CN100434534C - A method for assisted breeding of rapeseed varieties using artificially synthesized molecular markers of Brassica napus - Google Patents

Abstract

The invention belongs to the technical field of rapeseed breeding, in particular to the technical field of using molecular markers to assist in breeding new varieties of Brassica oleracea yellow seed. In the present invention, F1 plants are obtained by selecting suitable parents for hybridization, double haploid (DH) segregation populations are obtained by microspore culture, two gene pools of rape yellow seed group and black seed group are constructed, specific primers are designed, PCR and recovery , cloning, sequencing and other steps to screen and obtain the RAPD and AFLP marker fragments closely linked to the yellow seed gene of Brassica napus; the DNA fragments are named as two molecular markers SCS1130 and SCA1, and their nucleotide sequences are respectively shown in SEQ ID NO : 1 or 2 shown. The above molecular markers have been successfully applied to the population and linkage genetic analysis of Brassica napus. The invention also discloses the preparation of the molecular marker and its use in assisted breeding. The invention provides a new practical marker gene and a new utilization method for molecular breeding of rapeseed.

Description

A method for assisted breeding of rapeseed varieties using artificially synthesized molecular markers of Brassica napus

technical field

The invention belongs to the technical field of rapeseed breeding, and in particular relates to an artificially synthesized molecular marker of Brassica oleracea and using the molecular marker to perform marker-assisted selection to cultivate a genetically stable new variety of yellow-seed rape.

Background technique

A large number of studies have shown that, under the same genetic background, yellow seed rape has higher oil and protein content than black seed; thin seed coat (about half); The quality is clear and transparent, with less impurities; the cake has good palatability to livestock and poultry; the maturity is easy to identify, and it is convenient for timely harvesting and other advantages. There are natural yellow seed germplasm in Brassica napus, Brassica napus and Ethiopian mustard. However, the yellow seed resources of Brassica napus are lacking. Black seeds appear, the color is not bright. Many researchers actively create yellow seed germplasm of Brassica napus by means of distant hybridization, radiation mutagenesis and artificial synthesis. Excellent yellow-seed Brassica napus can be bred through directional selection of yellow-seed mutants of Brassica oleracea, hybridization between varieties and species, radiation mutagenesis, tissue culture and other methods and methods. Therefore, the yellow seed coat is combined with quality traits such as high oil content, low cellulose, low erucic acid, and low glucosinolates, combined with the use of heterosis and biotechnology, and comprehensive trait breeding is carried out to cultivate a new rapeseed variety. It has become an important way to increase the oil content of seeds, improve the quality of rapeseed, increase seed yield, and increase yield potential.

However, the inheritance of seed coat color of Brassica napus is extremely unstable, the genetic mechanism is very complex, and the environment and external factors are greatly affected. The traditional breeding method of phenotypic selection is affected by environmental conditions, and has many shortcomings and low efficiency. Molecular marker-assisted selection is an efficient breeding method. Compared with traditional phenotypic selection, it can be carried out at any growth stage, is not affected by environmental conditions, and can exclude interference caused by non-allelic interactions. Economical, high efficiency, strong accuracy and other advantages. In the process of Brassica napus breeding, the molecular marker technology was combined with backcross breeding, and the genotype of the yellow seed trait was directly and quickly selected by molecular markers, so as to eliminate the influence of environment and external factors. At the same time, the background is selected, which can speed up the recovery of the genetic background, shorten the breeding period and reduce the effect of linkage burden.

In Brassica napus, Van Deyzne et al. (Van Deynze et al., The identification of restriction fragment length polymorphisms linked to seed color gene in Brassicca napus., Genome, 1995, 38:: 534-542) identified an The RFLP mark. Somers et al. (Somers D J etc.,. Identification of a major gene and RAPD marker for yellow seed coat color in Brassica napus., Genome, 2001, 44:1077-1082) adopt RAPD and BSA (Michelmore et al., Identification of markers linked to disease -resistance genes by bulked segregant analysis: a rapid method detect markers in specific genomic regions by using segregating populations., Proc Natl Acad Sci USA, 1991, 88::: 9828-9832) combined method to find a major factor related to seed coat color Genes, 8 RAPD markers linked to seed coat color were obtained, one of which was co-segregated with pigment gene (black seed), and two were closely linked with yellow seed gene. These works laid the foundation for molecular marker-assisted selection of yellow-seed rape varieties. At present, molecular marker-assisted selection (MAS) is not widely used in Brassica napus, and there is no report of MAS in Brassica napus.

Contents of the invention

The purpose of the present invention is to find RAPD and AFLP markers closely linked with rapeseed yellow seed genes by using DNA molecular markers combined with BSA analysis method, and convert them into more stable dominant SCAR markers or co-dominant CAPS markers. The SCAR and CAPS markers were used to quickly and accurately select the yellow-seed gene, and the RAPD and AFLP markers were used to select its genetic background, so as to speed up the breeding of new varieties of yellow-seed rape.

The present invention is realized through the following technical solutions:

A method for preparing a rapeseed molecular marker closely linked to the Brassica napus yellow seed gene, comprising the following steps:

1) Obtaining double haploid (DH) segregation population: take Hui 5148-2 as the female parent (the strain was preserved in the China Center for Type Culture Collection on January 18, 2006, and the preservation number is CCTCC-P200601), and Yellow seed line NO.2127-17 (this line was preserved in the China Center for Type Culture Collection on January 18, 2006, and the preservation number is CCTCC-P200602) is the male parent, hybridized, and the pollen of the F1 plant is taken for microspore culture ( For microspore culture methods, please refer to literature, Yu Fengqun, etc., Research on Some Culture Factors to Improve the Seedling Rate of Brassica napus Microspore Embryoids, Acta Crops, 1997, 23(2): 165-168), to obtain double haploid (DH) Separating populations.

2) Construct the gene pool of DH segregation population:

Use group analysis (bulked seregation analysis, referred to as BSA, see Michelmore RW et al., Identification of markers linked todisease resistance gene by bulked segregant analysis: a rapid method to detect markers inspecific genomic regions using segregating population., Procad. ., USA, 88:9829-9832), constructing two gene pools of the yellow seed group and the black seed group.

3) Utilize the gene pool of the DH segregation population constructed in the above step 2) to screen the RAPD and AFLP markers closely linked to the Brassica napus yellow seed gene:

Use primers that differ between the two parents to perform PCR amplification on the two DNA pools constructed in the above 2, and perform PCR amplification and electrophoresis on the DNA samples of each individual plant between the pools with primers that show differences between the two pools detection. After the linkage marker of the trait is determined, PCR amplification and electrophoresis detection are performed on the DNA samples of each individual plant of the DH population.

4) Recover, clone, and sequence the RAPD and AFLP-marked fragments screened in the above 3 and closely linked to the yellow seed gene of Brassica napus.

5) According to the sequencing results in step 4) above, design specific primers (see the examples for primer sequences), and convert the screened RAPD and AFLP markers closely linked to the Brassica napus yellow seed gene into SCAR markers.

6) The SCAR markers that were not successfully transformed in the above step 5) were developed into CAPS markers.

When the polymorphism cannot be amplified by the candidate SCAR primers in the above step 5), CAPS digestion analysis is performed. The amplified product was digested with restriction endonuclease, and the electrophoresis separation of the digested product was the same as SCAR analysis.

7) Perform population analysis and linkage genetic analysis on the RAPD markers, AFLP markers, SCAR markers and CAPS markers in the above steps 3), 5), and 6).

8) Using the molecular markers and assisted selection methods in the above step 7) to introduce the yellow seed gene into the restorer line of Brassica napus polima cytoplasmic male sterility, and select the restorer line of yellow seed type rape.

A more detailed technical solution is as follows:

1. Rapeseed microspore culture method:

At the initial flowering stage of rapeseed, take the buds of about 3-4mm on the _F1 plant, put them into a sterilized small beaker (the required devices and liquids must be sterilized and operate under aseptic conditions), and add 70% alcohol to sterilize 1min, sterilized with 0.1% HgCl ₂ for 10min, and then rinsed with sterile water 3 times, 5min each time. Put the detoxified flower buds into a test tube, add 1ml of B ₅ -13 (B ₅ liquid medium, sucrose concentration is 13%) extract, grind it into a homogenate with a glass rod, pour it into a 0.44 μm nylon membrane Filter through a funnel and collect the filtrate in a 10ml centrifuge tube. Add _B5 extract to the desired scale, centrifuge for 5min at 1000rpm/min. Discard the supernatant, add B ₅ -13 extract to resuspend, centrifuge for 5 min at 500 rpm/min, repeat this step once. Discard the supernatant, add NLN-16 medium (containing 13mg/L colchicine) to re-suspend, and divide into petri dishes with a diameter of 6cm. Incubate in the dark for two days. Add 2ml of fresh NLN-13 culture medium to each dish, put it in 25°C and continue dark cultivation for 2-3 weeks. When the embryos are visible to the naked eye, place them on a constant temperature shaker at 25°C for 1 week with shaking at a speed of 55rpm/min. Embryos that reach the cotyledon stage are transferred to solid B ₅ -G (containing 0.1mg/LGA3) medium for subculture , Transplant in good time after seedlings grow.

2. Construct two DNA pools of Huangzi Group and Heizi Group: use the color difference meter CR-300 (produced by Japan Minolta Company, the main principle is that the instrument has a measuring head with a diameter of 8 mm, 6 highly sensitive 2 silicon photocells, and adopts Diffuse illumination and 0 degree observation angle, the pulsed arc lamp in the mixing chamber provides illumination for the sample surface, the dual-beam feedback system with the instrument is used to measure the incident light and reflected light, and give the corresponding brightness value), the DH population Seeds were subjected to testa color measurements, which ranged continuously from 17 to 50 for all samples. After measuring a large number of samples, referring to the results of previous studies on the classification of yellow seeds and combining with visual inspection, we divided all the samples into 6 grades from yellow to black. The brightness value of grade 3 is 35-40, the brightness value of grade 3 is 30-35, the brightness value of grade 4 is 25-30, the brightness value of grade 5 is 20-25, and the brightness value of grade 6 (the darkest) is less than 20 (accompanying drawing 1 ), with the same person using the same method and standard to divide individual plants and record the self-bred seed level. In the DH segregation population, according to the testa color measurement and color analysis results of each DH line, select 10 DH lines in level 1 and 10 DH lines in level 6, and mix their DNA in equal amounts to form the yellow seed gene Pool (Yellow-seeded bulk) and black seed gene pool (Black-seeded bulk).

3. RAPD analysis and AFLP analysis

RAPD analysis: RAPD primers were purchased from Shanghai Bioengineering Co., Ltd. (For methods, see Rohrer G A et al., The use of a randomly amplified polymorphic DNA marker RAPD in an analysis of susceptibility to hemonchus and coccidia infections in goats, journal of animal science, 69 2-3, 1991,). After optimization experiments, RAPD analysis determined the following optimal PCR reaction system: 1×PCR buffer, 1.35mmol/L MgCl ₂ , 0.08mmol/L dNTPs, 1.0U Taq polymerase (the above four are all MBI Fermentas, Lithuania), 0.45μmol /L 10-mer RAPD primer (Shanghai Bioengineering Co., Ltd.), 50ng DNA template, add ddH ₂ O to a final volume of 20 μl. The reaction was performed on a PTC-225 thermal cycler (MJ Research, USA). Thermal cycle parameters are: 94□3min; 94□30s, 40□45s, 72□60s, 38 cycles; 72□10min; 4□save. The amplified product was electrophoresed on a horizontal electrophoresis tank (Beijing Liuyi Instrument Co., Ltd.), using 1×TAE buffer, voltage 3V/cm, electrophoresis for about 2.5h, and separated by electrophoresis on 1.2% agarose gel (containing EB). HindIII+EcoRI marker estimates the size of the amplification product. After electrophoresis, take pictures and save them in the gel imaging system (UVP), and record the polymorphism results.

AFLP analysis: (1) Digestion and ligation of total DNA A reaction system was prepared according to Table 1: 200 ng of total DNA, 2U EcoRI and 2U MseI (MBI Fermentas, Lithuania) restriction enzymes, and the final volume was 20 μl. First put in a water bath at 37°C for 5hrs-8hrs, then transfer to a water bath at 65°C for 45min, add 5μl of ligation mixture after complete enzyme digestion, and place the enzyme digestion ligation mixture at 22°C for overnight ligation. After the ligation was completed, it was inactivated at 70°C for 10 min, and then the reaction solution was diluted 10 times with ddH ₂ O as the substrate for PCR pre-amplification; (2) AFLP adapter and primer sequence design were carried out according to the method reported by Vos et al. (1995). Synthesized by Shanghai Bioengineering Company. Linker preparation: First, add a certain amount of sterilized ddH ₂ O to the left and right primers (F, R) of the EcoR I and Mse I linkers according to the OD value, dissolve and dilute them uniformly to 100 μmol/L, and then mix equal volumes of the left and right primers (F, R) R) Mix in the same sterilized 0.5ml centrifuge tube for renaturation. The refolding parameters were 65□10 minutes, 37□10 minutes, 25□10 minutes, one cycle, carried out on a DNA-Thermal Cycler 480 PCR instrument (Perkin elmer Ltd., USA), then placed on ice for 10 minutes, and finally placed on ice. Save it at -20□ for future use.

EcoRI linker sequence:

Left primer (EF): 5′-CTC GTA GAC TGC GTA CC-3′

Right primer (ER): 3′-CTG ACG CAT GGT TAA-5′;

Mse I linker sequence:

Left primer (MF): 5′-GAC GAT GAG TCC TGA G-3′

Right primer (MR): 3′-TA CTC AGG ACT CAT-5′.

Table 1 DNA digestion of the present invention, connection system design

(3) Pre-amplification: In 20μl PCR reaction system, containing 0.125mmol/L dNTPs, 1U Taq polymerase, 1.35mmol/L MgCl ₂ , 1×PCR buffer (all are MBI Fermentas, Lithuania), 50ng EA and 50ng MC (or MG) pre-amplification primers, 3μl enzyme-cut ligation product (Table 2). The PCR cycle parameters are: 94□30s, 56□30s, 72□1min, 20 cycles, completed on the PTC-225 PCR instrument. Take 4 μl of the PCR product and test it on 1.0% agarose gel. If it is diffuse (Smear, 50bp-1000bp), it indicates that the pre-amplification effect is good. The remaining product was diluted 10-15 times and used as a template for the next step of PCR selective amplification.

The corresponding preamplification primer sequences are:

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: 15 μl PCR reaction system contains 3 μl pre-amplified diluted amplification products, 37.5ng EA+2 primers (add 2 selective bases for EA pre-amplification primers, similar to the following) and 37.5ng MC /MG+2 primers, 0.15mmol/L dNTPs, 0.75U Taqpolymerase, 0.45mmol/L MgCl ₂ , 1×PCR buffer (the last four are MBI Fermentas, Lithuania) (Table 3). The PCR cycle parameters are: first cycle 94°C for 30s, 65°C for 30s, 72°C

Table 2 Pre-amplification and selective amplification reaction system

Table 3 AFLP selective amplification primer design of the present invention

1min, after which the renaturation temperature decreased by 0.7°C for each cycle, a total of 13 cycles; then 94°C for 30s, 56°C for 30s, 72°C for 1min, a total of 23 cycles, completed on the PTC-225 PCR instrument. See Table 3 for the sequences and numbers of selective amplification primers.

(5) Electrophoretic detection of amplified products

Wash the electrophoretic glass thoroughly with detergent, first rinse with deionized water, then rinse with absolute ethanol and dry. Spread 2 ml of siliconizing solution (AMRFSCO) evenly on the long rubber plate with a smooth napkin. Apply 1ml desiliconization solution (95% ethanol, 0.5% glacial acetic acid, 2μl desiliconization agent) evenly on the short rubber plate, scrub gently with 95% ethanol after 5min, remove excess siliconization solution and desiliconization solution and dry for 5min . The glass is assembled and separated by side strips (0.4mm), and the electrophoresis system is assembled. Use a syringe to inject 80ml of denaturing gel solution (6% acrylamide, 7mol/L urea, 0.5×TBE buffer solution, add 300μl 10% ammonium persulfate and 30μl TEMED and mix quickly) from the small hole at the bottom of the gel-making clamp Slowly inject, and finally insert the comb and add a clip for protection, and electrophoresis can be performed after condensing for 2hrs.

sequencing cell(Bio-Rad，USA)。选择性扩增产物加入等体积的上样缓冲液(98％去离子甲酰胺，10mmol/L EDTA，0.005％二甲苯青FF，0.005％溴酚蓝)，95℃变性5min后立即冰浴冷却，点样2.2μl。85W左右电泳，当二甲苯青FF跑过2/3胶板时中止电泳(约需2hrs)。Remove the gel-making splint at the bottom of the electrophoresis tank, clean the outside of the glass of the electrophoresis tank, and fix it vertically on the base, add 1000ml and 500ml of 0.5×TBE buffer solution to the upper tank and lower tank respectively, and rinse the spot immediately after pulling out the comb For sample wells, turn on the power supply and preheat 120W electrophoresis for 30 minutes. The instrument used for electrophoresis is Sequi-

Sequencing cells (Bio-Rad, USA). Selectively amplified products were added to an equal volume of loading buffer (98% deionized formamide, 10mmol/L EDTA, 0.005% xylene cyanol FF, 0.005% bromophenol blue), denatured at 95°C for 5min, and immediately cooled in an ice bath. Spot 2.2 μl. Electrophoresis at about 85W, stop electrophoresis when xylene cyanol FF runs over 2/3 of the gel plate (about 2hrs).

After electrophoresis, carefully peel off the rubber sheet, immerse it in 2L of fixative solution (10% glacial acetic acid), shake gently for 30 minutes or until the indicator disappears, and then rinse the rubber sheet twice with deionized water, 5 minutes each time. After washing, transfer to 2L staining solution (0.1% AgNO ₃ , 0.056% HCHO) and gently shake for 30 min. Take out the rubber sheet, rinse it quickly in double distilled water for 10 seconds, and immediately transfer to 2L pre-cooled (10°C) developer solution (3% Na ₂ CO ₃ , 0.056% HCHO, 2mg/L Na ₂ S ₂ O ₃ 5H ₂ O ), shake gently until the bands are clearly visible, then take it out and put it back into the fixative solution (10% glacial acetic acid) to stop developing, then rinse with double-distilled water for 5 minutes, dry naturally at room temperature, and take pictures for preservation.

4. Recovery of target fragments: the amplified target fragments were recovered from 1% agarose gel with UNIQ-10 Column DNA Gel Recovery Kit (Shanghai Bioengineering Company). The operating procedure is according to the method provided in the kit: dig out the target fragment with a blade and put it into a 1.5ml centrifuge tube, add Bing Buffer, heat in a water bath at 50-60°C for 10 minutes, and mix every 2 minutes; transfer the melted gel Put it into the UNIQ-10 column set in the collection tube, place it at room temperature for 2 minutes, centrifuge at 8,000rmp for 1min; pour off the waste liquid in the collection tube, add 500μl Wash Solution, and centrifuge at 8,000rmp for 1min at room temperature, repeat the steps once; discard the collection tube Put the waste liquid in the column into the same collection tube, centrifuge at 12,000rmp for 15sec; put the UNIQ-10 column into a new 1.5ml centrifuge tube, add 30μl Elution Buffer or Water (pH>7.0), placed at room temperature or 37°C for 2min; centrifuged at 12,000rmp for 1min, the liquid in the centrifuge tube is the recovered DNA fragment, which can be used immediately or stored at -20°C for later use.

-easy载体(购自美国Promega公司，北京原平皓生物公司代理)。操作程序按试剂盒提供的方法：试剂盒中试剂在使用前先短暂离心将其收集在管底部；在0.5ml的离心管中建立如下连接反应体系：5. Cloning and sequencing of recovered fragments: take 3 μl of the recovered target fragments as a template, perform PCR amplification with corresponding primers, and check whether the amplified fragments are the desired target fragments in 1% agarose gel. If not, re-amplify and recover; if it is the desired target fragment, proceed to the next step of TA-cloning operation. Recycled target fragments are concatenated in

-easy carrier (purchased from Promega Company of the United States, agent of Beijing Yuanpinghao Biological Company). The operating procedure is according to the method provided in the kit: the reagents in the kit are centrifuged briefly before use to collect them at the bottom of the tube; the following connection reaction system is established in a 0.5ml centrifuge tube:

-easy载体的反应体系Table 4 Target Fragments Linked in

-easy carrier reaction system

Use a pipette to suck back and forth several times to mix well, and put it in a refrigerator at 4°C for overnight ligation reaction; prepare SOC medium (see the literature for the composition and dosage of the medium:) and LB medium (containing ampli, IPTG and X-Gal) ;Take out the competent cells from the -70°C refrigerator and put them on ice until they thaw slowly (about 5min); centrifuge to collect the ligation reaction solution, take 2μl of the reaction solution and add it to a sterilized 1.5ml centrifuge tube (on ice flick the bottom of the tube with competent cells to mix well, take 50μl of competent cells and add 2μl of the connection reaction solution to a 1.5ml centrifuge tube, flick with your fingers to mix well, put on ice for 20min ; Heat shock in a water bath at 42°C for 90sec (do not shake), then place on ice for 2min; add 950μl of SOC medium and culture at 37°C for 1.5hrs with vibration (150rmp/min); absorb 100μl of transformation solution after shaking culture Spread on sterile LB medium (containing ampli, IPTG and X-Gal), place at 37°C for 16-24hrs; screen with blue and white spots, select positive clones and shake culture in sterile liquid LB medium (containing ampli) 16-24hrs; draw 400μl of turbid bacterial solution on the ultra-clean workbench, of which 200μl of bacterial solution plus 200μl of 50% sterile glycerin is stored in a 1.5ml sterile centrifuge tube at -70°C, and the remaining 200μl of bacterial solution Collect the precipitate by centrifugation, add 200 μl of sterile water to mix, absorb 3 μl of the bacterial solution as a PCR template, use the corresponding primers to amplify, and check whether the target fragment is successfully transformed on a 1% agarose gel. If the transformation is successful, take the corresponding number and send it to the sequencing company for sequence determination.

6. Transformation of SCAR markers

SCAR primer design: According to the information of all sequences, use Primer3 primer design software ( http://redb.croplab.org/modules/redbtools/primer3.php ) to design different primers on the Internet, requiring GC content of 40%-70% , the Tm value is 60°C-70°C, there is no secondary structure in the primer, and the primers cannot be paired with each other. The length of the forward and reverse directions (Forward, Reverse) of the candidate SCAR primer is 20bp-25bp, synthesized by Shanghai Bioengineering Company.

SCAR marker analysis: PCR reaction system: 1×PCR buffer, 1.35mmol/L MgCl ₂ , 0.08mmol/L dNTPs, 1.0U Taqpolymerase (all four are MBI Fermentas, Lithuania), 50ng DNA, 0.45μmol/L forward and reverse Primers (Forward, Reverse), ddH ₂ O supplemented to a final volume of 20 μl. Thermal cycle parameters are: 94°C for 3 min; 94°C for 30 s, the optimal renaturation temperature for 45 s (two parents were used as DNA templates for the preliminary test to optimize the renaturation temperature), 72°C for 60 s, 38 cycles; 72°C for 10 min, 1 cycle; stored at 4°C, and the reaction was completed on a PTC-225 PCR instrument. The amplified products were separated on 1.2% agar gel (containing EB) on a horizontal electrophoresis tank, using 1×TAE buffer solution, voltage 3V/cm, electrophoresis for about 1.5hrs. After electrophoresis, the gel imaging system (UVP) took pictures and saved them, and recorded the polymorphism results.

7. Development of CAPS markers: When candidate SCAR primers cannot amplify polymorphisms, CAPS enzyme digestion analysis is performed. The system is: 10 μl SCAR-PCR amplification product, 1.5 μl 10×digested buffer (MBI Fermentas, Lithuania), 1.5U restriction endonucleases recognizing 4 bases (Bsh1236I, HaeIII, RsaI, Sau3AI, AluI, MseI, MBI Fermentas, Lithuania), digest at 37°C for 3hrs-6hrs. Electrophoretic separation of digested products is the same as SCAR analysis

8. Marker population analysis and linkage genetic analysis: use RAPD markers, AFLP markers, SCAR markers and CAPS markers to perform PCR amplification and electrophoresis detection on the DNA samples in the DH segregation population one by one, calculate the genetic map distance and analyze the linkage relationship. In this study, the color of the seed coat showed a single gene difference. According to the requirements of the MAPMAKER software, the yellow seed trait was assigned the value "A", and the black seed trait was assigned the value "B" to participate in the construction of the linkage map. Theoretically, molecular markers can only have two band patterns in the offspring of the DH population, that is, the band pattern of one of the parents, and the band patterns corresponding to the parents are recorded as "A" and "B", respectively, and the band patterns that are difficult to interpret or are missing are recorded as "- ". Data analysis was carried out with MAPMAKER/EXP Version 3.0 (Lander et al, 1987; Lincoln et al, 1992) software, the recombination value and genetic map distance between testa genes and molecular markers were calculated by two-point test, the linkage map of markers and genes and The sequence was performed with a three-point or multi-point test (LOD=4.0, r=0.3), and the genetic map distance was transformed with the Kosambi function (Kosambi, 1944). The results showed that there was a close linkage between markers and genes, and the recombination value between markers and genes was 2.4%-9.4%. The linkage map covers a genome size of 53.8cM with an average distance of 5.4Cm. The order and distance between markers and genes are shown in Figure 2

9. Field hybridization and molecular marker-assisted selection: based on molecular marker technology, combined with traditional phenotypic selection through MAS, supplemented by field phenotype observation and quality analysis, through the hybridization-backcrossing-selfing method, the The yellow seed gene of NO.2127-17 was introduced into Hui 5148-2, and the yellow seed restorer line was bred. The specific process was carried out according to the molecular marker-assisted selection backcross breeding scheme shown in Figure 3, and three foreground selections and three background selections were performed. The whole process includes 3 times of crossing, 2 times of backcrossing and 1 time of selfing. First, 1141A (the strain was preserved in the China Center for Type Culture Collection on January 18, 2006, with the preservation number CCTCC-P200603) was crossed with Hui 5148-2, and then its F ₁ hybrid was used as the female parent, and Hui 5148 -2×NO.2127-17 DH line with yellow seed coat cultured by microspores (selected by MAS and the background is partial to 5148-2) is used as the male parent to cross and prepare a three-cross combination to ensure that the selected single plants in each generation have Restoring genes. Sanjiao F ₁ had two backcrosses with Hui 5148-2 and one selfcross. In this program, each individual plant of each backcross generation is subjected to PCR analysis and species test analysis at harvest time, and the yellow seed individual plant is selected for planting.

When preparing the three-cross combination, no molecular marker linked to the yellow seed gene has been found, so we can only select the three-cross combination with more DH lines with excellent agronomic characteristics and performance most similar to Hui 5148-2 in the field. After mature harvest, conduct seed color test and quality analysis on the DH lines one by one, select 12 high-quality yellow-seeded DH lines for background selection, select two excellent DH lines from them, and plant them in three-cross combinations accordingly, and the rest of the combinations Be eliminated. In BC ₁ F ₁ , the yellow seed gene was used to specifically mark S1129 and S1130. In BC ₂ F ₁ , SCS1130, S1130 and S1129 were used to select the yellow seed gene (foreground selection) for each individual plant. Combined with field investigations in each generation, about half of them were eliminated Non-target individual plants (black seed individual plants). A two-step background selection method was used for the selected single plants, in which BC ₁ F ₁ was analyzed with RAPD markers, and BC ₂ F ₁ was analyzed with AFLP markers. Finally, 3 single plants were selected in each generation and bagged and selfed at the same time. According to the field performance of each combination, one of the combinations was selected for MAS analysis. Yellow seed trait segregation existed in BC ₂ F ₂ , and the codominant marker SCA1 was used to select homozygous yellow seed plants. The selected single plant and 1141A were prepared and combined for comparative test

Positive effect of the present invention:

The present invention finds RAPD and AFLP molecular markers that are closely linked to yellow seed traits, and converts two of them into stable dominant SCAR markers and co-dominant CAPS markers, and uses the molecular markers to carry out assisted breeding of yellow rapeseed , can overcome the shortcomings of selection based on phenotype in the breeding process of yellow rapeseed, greatly reduce the workload, shorten the breeding period, and quickly cultivate new varieties.

Description of drawings

Figure 1 Rapeseed seed coat color is divided into 6 grades, the value in brackets in the figure is the chromaticity value

Fig. 2 Genetic linkage map of RAPD, AFLP, SCAR and CAPS markers of Brassica napus seed coat genes. The left side is the map distance, expressed in cM, and the right side is the mark

Figure 3 The molecular marker-assisted selection breeding scheme for the yellow seed trait backcrossing into Hui 5148-2

Fig. 4 Amplification effects of S1130 (RAPD, A) and EA07MC13 (AFLP, B) on each DH line constituting the gene pool. Lane 1 is No2127-17, lane 2 is the yellow seed gene pool, lane 3 is the black seed gene pool, lane 4 is Hui5148-2, lanes 5-14 are the DH lines that constitute the yellow seed gene pool, and lane 15- 24 are the DH lines constituting the black seed gene pool, M indicates molecular weight, arrows indicate molecular markers, ^* indicates exchange DH lines

Fig. 5 SCS1130 (SCAR, A) and SAC1 (CAPS, B) marker amplification effects on each DH line constituting the gene pool. Lane 1 is NO.2127-17, lane 2 is the yellow seed gene pool, lane 3 is the black seed gene pool, lane 4 is Hui 5148-2, and lanes 5-14 are the DH lines that constitute the yellow seed gene pool. 15-24 are the DH lines that constitute the black seed gene pool, and M indicates the molecular weight

Fig. 6 Effect diagram of background selection of RAPD primer S86 in BC ₁ F ₁ population. P ₁ is the yellow seed parent NO.2127-17, P ₂ is the black seed parent Hui 5148-2, and the rest are different individual plants. The arrows indicate polymorphic bands

Fig. 7 Effect diagram of foreground selection of SCS1130 in a part of BC ₂ F ₁ population. P ₁ is the parent NO.2127-17 of the yellow seed, P ₂ is the parent of the black seed Hui 5148-2, and the rest are different individual plants. The arrow shows the specific band of the yellow seed individual plant

Fig. 8 Partial individual plant selection results of SCA1 in BC ₂ F ₂ population. P ₁ is the parent No.2127-17 of Huangzi, P ₂ is the parent of Hui5148-2 of Heizi, and the rest are different individuals. The arrows indicate homozygous Huangzi individuals.

Detailed ways

Example 1

1) Establishment of a double haploid (DH) population: In this example, Hui 5148-2 was used as the female parent, and Brassica napus NO.2127-17 was used as the male parent for crossing to obtain F1, and _F1 plants were used pollen cultured by microspores (see literature for microspore culture methods, Yu Fengqun, etc., research on some culture factors for improving the rate of microspore embryoid body formation in Brassica napus, Acta Crops, 1997, 23 (2): 165-168 ) to obtain 127 double haploid (DH for short) populations.

2) Extraction of DNA and establishment of gene pool: Extract and purify DNA from the seedlings in the population obtained in step 1. For the specific preparation method, refer to Li Jia et al. (Li Jia et al., A method for effectively extracting total DNA from rape leaves, Huazhong The method reported in Journal of Agricultural University, 1994, 13(5): 521-523). For the construction methods of the yellow seed gene pool and the black seed gene pool, please refer to 2 in the above detailed technical scheme.

3) Combining group analysis (BSA) with RPAD and AFLP techniques to screen molecular markers closely linked to rapeseed yellow seed genes: among 810 RAPD primers (S1-S410, S1000-S1400, purchased from Shanghai Bioengineering Co., Ltd.) There are 240 (29.6%) articles showing polymorphism, 240 RAPD primers and 512 pairs of AFLP primer combinations (EA/MC and EA/MG combinations, AFLP adapter and primer sequence design are all according to Vos et al. (Vos et al.AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res, 1995, 23: 4407-4414) reported method, synthesized by Shanghai Bioengineering Co., Ltd.) Screening between parents and gene pools showed that 4 RAPD and 16 AFLP combinations in Polymorphisms were exhibited between the two gene pools. Further analysis showed that 2 RAPD and 8 AFLP markers were closely linked to the yellow seed gene, and the recombination value on the 20 DH lines constituting the gene pool was less than 15% (see Figure 4). Linkage analysis of 10 markers on the population of 127 DH lines showed that 10 markers were on the same linkage group, of which 2 RAPD and 5 AFLP were reciprocally linked with the yellow seed gene, and the other 3 AFLP markers were repelled to it Linkage (accompanying drawing 2), the characteristics of each marker are shown in Table 5.

Table 5 Primer sequences, phases, sizes and genetic map distances of RAPD and AFLP markers linked to the yellow seed gene

E=EcoRI primer, 5'-GACTGCGTACCAATTC-3';

M=MseI primer, 5'-GATGAGTCCTGAGTAA-3

4) The RAPD and AFLP markers closely linked to the yellow seed gene screened in the above step 3) were converted into SCAR markers and CAPS markers: the AFLP differential fragments were first recovered from the respective parental bands on the PAGE gel. The specific fragments of S1129 and S1130 and the reamplification products of 8 AFLP markers were separated on agarose gel, and the SK1131 kit (purchased from Shanghai Bioengineering Co., Ltd.) was recovered and purified. The purified recovered product was directly ligated with pGEMT-Easy vector (Promega Corp, ^# A1360 Madison, Wis) to transform into Escherichia coli, spread on LB (containing amp) solid medium plate, and cultured overnight at 37°C. Pick 6-10 white single colonies for each fragment with a dental guide, inoculate them on LB (containing amp) liquid culture overnight, take 5 μl of culture liquid as DNA template, and perform PCR with SP6/T7 primers (purchased from Shanghai Bioengineering Co., Ltd.) The results showed that about 90% of the 10 marker fragments were positive clones, and positive recombinants were obtained. For each fragment, two samples of positive clones were selected and sent to Shanghai Bioengineering Co., Ltd. for sequencing. Sequencing results showed that the 5' and 3' ends of each AFLP marker sequence contained Mse I and EcoR I restriction sites and the complete AFLP primer sequence respectively; the two ends of the two RAPD marker sequences contained corresponding primer sequences or complementary sequences .

According to their sequence sequencing results and based on the entire sequence information, a pair of optimal primers were designed with Primer3 software ( http://redb.croplab.org/modules/redbtools/primer3.php ). Each primer was first amplified between the parent NO.2127-17 and Hui5148-2, the PCR reaction conditions were optimized, the annealing temperature was determined, and the optimal reaction system was established. Then between the two parents and the two gene pools, PCR amplification was performed under respective optimal conditions to find polymorphisms. The results showed that the SCAR primer of S1130 showed polymorphisms in the two parents and the two gene pools (accompanying drawing 5) , indicating that the S1130 marker developed into a dominant SCAR marker named SCS1130.

The PCR products of primers that did not develop into SCAR markers were digested with the corresponding endonucleases (MBI Fermentas, Lithuania) in the two parents and the two gene pools to find differences. The results of enzyme digestion showed that only the product of the EA05MC12/Bsh1236I primer enzyme combination revealed polymorphisms (Fig. 5), and the EA05MC12 marker was developed into a co-dominant CAPS marker, named SCA1. The characteristics of SCS1130 and SCA1 are shown in Table 6.

Table 6 Characteristics of SCAR and CAPS markers

The DNA sequence of the above molecular marker SCS1130 is shown in the sequence listing SEQ ID NO: 1; the DNA sequence of the marker SCA1 is shown in the sequence listing SEQ ID NO: 2.

Linkage analysis using SCS1130 and SCA1 on the mapping population showed that they were still on both sides of the yellow seed gene, and the genetic distances were 3.2cM and 3.9cM, respectively. The MAPMAKER/EXP 3.0 program mapped these two markers to the same linkage group as the original 10 RAPD and AFLP markers. See Figure 2 for the sequence and distance on the linkage group

A BC ₁ F ₁ backcross population with 353 individual plants (169 yellow-seeded and 184 black-seeded) was selected to evaluate SCS1130 and SCA1. It shows that these two markers have strong ability to identify the genotypes of Brassica napus and can accurately select the genotypes of yellow seeds (Table 7).

Table 7 Analysis results of SCS1130 and SCA1 markers in BCF ₁

Note: +/-, the presence or absence of specific bands

Example 2

The parental materials were Brassica napus Hui 5148-2, NO.2127-17, and Pol cms male sterile line 1141A (sources as indicated above). The DNA of each individual plant was extracted by a small amount method.

In the process of backcrossing the yellow seed gene, SCS1130 and SCA1 were used for foreground selection (tracking of the yellow seed gene), and RAPD and AFLP markers were used for genetic background selection. The specific process was carried out according to the molecular marker-assisted selection backcross breeding scheme shown in Figure 3, and three foreground selections and three background selections were performed. During the whole growth period, the agronomic traits of each combination and single plant were observed, mainly the growth appearance, plant and leaf shape, uniformity, growth period, etc., and several important lines were tested indoors, and the selected plants were compared with those of Hui 5148-2. The similarities and differences as well as the similarities and differences between its preparation combination and the combination of Hui 5148-2. The quality analysis was completed by the quality analysis branch of the Rapeseed Research Laboratory of Huazhong Agricultural University, using the near-infrared analysis method.

Each individual plant is assigned a value of 1 and 0 according to the band type. According to the method of Nei and Li (Nei et al. Mathematical model for studying genetic variation interms of restriction endonucleases. Proc Natl Acad Sci USA, 1979, 76: 269-573), the similarity coefficient and genetic distance. Similarity analysis UPGMA (Unweighted pair group witharithmetric average, arithmetic mean unweighted class average method) method for cluster analysis.

Specific steps are as follows:

1) Three-cross combination: 1141A as the female parent and Hui 5148-2 as the male parent were crossed to obtain F1, and 65 DH lines in the DH (Hui 5148-2×No.2127-17) population were selected as male parents and F1 Hybridize to obtain a three-cross combination.

2) RAPD primers were used for background selection of the DH lines prepared with three-cross combination in the above 1: select 142 polymorphic RAPD primers with the best polymorphism, and prepare three-cross The combined yellow DH line was analyzed for the similarity between its genetic background and Hui 5148-2. The results showed that a total of 830 bands were amplified, and 314 polymorphic bands were amplified. After cluster analysis, two DH lines DH21 and DH146 with the smallest genetic distance to Hui 5148-2 were screened out. Field observations found that the two DH lines The performance of the line is most similar to that of Hui 5148-2, which is consistent with the background selection results.

3) The three-cross combination prepared by the two DH lines selected in the above 2 is backcrossed and self-crossed: the three-cross combination prepared by the two DH lines (DH21 and DH146) is planted in the field, and at the bud stage, select fertile A single plant was backcrossed with Hui 5148-2 to obtain two backcross BC ₁ F ₁ (BC _1-21 F ₁ , BC _1-146 F ₁ ) populations, and the three-cross combination was self-crossed to obtain a three-cross combination F ₂ (F _2-21 and F _2-146 ).

4) Use SCS1130 and SCA1 to perform foreground selection on the combinations in the above 3: use RAPD primers to perform background selection on the combinations in the above 3: harvest the combinations in the above 3, and the test results show that the three-cross combination F ₂ (F _{2- 21} , F _2-146 ) are both yellow in seed coat color, and the quality analysis shows that the two are of good quality and high oil content. The quality analysis of two backcross contemporary seeds (BC _1-21 F ₀ , BC _1-146 F ₀ ) Better, according to the quality analysis results, combined with the performance of two BC ₁ F ₁ combinations in the field such as growth appearance, leaf color and shape, uniformity, etc., select one of the combinations BC _1-146 F ₁ that is most similar to Hui 5148-2 to carry out MAS selection. SCS1130 and SCA1 were used to select the individual plants of BC _1-146 F ₁ for the trait of yellow seeds. The test results: 175 positive yellow seed plants were detected by S1129, and 170 positive yellow seed individual plants were detected by S1130. A total of 169 positive individual plants were detected (see Table 10).

5) Use RAPD primers to carry out background selection on the combination in the above 3: Combined with the growth of individual plants in the field, select 142 positive individual plants with strong growth and vigorous growth among the 169 individual plants selected in the above 4 for background analysis. 30 RAPD primers with the best polymorphisms were used to perform background selection on the 142 positive individual plants (see Figure 6), and cluster analysis was performed based on the data, and 46 genetic distances closest to Hui 5148-2 were screened out. single plant. Another 58 RAPD primers were added for the next step of background selection. Combined with the growth conditions in the field, finally select three individual plants 25 ^# , 85 ^# , and 49 ^# that grow robustly and have the smallest distance with Hui 5148-2 (Table 8).

6) Backcrossing and selfing of the individual plants selected in the above 4: backcross the individual plants 25 ^# , 85 ^# , 49 ^# selected in the above 4 with Hui 5148-2 to obtain a BC ₂ F ₁ population (BC _2-25 F ₁ , BC _2-85 F ₁ , BC _2-49 F ₁ ), and at the same time three plants were selfed to obtain BC ₁ F ₂ (BC ₁ F _2-25 , BC ₁ F _2-85 , BC ₁ F _2-49 ) . The seed color of 3 BC ₁ F ₂ and the current generation of backcross (BC ₂ F ₀ ) was yellow, consistent with the result of marker selection, indicating that the selected combination contained yellow seed gene.

7) Foreground selection is carried out on the 6 seeds harvested in the above 5, and the quality analysis is carried out on the 6 seeds harvested in the above 5, combined with the performance of the growth, uniformity, plant and leaf morphology of the 3 BC ₂ F ₁ populations in the field, The BC _2-85 F ₁ population was selected, and SCS1130 and SCA1 were used for a new round of MAS prospect selection (Fig. 7), and individual plants containing the yellow seed gene were screened. The selection results showed that 107 positive yellow seed plants were selected by SCS1130, S1130 detected 108 strains, and S1129 detected 109 strains. There was no double exchange between the three, and 107 single strains were detected together. SCS1130 is developed from S1130, and the selection result has greater identity with S1130, with high specificity and low misselection rate (Table 10). Combined with the growth of each individual plant in the field, 94 robust yellow seed positive individual plants were selected for MAS background analysis.

8) Use AFLP primers to perform background selection on the individual plants selected in the above 6: use 20 pairs of AFLP primers to perform background analysis on the 94 individual plants selected in the above 6, and screen out 46 individual plants with the closest genetic distance to Hui 5148-2. strain. In addition, 40 pairs of AFLP primers were added, and the next background selection was carried out on these 46 individual plants. Combined with the growth conditions in the field, 3 individual plants 278 ^# , 248 ^# , and 243 ^# with the smallest genetic distance from Hui 5148-2 were selected (Table 8 ).

9) Selfing and hybridization of the individual plants selected in the above 7: Bagging and selfing of the three individual plants 278 ^# , 248 ^# , and 243 ^# selected in the above 7 to obtain BC ₂ F ₂ (BC ₂ F _2-278 , BC ₂ F _2-248 , BC ₂ F _2-243 ), and prepare a hybrid combination with 1141A for preliminary comparative experiments.

Table 8 The 10 individuals with the smallest genetic distance to Hui 5148-2 in BC ₁ F ₁ and BC ₂ F ₁

10) Foreground selection of the self-crossing and crossing combination in the above step 9): after performing quality analysis and comparison of seed coat color on the seeds of the three BC ₂ F ₂ in the above step 9), according to the three BC ₂ F ₂ The uniformity, growth vigor, plant and leaf morphology and other field performances of the population were analyzed by MAS using the co-dominant marker SCA1 on one of the BC ₂ F _2-248 populations that were uniform, vigorous, and of high quality (see Figure 8 ). The analysis results of 49 individual plants of BC ₂ F _2-248 population with the co-dominant marker SCA1 showed that the ratio of dominant homozygous (yellow seeds): heterozygous (yellow seeds) : recessive homozygous (black seeds) was 9:23:17, the chi square value is 2.79 (χ ² _{0.05, 2} =5.99), which is in line with the expected segregation ratio of 1:2:1, basically consistent with the phenotype identification results of BC ₂ F ₂ offspring, and the probability of misselection is 6.12% (Table 10) The results showed that there were 9 strains of obvious homozygosity (yellow seeds), of which 2 strains were not resistant to disease in the later stage and had poor traits and were eliminated.

11) Selfing and hybridization of the individual plants selected in the above step 10): the 7 homozygous individual plants selected in the above 9 (402 ^# , 405 ^# , 422 ^# , 433 ^# , 440 ^# , 445 ^# , 447 ^# ) were self-bred in bagging, and prepared a hybrid combination (including Hui 5148-2) with 1141A, and carried out the next comparison experiment. The BC ₂ F _2:3 of 7 homozygous individual plants were harvested, and the quality analysis and testa color analysis of the corresponding cross combinations showed that the testa color of the 7 individual plants was darker than that of the parent NO.2127-17, while all The color of the hybrid combination is darker than that of the selected single plant, and the oil content is higher than that of Hui 5148-2, which is equivalent to that of No.2127-17. The average value, medium sulfur level, indicated that the yellow seed gene of NO.2127-17 had been successfully introduced into Hui5148-2, and the glucosinolate level needed to be further improved (see Table 9 for the analysis of quality traits). The 7 hybrid combinations are undergoing comparative experiments for more than a year, and will finally determine the selected individual plants.

Table 9 Analysis table of quality traits of different materials

Comparison of foreground selection accuracy for each marker

Comparing the selection results of various molecular markers with the results of phenotypic identification in the field, it was found that with the increase of generations, the selection accuracy rate of each marker decreased, and the misselection rate was higher than the marker recombination value, which was extremely inconsistent. Multiple unilateral markers did not greatly improve the accuracy rate, and the smaller the distance between the marker and the genetic map of the target gene, the higher the selection accuracy rate; the selection accuracy rate of double-sided markers was much higher than that of unilateral markers. The accuracy of foreground selection mainly depends on the degree of linkage between the marker and the target gene. The closer the linkage between the marker and the gene, the higher the reliability of selection based on the marker. Therefore, in practical applications, it is preferred to select two genes that are closest to both sides of the gene. Molecular markers, which can not only improve the accuracy, but also increase the homogeneity of the genetic background between the selected individual and the recipient's parents (see Table 10).

Table 10 Each marker of the present invention compares the selection accuracy of each generation

Note: ^{1) 2)} The data comes from molecular marker evaluation experiments; +/-, the presence or absence of specific bands

Note: ^{1) 2)} the data are from the two makers' validation experiment:

+/-, presence/absence of the specific fragment

The above-mentioned test molecular markers are all cloned in the present invention.

sequence listing

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Claims (1)

1, a kind of method that utilizes artificially synthesized brassica oleracea molecular markers to assist in the selection of rapeseed varieties, it comprises the following steps: 1) Hybridizing the Brassica napus line Hui 5148-2 as the female parent and the yellow-seed rape line NO.2127-17 as the male parent to obtain F1 seeds, sowing the F1 seeds to obtain F1 rapeseed plants, and taking pollen from the plants Perform microspore culture to obtain double haploid (DH) segregation population; 2) Two gene pools of rapeseed yellow seed group and black seed group were constructed by group analysis method; 3) Design differential primers between the two parents to perform PCR amplification on the two gene pools constructed in the above step 2), select primers that show differences, and then perform PCR amplification and electrophoresis detection on the DNA samples of each individual plant between the gene pools After determining that its character is a linkage marker, PCR amplification and electrophoresis detection are performed on the DNA samples of each individual plant of the DH population; 4) Steps of recovering, cloning, and sequencing 3) Screening the RAPD and AFLP-marked fragments closely linked to the yellow seed gene of Brassica napus; 5) Design specific primers according to the sequencing results of step 4), and convert the screened RAPD and AFLP markers closely linked to the yellow seed gene of Brassica napus into SCAR markers; 6) SCAR markers that are not successfully transformed in step 5) are developed into CAPS markers; 7) RAPD markers, AFLP markers, SCAR markers and CAPS markers in steps 3), 5) and 6 are subjected to population analysis and linkage genetic analysis; 8) Using the molecular markers and assisted selection in step 7) to introduce the rapeseed yellow seed gene into the restorer line of Brassica napus polima cytoplasmic male sterility, and to obtain the restorer line of yellow seeded rapeseed.

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