CN102952797B - SCAR marker closely linked with onion male sterility gene ms and application thereof - Google Patents

Abstract

The invention discloses an SCAR marker closely linked with an onion male sterility gene ms. The SCAR marker has a specific fragment length of 357 bp, the nucleotide sequence thereof being shown in SEQ ID NO. 1. A forward primer of the primers of the SCAR marker is 5'-TCAGTATCAATAGAAGGAATCAC-3', and a reverse primer of the primers of the SCAR marker is 5'-GTATACCATTGGTACTTGATGCA-3'. The SCAR marker provided by the invention can be utilized to rapidly and accurately determine presence or absence of the onion male sterility gene, thereby being with great significance of accelerating breeding process of the onion and building an onion breeding technology system assisted by molecule marker. The application of the SCAR marker can substantially shorten breeding years, prevent blindness of conventional breeding methods, and greatly increase selection efficiency.

Description

With the closely linked SCAR mark of onion male sterility gene ms and application thereof

Technical field

The present invention relates to a kind of SCAR mark and application thereof, it relates to a kind of and the closely linked SCAR mark of onion male sterility gene ms and application thereof, in onion breeding, can utilize this mark to come assisted Selection onion male sterile line and supporting maintenance line, belong to biological technical field.

Background technology

Onion Allium cepaL.), be one of Main Vegetable Species Suitable For Culture in the world, have the laudatory title of protective foods.According to FAO (2009) statistics, 3,700,000 hectares of world's onion cultivated areas, 7,230 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 1,000,000 hectares, and 2,107 ten thousand tons of ultimate productions, account for 30% of worldwide production.Onion not only can be eaten raw, also, in a large number for fabricated product, 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, H.A.and S.L.Emsweller.1936.A male sterile onion.Proc.Amer.Soc.Hort.Sci.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, H.A and A.Clarke.1943.Inheritance of male sterility in the onion and the production of hybrid seed.Proc Amer.Soc.Hort.Sci.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.) .TheorAppl 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.JAmer Soc Hort Sci, 2002,127 (4): 576-582] utilize AFLP and the nuclear MS of RFLP method mark onion site, built 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 development 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 or the assisted Selection of the chain mark of nuclear gene, improve breeding efficiency.Utilize and the closely linked mark of nucleus msji gene, the material that contains nucleus sterile gene can be detected, eliminate the dominant onion material isozygotying, only from hold the individual plant of male sterility gene ms, select maintenance line, guarantee the reliability of selecting, thereby reduce test cross number of combinations and selfing strain number, reduce workload, avoid blindness, improve and select effect.Therefore, obtain with onion nucleus ms because of closely linked molecule marker, 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, invent a kind of SCAR mark that can be used for assist-breeding onion male sterile line and supporting maintenance line.

The present invention is achieved by the following technical solutions:

One and the closely linked SCAR mark of onion male sterility gene ms, its specific fragment length is 357bp, its nucleotide sequence is as shown in SEQ ID NO.1.

Described SCAR labeled primer is:

Forward primer: 5 '-TCAGTATCAATAGAAGGAATCAC-3 ';

Reverse primer: 5 '-GTATACCATTGGTACTTGATGCA-3 '; As shown in SEQ ID NO.2, SEQ ID NO.3.

The present invention utilizes AFLP marking method take onion sterile line 118 S (msms) and first backcross generation BC1[118 × (118 × 12-12) of Fertile material 12-12 S (MsMs) with different genetic backgrounds] arrive and the closely linked AFLP molecule marker of onion fertility restorer gene Ms gene as material screening, by Tail-PCR technology, this flag sequence is carried out after chromosome walking, successfully obtain the closely linked SCAR mark with onion male sterility gene ms, by its called after OSG-SCAR.Utilize OSG-SCAR mark can judge fast the nuclear gene of onion candidate maintenance line material, eliminate the individuality without this band (MsMs), remain with the individuality of band (msms or Msms), thereby in assurance candidate material, there is the gene of nuclear male sterility ms, reduce the blindness of conventional breeding, to accelerate the process of maintenance line of seed selection onion, and then set up onion molecular mark technical system.

The screening process of described SCAR mark is as follows:

(1) hybridize with male parent self-mating system 12-12 S (MsMs) as maternal take onion male sterile line 118 S (msms), the F1 generation S that obtains of institute (Msms) backcrosses for male parent and maternal sterile line 118 S (msms), obtain four backcross progeny segregating population 07-11812,07-11012 and 08-11812,08-11012, 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.

(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) obtain after AFLP mark, by Tail-PCR technology, this flag sequence is carried out after chromosome walking, acquisition can be identified the SCAR mark of onion male sterility gene ms.

(5), through genetic analysis and verify the feasibility of this mark, obtained with onion male sterility gene ms and be divided into the mark from OSG-SCAR.

Described SCAR mark 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 the primer pair individuality to be measured of SCAR mark to carry out pcr amplification, detect the fragment that whether amplifies big or small 357bp, if there is no amplified production, the cell nucleus gene type of this individuality to be measured is MsMs, can be used as male parent system (recovery fertility), if there is amplified production, in this individuality to be measured, contain ms, its genotype is the material that Msms or msms can be used as screening sterile line or maintenance line, the judgement cytoplasm fertility technology (granted patent number: ZL200910014679 of developing before this in conjunction with applicant, publication number: CN 101492738A), can be applicable to 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 is problem in the urgent need to address in breeding work.The present invention utilizes the technology of molecule marker to obtain the closely linked OSG-SCAR mark with onion male sterility gene ms.Utilize this mark can screen the individual plant with 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:

The present invention obtain with the closely linked OSG-SCAR mark of onion male sterility gene ms, result is stable, accurate, easy and simple to handle, it can precise Identification onion male sterility gene.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, there is not yet the report with onion male sterility gene ms close linkage (be divided into from) mark.This research obtain OSG-SCAR and onion male sterility gene be divided into from, be therefore applicable to different genetic background materials widely, be a ubiquity mark.

3. can Direct Identification there is the male sterility gene ms of different genetic background materials due to OSG-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: the electrophoretogram of OSG-SCAR mark checking; Wherein: M is Marker; In figure, 1-12 is the selfing individual plant of male parent (MsMs).12-24 is the sterile individual plant (msms) in segregating population.

Fig. 4: the part electrophoretogram that onion segregating population 07-11812,08-11812 and 07-11012,08-11012 colony are carried out to the checking of OSG-SCAR mark; Wherein: M is Marker; 1,16 is MsMs contrast; 2-15 is the individual plant that onion cell caryogram is msms, and 17-30 is the individual plant that onion cell nucleus gene type is Msms.

Fig. 5: the part electrophoretogram that carries out the checking of OSG-SCAR mark to deriving from known type (MsMs) material of different genetic backgrounds; Wherein: M is Marker; 1 is msms contrast, and 2-5 is PR146 individual plant, and 6-9 is PR149 individual plant, and 10-13 is PR153 individual plant, and 14-17 is PR156 individual plant.

Fig. 6: to deriving from the onion male sterile line S (msms) of different genetic backgrounds and the part electrophoretogram that maintenance line N (msms) material carries out the checking of OSG-SCAR mark.1,18 is MsMs contrast, and 2-17 is different sterile line material.19-30 is different maintenance line material.

Fig. 7: the part electrophoretogram that the onion cross-fertilize seed material of part different sources is carried out to the checking of OSG-SCAR mark.Wherein, M is Marker; Being for No. 1 and No. 13 male parent 12-12 (MsMs) individual plant, is for No. 2 and No. 14 can educate individual plant (Msms) in classification colony, is for No. 3 and No. 15 sterile individual plant (msms) in classification colony; 4-6,7-9,10-12, is respectively onion cross-fertilize seed タ mono-ボ of Japanese Long Jing seedling Co., Ltd., ネ オ ア mono-ス, ア ト Application, 16-18,19-21,22-24, is respectively No. 3, the onion cross-fertilize seed も body じ of Japanese Sapporo seedling Co., Ltd., タ mono-ザ Application, ア De Application ス.

Embodiment

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

The screening and application of embodiment 1SCAR mark

(1) materials and methods

1. the extraction of genome DNA and detection

07-11812, the 07-11012 of four first backcross generation colonies of onion and the genomic dna of 08-11812,08-11012 adopt genome rapid extraction test kit (Beijing, TIANGEN) method is extracted, 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 07-11812 are distinguished to balanced mix, form the sterile gene pond of onion and can educate gene pool.

Synthesizing of 3.AFLP joint and primer

The enzyme of genomic dna 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 16EcoR I and 16Mse I selective amplification primer, 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 LMse 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 at above-mentioned enzyme, add 5 μ MEcoRI joints and the each 0.5 μ L of 50 μ MMse I joint, T ₄dNA ligase (5U/ μ L) 1 μ L, 10 × T ₄dNALigase Buffer3.0 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 × PCRBuffer (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 of pre-amplimer (30ng/ μ L) of EcoR I and Mse I, Taq DNA polymerase 0.2 μ L (5U/ μ L), dNTPs (each 2.5mM) 2.0 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⁺) 2.0 μ L, dNTPs (each2.5mM) 1.6 μ L, Taq DNA polymerase (5U/ μ L) 0.2 μ L, the each 1 μ L of selective amplification primer (30ng/ μ L) 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 60sec (each cycle annealing temperature reduces 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 in selective amplification sample, 10mM EDTA, 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene green grass or young crops), after 95 ℃ of sex change 5min, be placed in immediately cooled on ice.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% concentrated 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 L 10mg/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 SEQID NO.4.

7. the SCAR of mark transforms

According to sequencing result, utilize PrimerPremier 5.0 software design primers, obtain the unknown nucleotide sequence of known dna sequence flank by Tail-PCR chromosome walking, recycling Primer Premier 5.0 software design SCAR primers.

8. linkage analysis

Utilize SCAR mark, the genomic dna of 07-11812,07-11012 and 08-11812,08-11012 first backcross generation colony is carried out to pcr amplification and analyze whether there is the individuality with crossover value, detect the stability of OSG-SCAR mark and the genetic distance with male sterility gene ms.

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

Utilize cross-fertilize seed material Japan's Long Jing seedling Co., Ltd.'s kind (タ mono-ボ, ネ オ ア mono-ス, ア ト Application) of different sterile lines, maintenance line, self-mating system and known type, Sapporo seedling Co., Ltd. (No. 3, も body じ, Sapporo are sweet 70, タ mono-ザ Application, ア De バ Application ス), and the broad spectrum of fertility restorer material (PR146, PR149, PR153, PR156) checking OSG-SCAR mark and the feasibility of molecular marker assisted selection.Derive from Japanese different onion kinds (cross-fertilize seed), nuclear gene type is MsMs, form by test cross, the method seed selection of backcrossing, selection is referring to [Pike, L.M.1986.Onion breeding.In:Basset, M.J. (ed.) Breeding Vegetable Crops.AVI Publishing Co., Connecticut, pp, 357-394].

(2) results and analysis

1. utilize the detection analysis of the genomic dna of four groups of 07-11812,07-11012 and 08-11812,08-11012 first backcross generation colony

Adopt the method for genome rapid extraction test kit (Beijing, TIANGEN) to extract onion blade genome DNA, show (seeing Fig. 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 EcoRI 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 Premier 5.0 software designs, obtain the flank unknown nucleotide sequence of onion AGG/CGT fragment by Tail-PCR chromosome walking, recycling Primer Premier 5.0 software design SCAR primers, primer sequence is: forward primer: 5 '-TCAGTATCAATAGAAGGAATCAC-3 ' and reverse primer: 5 '-GTATACCATTGGTACTTGATGCA-3 ' is (as SEQ ID NO.2, shown in SEQ ID NO.3), utilize the genomic dna of this SCAR primer pair male parent self-mating system 12-12 (MsMs) and the sterile individual plant of segregating population (msms) material to carry out pcr amplification, the DNA that all cells nuclear gene type is msms all amplifies the fragment (as shown in SEQ ID NO.1) of big or small 357bp, and the DNA that all cells nuclear gene type is MsMs does not have amplified production (seeing Fig. 3).

4. linksystem analytical results

To first backcross generation segregating population 07-11812,07-11012 and 08-11812, the 08-11012 genomic dna of totally 472 individual plants carries out pcr amplification, and the DNA of all individualities all amplifies the fragment (as shown in SEQ ID NO.1) of big or small 357bp, (Fig. 4).Do not find to there is crossover value individuality, illustrate OSG-SCAR mark and onion male sterility gene ms be close linkage be divided into from, its genetic distance is zero.

5.OSG-SCAR is marked at the result in known type self-mating system (kind, cross-fertilize seed) to many groups sterile line S (msms) and maintenance line N (msms), the Japanese Long Jing seedling of cross-fertilize seed S (Msms) Co., Ltd. kind (タ mono-ボ, ネ オ ア mono-ス, ア ト Application), the DNA of Sapporo seedling Co., Ltd. (No. 3, も body じ, タ mono-ザ Application, ア De バ Application ス) detects, and result all amplifies OSG-SCAR labeled fragment (table 2, Fig. 6,7).DNA to four groups of known type self-mating system (PR146, PR149, PR153, PR156) S/N (MsMs) detects, and does not all amplify the fragment (in table 2, Fig. 5) of 357bp.This presentation of results OSG-SCAR be marked in different genetic background materials too with male sterility gene ms close linkage be divided into from.

Table 1 various combination BC1 is for fertility separating ratio result

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

+ indicate band (ms);-indicate without band (Ms)

Claims (4)

1. with the closely linked SCAR mark of onion male sterility gene ms, it is characterized in that: the spy of described SCAR mark

Heteroleptic length is 357bp, and its nucleotide sequence is as shown in SEQ ID NO.1.

2. the according to claim 1 and closely linked SCAR mark of onion male sterility gene ms, is characterized in that: institute

The primer of stating SCAR mark is:

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

Reverse primer RN1:5'-GTATACCATTGGTACTTGATGCA-3'.

3. described in claim 1 or 2, be marked at assist-breeding onion hero with the closely linked SCAR of onion male sterility gene ms

Application in property sterile line and supporting maintenance line.

4. application according to claim 3, is characterized in that: concrete application mode is, utilizes the SCAR described in claim 2

The genomic dna of the primer pair individuality to be measured of mark carries out pcr amplification, detects the fragment that whether amplifies big or small 357bp,

If there is no amplified production, the cell nucleus gene type of this individuality to be measured is MsMs, if there is this amplified production, and this to be measured

In body genotype, contain ms, its genotype is Msms or msms.

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