure in the primer can not be matched the positive and negative both direction (Forward of candidate SCAR primer mutually between primer, Reverse) length 20bp-25bp, synthetic by Shanghai bio-engineering corporation.
SCAR labeled analysis: PCR reaction system: 1 * PCR buffer, 1.35mmol/L MgCl
2, 0.08mmol/L dNTPs, and 1.0U Taqpolymerase (four are MBI Fermentas, Lithuania), and 50ng DNA, the forward and reverse primer of 0.45 μ mol/L (Forward, Reverse), ddH
2O is supplemented to final volume 20 μ l.Thermal circulation parameters is: 94 ℃ of 3min; 94 ℃ of 30s, optimum renaturation temperature 45s (do pilot study with two parents as dna profiling, optimize the renaturation temperature), 72 ℃ of 60s, 38 circulations; 72 ℃ of 10min, 1 circulation; 4 ℃ of preservations are reflected on the PTC-225PCR instrument and finish.Amplified production 1.2% agar gel (containing EB) on the horizontal strip electrophoresis groove separates, and uses 1 * TAE damping fluid, and voltage 3V/cm is about electrophoresis 1.5hrs.Electrophoresis finishes, gel imaging system (UVP) preservation of taking pictures, record polymorphism result.
7, the development of CAPS mark: when candidate's SCAR primer can not amplify polymorphism, then carry out the CAPS restriction analysis, system is: 10 μ lSCAR-PCR amplified productions, 1.5 μ l 10 * digested buffer (MBI Fermentas, Lithuania), 1.5U discerns the restriction enzyme (Bsh1236I of 4 bases, HaeIII, RsaI, Sau3AI, AluI, MseI, MBI Fermentas, Lithuania), 37 ℃ of enzymes are cut 3hrs-6hrs.Enzyme is cut the product electrophoretic separation and is analyzed with SCAR
8, the population analysis of mark and linkage inheritance analysis: use the RAPD mark, the AFLP mark, SCAR mark and CAPS mark carry out pcr amplification and electrophoresis detection one by one to the DNA sample in the DH segregating population, calculate genetic distance and analyze linkage relationship.Kernel seed coat colour shows as single-gene difference in this research, according to the MAPMAKER software requirement, be " A " with yellow seed proterties assignment, and black seed assignment is " B ", participates in linkage maps.In theory, molecule marker 2 kinds of banding patterns can only occur the offspring of DH colony, i.e. one of parents banding pattern, and corresponding parents' banding pattern is recorded as " A " and " B " respectively, and the banding pattern that is difficult to interpretation or disappearance is designated as "-".MAPMAKER/EXP Version 3.0 (Lander et al, 1987 are used in data analysis; Lincoln et al, 1992) software carries out, recombination value and genetic distance calculate with 2 tests between seed coat gene and molecule marker, the linkage map of mark and gene and order are test with 3 or multiple spot and are carried out (LOD=4.0, r=0.3), genetic distance is changed (Kosambi, 1944) with the Kosambi function.The result shows: be the close linkage relation between mark and gene, each mark and intergenic recombination value are at 2.4%-9.4%.The size of linkage map covering gene group is 53.8cM, and mean distance is 5.4Cm.Mark and intergenic order and distance see accompanying drawing 2 for details
9, field hybridization and molecular marker assisted selection: based on molecular marking technique, combine with traditional Phenotypic Selection by MAS, being aided with the field phenotype observes and attributional analysis, by hybridizing-backcross-method of selfing, the yellow seed gene of NO.2127-17 is imported to extensive 5148-2, select yellow seed and recover system.Detailed process is undertaken by the molecular marker assisted selection back cross breeding scheme shown in the accompanying drawing 3, carries out three foreground selection and three backgrounds and selects.Whole process comprises 3 hybridization, backcrosses for 2 times and 1 selfing.At first with 1141A (these product lie in and were deposited in Chinese typical culture collection center on January 18th, 2006, and deposit number is CCTCC-P200603) and extensive 5148-2 hybridization, again with its F
1Hybrid is maternal, with by extensive 5148-2 * NO.2127-17 through microspores culture and the yellow seed coat DH that comes system (selecting the extensive partially 5148-2 of background through MAS) makes paternal hybrid, combination is handed in preparation three, guarantees that each Dai Zhongzhong menu strain all has the recovery gene.Three hand over F
1Carry out backcrossing for twice with extensive 5148-2, and selfing once.In this program, each each individual plant from generation to generation of backcrossing all carries out the species test analysis of pcr analysis and harvesting time, selects yellow seed single-strain planting and goes down.
When combination is handed in preparation three, also do not find the molecule marker with yellow seed gene linkage,, show the DH system the most similar and prepare three many friendships and make up to extensive 5148-2 so can only select economical character good in the field.After ripe the results, DH system is carried out a look species test and an attributional analysis one by one, select the yellow seed DH of 12 high-qualitys system to carry out background and select, therefrom select two good DH to be, correspondingly hand over combined grows to go down its preparation three, all the other combinations are eliminated.BC
1F
1Middle with yellow seed gene specific mark S1129 and S1130, BC
2F
1In with SCS1130, S1130 and S1129 each individual plant is carried out yellow seed gene Selection (foreground selection), in each generation, investigated in conjunction with the field, eliminates only about half of non-target individual plant (deceiving the seed individual plant).The strain of centering menu is adopted two-step approach to carry out background and is selected, wherein BC
1F
1Use the RAPD mark, BC
2F
1Analyze with the AFLP mark, 3 individual plants of last every Dai Zhongxuan, the bagging selfing is carried out attributional analysis to menu strain in these and cross-pollinated seed simultaneously, according to the field performance of each combination, selects one of them combination to carry out MAS and analyzes.BC
2F
2In exist yellow seed proterties and separate, with the isozygoty selection of yellow seed individual plant of codominant marker SCA1.Middle menu strain and 1141A preparation combination compare test
Positively effect of the present invention:
The present invention searches out and closely linked RAPD of yellow seed proterties and AFLP molecule marker, and will be wherein two change into stable dominant SCAR mark and codominant CAPS mark, utilize this molecule marker to carry out the assistant breeding of yellow seed rape, can overcome in the yellow seed rape breeding process shortcoming according to Phenotypic Selection, reduce workload greatly, shortening the breeding cycle, cultivate new variety fast.
Description of drawings
Fig. 1 rape seed kernel seed coat colour is divided into 6 ranks, and the value in the figure bracket is a colourimetric number
The RAPD of Fig. 2 yellow-seeded brassica napus seed coat gene, AFLP, the genetic linkage map of SCAR and CAPS mark.The left side is a map distance, represents with cM, and the right is a mark
The molecular marker assisted selection seed selection scheme that the yellow seed proterties of Fig. 3 is backcrossed and imported to extensive 5148-2
Fig. 4 S1130 (RAPD, A) and EA07MC13 (AFLP, B) the expanding effect figure that fastens at each DH that constitutes gene pool.Swimming lane 1 is No2127-17, swimming lane 2 is yellow seed gene pool, swimming lane 3 is black seed gene pool, swimming lane 4 is extensive 5148-2, swimming lane 5-14 is respectively each DH system that constitutes yellow seed gene pool, and swimming lane 15-24 is respectively each DH system that constitutes black seed gene pool, and M shows the molecular weight size, arrow is depicted as molecule marker
*Show exchange DH system
Fig. 5 SCS1130 (SCAR, A) and SAC1 (CAPS B) is marked at the expanding effect figure that each DH of constituting gene pool fastens.Swimming lane 1 is NO.2127-17, and swimming lane 2 is yellow seed gene pool, and swimming lane 3 is black seed gene pool, swimming lane 4 is extensive 5148-2, swimming lane 5-14 is respectively each DH system that constitutes yellow seed gene pool, and swimming lane 15-24 is respectively each DH system that constitutes black seed gene pool, and M shows the molecular weight size
Fig. 6 RAPD primer S86 is at BC
1F
1Background in the colony is selected design sketch.P
1Be yellow seed parent NO.2127-17, P
2Be the black extensive 5148-2 of seed parent, all the other are different individual plants, and arrow is depicted as polymorphic band
Fig. 7 SCS1130 at part BC
2F
1The design sketch of the individual plant foreground selection of colony.P
1Be yellow seed parent NO.2127-17, P
2Be the black extensive 5148-2 of seed parent, all the other are different individual plants, and arrow is depicted as the special band of yellow seed individual plant
Fig. 8 SCA1 is at BC
2F
2Part individual plant selection result in the colony.P
1Be yellow seed parent NO.2127-17, P
2Be the black extensive 5148-2 of seed parent, all the other are different individual plants, and arrow is depicted as the yellow seed individual plant that isozygotys.
Embodiment
1) set up double haploid (DH) colony: in the present embodiment, make female parent with extensive 5148-2, yellow-seeded brassica napus NO.2127-17 is hybridized as male parent, obtains F1, utilizes F
1(the microspores culture method is referring to document through microspores culture for the pollen of plant, lingering remnants of past customs groups etc. improve some cultivation factor research that swede type rape sporule embryoid becomes the bacterium rate, Acta Agronomica Sinica, 1997,23 (2): 165-168) obtain 127 double haploids (being called for short DH) colony.
2) foundation of the extraction of DNA and gene pool: extract and purify DNA in the seedling from the colony that step 1 obtains, 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.The construction process of yellow seed gene pool and black seed gene pool is referring to described in 2 in the above-mentioned detailed technical scheme.
3) group's analytical method (BSA) combines with RPAD and AFLP technology and screens and the closely linked molecule marker of rape yellow seed gene: at 810 RAPD primer (S1-S410, S1000-S1400, available from Shanghai biotechnology company limited) in have 240 (29.6%) bars to show polymorphism, article 240, RAPD primer and 512 pairs of AFLP combination of primers (EA/MC and EA/MG combinations, AFLP joint and primer sequence design are all by (Vos et al.AFLP:a new technique for DNA fingerprinting.Nucleic Acids Res such as Vos, 1995,23:4407-4414) the report method is carried out, synthetic by Shanghai biotechnology company limited) between parents and gene pool, screen and show that 4 RAPD and 16 AFLP are combined between two gene pools and show polymorphism.Further analysis revealed, 2 RAPD and 8 AFLP marks and yellow seed gene close linkage are fastened recombination value less than 15% (see figure 4) at 20 DH of formation gene pool.Linkage analysis shows in 10 colonies that are marked at 127 DH system, 10 are marked on the same linkage group, wherein 2 RAPD and 5 AFLP and yellow seed gene are coupling linkage, and other 3 AFLP marks are repulsion linkage (accompanying drawing 2) with it, and the feature of each mark sees Table 5.
Table 5 and the RAPD of yellow seed gene linkage, primer sequence, phase place, size and the genetic distance of AFLP mark
E＝EcoRI?primer，5’-GACTGCGTACCAATTC-3’；
M＝MseI?primer，5’-GATGAGTCCTGAGTAA-3
4) with above-mentioned steps 3) screen with the closely linked RAPD of yellow seed gene, the AFLP mark is converted into SCAR mark and CAPS mark: with the recovery separately parent is with from PAGE glue earlier of AFLP differential fragment.The specific fragment of S1129 and S1130 separates on agarose gel with the heavy amplified production of 8 AFLP marks, and SK1131 test kit (available from Shanghai biotechnology company limited) advances to reclaim purifying.Directly purifying is reclaimed product and pGEMT-Easy carrier (Promega Corp,
#A1360Madison Wis) connects transformed into escherichia coli, coats on LB (containing amp) the solid medium flat board 37 ℃ of overnight incubation.Each fragment is chosen 6-10 white single bacterium colony with the tooth mirror, be seeded on LB (the containing amp) liquid culture and spend the night, get 5 μ l nutrient solutions and make dna profiling, SP6/T7 primer (available from Shanghai biotechnology company limited) carries out PCR and detects, the result shows, 10 labeled fragments all have the positive colony about 90%, have obtained positive recombinant.Each fragment selects the bacterium sample of two parts of positive colonies to deliver to the order-checking of Shanghai biotechnology company limited.Sequencing result shows that each AFLP flag sequence 5 ' end and 3 ' end includes Mse I and EcoR I restriction enzyme site and complete AFLP primer sequence respectively; Corresponding primer sequence or complementary sequence are contained in the two ends of two RAPD flags sequence.
According to they sequence sequencing results, on whole sequence information basis, usefulness Primer3 software (
Http:// redb.croplab.org/modules/redbtools/primer3.php) respectively design a pair of optimum primer.Each primer increases between parent NO.2127-17 and extensive 5148-2 earlier, optimizes the PCR reaction conditions, determines annealing temperature, sets up optimum reaction system.Again between two parents and two gene pools, and under optimal conditions separately pcr amplification, seek polymorphism, the result shows, the SCAR primer of S1130 shows polymorphism (accompanying drawing 5) two parents and two gene pools, shows that the S1130 mark has developed into a dominant SCAR mark, called after SCS1130.
The PCR product that does not develop into the primer of SCAR mark is used corresponding restriction endonuclease respectively, and (MBI Fermentas Lithuania) carries out enzyme at two parents and two gene pools and cuts, and seeks difference.Enzyme is cut the result and is shown, has only the product of EA05MC12/Bsh1236I primase combination to disclose polymorphism (accompanying drawing 5), and the EA05MC12 mark is developed into a codominant CAPS mark, called after SCA1.The feature of SCS1130 and SCA1 is shown in Table 6.
The feature of table 6SCAR and CAPS mark
The dna sequence dna of above-mentioned molecule marker SCS1130 is shown in sequence table SEQ ID NO:1; The dna sequence dna of mark SCA1 is shown in sequence table SEQ ID NO:2.
Show as linkage analysis in target group that with SCS1130 and SCA1 they still are in yellow seed gene both sides, genetic distance is respectively 3.2cM and 3.9cM.MAPMAKER/EXP 3.0 programs navigate to same linkage group with these two marks and original 10 RAPD and AFLP mark, and order on linkage group and distance are seen accompanying drawing 2
Select a BC that 353 individual plants (yellow seed of 169 strains and the black seed of 184 strains) arranged for use
1F
1Backcross population is estimated SCS1130 and SCA1.Show that these two are marked at strong ability is arranged in the genotypic evaluation of yellow-seeded brassica napus, can accurately select yellow seed genotype (table 7).
Table 7SCS1130 and SCA1 are marked at BCF
1The analytical results table
Annotate :+/-, the having or not of special band
Parent material is respectively the extensive 5148-2 of swede type rape, NO.2127-17, Pol cms sterile line 1141A (source as previously shown).Each individual plant DNA adopts method extraction in a small amount.
In yellow seed gene backcross transformation process, carry out foreground selection (tracking of yellow seed gene) with SCS1130, SCA1, RAPD and AFLP mark are selected in the selection of genetic background for use.Detailed process is undertaken by the molecular marker assisted selection back cross breeding scheme shown in the accompanying drawing 3, carries out three foreground selection and three backgrounds and selects.Carrying out each combination and individual plant economical character in the whole growing observes, main investigation growing way appearance, the leaf attitude of strain, reguarity, breeding time etc., several important strains system is carried out indoor species test, relatively in roguing and extensive 5148-2 the similarities and differences with and the similarities and differences that make up of preparation combination and extensive 5148-2.Attributional analysis is finished by Hua Zhong Agriculture University's rape research department attributional analysis locellus, adopts the near-infrared analysis method.
Each individual plant has or not respectively according to banding pattern, and assignment is 1 and 0.Press Nei and Li (Nei et al.Mathematical model for studying geneticvariation interms of restriction endonucleases.Proc Natl Acad Sci USA, 1979, method 76:269-573) is calculated similarity factor and the genetic distance of each material and the extensive 5148-2 of recurrent parent.Similarity analysis adopts UPGMA (Unweighted pair group witharithmetric average, the non-weighting class of arithmetical mean statistical method) method to carry out cluster analysis.
Concrete steps are as follows:
1) system three is handed over combination: is maternal with 1141A, extensive 5148-2 is that paternal hybrid gets F1, and (male parent and F1 hybridization are done by 65 DH systems in the colony of extensive 5148-2 * No.2127-17), obtain three and hand over and make up to select DH.
2) the RAPD primer is handed over the DH system of combination to carry out background to preparation in above-mentioned 1 three to select: select 142 best polymorphism RAPD primers of polymorphism for use, quality is better, seed is full, grain is big and prepare three to hand over the yellow DH that makes up be the similarity analysis that carries out genetic background and extensive 5148-2 to 12.The result shows that coamplification goes out 830 bands, 314 on polymorphism band, and carry out cluster analysis, filtering out two DH with extensive 5148-2 genetic distance minimum is DH21 and DH146, and field observation finds that this two DH systems performance is the most similar to extensive 5148-2, and is consistent with the background selection result.
Three of two DH systems preparation of choosing in 3) in above-mentioned 2 hands over combination to backcross and selfing: hand over combined grows to the field three of this two DH systems (DH21 and DH146) preparation, at bud stage, choose and can educate individual plant, backcross with it with extensive 5148-2, obtain two BC that backcross
1F
1(BC
1-21F
1, BC
1-146F
1) colony, and hand over the combination selfing to obtain three with three and hand over combination F
2(F
2-21And F
2-146).
4) with SCS1130 and SCA1 foreground selection is carried out in the combination in above-mentioned 3: with the RAPD primer background is carried out in the combination in above-mentioned 3 and select: gather in the crops the combination in above-mentioned 3, species test is the result show, three hand over combination F
2(F
2-21, F
2-146) kernel seed coat colour is yellow, attributional analysis shows that the two quality is better, oleaginousness height, two contemporary seed (BC that backcross
1-21F
0, BC
1-146F
0) attributional analysis is better, according to the attributional analysis result, in conjunction with two BC in field
1F
1Combination growing way appearance, leaf color and The Characters such as shape, reguarity are selected one of them combination BC the most similar to extensive 5148-2
1-146F
1Carrying out MAS selects.With SCS1130 and SCA1 to BC
1-146F
1Individual plant carry out the selection of yellow seed proterties, detected result: S1129 detects 175 yellow seed individual plants of the positive altogether, and S1130 detects the yellow seed individual plants of 170 positive strains, the two all detected positive individual plant totally 169 strain (seeing Table 10) altogether.
5) with the RAPD primer background being carried out in the combination in above-mentioned 3 selects: in conjunction with field individual plant growing state, select robust growth in above-mentioned 4 169 individual plants selecting, the vigorous positive individual plant of 142 strains of growth potential is made context analyzer.Earlier these 142 positive individual plants are carried out background and select (accompanying drawing 6), carry out cluster analysis as data with 30 best RAPD primers of polymorphism, filter out 46 with the nearest individual plant of extensive 5148-2 genetic distance.Other increases by 58 RAPD primers and carries out next step background selection.In conjunction with the field growing situation, select robust growth at last, with 3 individual plants 25 of extensive 5148-2 apart from minimum
#, 85
#, 49
#(table 8).
6) individual plant of selecting in above-mentioned 4 is backcrossed and selfing: middle menu strain 25 in will above-mentioned 4
#, 85
#, 49
#Backcross with extensive 5148-2, obtain BC
2F
1(the BC of colony
2-25F
1, BC
2-85F
1, BC
2-49F
1), simultaneously three strain selfings are obtained BC
1F
2(BC
1F
2-25, BC
1F
2-85, BC
1F
2-49).3 BC
1F
2With (the BC that backcrosses the present age
2F
0) seed color be yellow, consistent with the mark selection result, illustrate that selected combination contains yellow seed gene.
7) 6 parts of seeds to results in above-mentioned 5 carry out foreground selection, 6 parts of seeds of results in above-mentioned 5 are carried out attributional analysis, in conjunction with 3 BC in field
2F
1BC is chosen in performances such as the growing state of colony, reguarity, the leaf attitude of strain
2-85F
1Colony, carry out new round MAS foreground selection (accompanying drawing 7) with SCS1130 and SCA1, screening contains the individual plant of yellow seed gene, selection result shows: SCS1130 chooses the positive yellow seed individual plant of 107 strains, S1130 detects 108 strains, S1129 detects 109 strains, and no double exchange takes place between the three, detects 107 strain individual plants jointly.SCS1130 is by the S1130 development, and selection result and S1130 have big identity, the specificity height, and mistake is selected rate low (table 10).And, select the positive individual plant of the yellow seed of 94 strains of robust growth to make the MAS context analyzer in conjunction with each individual plant growing state of field.
8) with the AFLP primer individual plant of selecting in above-mentioned 6 being carried out background selects: select for use 20 pairs of AFLP primers that 94 individual plants of choosing in above-mentioned 6 are carried out context analyzer, filter out 46 individual plants nearest with extensive 5148-2 genetic distance.Increase by 40 pairs of AFLP primers in addition, these 46 individual plants are carried out next step background select,, select the individual plant 278 of 3 strains and extensive 5148-2 genetic distance minimum in conjunction with the field growing situation
#, 248
#, 243
#(table 8).
9) individual plant of choosing carries out selfing and hybridization in above-mentioned 7: to 3 individual plants 278 of choosing in above-mentioned 7
#, 248
#, 243
#The bagging selfing obtains BC
2F
2(BC
2F
2-278, BC
2F
2-248, BC
2F
2-243), and with 1141A preparing hybrid combination, carry out preliminary comparative experiments.
Table 8BC
1F
1And BC
2F
1In with 10 individual plants of extensive 5148-2 genetic distance minimum
10) to above-mentioned steps 9) in selfing and cross combination carry out foreground selection: to above-mentioned steps 9) in 3 BC
2F
2Seed carry out attributional analysis and kernel seed coat colour relatively after, according to 3 BC
2F
2The reguarity of colony, growth potential, fields such as the leaf attitude of strain performances, with codominant marker SCA1 to one of them neat and consistent, growth potential is vigorous, quality is more excellent BC
2F
2-248Colony carries out MAS and analyzes (seeing accompanying drawing 8).With codominant marker SCA1 to BC
2F
2-24849 individual plants of colony carry out analytical results and show: dominance is isozygotied (yellow seed): heterozygosis (yellow seed): recessiveness (black seed) ratio of isozygotying is 9: 23: 17, and the card square value is 2.79 (χ
2 0.05,2=5.99), the expection that meets 1: 2: 1 separates ratio, with BC
2F
2Offspring's phenotypic evaluation is basically identical as a result, mistake select probability be 6.12% (table 10) result show dominance isozygoty (yellow seed) have 9 strains, wherein not disease-resistant, proterties difference was eliminated 2 strain later stages.
11) to above-mentioned steps 10) in the individual plant selected carry out selfing and hybridization: to 7 individual plants (402 that isozygoty of choosing in above-mentioned 9
#, 405
#, 422
#, 433
#, 440
#, 445
#, 447
#) the bagging selfing, and with 1141A preparing hybrid combination (comprising extensive 5148-2), carry out next step comparative experiments.The BC of 7 individual plants that isozygoty of results
2F
2:3And attributional analysis is carried out in corresponding cross combination and the kernel seed coat colour analytical results shows, 7 individual plant kernel seed coat colours are all dark than parent NO.2127-17, and the institute join the cross combination color than in menu strain color dark, the more extensive 5148-2 of oleaginousness all is improved, suitable with NO.2127-17, erucic acid drops to zero mustard level, sulphur glycosides content increases than recurrent parent, be about two parent's averages, in the sulphur level, show that successfully the yellow seed gene with NO.2127-17 imports among the extensive 5148-2, sulphur glycosides level need be done further improvement (quality trait analysis in table 9).Comparative experiments is being put in 7 cross combinations more than a year, with the elected individual plant of final decision.
The quality trait analytical table of table 9 differing materials
Each mark foreground selection accuracy relatively
Various molecule marker selection results and field compare discovery according to the phenotypic evaluation result: along with increasing from generation to generation, each mark selects accuracy rate to reduce, and mistake selects rate all big than mark recombination value, and is extremely inconsistent.A plurality of one-sided marks are also failed bigger raising accuracy rate, and mark and target gene genetic distance are more little, and it selects accuracy rate high more; The bilateral mark selects accuracy rate more much higher than the accuracy rate of one-sided mark.The accuracy of foreground selection depends primarily on the linkage degree of mark and target gene, healing closely of mark and gene linkage, the reliability of selecting according to mark is higher, therefore in actual applications, pay the utmost attention to 2 of selections and be positioned at the nearest molecule marker in gene both sides, so not only can improve accuracy rate, and the individual and receptor parent genetic background homogeneity (seeing Table 10) of choosing in can increasing.
Table 10 each mark of the present invention is to each selection accuracy comparison sheet from generation to generation
Annotate:
1) 2)Data come from the molecule marker evaluation experimental; +/-, the having or not of special band
Note：
1)2)the?data?are?from?the?two?makers’validation?experiment：
+/-，presence/absence?of?the?specific?fragmen
Above-mentioned test molecule mark is that the present invention clones.
Sequence table
＜110〉Hua Zhong Agriculture University
＜120〉molecule marker of the yellow-seeded brassica napus of synthetic and application thereof
<130>
<141>2006-01-19
<160>2
<170>PatentIn?version?3.1
<210>1
<211>338
<212>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>gene
<222>(1)..(338)
<223>
<220>
<221>primer_bind
<222>(319)..(338)
<223>
<220>
<221>primer_bind
<222>(1)..(21)
<223>
<400>1
gtggcgatgt?ctttcctaaa?tcctacattg?attggtgatt?ttgaccgacc?taaaagtaaa 60
atataaaaca?acattattct?gattatgata?taratgggta?atgtattgtt?aggaatttga 120
ttagcacact?cgaggtcggt?ttaatatttt?tttatggatt?tgggatattt?ttttccctaa 180
acttttaaaa?tttatatttt?aaaatggcta?ctaatttttt?aagcaatatt?ataggtttaa 240
tctaatctat?taaagtttaa?atttttgtat?agtatatata?actaaatttt?aataattaag 300
acattgaaac?ataaggtcag?gttttggtcg?tgagcaca 338
<210>2
<211>465
<112>DNA
＜213〉swede type rape (Brassica napus)
<220>
<221>gene
<222>(1)..(465)
<223>
<220>
<221>primer_bind
<222>(443)..(465)
<223>
<220>
<221>primer_bind
<222>(1)..(22)
<223>
<400>2
ttgctcaatg?tgactcttgc?tcccctcgag?accccaaaac?caagattgcc?tcttttgtca 60
agatctatag?acctttccaa?aatcttcaag?gagcaaatgt?tttttgggtt?ctccgggagc 120
acgggtacga?ccagaagtca?tcaatatatt?cttggttggt?cactggctat?aggcggaaaa 180
gcgcaaagcc?ttgacatttc?tcaggtaatg?gatctccctc?gaccgccacc?tgatcactta 240
ccgcttatac?tcgcggttgc?atcagtagtt?gctttcctga?taatagcagg?cggaatagcg 300
tacctttacc?aacggaatag?gtatgcggaa?gtgtttgaac?aatgggaatt?acaatacagc 360
ccccagagat?tctccttcag?aaccctgtat?aaagcaacca?aaggtttcaa?ggagaataga 420
gtgcttggag?ctggaggttt?cggcaaggtt?tacagaggag?agctt 465
Claims (1)
1, a kind of method of utilizing the yellow-seeded brassica napus molecular marking supplementary breeding rape variety of synthetic, it comprises the following steps:
1) be female parent with the extensive 5148-2 of swede type rape strain, NO.2127-17 is a paternal hybrid with the yellow seed rape strain, obtains the F1 seed, and described F1 planting seed is obtained F1 rape plant, get this plant pollen and carry out microspores culture, obtain double haploid (DH) segregating population;
2) adopt group's analytical method to make up rape yellow seed group and two gene pools of black seed group;
3) between design two parents discrepant primer to above-mentioned steps 2) two gene pools making up carry out pcr amplification; the discrepant primer of choosing performance carries out pcr amplification and electrophoresis detection to the DNA sample of each individual plant between gene pool again; after being defined as its proterties and being linked marker, and the DNA sample to each individual plant of DH colony carries out pcr amplification and electrophoresis detection again;
4) recovery, clone, the screening of order-checking step 3) that obtain and the closely linked RAPD of yellow seed gene swede type rape and the fragment of AFLP mark;
5) according to the sequencing result design special primer of step 4), be converted into the SCAR mark with closely linked RAPD of cabbage type rape yellow seed gene and AFLP mark with what screen;
6) the not successful SCAR mark that transforms in the step 5) is developed into the CAPS mark;
7) with step 3), 5) and 6 in RAPD mark, AFLP mark, SCAR mark and CAPS mark carry out population analysis and linkage inheritance analysis;
8) utilize the molecule marker of step 7) and assisted Selection that the rape yellow seed gene is imported in the swede type rape pol cytoplasmic male sterility (pol cms) recovery system, seed selection obtains yellow seed type rape and recovers system.
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| CN101250524B (en) * | 2008-04-07 | 2011-05-11 | 华中农业大学 | Molecule marker of brassica napus self-incompatible maintenance line as well as preparation and uses thereof |
| CN101824472B (en) * | 2009-12-28 | 2012-02-08 | 华中农业大学 | Cabbage type rape high oleic acid molecular marker, preparation method and application thereof |
| CN102021235B (en) * | 2010-03-10 | 2013-03-27 | 华中农业大学 | Brassica napus L. grain weight major QTLs molecular marker and application thereof |
| CN102224801B (en) * | 2011-04-19 | 2012-09-26 | 江苏省农业科学院 | Rapid multi-target property polymerization breeding method for rape |
| CN102499056B (en) * | 2011-10-25 | 2014-08-13 | 西北农林科技大学 | Method for breeding new line of Brassica napus by utilizing biotechnology |
| CN102586274B (en) * | 2012-01-19 | 2013-04-24 | 西南大学 | Wild cabbage TT16 gene family and application thereof |
| CN109536630B (en) * | 2018-12-12 | 2019-10-01 | 广东省农业科学院蔬菜研究所 | The molecular labeling isolated with cabbage mustard petal color gene and its application |
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| 人工合成甘蓝型黄籽油菜的分子标记和利用研究. 刘志文,,，40-80以及104-105页，华中农业大学. 2004 * |
| 人工合成甘蓝型黄籽油菜的分子标记和利用研究. 刘志文，40-80以及104-105页，华中农业大学. 2004 * |
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