CN109593878A - Molecular labeling and its application with capsicum genic male sterile gene close linkage - Google Patents
Molecular labeling and its application with capsicum genic male sterile gene close linkage Download PDFInfo
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- CN109593878A CN109593878A CN201910094416.7A CN201910094416A CN109593878A CN 109593878 A CN109593878 A CN 109593878A CN 201910094416 A CN201910094416 A CN 201910094416A CN 109593878 A CN109593878 A CN 109593878A
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Abstract
The present invention relates to a kind of and capsicum genic male sterile gene close linkage molecular labeling, the primer sequences of the molecular labeling P-05-112 are as follows: P-05-112F:5 '-ACTGGGGAACACTTACGGC-3 '；P-05-112R:5 '-TCTCAGCAAGGGATTAAAGGC-3 '；The invention also discloses its application methods.The invention has the benefit that utilization efficiency of the capsicum nuclear male sterility in breeding not only can be improved, the accuracy and efficiency selected in breeding is improved, significantly shortening breeding cycle, moreover it is possible to reduce breeding breeding cost.
Description
Technical field
The present invention relates to genetic breedings and molecular biology field, it particularly relates to a kind of and capsicum nuclear male
The molecular labeling of sterile gene close linkage and its application.
Background technique
Capsicum is that Solanaceae (Solanaceae) Capsicum (Capsicum) is annual or herbaceos perennial.Cultivation extensively
In all parts of the world, be in vegetable crop and the people's dietetic life with Important Economic value in a kind of world wide very
A kind of important flavouring.According to the relevant information that the capsicum fair of Chinese Zun Yi Second world is issued, China pepper planting face
The 35% of the world the Ji Zhan pepper planting gross area；Nearly 28,000,000 tons of 3 annual annual output, account for world's capsicum total output
46%；Annual value of production exceedes 70,000,000,000 yuan, and capsicum industry has become the maximum vegetables industry in China.
Pepper hybrid advantage clearly, using the male sterile line production of hybrid seeds can save artificial emasculation, be effectively reduced miscellaneous
Kind seed production cost, guarantees the purity of seed, and is conducive to protect intellectual property.The hero of most vegetables crop natural mutation
Property infertility only has a kind of hereditary form, but cell Genetic Sterility type and nucleo-cytoplasmic interreaction infertility type (cytoplasm are existed simultaneously on capsicum
Infertility), therefore capsicum is the research good test material of plants male sterility Fertility Expression.Cytoplasmic male sterility
The fertility of (cytoplasmic male sterility, CMS) can be bred as by cytoplasm and cell nucleus gene co- controlling
The male sterile line of 100% infertility, utilization is more convenient, but the restoring gene of male parent is mainly distributed in small fruit type capsicum, generally
Restorer is more difficult in the large fruit capsicum of cultivation finds, and the excellent Hybrid of breeding is more difficult；Capsicum nuclear male sterility
(genic male sterility, GMS) fertility is only controlled by cell nucleus gene, is not influenced by cytoplasm, and that reports at present is big
Mostly 1 pair of recessive nuclear gene control, can only be bred as male sterile two-purpose line, the fertile plant except 50% need to be dialled in the production of hybrid seeds, but
Capsicum nuclear male sterility fertility is more stable, and heredity is simply convenient for utilizing, and common self-mating system can make the fertility of hybrid
Full recovery, paternal origin is extensive in breeding, convenient for being bred as excellent Hybrid.
Nearly 20 of the capsicum genic male sterile gene that up to the present, domestic and international researcher has found (ms1 --
Ms15, msk, msc-1, msc-2), wherein msc-1 and msc-2 is the sterile gene for being found and being utilized by domestic scholars, but
It is whether relationship between these genes, gene are mutually same unclear.Only ms1 and msc-1 fertility controls gene quilt at present
Clone come, in terms of linked marker exploitation, domestic and international researcher to 5 ms genes (ms1, ms3, ms8, ms10,
Msc-1 it) has carried out positioning or developing linked marker.Other than msc-1 develops a genetic marker, remaining and ms gene
Chain molecular labeling either linkage distance farther out otherwise label higher operating costs, need to be improved in applicability.
For the problems in the relevant technologies, currently no effective solution has been proposed.
Summary of the invention
For above-mentioned technical problem in the related technology, the object of the present invention is to provide a kind of and capsicum nuclear males not
Molecular labeling and its application for educating gene close linkage overcome the shortcomings of above-mentioned aspect in existing product.
The purpose of the present invention is be achieved through the following technical solutions:
A kind of molecular labeling with capsicum genic male sterile gene close linkage, the molecular labeling P-05-112's draws
Object sequence are as follows:
P-05-112F:5 '-ACTGGGGAACACTTACGGC-3 '；
P-05-112R:5 '-TCTCAGCAAGGGATTAAAGGC-3 '.
A kind of application with the molecular labeling P-05-112 of capsicum genic male sterile gene close linkage, the molecule
Apply the tag to the assisted selection of capsicum nuclear male sterility strain.
Further, molecular labeling application method the following steps are included:
1) DNA of sample to be tested is extracted；
2) PCR amplification is carried out using molecular labeling；
3) electrophoresis test is carried out.
Further, pcr amplification reaction system in the step 2 are as follows: 2 μ L of DNA working solution, 10 × Buffer buffer 2
μ L, 0.8 dNTPs μ L, 0.4 μ L of archaeal dna polymerase, forward primer and each 0.2 μ L of reverse primer, will be overall with 14.4 μ L ddH2O
Polishing is accumulated to 20 μ L；Pcr amplification reaction program: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, 72 DEG C
Extend 30 seconds, 35 circulations；72 DEG C extend 7 minutes.
Further, electrophoresis is tested and carries out electrophoresis, silver nitrate with 7% non-denaturing polyacrylamide gel in the step 3)
It is observed after dyeing.It can detect that 243,256 and 258bp segment is male-fertile capsicum strain, and clip size is 256 Hes
258bp segment is then male sterile capsicum strain.
Further: can detect that three counterband tape segments are male-fertile capsicum strain in the step 3), and detect
Two counterband tapes are then male sterile capsicum strains out.
The invention has the benefit that capsicum nuclear male sterility, which not only can be improved, utilizes effect in breeding
Rate improves the accuracy and efficiency selected in breeding, significantly shortening breeding cycle, moreover it is possible to reduce breeding breeding cost.
Detailed description of the invention
Below according to attached drawing, invention is further described in detail.
Fig. 1 is amplification figure of molecular labeling of the embodiment of the present invention P-05-112 in fertile and sterile pond, in figure: S: no
Educate pond；F: fertile pond；
Fig. 2 is the genetic distance figure of molecular labeling of embodiment of the present invention P-05-112 and sterile gene；
Fig. 3 is test result figure of molecular labeling of the embodiment of the present invention P-05-112 to known profile material.
Specific embodiment
A kind of molecular labeling with capsicum genic male sterile gene close linkage described in the embodiment of the present invention and its
Using.
Embodiment 1: providing a kind of and capsicum genic male sterile gene close linkage molecular labeling P-05-112,
Primer sequence are as follows:
P-05-112F:5 '-ACTGGGGAACACTTACGGC-3 '；
P-05-112R:5 '-TCTCAGCAAGGGATTAAAGGC-3 '.
The building of target group
This test dual purpose lines sterile source by Ji grind No. 16 it is isolated, male parent be this laboratory homozygous inbred lines；It is thin with capsicum
Karyon male sterile two-purpose line group 18C2655 is test material, obtains the dual purpose lines group comprising 280 single plants, single
Strain statistics pollen fertility.
Molecular markers development
Developing SSR marks on No. 5 specific chromosome regions, and totally 98 pairs.
Amplification of the molecular labeling P-05-112 in fertile and sterile pond is as shown in Figure 1.
Fig. 1 shows: it is 243,256 and that the fertile pond genomic DNA amplification product of molecular labeling P-05-112, which contains size,
The segment of 258bp contains the segment that size is 256 and 258bp in sterile pond DNA cloning product.
Utilize the gene for 280 single plants for screening resulting polymorphic molecular marker P-05-112 analysis dual purpose lines group
Type, and combine the field Fertility identification of group's single plant as a result, utilizing JoinMap4.0 Software on Drawing capsicum cell genic male sterile gene
Genetic linkage maps；Map is as shown in Figure 2；
Fig. 2 is the result shows that the genetic distance of molecular labeling P-05-112 and sterile gene is 1.5cM.
Embodiment 2: a kind of application with the molecular labeling of capsicum genic male sterile gene close linkage is provided, to 41
The capsicum self-mating system and kind of part known type (MsMs) carry out genotype detection, the specific steps are as follows:
1) capsicum genomic DNA is extracted using modified CTAB method；
2) PCR amplification, pcr amplification reaction system are as follows: 2 μ L of DNA working solution, 10 × Buffer buffer are carried out using molecular labeling
2 μ L, 0.8 dNTPs μ L, 0.4 μ L of archaeal dna polymerase, forward primer and each 0.2 μ L(10uM/L of reverse primer), with 14.4 μ L
DdH2O is by total volume polishing to 20 μ L；Pcr amplification reaction program: 94 DEG C of 5 min of initial denaturation；94 DEG C of 30 s of denaturation, 55 DEG C of annealing
30s, 72 DEG C of extension 30s, 35 circulations；72 DEG C of 7 min of extension.
3) 7% native polyacrylamide gel electrophoresis of amplified production is observed photograph after cma staining, and is expanded PCR
Volume increase object be sequenced, testing result as shown in figure 3, the results show that the success rate of P-05-112 be 78%, show this label
Strong applicability, Rapid identification can be carried out to fertility in Breeding Application.
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the inspiration of the present invention
The product of form, however, make any variation in its shape or structure, it is all that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
<110>Lv Heng Science and Technology Co., Ltd.
<120>with the molecular labeling of capsicum genic male sterile gene close linkage and its application
<130> 2019
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
actggggaac acttacggc 19
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
tctcagcaag ggattaaagg c 21
Claims (6)
1. a kind of and capsicum genic male sterile gene close linkage molecular labeling, it is characterised in that: the molecule mark
Remember the primer sequence of P-05-112 are as follows:
P-05-112F:5 '-ACTGGGGAACACTTACGGC-3 '；
P-05-112R:5 '-TCTCAGCAAGGGATTAAAGGC-3 '.
2. a kind of application described in claim 1 with the molecular labeling of capsicum genic male sterile gene close linkage,
Be characterized in that: the molecular labeling is applied to the assisted selection of capsicum nuclear male sterility strain.
3. the application of molecular labeling as claimed in claim 2, it is characterised in that: specific method the following steps are included:
1) DNA of sample to be tested is extracted；
2) PCR amplification is carried out using molecular labeling；
3) electrophoresis test is carried out.
4. the application of molecular labeling according to claim 3, it is characterised in that: pcr amplification reaction body in the step 2
System are as follows: 2 μ L of DNA working solution, 10 × Buffer buffer, 2 μ L, 0.8 dNTPs μ L, 0.4 μ L of archaeal dna polymerase, forward primer and anti-
To each 0.2 μ L of primer, with 14.4 μ L ddH2O is by total volume polishing to 20 μ L；Pcr amplification reaction program: 94 DEG C of initial denaturations 5 are divided
Clock；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds, 35 circulations；72 DEG C extend 7 minutes.
5. the application of molecular labeling according to claim 3, it is characterised in that: electrophoresis test is non-with 7% in the step 3)
Denaturing polyacrylamide gel carries out electrophoresis, observes after cma staining.
6. the application of molecular labeling according to claim 3, it is characterised in that: can detect three tapes in the step 3)
It is male-fertile capsicum strain with segment, and detecting two counterband tapes then is male sterile capsicum strain.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910094416.7A CN109593878A (en) | 2019-01-31 | 2019-01-31 | Molecular labeling and its application with capsicum genic male sterile gene close linkage |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148920A (en) * | 2017-12-19 | 2018-06-12 | 中国农业大学 | The functional form molecular labeling of capsicum nuclear male sterility related gene and its application |
| CN108330208A (en) * | 2018-05-09 | 2018-07-27 | 绿亨科技股份有限公司 | A kind of molecular labeling of detection capsicum cytoplasmic male sterility restoring gene |
-
2019
- 2019-01-31 CN CN201910094416.7A patent/CN109593878A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148920A (en) * | 2017-12-19 | 2018-06-12 | 中国农业大学 | The functional form molecular labeling of capsicum nuclear male sterility related gene and its application |
| CN108330208A (en) * | 2018-05-09 | 2018-07-27 | 绿亨科技股份有限公司 | A kind of molecular labeling of detection capsicum cytoplasmic male sterility restoring gene |
Non-Patent Citations (1)
| Title |
|---|
| PONNAM NARESH ET AL.: "Molecular Markers Associated to Two Non-allelic Genic Male Sterility Genes in Peppers ( Capsicum annuum L.)", 《FRONT PLANT SCI》 * |
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Address after: 511453 South Company 663, Dongyong Town, Nansha District, Guangzhou City, Guangdong Province, Self-compiled Building A, [4] Store L4-10-11-12-13-14-15] Applicant after: Lvheng Technology Group Co.,Ltd. Address before: 511453 South Company 663, Dongyong Town, Nansha District, Guangzhou City, Guangdong Province, Self-compiled Building A, [4] Store L4-10-11-12-13-14-15] Applicant before: LUHENG TECHNOLOGY Co.,Ltd. |
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