CN103184293B - Codominant SCAR marker for identifying onion male sterility gene and application thereof - Google Patents

Abstract

The invention discloses a codominant SCAR marker for identifying an onion male sterility gene and an application thereof. Specific fragment length is divided into 886bp and 621bp, wherein the marker with specific fragment length as 886bp is closely linked with an onion restorer gene Ms, and nucleotide sequence is shown in SEQ ID NO.1; and the marker with specific fragment length as 621bp is closely linked with an onion sterile gene ms, and nucleotide sequence is shown in SEQ ID NO.2. The codominant marker can be, as a molecular marker, applied to auxiliary breeding of an onion male sterile line and a mating maintainer line. The codominant marker for identifying the onion male sterility gene can be obtained through a molecular marker technology. The marker can rapidly and accurately judge genotype of the onion male sterility gene, which is important for accelerating breeding progress of the onion and for establishing an onion molecular marker auxiliary breeding technical system.

Description

A kind of codominance SCAR mark and application of identifying onion male sterility gene

Technical field

The present invention relates to a kind of codominant SCAR mark and application thereof, relate in particular to a kind of and onion male sterility gene and the recovery closely linked codominant marker of gene and application, apply this mark and can distinguish three kinds of nuclear gene types of onion, can directly apply to molecular mark and select onion male sterile line and supporting maintenance line, belong to biological technical field.

Background technology

Onion (Allium cepa L.), is one of Main Vegetable Species Suitable For Culture in the world, have the laudatory title of protective foods.According to FAO(2011) statistics, 4,290,000 hectares of world's onion cultivated areas, 8,538 ten thousand tons of output, in all vegetable crops, its cultivated area occupies second, and output occupies the 3rd.Wherein, China has onion the biggest in the world and produces area and output, produces area and exceedes 1,020,000 hectares, and 2,476 ten thousand tons of ultimate productions, account for 29% of worldwide production.Onion not only can be eaten raw, also can be in a large number for fabricated product, and being rich in multiple sulfide and carbohydrate is its major cause with peculiar flavour.From health care angle, onion has antithrombotic, stimulates circulation, the effect such as anticancer, and its nutrition and medical value are generally acknowledged by the whole world gradually.

The Jones of the U.S. in 1936 and Emsweller(Jones HA.and Emsweller, SL.A male sterile onion.Proc.Amer.Soc.Hort.Sci.1936,34:582-585.) in " Italian red " (Italian Red) kind, find male sterile plant first, its sterility is by single core gene and tenuigenin co-controlling.Nineteen forty-three Jones and Clarke(Jones, HA and Clarke A.Inheritance of male sterility in the onion and the production of hybrid seed.Proc Amer.Soc.Hort.Sci.1943,43:189-194.) find again male sterile line.Onion becomes and utilizes the earliest in the world one of heterotic vegetable crop subsequently.The fertility of nucleo-cytoplasmic interaction control onion, produces and has great advantage for onion cross-fertilize seed.But, due to Ms gene or the high-frequency existence of S type tenuigenin, be difficult to successfully be maintained and be from some Cultivars, such as: " Texas1015Y " (United States), " SapporoKi " (Japan), " Pukekohe Longkeeper " (New Zealand).Therefore, the seed selection of onion male sterile maintainer line is a job of wasting time and energy very much.

Onion originates from Central Asia, and the history of importing China into is shorter.Due to the onion breeding time limit be the vegetables such as Chinese cabbage, radish 2-4 doubly, and the germ plasm resource of China onion is relatively deficient, the utilization of malesterile technique on vegetables also far lags behind other developed countries, the market of cross-fertilize seed is almost monopolized by external kind.Along with the adjustment of agricultural structure, demand to onion improved seeds increases day by day, in production mainly take kind introduce and conventional variety seed selection as main, lack the combination with China's independent intellectual property right, need to spend a large amount of foreign exchanges from external import seed, make the production cost of onion high.The seed selection of good male sterile line and maintenance line is key link urgently to be resolved hurrily in onion breeding, is also the source work of onion industrialization.Therefore, change the backward situation of China on onion breeding field, key is extensively to collect as early as possible variety source, walk the road that modern molecular biology technique combines with conventional breeding, accelerate the research that molecular marker assisted selection and male sterile line utilize, thereby shortening the breeding cycle, select as early as possible and there is onion cross-fertilize seed China's independent intellectual property right and that meet the need of market.

In recent years, the molecular biological plant genetic mark that develops into provides a kind of new tool based on DNA variation, i.e. molecular marking technique.It directly occurs with DNA form, all can detect at each tissue of plant materials, each developmental stage, is not subject to season, environmental limit, does not have expression whether problem.Multinomial technology that it has comprised molecular biology research is as DNA restriction enzyme digestion, Southern transfer, molecular hybridization, round pcr etc.DNA molecular marker is the direct reflection of genetic diversity on DNA level, mainly contain at present following several marking method: Restriction fragment length polymorphism markers (Restriction fragment length polymorphisms, RFLP), random amplified polymorphism mark (random amplified polymorphic DNA, RAPD), simple sequence repeating label (simple sequence repeats, SSR), amplified fragment length polymorphism mark (Amplified fragment length polymorphisms, AFLP).RFLP mark needs Southern hybridization, operate comparatively loaded down with trivial details, and RAPD mark Stability and veracity is poor, all be not suitable for the evaluation work of large-scale field material, AFLP combines RFLP and RAPD advantage separately, and fast and easy only needs minute quantity DNA material, just can obtain fast bulk information, and experimental result is reliable and stable.SCAR marking operation is easy, accuracy is high, stability is high, does not have the shortcoming of above-mentioned mark, becomes first-selected molecule marking method.So-called SCAR mark just refers to that, by the order-checking of specificity segment two ends, synthetic Auele Specific Primer, carries out the amplification of specificity segment.Havey(Havey MJ.Diversity among male-sterility-inducing and male-fertile cytoplasms of onion.Theor.Appl.Genet.2000,101:778-782) utilize RFLP molecule marker means to find out the difference between onion male maintenance line system and sterile line Mitochondrial Genome Overview.[the Engelke T such as Engelk, Terefe D, Tatlioglu T.A PCR-based marker system monitoring CMS-(S), CMS-(T) and (N)-cytoplasm in the onion (Allium cepa L.) .Theor.Appl.Genet.2003,107:162-167.] obtain evaluation onion S type, N-type and T-shaped cytoplasmic molecule marker.

deng [ aF, McCallum J, Sato Y, Havey MJ.Molecular tagging of the Ms locus in Onion.J.Amer.Soc.Hort.Sci.2002,127 (4): 576-582.] utilize AFLP and the nuclear Ms of RFLP method mark onion site, build linkage map, and obtained the RFLP mark that genetic distance is 0.9cM.

By to the male sterile research of onion, obtain the molecule marker of the stable identification of cell matter type of PCR-based.In the evaluation of cell nucleus gene type, also obtained some RFLP marks, compared with the method such as traditional " test cross, backcross, selfing ", this technology can shorten the seed selection time limit of maintenance line greatly, improves breeding efficiency.But, because RFLP molecule marker is comparatively loaded down with trivial details in operation, and acquired RFLP is marked in the evaluation scope of material and has certain limitation, and can above-mentioned achievement in research directly apply to the seed selection of the onion cross-fertilize seed of China, it be not immediately clear, need to carry out a large amount of research work.The object of genetic marker is to realize molecular mark (Marker Assisted Selection, MAS).Be all can reduce workload with tenuigenin site or with the assisted Selection of the chain mark of nuclear gene, improve breeding efficiency.Utilize and the closely linked mark of onion male sterility gene, can identify the nucleus type of individual plant, eliminate the nucleus type that can educate in onion material, the individual plant of the nucleus type of only isozygotying from recessiveness, select maintenance line, thereby reduce test cross number of combinations and selfing strain number, reduce workload, avoid blindness, improve and select effect.We have developed the closely linked SCAR mark (number of patent application: 201110226241.4) with onion fertility restorer gene Ms, but because this is labeled as dominant marker, in actual Breeding Application, can be because thereby failed PCR cannot obtain the classification that amplified production leads to errors, codominant marker is optimal mark in molecular mark at present.Therefore, obtain and the closely linked codominant marker of onion male sterility gene, SCAR mark especially easy and simple to handle, accuracy stability is high, supporting for onion sterile line and maintenance line, seed selection onion cross-fertilize seed is significant.

Summary of the invention

For the difficulty existing in current onion breeding, the invention provides a kind of codominant marker who can be used for assist-breeding onion male sterile line and supporting maintenance line.Utilize this mark to judge fast and accurately the genotype of onion male sterility gene, this,, for the breeding process of accelerating onion, sets up onion molecular mark technical system significant.

The present invention is achieved by the following technical solutions: a kind of codominance SCAR mark of identifying onion male sterility gene, it is characterized in that, its specific fragment length is respectively 886bp and 621bp, wherein, specific fragment length is that mark and the onion of 886bp recovers gene M s close linkage, and its nucleotide sequence is as shown in SEQ ID NO.1; Specific fragment length is mark and the onion sterile gene ms close linkage of 621bp, and its nucleotide sequence is as shown in SEQ ID NO.2.

Described codominant marker's primer is:

Forward primer FN1:5'-ATACACAGCTTCTAGCTGAATTTTTA-3'(SEQ ID NO.3);

FN2：5'-TGTGTGTGTAATTTCTCTGTGCG-3'（SEQ?ID?NO.4）。

Reverse primer RN1:5'-ACAGAGTGAGAAATTTTATATATATAGGAAT-3'(SEQ ID NO.5);

RN2：5'-CGGAAGATTAATATTTTGCGTATACAT-3'（SEQ?ID?NO.6）。

The present invention utilizes AFLP marking method take onion sterile line 118S (msms) and first backcross generation BC1[118 × (118 × 12-12) of Fertile material 12-12S (MsMs) with different genetic backgrounds] arrive and the closely linked AFLP molecule marker of the male recovery gene M of onion s gene as material screening, and be translated into codominant marker, called after OMS-SCAR.Utilize OMS-SCAR mark can carry out fast the nuclear gene type of onion candidate maintenance line material and judgement accurately, by a pcr amplification reaction, eliminate the individuality (MsMs) of the 886bp band that only increases and the individuality (Msms) of amplification 866bp and 621bp, retain the individuality of 621bp band (msms) that only increase, thereby guarantee to screen the individuality of nuclear male sterility homozygous genotype, to accelerate the process of maintenance line of seed selection onion, and then set up onion molecular mark technical system.

Described codominant marker's screening process is as follows:

(1) hybridize with male parent self-mating system 12-12S (MsMs) as maternal take onion male sterile line 118S (msms), the F1 generation S that obtains of institute (Msms) backcrosses for male parent and maternal sterile line 118S (msms), obtain backcross progeny segregating population 11812, after blooming, be divided into and can educate colony and sterile population according to fertility judged result, it can be educated with sterile strain and count ratio in table 1.

(2) extract test kit by the rapid gene group of Beijing TIANGEN company and extract onion genome DNA.

(3) adopt the screening of AFLP molecule marking method and onion to recover the closely linked mark of gene M s.

(4) filter out an AFLP mark, carry out the unknown nucleotide sequence of chromosome walking acquisition known dna sequence flank by Tail-PCR, utilize the flanking sequence design primer obtaining, thereby it is converted into codominant marker by AFLP mark, through genetic analysis, verify the feasibility of this mark, obtained a kind of codominant marker who identifies onion male sterility gene.

Described codominant marker can be used as molecule marker and is applied to assist-breeding onion male sterile line and supporting maintenance line, concrete application mode is: utilize the genomic dna of codominant marker's primer pair individuality to be measured to carry out pcr amplification, if individuality to be measured only amplifies the fragment of 886bp, the educated individuality that this individual cells nuclear gene type to be measured is MsMs, can be used as male parent system (recovery fertility); If amplify 886bp and 621bp two bar segment, the cell nucleus gene type of individuality to be measured is Msms; If only amplify the fragment of 621bp, the cell nucleus gene type of this individuality to be measured is msms, in conjunction with the judgement cytoplasm fertility technology (granted patent number: ZL200910014679, publication number: CN101492738A) of exploitation before applicant, can directly screen onion male sterile line and maintenance line.

The invention has the beneficial effects as follows: because onion is 2 years raw plants, the breeding time limit is considerably beyond annual vegetables.Onion breeding year limit for length's problem is problem in the urgent need to address in breeding work.The present invention utilizes the technology of molecule marker to obtain the codominant marker who identifies onion male sterility gene.Utilize this mark to judge fast and accurately the genotype of onion male sterility gene, this,, for the breeding process of accelerating onion, sets up onion molecular mark technical system significant.Its advantage is specific as follows:

1. the codominant marker of the evaluation onion male sterility gene that the present invention obtains, result is stable, accurate, easy and simple to handle, and it can go out by a round pcr precise Identification cell nucleus gene type of onion.Application of the present invention shortening the breeding cycle greatly, avoid the blindness in conventional breeding method, greatly improve efficiency of selection.

2. aspect onion male sterility gene mark, remove

deng the AOB272(genetic distance 0.9cM of (2002) report) beyond mark, there is not yet the report of close linkage mark more.Although AOB272 mark and onion male sterility gene site close linkage, owing to there being the crossover value of 0.9cM, have significant limitation to other different genetic background materials.This research obtains the codominant marker who identifies onion male sterility gene, is applicable to different genetic background materials widely, is a ubiquity mark.

3. can Direct Identification there is the cell nucleus gene type of different genetic background materials due to OMS-SCAR mark, therefore the onion molecular mark technical system of utilizing this mark to set up to be of universal significance, effectively accelerates the seed selection process of male sterile line and maintenance line.

Accompanying drawing explanation

Fig. 1: the electrophoretogram of the genome DNA of onion first backcross generation segregating population; Wherein: S1-S8 is that cell nucleus gene type is the sterile individual plant of msms, and N1-N8 is that cell nucleus gene type is the educated individual plant of Msms.

Fig. 2: the sterile pond of onion, can educate the AFLP electrophoretic band figure of the individual plant genome DNA in pond and constitutive gene pond; Wherein: S pond is onion nucleus sterile gene pond, S1-S10 is ten individual plants that form sterile gene pond; N pond is that onion nucleus can be educated pond, and N1-N10 forms ten individual plants can educating gene pool.Arrow represents to educate the specific fragment amplifying in pond.

Fig. 3: to the electrophoretogram of onion 11812 onion gene pool individual plant codominant marker checkings; Wherein: the Marker that M is DL100bp; 1 is onion nucleus sterile gene pond, and 1-11 is ten individual plants that form sterile gene pond; 12 ponds are that onion nucleus can be educated pond, and 13-22 forms ten individual plants can educating gene pool.

Fig. 4: the part electrophoretogram that onion 11812 and 11012 colonies are carried out to codominant marker's checking; Wherein: M is Marker; 1-17 is the sterile individual plant of onion nucleus, and 18-34 is that onion nucleus can be educated individual plant.

Fig. 5: the part electrophoretogram of verifying deriving from the known shaped material of different genetic backgrounds; Wherein, M is Marker; 1-5, the sterile line material of the different genetic backgrounds of 5 strains; 6-10, derives from the Japanese onion cross-fertilize seed of different company, 11-15, and 5 strains derive from the onion male parent based material of different genetic backgrounds.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated.

Embodiment 1 codominant marker's screening and application

(1) materials and methods

1. the extraction of genome DNA and detection

Hybridize with male parent self-mating system 12-12S (MsMs) as maternal take onion male sterile line 118 and 110S (msms), the F1 generation S that obtains of institute (Msms) backcrosses for male parent and maternal sterile line 118 and 110S (msms), obtain backcross progeny segregating population 11812 and 11012, after blooming, be divided into and can educate colony and sterile population according to fertility judged result, it can be educated with sterile strain and count ratio in table 1.11812 and 11012 genomic dna of onion Liang Ge first backcross generation colony adopts the method for genome rapid extraction test kit (Beijing, TIANGEN) to extract, and utilizes agarose gel electrophoresis and spectrophotometer to detect the quality of DNA.

2. the foundation of gene pool

Application segregating population fractional analysis method (Bulked Segregation Analysis) is BSA method, 10 sterile individual plants and 10 the DNA samples that can educate individual plant of backcross population 11812 are distinguished to balanced mix, form the sterile gene pond of onion and can educate gene pool.

Synthesizing of 3.AFLP joint and primer

Above-mentioned sterile gene pond and the enzyme that carries out of the genomic dna that can educate gene pool are cut, and enzyme is cut the restriction enzyme EcoR I and the Mse I that adopt NEB company, and its corresponding joint and primer are synthetic by Beijing Bo Shang Bioisystech Co., Ltd, PAGE purifying.

4.AFLP analyzes

Aflp analysis program, with reference to the method for Vos et al. (1995), improves to some extent.Adopt 16 EcoR I and 16 Mse I selective amplification primers, form altogether 256 pairs of combination of primers.

(1) enzyme of genomic dna is cut: in the centrifuge tube of 0.5mL, add 2.5 μ L10 × 4Buffer, 0.25 μ L100 × BSA, template DNA (200-300ng), 0.5 μ LEcoR I and 0.5 μ L Mse I (10U/ μ L), ddH ₂o supplies cumulative volume to 25 μ L.Fully mix, of short duration centrifugal, 37 ℃ of water-baths 6 hours, 65 ℃ of 15min, inactivator.

(2) enzyme is cut being connected of product and manual splice: cut in mixture (sterile gene pond and the enzyme that can educate the DNA in gene pool are cut the mixture of product) at above-mentioned enzyme, add 5 μ M EcoR I joints and the each 0.5 μ L of 50 μ M Mse I joint, T ₄dNA ligase (5U/ μ L) 1 μ L, 10 × T ₄dNALigase Buffer3 μ L.Fully mix, of short duration centrifugal, 16 ℃ of connections are spent the night, 70 ℃ of 15min, inactivator.By TE damping fluid template as pre-amplified reaction after connection product dilutes 10 times.

(3) amplification in advance: add 10 × PCR Buffer (with Mg in the reaction system of 25 μ L ²⁺) 2.5 μ L, the connection product 1 μ L of dilution, the each 1 μ L(30ng/ μ L of pre-amplimer of EcoR I and Mse I), Taq DNA polymerase0.2 μ L(5U/ μ L), dNTPs (each2.5mM) 2 μ L, ddH ₂o17.3 μ L.Pcr amplification reaction program is 94 ℃ of denaturation 5min, [72 ℃ are extended 60sec for 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec], 30 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations.Using the template as selective amplification after 50 times of pre-amplified production dilutions.

(4) selective amplification: 20 μ L reaction systems, wherein 10 × PCR Buffer (with Mg ₂₊) 2 μ L, dNTPs (each2.5mM) 1.6 μ L, Taq DNA polymerase (5U/ μ L) 0.2 μ L, the each 1 μ L(30ng/ μ L of selective amplification primer of EcoR I and Mse I), template 1 μ L, ddH ₂o supplies cumulative volume to 20 μ L, response procedures is 94 ℃ of denaturation 5min, [94 ℃ of sex change 30sec, 65 ℃ of annealing 30sec, 72 ℃ are extended the each cycle annealing temperature of 60sec(and reduce by 0.7 ℃), 13 circulations], 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec, 72 ℃ are extended 60sec, 23 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations.

5. polyacrylamide gel electrophoresis

(1) polyacrylamide gel electrophoresis: add isopyknic sample-loading buffer (98% methane amide, 10mM EDTA, 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene green grass or young crops) in selective amplification sample, be placed in immediately cooled on ice after 95 ℃ of sex change 5min.Every duplicate samples is got 5 μ L electrophoresis, and the permanent power of 50W to the blue or green indicator of dimethylbenzene is that Jiao2/3Chu stops electrophoresis.

(2) silver dyes colour developing:

1. fixing: after electrophoresis finishes, the short slab that has glue to be put into stationary liquid (900mLddH ₂in O, add 100mL Glacial acetic acid) middle jog 10min termination reaction, then use ddH ₂o rinses 1min.

2. after washing 3min with 1.5% nitric acid, use ddH ₂o rinses 1min.

3. dyeing: with 0.2% cma staining liquid dyeing 20min.

4. colour developing: by the short slab ddH after cma staining ₂after O flushing 30s, be placed in rapidly the nitrite ion (30gNa of precooling ₂cO ₃be dissolved in 1LddH ₂in O, and add the formaldehyde of 540 μ L37% and the Na of 200 μ L10mg/mL ₂s ₂o ₃) middle 4-7min, then use ddH ₂o rinses 1min.

5. fixing: in 5% glacial acetic acid solution, jog 5min, with termination reaction, then uses ddH ₂o rinses 1min.

6. dry glue: naturally dry under room temperature.

6. the recovery of polymorphic DNA fragment, purifying, Clone and sequence on polyacrylamide gel

(1) recovery of polymorphic DNA fragment and purifying

Extract the differential band on polyacrylamide gel with the scalpel after sterilizing, be dissolved in 20 μ LddH ₂in O, of short duration centrifugal after 95 ℃ of water-bath 10min, as the template of differential fragment PCR reaction.PCR reaction system and the response procedures of differential fragment amplification are identical with the reaction conditions of selective amplification.Differential band pcr amplification product reclaims purifying specific band according to Biospin Gel Extraction Kit operation instruction after agarose gel electrophoresis detects.

(2) clone of polymorphic DNA fragment, order-checking

The polymorphic DNA fragment that reclaims purifying is carried out to molecular cloning according to the method for molecular cloning II, enzyme is cut with the rear picking positive colony of bacterium liquid PCR detection and is entrusted Beijing Bo Shang Bioisystech Co., Ltd to carry out the mensuration of DNA sequence dna, and sequencing result is as shown in SEQ ID NO.7.

7. the SCAR of mark transforms

According to sequencing result, utilize Primer Premier5.0 software design Tail-PCR primer, with sterile gene pond with can educate gene pool and carry out chromosome walking as template, obtain the flank unknown nucleotide sequence of onion AGG/CGT fragment, utilize the primers newly obtaining, thereby obtain codominance SCAR mark, called after OMS-SCAR.

8. linkage analysis

Utilize codominant marker, the genomic dna of 11812 and 11012 first backcross generation colonies is carried out to pcr amplification and analyze whether there is the individuality with crossover value, detect the stability of OMS-SCAR mark.

9.OMS-SCAR is marked at the checking in the known shaped material that derives from different genetic backgrounds

Be used to come from the cross-fertilize seed material of sterile line, male parent system and the known type of different genetic backgrounds, Japan's Long Jing seedling Co., Ltd.'s kind (タ ー ボ, ネ オ ア ー ス, ア ト Application), Sapporo seedling Co., Ltd. (No. 3, も body じ, the sweet 70, タ ー ザ Application of Sapporo, ア De バ Application ス), the broad spectrum of checking OMS-SCAR mark and the feasibility of molecular marker assisted selection.

(2) results and analysis

1. utilize the detection analysis of the genomic dna of two group 11812 and 11012 first backcross generation colonies

Adopt the method for genome rapid extraction test kit (Beijing, TIANGEN) to extract onion blade genome DNA, show (see figure 1), the DNAOD of extraction through spectrophotometer and 0.8% agarose gel electrophoresis detected result ₂₆₀/ OD ₂₈₀ratio is between 1.8-2.0, and electrophoresis detection result demonstration polysaccharide and protein content are low, without RNA, and structural integrity, banding pattern neat and consistent, without signs of degradation, can be for further aflp analysis and pcr amplification.

2.AFLP interpretation of result

Select 16 EcoR I selective amplification primers and 256 pairs of combination of primers of 16 Mse I selective amplification primers composition to backcross 1 generation colony sterile Chi Hekeyuchi carry out aflp analysis, found that most of combination of primers all can amplify stable and band clearly, every pair of combination of primers 60-70 bar band clearly that on average increases.Through twice revision test and carry out individual plant checking (result is as shown in Figure 2) in He Keyu pond, sterile pond independently, determine a stable AFLP polymorphic bands, produced called after AGG/CGT by combination of primers E-AGG/M-CGT amplification.

The SCAR of 3.AFLP mark transforms

Polymorphic DNA fragment is reclaimed, purifying, after Clone and sequence, according to the sequence of AGG/CGT fragment, the required primer of Tail-PCR that utilized Primer Premier5.0 software design, with sterile gene pond with can educate gene pool and carry out chromosome walking as template, obtaining respectively length is that 2440bp(is as shown in SEQ ID NO.8) and 2403bp(as shown in SEQ ID NO.9) the flanking sequence of AGG/CGT fragment, wherein length is that the fragment of 2440bp and 2403bp has amplification in can educating gene pool, and in sterile gene pond, only contain the fragment of 2440bp, recycling Primer Premier5.0 software design codominant marker's SCAR primer, primer sequence is: forward primer: 5'-ATACACAGCTTCTAGCTGAATTTTTA-3'(is as shown in SEQ ID NO.3) and 5'-TGTGTGTGTAATTTCTCTGTGCG-3'(as shown in SEQ ID NO.4), reverse primer: 5'-ACAGAGTGAGAAATTTTATATATATAGGAAT-3'(is as shown in SEQ ID NO.5) and 5'-CGGAAGATTAATATTTTGCGTATACAT-3'.(as shown in SEQ ID NO.6), utilize this SCAR primer pair sterile gene pond and the individual plant DNA that can educate gene pool and formation gene pool to carry out pcr amplification, all DNA that educate individual plant and can educate gene pool (genotype is Msms) all amplify the fragment (as shown in SEQ ID NO.1-2) of big or small 886bp and 621bp, and all DNA of sterile gene pond and sterile individual plant only amplify the fragment (see figure 3) that size is 621bp.

4. linksystem analytical results

To first backcross generation segregating population 11812 and 11012 genomic dna of totally 472 individual plants carry out pcr amplification, all DNA that educate individuality (genotype is Msms) all amplify the fragment (as shown in SEQ ID NO.1-2) of big or small 886bp and 621bp, and all DNA of sterile individuality (genotype is msms) only amplify the fragment (in table 1, Fig. 4) of size for 621bp.In this presentation of results codominant marker, the fragment of 886bp and onion fertility restorer gene Ms are close linkages, and the fragment onion male sterility gene ms of 621bp is closely linked, and its genetic distance is zero.

The linkage analysis of table 1OMS-SCAR mark and onion fertility restorer gene Ms

+ indicate band;-indicate without band

5.OMS-SCAR is marked at the result in known type individuality (kind)

The sterile lines of many groups and maintenance line are detected, and result all only amplifies the fragment of 621bp.To the Japanese Long Jing seedling of six cross-fertilize seed S (Msms) Co., Ltd. kind (タ ー ボ, ネ オ ア ー ス, ア ト Application), Sapporo seedling Co., Ltd. (No. 3, も body じ, the sweet 70, タ ー ザ Application of Sapporo, ア De バ Application ス) is detected, and result all amplifies the fragment of 886bp and 621bp.Four groups of known type self-mating system (PR146, PR149, PR153, PR156) S/N (MsMs) are detected, and result only amplifies the fragment (in table 2, Fig. 5) of 886bp.This presentation of results OMS-SCAR mark is applicable to the material of different genetic backgrounds.

The different genetic background material of table 2 onion nuclear gene type individual plant the result

Numbering

Systematic name

Type

Checking strain number

Nuclear gene type

886bp

621bp

01

PF110

Sterile line

10

msms

-

+

02

PF118

Sterile line

10

msms

-

+

03

502F

Sterile line

10

msms

-

+

04

503F

Sterile line

10

msms

-

+

05

PF10-8

Sterile line

10

msms

-

+

06

PF17-5

Sterile line

10

msms

-

+

07

PF17-11-1

Sterile line

10

msms

-

+

08

PF17-10-1

Sterile line

10

msms

-

+

09

PF17-10-2

Sterile line

10

msms

-

+

10

PF17-10-3

Sterile line

10

msms

-

+

11

PM210

Maintenance line

10

msms

-

+

12

PM218

Maintenance line

10

msms

-

+

13

502M

Maintenance line

10

msms

-

+

14

52503M

Maintenance line

10

msms

-

+

15

PM10-11M

Maintenance line

10

msms

-

+

16

PM10-13M

Maintenance line

10

msms

-

+

17

PM17-5

Maintenance line

10

msms

-

+

18

PM17-11-1

Maintenance line

10

msms

-

+

19

PM17-10-3

Maintenance line

10

msms

-

+

20

PM17-10-1

Maintenance line

10

msms

-

+

21

PM17-10-2

Maintenance line

10

msms

-

+

22

ターボ

Cross-fertilize seed

10

Msms

+

+

23

ネオアース

Cross-fertilize seed

10

Msms

+

+

24

アトン

Cross-fertilize seed

10

Msms

+

+

25

No. 3, も body じ

Cross-fertilize seed

10

Msms

+

+

26

Sapporo sweet 70

Cross-fertilize seed

10

Msms

+

+

27

ターザン

Cross-fertilize seed

10

Msms

+

+

28

アドバンス

Cross-fertilize seed

10

Msms

+

+

29

PR146

Self-mating system

10

MsMs

+

-

30

PR149

Self-mating system

10

MsMs

+

-

31

PR153

Self-mating system

10

MsMs

+

-

32

PR156

Self-mating system

10

MsMs

+

-

+ indicate band;-indicate without band

Claims (3)

1. identify the codominance SCAR mark of onion male sterility gene for one kind, it is characterized in that, its specific fragment length is respectively 886bp and 621bp, wherein, specific fragment length is that mark and the onion of 886bp recovers gene M s close linkage, and its nucleotide sequence is as shown in SEQ ID NO.1; Specific fragment length is mark and the onion sterile gene ms close linkage of 621bp, and its nucleotide sequence is as shown in SEQ ID NO.2; The primer of described codominance SCAR mark is:

Forward primer FN1:5'-ATACACAGCTTCTAGCTGAATTTTTA-3';

FN2：5'-TGTGTGTGTAATTTCTCTGTGCG-3'；

Reverse primer RN1:5'-ACAGAGTGAGAAATTTTATATATATAGGAAT-3';

RN2：5'-CGGAAGATTAATATTTTGCGTATACAT-3'。

2. the codominance SCAR of evaluation onion male sterility gene claimed in claim 1 is marked at the application of assist-breeding onion male sterile line and supporting maintenance line aspect.

3. application as claimed in claim 2, it is characterized in that, its application method is: utilize the genomic dna of the primer pair individuality to be measured described in claim 1 to carry out pcr amplification, if individuality to be measured only amplifies the fragment of 886bp, and the educated individuality that this individual cells nuclear gene type to be measured is MsMs; If amplify 886bp and 621bp two bar segment, the cell nucleus gene type of individuality to be measured is Msms; If only amplify the fragment of 621bp, the cell nucleus gene type of this individuality to be measured is msms.

Images