CN101492738A - Onion cytoplasmic male sterility SCAR mark and uses thereof - Google Patents
Onion cytoplasmic male sterility SCAR mark and uses thereof Download PDFInfo
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Abstract
The invention discloses an onion cytoplasm male sterility SCAR marker, wherein the length of the specific fragment of the SCAR marker is 339bp, and the nucleotide sequence thereof is shown as SEQ ID No.2. The invention further discloses a primer of the SCAR maker, wherein the forward primer is Zt339-u:5'-GTTCTCAAGCAGATTCCGCAC-3', and the reverse primer is Zt339-d:5'-AGGACCAAACCGAGAGTGAGC-3'. Quick and accurate selection for onion cytoplasm fertility can be realized by using the obtained SCAR marker, thereby quickening the seed selection for new breed strain of onion cytoplasm male sterility, and establishing novel onion coenospecies parent selection system; first-filial generation breeds with independent intellectual property are selected and bred, thereby solving the difficult problems that conventional seeds are mainly used in the current production in China and large sums of foreign exchange is used for buying foreign coenospecies.
Description
Technical field
The present invention relates to a kind of SCAR mark and application thereof, relate in particular to a kind of onion cytoplasmic male sterility SCAR mark and application thereof, belong to biological technical field.
Background technology
Onion (Allium cepa.L), claim round onions, onion again, Liliaceae, allium, biennial plant, the cultivation history in existing more than 5000 year, it is main in the world vegetable species, have the laudatory title of protective foods, be China main Cultivar and export varieties [Hou XL, Wu ZX.Cultivation, storage and processing of onion in China.In:the international symposyium on the utilization and processing of onions, 1997,11:107-121].According to the preliminary statistics, China onion cultivated area reaches 22.6 ten thousand hm
2(1999), occupy the second place of the world, ultimate production 462.9 ten thousand t, rank first in the world [Su Baole. the Vegetables Exportation of earning foreign exchange guide. Beijing: Chinese agriculture science and technology press, 1999:180-195], 2000 are only the onion output value produced in USA just surpasses 1,000,000,000 dollars, is listed as the 3rd in vegetables, and its output value is with the speed increase in every year 5%.Onion not only can be eaten raw, also be used for fabricated product in a large number, be rich in the main component that multiple sulfide and carbohydrate are its peculiar flavours, have and reduce and the prevention thrombosis, the effect of reducing blood-fat, hypotensive, arteriosclerosis, prevention myocardial infarction is well received by consumers.
China's the eighties in last century is introduced a fine variety Hybrid from countries such as Japan, the U.S., its quality, output and consistence all obviously are better than conventional [Berninger E.Contribution a I ' etude de Ia sterilite male de I ' oignon (Allium ceps L) the .Ann Amehor Plantes of kind, 1965,15:183-199].From nineteen twenty-five Jones[Jones H, ClarkeA.Inheritance of male sterility in the onion and the production of hybrid seed.Proc Am SocHortic Sci, 1943,43:189-194] at first in " Italian red " (Italian Red) kind, find male sterile plant after, just begun the research of onion heterosis utilization abroad, last century the '30s to the improved crossing kind of the forties success, started the precedent of vegetable variety cross-breeding.Present paper studies relates generally to conventional tag, biochemical marker, molecule marker.[Havey MJ.Identification of cytoplasms using the polymerase chainreaction to aid in the extraction of maintainer lines from open-pollinated populations of onionTheor Appl Genet such as Havey, 1995,90:263-268] utilize the evaluation of PCR kytoplasm from style of opening pollination onion population, to isolate maintenance line fast, and utilize round pcr that onion cpDNA is carried out specific amplified, find that the fragment of 100bp can be distinguished sterile line, maintenance line.Afterwards, utilize RFLP molecule marker means to find out difference [Havey MJ.Diversity among male-sterility-inducing and male-fertilecytoplasms of onion Theor Appl Genet between onion male-fertile system and the sterile line Mitochondrial Genome Overview, 2000,101:778-782].[Engelke T such as Engelk, Tatlioglu T.Mitochondrial genome diversity in connection with male sterile in Alliun schoenoprasum L.Theor Appl Genet, 2000,100:942-948] find that also onion male sterile is relevant with Mitochondrial Genome Overview difference.Sato[Sato Y.PCR amplification of CMS-specific mitochondrial nucleotide sequences toidentify cytoplasmic genotypes of onion (Allium cepa L.) Theor Appl Genet, 1998,96:367-370] utilize the pcr amplification of CMS distinct line plastochondria nucleotide sequence to identify onion cytogene type.Holford[Holford P, Croft JH, Newbury HJ.Differences between, and possible origins of, the cytoplasms found infertile and male-sterile onions (Allium cepa L.) [J] .Theor Appl Genet, 1991,82:737-744] utilize BamHI, HindIII enzyme to cut onion mtDNA, EcoRI, HindIII, the Xbal enzyme is cut cpDNA, has successfully distinguished sterile line, maintenance line.As seen, onion male infertility and chloroplast gene group and Mitochondrial Genome Overview all exist and get in touch.By research onion cytoplasmic sterility, obtained a large amount of RFLP marks, the part mark has been successfully applied to screening, has differentiated S kytoplasm and N kytoplasm.Compare with traditional methods such as " test cross-backcross, selfings ", this technology can shorten the two seed selection time limits that are greatly, improves breeding efficiency.Yet, because the dna sequence dna in the tenuigenin produces variation phenomenon, the difference of adding genetic background, artificial selection and natural selections etc. such as disappearance, insertion, 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 carry out number of research projects.
China's onion cultivation history is shorter, and variety source is deficient.At present the kind of plantation can be divided into red skin, Calusena lansium and silver skin onion, and Cultivar is for the conventional variety of open pollination, along with the adjustment of agricultural structure, the demand of onion improved seeds is increased day by day.Because the cycle of onion breed breeding is long, be common vegetable crop Chinese cabbage, the 2-4 of radish doubly, mainly introduce and the conventional variety seed selection in the production based on kind, lack the first-filial generation of independent intellectual property right kind is arranged, need a large amount of foreign exchange of cost from external import seed, make foreign exchange earning vegetables production cost high, 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, shortening the breeding cycle, accelerate the research that molecular marker assisted selection and male sterile line utilize.Therefore, the evaluation work of sterile material and Fertile material has become a difficult problem that needs to be resolved hurrily at present, is the source work of onion industrialization.
In recent years, development of molecular biology provides a kind of new technical means based on the DNA variation, i.e. molecular marking technique for the plant genetic mark.Compare with other marking methods, molecule marker has incomparable superiority.It directly occurs with dna form, all can detect at each tissue, each developmental stage of plant materials, is not subjected to season, environmental limit, does not have expression whether problem; Quantity is many, spreads all over whole genome; The polymorphism height utilizes a large amount of primers, probe can finish the analysis of covering gene group; Neutral mark does not promptly influence the expression of objective trait, does not have the chain of certainty with bad proterties; The codominant marker can identify the genotype of isozygotying and the genotype of heterozygosis, and complete genetic information is provided.RFLP mark, AFLP marking operation are comparatively loaded down with trivial details, and RAPD mark accuracy and less stable, all are not suitable for the evaluation work of large-scale field material; The shortcoming that the SCAR marking operation is easy, accuracy is high, stable height has overcome above-mentioned mark becomes first-selected molecule marking method.So-called SCAR mark just is meant the order-checking of specificity segment two ends, and synthetic Auele Specific Primer carries out the amplification of specificity segment, and its advantage is accuracy height, good stability.
Comparative genomics is by a kind of research of biophase correlation gene group being understood, annotated another kind of biological genome.Obtain the similar sequences of another species according to the known sequence, thereby carry out the genetic comparison analysis, for we excavate homologous gene, research complex physical and pathologic process, thus evolutionary relationship between the difference of genetic background and species in the ubiquity of cognitive biology mechanism and the analysator, relatively the research of aspect such as mapping all has using value.Comparative genomics is applied to molecule marking research, has important practice significance and using value equally.
The purpose of genetic marker be realize molecular mark (Marker Assisted Selection, MAS).Be all can reduce workload, improve breeding efficiency with the tenuigenin site or with the assisted Selection of the chain mark of nuclear gene.Utilize and the chain mark in tenuigenin site, can identify the cytoplasm type of individual plant, eliminate S type tenuigenin in the Fertile material, only from the individual plant of N type cytoplasm type, select maintenance line, thereby reduce test cross number of combinations and selfing strain number, reduce workload, avoid blindness, improved the selection effect.Theoretically, if judged the tenuigenin fertility, the probability that the sterile type nuclear gene of homozygote msms occurs is 25%, but i.e. test cross 10 strains just might obtain the maternal plant of 2-3 strain maintenance line.Therefore, obtain onion cytoplasmic fertility mark, supporting for onion sterile line and maintenance line, seed selection onion cross-fertilize seed is significant.
Summary of the invention
The objective of the invention is to utilize the special primer of existing Herba Allii Schoenoprasi cytoplasmic male sterility, onion cytoplasmic male sterility system and corresponding maintenance line thereof are carried out pcr amplification, and the segment that obtains checked order, according to the SCAR mark of sequencing result exploitation at onion cytoplasmic male sterility system, the SCAR mark that utilization obtains carries out selecting fast and accurately to onion maintenance line material, accelerate the seed selection of onion cytoplasmic male sterility new lines, set up novel onion hybrid parents apolegamy system.
Onion cytoplasmic male sterility SCAR mark of the present invention is characterized in that: the specific fragment of described SCAR mark is long to be 339bp, and its nucleotide sequence is shown in SEQ ID No.2.
Above-mentioned onion cytoplasmic male sterility SCAR mark primer is:
Forward primer: Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 '
Reverse primer: Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 '.
The present invention is according to Engelke and Tatlioglu[Engelke T, Tatlioglu TA.PCR-marker for theCMS1-inducing cytoplasm in chives derived from recombination events effecting themitochondrial gene atp9.Theor Appl Genet, 2002,104:698-702] special primer (5 '-ATGGCTCGCCTTGAAAGAGAGC-3 ' and 5 '-CCAAGCATTTGGCGCTGAC-3 ') of discriminating Herba Allii Schoenoprasi cytoplasmic male sterility of report is S110 to onion cytoplasmic male sterility, total DNA of S118 and corresponding maintenance line N210 and N218 carries out pcr amplification, the result only amplifies size and is the fragment of 472kb in the partial sterility based material, can not accurately distinguish the shortcoming of onion cytoplasmic fertility at it, the inventor carries out sequencing analysis to this fragment, and its nucleotide sequence is shown in SEQ ID No.1; According to sequencing result, use Premier 5.0 inventor and therefrom design a pair of SCAR labeled primer, forward primer: Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 '; Reverse primer: Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 '.Synthetic by Beijing Bo Shang Bioisystech Co., Ltd, the PAGE purifying.
Use forward primer Zt339-u and reverse primer Zt339-d, to onion cytoplasmic male sterility is that total DNA of S110, S118 and corresponding maintenance line N210 and N218 carries out pcr amplification, sterile line S110, S118 have all amplified the specific band of a 339bp, and corresponding maintenance line N210 and N218 do not amplify this band.
340 parts of materials to known fertility carry out PCR checking (seeing Table 1), all amplify size in all sterile lines and are the fragment of 339bp, do not have the amplified production (see figure 1) in the maintenance line, and PCR result and field judged result are coincide.Illustrate that using this can differentiate the onion cytoplasmic male sterility type fully to primer, can learn that in view of the above the 339bp fragment and the male sterile that amplify have certain getting in touch.
To above onion cytoplasmic male sterility is the specific fragment that amplification is come out among S110 and the S118, through reclaiming, transform, clone and order-checking, the sequencing result that obtains S110 and S118 is in full accord, sequence total length 339bp, its nucleotide sequence is shown in SEQ ID No.2, primer sequence is contained at two ends, and its based composition is A+T=52%, C+G=48%.This section sequence and Genbank and DDBJ database are carried out sequence alignment, and homology shows that less than 40% this is one section new sequence.
Translate into albumen and carry out the tBlastx contrast, find Mitochondrial Genome Overview (DQ490951.2) with maize cell matter male sterile line, gama grass Mitochondrial Genome Overview (DQ984517.1), corn mitochondrial ATP 6 genes (MZEMTATP6.1), corn C type cytoplasmic sterility (DQ645536.1) and T type cytoplasmic sterility (DQ490953.1) Mitochondrial Genome Overview, Chinese sorghum Mitochondrial Genome Overview (DQ984518.1) and two look shizandra berry mitochondrial ATP 6 genes (X57100.1) etc. have certain homology, can think onion cytoplasmic sterile with Mitochondrial Genome Overview certain getting in touch arranged.Therefore, from now on to onion cytoplasmic the dependency between the sterile and Mitochondrial Genome Overview need further study.
The screening process of above-mentioned SCAR labeled primer is as follows:
(1) selecting onion cytoplasmic male sterility for use is S110, S118 and corresponding maintenance line N210, N218;
(2) the CTAB method is extracted the onion genome DNA;
(3) utilize the total DNA of Herba Allii Schoenoprasi cytoplasmic male sterility primer amplified onion;
(4) from sterile line, reclaim the specificity segment, after connecting, transform, checking order, design SCAR labeled primer;
(5) utilize the SCAR primer that sterile line S110, S118 and maintenance line N210, N218 are carried out authenticate reverse;
(6) utilize the SCAR primer to other individual materials of known type totally 340 parts verify.
The onion DNA extraction adopts modified CTAB method; Pcr amplification carries out on U.S. Bole's TC-XP-D type gene-amplificative instrament, 25 μ L systems, wherein 10 * PCR Buffer (with Mg
2+) 2.5 μ L, dNTPs (each 2.5mM) 2.0 μ L, each 1.0 μ L of Primer1,2 (0.5 μ mol/L), Taq DNA polymerase (5U/ μ L) Taq DNApolymerase (5U/ μ L) 0.3 μ L, ddH
2O17.2 μ L, response procedures are 94 ℃ of pre-sex change 4min, [72 ℃ are extended 30sec for 94 ℃ of sex change 30sec, 65 ℃ of annealing 30sec], 30 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations; The PCR product carries out the electrophoretic separation analysis in 1.0% sepharose, bromination second pyridine dyeing, gel imaging system automated imaging; Specific band reclaims purifying, according to Biospin Gel Extraction Kit operation instruction; Connect, transform, identify the method that adopts molecular cloning II; Determined dna sequence entrusts Beijing Bo Shang Bioisystech Co., Ltd to finish.Its result is, use forward primer Zt339-u and reverse primer Zt339-d, to onion cytoplasmic male sterility is that total DNA of S110, S118 and corresponding maintenance line N210 and N218 carries out pcr amplification, sterile line S110, S118 have all amplified the specific band of a 339bp, and corresponding maintenance line N210 and N218 do not amplify this band.
The present invention has obtained a stable SCAR mark of onion cytoplasmic male sterility genes involved.Onion cytoplasmic male sterility SCAR mark of the present invention has widespread use in assist-breeding onion cytoplasmic male sterility new lines.
Beneficial effect of the present invention: it is S110 that the present invention selects onion cytoplasmic male sterility for use, total DNA of S118 and corresponding maintenance line N210 and N218 carries out pcr amplification, having obtained to distinguish two is cytoplasmic stable SCAR mark, be laying a good foundation of onion molecular marker assisted selection breeding system, compare with present technology, its advantage is: (1) mark is stable: it is S110 that the present invention selects onion cytoplasmic male sterility for use, total DNA of S118 and corresponding maintenance line N210 and N218 carries out pcr amplification, the two stable SCAR marks that are have been obtained to distinguish, and 340 parts of materials have been carried out widely checking, its phenotype and molecular markers for identification result are in full accord.(2) molecular marker assisted selection is easy and simple to handle, and save cost: onion is 2 years living plants, and the 2-4 that its breeding time limit is the vegetable breeding time limits such as Chinese cabbage, radish times, conventional selection, the cycle is long, cost height, time-consuming effort again.By the present invention, only need the simple total DNA of onion that extracts, carry out simple PCR amplification, agarose gel electrophoresis then, get final product the fertility of effective expert evidence.Not only avoid the loaded down with trivial details screening process of ordinary method, also avoided extracting the complex operations of Mitochondrial DNA, made its operating process simpler, effective.
Description of drawings
The molecule checking of Fig. 1 onion male sterile line.
Wherein: M is Marker, and S is sterile individual plant, and N is for can educate individual plant.
The DNA electrophoretogram of Fig. 2 sterile line and maintenance line thereof.
Wherein: M is Marker, and S is sterile individual plant, and N is for can educate individual plant.
Fig. 3 primer Zt339-u, Zt339-d is to the pcr amplification result of onion sterile line and maintenance line thereof.
Wherein: M is Marker, and S is sterile individual plant, and N is for can educate individual plant.
Embodiment
Embodiment 1
(1) materials and methods
The extraction of 1 DNA and detection
Onion cytoplasmic male sterility system and corresponding maintenance line genome DNA extracting method thereof adopt modified CTAB method, and agarose gel electrophoresis and spectrophotometer detect.
The foundation of 2 gene pools
Using segregating population fractional analysis method (Bulked Segregation Analysis) is the BSA method, with 10 individual plants of sterile line and the DNA sample balanced mix respectively of corresponding 10 individual plants of maintenance line with it, form the gene pool of onion cytoplasmic male sterility system and maintenance line thereof.
3 primer design are with synthetic
Special primer 5 '-ATGGCTCGCCTTGAAAGAGAGC-3 ' and 5 '-CCAAGCATTTGGCGCTGAC-3 ' according to the discriminating Herba Allii Schoenoprasi cytoplasmic male sterility of Engelke and Tatlioglu report is S110 to onion cytoplasmic male sterility, S118 and corresponding maintenance line N210 thereof and N218 carry out pcr amplification, the result only amplifies size and is the fragment of 472kb in the partial sterility based material, can not accurately distinguish the shortcoming of onion cytoplasmic male sterility system and maintenance line at it, this fragment is carried out sequencing analysis, according to sequencing result, use Primer Premier 5.0 and therefrom design a pair of primer, forward primer: Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 '; Reverse primer: Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 '.Synthetic by Beijing Bo Shang Bioisystech Co., Ltd, the PAGE purifying.
4 pcr amplifications and detection
Pcr amplification carries out on U.S. Bole's TC-XP-D type gene-amplificative instrament, 25 μ L systems, wherein 10 * PCRBuffer (with Mg
2+) 2.5 μ L, dNTPs (each 2.5mM) 2.0 μ L, each 1.0 μ L of Primer1,2 (0.5 μ mol/L), TaqDNA polymerase (5U/ μ L) Taq DNA polymerase (5U/ μ L) 0.3 μ L, ddH
2O17.2 μ L, response procedures are 94 ℃ of pre-sex change 4min, [72 ℃ are extended 30sec for 94 ℃ of sex change 30sec, 65 ℃ of annealing 30sec], 30 circulations, 72 ℃ are extended 5min, 4 ℃ of preservations; The PCR product carries out the electrophoretic separation analysis in 1.0% sepharose, bromination second pyridine dyeing, gel imaging system automated imaging.
5 specific bands reclaim purifying, clone and order-checking
Reclaim the purifying specific band according to Biospin Gel Extraction Kit operation instruction;
The molecular cloning of specific band adopts the method for molecular cloning II;
Determined dna sequence entrusts Beijing Bo Shang Bioisystech Co., Ltd to finish.
The checking of 6 individual plants
Judge the fertility of each strain system by field observation, thereby obtain 17 combinations of sterile line and maintenance line, each combination is respectively randomly drawed 10 strain materials from sterile strain with educating the strain, extract genome DNA, use primer Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 ' and primer Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 ' that 340 parts of materials of these known fertility are carried out the pcr amplification checking, reaction system and program are with 4.
7 dna sequence analysis
Sequence enters ncbi database and carries out the BLAST analysis.
(2) result and analysis
The check and analysis of 1 sterile line and corresponding maintenance line genomic dna thereof
The result that using modified CTAB method is extracted the onion bulb stem genome DNA shows that the dna solution that is extracted is limpid, no browning.0.8% agarose gel electrophoresis the results are shown in Figure 2, and electrophoresis result shows that the DNA that is extracted is significantly not residual at place, point sample hole, illustrates that polysaccharide and protein content are not high; Rna content is very few; DNA band is positioned at same position, and clear, no disperse, no obvious degradation.So the high quality onion bulb stem DNA that extracts is applicable to biological tests such as pcr amplification, AFLP molecule marker.
2 pcr amplification results
Using special primer 5 '-ATGGCTCGCCTTGAAAGAGAGC-3 ' and the 5 '-CCAAGCATTTGGCGCTGAC-3 ' that differentiates Herba Allii Schoenoprasi cytoplasmic male sterility fertility is that S110, S118 and corresponding maintenance line N210, N218 increase to onion cytoplasmic male sterility, the result amplifies the fragment that length is 472kb in S110 and S118 part individuality, this fragment is checked order, and its nucleotide sequence is shown in SEQ ID No.1.
According to the nucleotide sequence shown in the SEQ ID No.1, use Primer Premier 5.0 design primer: Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 ', Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 '.Using Zt-u and Zt-d primer is that the gene pool of S110, S118 and corresponding maintenance line N210 and N218 is that template is carried out pcr amplification to onion cytoplasmic male sterility, the results are shown in Figure 3, can be clearly seen that among the figure, in onion sterile line S110, S118, amplify a tangible fragment, size is 339bp, does not have amplified production in corresponding maintenance line N210 and N218.
3 individual checkings
To carrying out PCR checking (seeing Table 1) by observing 340 parts of bill of material judging the onion cytoplasmic fertility, all amplify size in all sterile lines and be the fragment of 339bp, there is not the amplified production (see figure 1) in the maintenance line, PCR result and field judged result are coincide.Only in the sterile line individual plant, amplified production is arranged, and there is not amplified production in the maintenance line individual plant, illustrate that using this can differentiate this seminar onion cytoplasmic male sterility type fully to primer, can learn that in view of the above the 339bp fragment and the onion fertility that amplify have certain getting in touch.
Table 1 onion sterile line and maintenance line individual plant checking result
* :+expression has band (sterile); * :-expression does not have band (can educate)
4 onion cytoplasmic male sterilities are the specific fragment sequential analysis of S110, S118
To above onion cytoplasmic male sterility is the specific fragment that amplification is come out among S110 and the S118, through reclaiming, transform, clone and order-checking, the sequencing result that obtains S110 and S118 is in full accord, sequence total length 339bp, its nucleotide sequence is shown in SEQ ID No.2, primer sequence is contained at two ends, and its based composition is A+T=52%, C+G=48%.SEQ ID No.2 sequence and Genbank database are carried out sequence alignment, and homology shows that less than 40% this is one section new sequence.
Carry out the tBlastx comparison, find Mitochondrial Genome Overview (DQ490951.2) with maize cell matter male sterile line, gama grass Mitochondrial Genome Overview (DQ984517.1), corn mitochondrial ATP 6 genes (MZEMTATP6.1), corn C type cytoplasmic sterility (DQ645536.1) and T type cytoplasmic sterility (DQ490953.1) Mitochondrial Genome Overview, Chinese sorghum Mitochondrial Genome Overview (DQ984518.1) and two look shizandra berry mitochondrial ATP 6 genes (X57100.1) etc. have certain homology, can think that the SEQ ID No.2 sequence that the present invention obtains is an onion Mitochondrial Genome Overview sequence, onion cytoplasmic is sterile to have certain getting in touch with Mitochondrial Genome Overview.
Sequence table
＜110〉Vegetable Research Institute, Shandong Academy of Agricultural Sciences
＜120〉a kind of onion cytoplasmic male sterility SCAR mark and application thereof
<141>2009-3-4
<160>2
<210>1
<211>472
<212>DNA
＜213〉artificial sequence
＜221〉onion specific DNA fragment
<222>(1)...(472)
<400>1
atggctcgcc?ttgaaagaga?gctgctctct?tacaagaaaa?gtggaattcc?tcgttctggg 60
gctttctatg?cttgttctca?agcagattgg?cgaccttgtt?tatcgtcttg?ttcgcttcct 120
ttttactgct?tttgtgggga?catttagtac?caatttggta?ctacagcgag?cggctttccg 180
ttgccatgga?ttttttgatg?tcgaaaggaa?ttccttttga?gttggagttc?gggtgggaca 240
aagtggtgat?tcgcccagtt?cagtcggtgg?caacttttct?caaagcaggg?gaagtacctc 300
ctgttccacc?agtgacgggt?atagtaccac?ccgtagaatc?ccactgccca?ctagagtgcc 360
acttattcac?aaatccatcc?ttgtctatgc?tgctcactct?cggtttggtc?ctactgatga 420
tttttttttt?gttacgaaaa?agggaggggg?aaagtcagcg?ccaaatgctt?gg
<210>2
<211>339
<212>DNA
＜213〉artificial sequence
＜221〉specific DNA fragment of onion cytoplasmic male sterility SCAR mark
<222>(1)...(339)
<400>1
gttctcaagc?agattggcga?ccttgtttat?cgtcttgttc?gcttcctttt?tactgctttt 60
gtggggacat?ttagtaccaa?tttggtacta?cagcgagcgg?ctttccgttg?ccatggattt 120
tttgatgtcg?aaaggaattc?cttttgagtt?ggagttcggg?tgggacaaag?tggtgattcg 180
cccagttcag?tcggtggcaa?cttttctcaa?agcaggggaa?gtacctcctg?ttccaccagt 240
gacgggtata?gtaccacccg?tagaatccca?ctgcccacta?gagtgccact?tattcacaaa 300
tccatccttg?tctatgctgc?tcactctcgg?tttggtcct
Claims (3)
1. onion cytoplasmic male sterility SCAR mark is characterized in that: the specific fragment of described SCAR mark is long to be 339bp, and its nucleotide sequence is shown in SEQ ID No.2.
2. onion cytoplasmic male sterility SCAR mark according to claim 1 is characterized in that: described SCAR labeled primer is:
Forward primer: Zt339-u:5 '-GTTCTCAAGCAGATTCCGCAC-3 '
Reverse primer: Zt339-d:5 '-AGGACCAAACCGAGAGTGAGC-3 '.
3. the application of the described onion cytoplasmic male sterility SCAR mark of claim 1 in assist-breeding onion cytoplasmic male sterility new lines.
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| CN 200910014679 CN101492738B (en) | 2009-03-09 | 2009-03-09 | Onion cytoplasmic male sterility SCAR mark and uses thereof |
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| CN102851379A (en) * | 2012-09-14 | 2013-01-02 | 山东省农业科学院蔬菜研究所 | Kit for detecting cytoplasmic male sterility of green Chinese onion and application thereof |
| CN102925431A (en) * | 2011-08-08 | 2013-02-13 | 山东省农业科学院蔬菜研究所 | SCAR marker closely linked to onion male sterile restoring gene Ms and application thereof |
| CN102952797A (en) * | 2011-08-24 | 2013-03-06 | 山东省农业科学院蔬菜研究所 | SCAR marker closely linked with onion male sterility gene ms and application thereof |
| CN103981281A (en) * | 2014-06-10 | 2014-08-13 | 山东省农业科学院蔬菜花卉研究所 | Method for breeding onion male sterile line and maintainer line by utilizing molecular markers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100435152B1 (en) * | 2001-06-25 | 2004-06-14 | 대한민국 | Identification method for cytoplasmic male sterility factor using PCR-RFLP in onion |
| CN100371343C (en) * | 2005-05-13 | 2008-02-27 | 浙江大学 | Molecule marking method for discriminating cytoplasmic male sterile gene of leaf mustard |
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| CN114686600A (en) * | 2022-02-24 | 2022-07-01 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
| CN114686600B (en) * | 2022-02-24 | 2023-12-12 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
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