CN103993070B - A kind of SSR primer for exquisite joint melon hybrid seed purity qualification and method - Google Patents
A kind of SSR primer for exquisite joint melon hybrid seed purity qualification and method Download PDFInfo
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- 241000219112 Cucumis Species 0.000 title claims abstract description 39
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 title claims abstract description 39
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012797 qualification Methods 0.000 title claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 7
- 230000008774 maternal effect Effects 0.000 claims description 20
- 239000003550 marker Substances 0.000 claims description 14
- 238000012408 PCR amplification Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- 238000001502 gel electrophoresis Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 235000009854 Cucurbita moschata Nutrition 0.000 description 4
- 240000001980 Cucurbita pepo Species 0.000 description 4
- 235000009852 Cucurbita pepo Nutrition 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 235000020354 squash Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 244000036905 Benincasa cerifera Species 0.000 description 2
- 235000011274 Benincasa cerifera Nutrition 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108020005120 Plant DNA Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000013094 purity test Methods 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
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Abstract
The invention discloses a kind of SSR primer for exquisite joint melon hybrid seed purity qualification, described primer comprises upstream primer ChQSSR304-F and the downstream primer ChQSSR304-R of primer ChQSSR304, is the nucleotide sequence of described upstream primer ChQSSR304-F as SEQ? ID? is the nucleotide sequence of described downstream primer ChQSSR304-R as SEQ shown in NO.1? ID? shown in NO.2.Also disclose a kind of authentication method of exquisite joint melon hybrid seed purity, this primer and method effectively can be distinguished exquisite joint melon cenospecies and female parent thereof, male parent seed, detect hybrid seed purity fast, accurately, there is quick, accurate, low cost, simple operation and other advantages simultaneously.
Description
Technical field
The invention belongs to hybrid seed purity authenticate technology field, be specifically related to a kind of SSR primer of qualification of exquisite joint melon hybrid seed purity and identify the method for exquisite joint melon hybrid seed purity.
Background technology
Seed purity is one of topmost quality index of seed testing.The test (being called for short DSU test) of new variety of plant specificity (Distinctness), consistence (Uniformity) and stability (Stability) is that Plant new variety protection office of the Ministry of Agriculture is to applying for that kind is authorized new variety power and carried out the most important action of examination as to substances.
Exquisite joint melon is first-filial generation joint melon kind.During the production of hybrid seeds, joint melon, the wax gourd material of parent's (male parent and female parent) multiplication fields, half-blood seed farm and other kind or type must strictly be isolated, isolation distance more than 1000 meters.Maternal and male parent branch's plantation according to a certain percentage during the production of hybrid seeds, before maternal female flower is bloomed, maternal plant is emasculation one by one.Utilize flowering period the method for insect vector or human assistance to be awarded on the female chapiter of female parent by the pollen of male parent, thus produce cenospecies.If maternal emasculation is not thorough, maternal pollen is just likely fallen on maternal female chapiter and is produced selfed seed, and occur pseudostationary, this is the main reason that production of hybrid seeds process causes seed impure; In addition, if male parent row plant fails to root out in time, also likely male parent fruit is mixed into hybridization fruit, causes and mix.Therefore cenospecies must do Purity at before sales, meets country and just can be sold to client to hybrid seed purity standard.
Traditional joint melon hybrids seed purity test is all carry out in field, morphological specificity according to plant carries out purity identification to hybrid population, because most characteristic trait needs could identify at fruiting period, hybrids seed purity test needs to complete usually for 2-3 month, the required cycle is long, and need land occupation and manpower, and qualification result is greatly affected by environment, bad climate, sick worm are caused harm and cultivation level all can make Purity accuracy reduce.20 th century later, develop rapidly along with molecular biological, make to carry out gene identification to variety DNA molecular level and analysis becomes possibility.Adopting molecular marking technique to carry out Purity Identification is a kind of quick, convenient, accurate and effective method.At present, do not set up yet in prior art joint melon hybrid seed purity authentication method.
Summary of the invention
First object of the present invention is to provide a kind of SSR primer for exquisite joint melon hybrid seed purity qualification, and this primer can produce maternal specific marker and male parent specific marker, high specificity.
The present invention also aims to the authentication method providing a kind of exquisite joint melon hybrid seed purity, the method utilizes above-mentioned SSR primer, qualification exquisite joint melon hybrid seed purity that can be quick, convenient, accurate and effective.
First object of the present invention is achieved by the following technical solution: a kind of SSR primer for exquisite joint melon hybrid seed purity qualification, described primer comprises upstream primer ChQSSR304-F and the downstream primer ChQSSR304-R of primer ChQSSR304, the nucleotide sequence of described upstream primer ChQSSR304-F is as shown in SEQIDNO.1, and the nucleotide sequence of described downstream primer ChQSSR304-R is as shown in SEQIDNO.2.
The upstream primer ChQSSR304-F of primer ChQSSR304 and the concrete nucleotide sequence of downstream primer ChQSSR304-R as follows:
ChQSSR304-F:5’-GGGGCCTGAAAATGAAGTAA-3’(SEQIDNO.1)；
ChQSSR304-R:5’-AAAACATGGTCTAGGGCTATGC-3’(SEQIDNO.2)。
This marker bands is clear, reproducible, when gel electrophoresis is carried out to pcr amplification product, electrophoresis result shows, and primer ChQSSR304 can produce the male parent specific marker SSR304185 (sequence is as shown in SEQIDNO.3) of 185bp and the maternal specific marker SSR304156 (sequence is as shown in SEQIDNO.4) of 156bp.
Second object of the present invention is achieved through the following technical solutions: a kind of authentication method of exquisite joint melon hybrid seed purity, containing following steps:
(1) genomic dna of joint melon seedling is extracted;
(2) to save melon genomic dna for template, above-mentioned SSR primer ChQSSR304 is used to carry out pcr amplification;
(3) gel electrophoresis is carried out to amplified production;
(4) electrophoresis result is analyzed, only have the individual plant simultaneously with parent's specific band to be just real cross-fertilize seed, any band lacked wherein is designated as pseudostationary, calculates seed purity, wherein, SSR primer ChQSSR304 can produce the male parent specific marker ChQSSR304 of 185bp
185and the maternal specific marker ChQSSR304 of 156bp
156.
In above steps:
The reaction system adopted during pcr amplification in step of the present invention (2) is preferably: genomic dna 10ng, 10 × PCRbuffer is (containing Mg
2+): 2 μ L, 2.5MmdNTP:1.5 μ L, SSR primer ChQSSR3040.25mmolL
-1, Taq enzyme 1U, ddH
2o complements to 20 μ L.
In step of the present invention (2), during pcr amplification, amplification program is preferably: after 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, and after 35 circulations, 72 DEG C of ends extend 7min, are placed in 4 DEG C of preservations.
8% native polyacrylamide gel electrophoresis is preferably adopted during gel electrophoresis in step of the present invention (3).
Tool of the present invention has the following advantages:
(1) the SSR primer ChQSSR304 in the present invention can produce male parent specific marker and maternal specific marker, high specificity;
(2) exquisite joint melon cenospecies and its Parent can make a distinction by method of the present invention, and rapid detection goes out the purity of cenospecies;
(3) the inventive method have fast, accurately, low cost, simple operation and other advantages, can replace the method for conventional hybridization Purity Identification, has higher commercial application value.
Accompanying drawing explanation
Fig. 1 is for being exquisite hairy squash seed Purity PCR primer polyacrylamide gel electrophoresis collection of illustrative plates (M:marker1 molecular weight standard in embodiment 1; 1: the male parent middle of the river saves No. 6; 2: No. 4, maternal More female lines; 3: the exquisite joint melon of commodity kind; Band in circle is SEQIDNO:3, and the band in rectangle is SEQIDNO:4);
Fig. 2 is partial results (the M:marker1 molecular weight standard of planting seedling seed purity sampling qualification in embodiment 2 in laboratory; 1: the male parent middle of the river saves No. 6; 2: No. 4, maternal More female lines; 21,31: pseudostationary; 3-20,22-30,32: the exquisite joint melon of sampling commodity kind);
Fig. 3 is partial results (the M:marker1 molecular weight standard of the exquisite hairy squash seed purity sampling in great Feng base in embodiment 3; 1: the male parent middle of the river saves No. 6; 2: No. 4, maternal More female lines; 3-30: the exquisite joint melon of sampling commodity kind).
Embodiment
The foundation of the exquisite hairy squash seed method for detecting purity of embodiment 1 cross-fertilize seed
1, the SSR primer of Purity is screened
Some to parent, (the male parent middle of the river saves No. 6 according to the primer of wax gourd transcript profile sequencing result design, maternal No. 4, More female lines) and filial generation between screen, select the primer ChQSSR304 of codominance difference mark band, this marker bands is clear, reproducible, and sequence is as follows:
ChQSSR304-F:5’-GGGGCCTGAAAATGAAGTAA-3’(SEQIDNO:1)
ChQSSR304-R:5’-AAAACATGGTCTAGGGCTATGC-3’(SEQIDNO:2)。
To save melon genomic dna for template, use above-mentioned SSR primer ChQSSR304 to carry out pcr amplification, carry out gel electrophoresis to amplified production, electrophoresis result shows, and SSR primer ChQSSR304 can produce the male parent specific marker ChQSSR304 of 185bp
185and the maternal specific marker ChQSSR304 of 156bp
156.
2, above-mentioned Auele Specific Primer is utilized to carry out Purity to the exquisite hairy squash seed of hybridization joint melon
The extraction of 2.1 joint melon DNA
Experiment material is tested for the exquisite joint melon of joint melon and is planted and male parent (middle of the river saves No. 6), maternal (No. 4, More female lines) cotyledon DNA, and step is as follows:
1. get a slice cotyledon and put into 2mL centrifuge tube, add the grinding of liquid nitrogen grinding pestle, add rapidly 1000 μ L2%CTAB extracting solutions when the fast evaporate to dryness of liquid nitrogen, mixing is placed on 65 DEG C of water-bath 50min (shaking up once every 10min);
2. leave standstill to room temperature centrifugal, 12000rmp, 10min, get supernatant (about 800 μ L) and be transferred to new 2mL centrifuge tube;
3. add phenol isopyknic with supernatant liquor in step 2: chloroform: iso pentane alcohol mixture, in this mixture, the volume ratio of each component is 25:24:1; Concuss, leaves standstill 5min, 12000rmp, 10min, gets supernatant liquor (about 510 μ L) and proceed in 1.5mL centrifuge tube;
4. add the Virahol of 2/3 volume 340 μ L precooling, slowly shake up (slowly putting upside down 20 times), at being placed in-20 DEG C, cultivate 1h;
5. centrifugal, 4 DEG C, 13000rmp, 10min, abandon supernatant, washs DNA1-2 time, be placed on Bechtop and dry up with 75% ethanol (volume percent) of precooling;
6. add 100 μ LTE to dissolve.
2.2SSR-PCR amplification
PCR system (20 μ L)
The program of pcr amplification is: after 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, and after 35 circulations, 72 DEG C of ends extend 7min, are placed in 4 DEG C of preservations.
2.3PAGE detected through gel electrophoresis
1. PCR primer is added 4 μ L6 × Loadingbuffer, get rid of lower rear vortex mixing;
2. 2 μ L amplified production 8% Native PAGE gel electrophoresis are got, 150V voltage stabilizing 60min;
3. PAGE glue is pulled down, use 0.1%AgNO
3solution-dyed 10min;
4. 2%NaOH, 0.04%Na is used
2cO
3, 0.4% formaldehyde colour developing 10min, after colour developing on lamp box photographic analysis.
2.4 amplification
Exquisite joint melon commodity kind amplifies 156bp and 185bp two band; The band that No. 6 amplify 185bp is saved in the male parent middle of the river; Maternal No. 4, More female lines amplifies the band (see Fig. 1) of 156bp.
Reclaim specific band, send handsome company to check order.The sequence of 185bp band is as shown in SEQIDNO:3, and in cenospecies, the sequence of 156bp band is as shown in SEQIDNO:4, conforms to the sequence of male parent, maternal amplified production.
Embodiment 2
Adopt the exquisite joint melon of 100 strain that the method for embodiment 1 is got the Purity Identification field from great Feng base, its individual plant is numbered, extract individual plant DNA to carry out detecting (see Fig. 2), detected result: cenospecies 90 strain, maternal 10 strains, seed purity is 90%, consistent with Fields detection result.
Embodiment 3
Adopt the exquisite joint melon of 100 strain that the method for embodiment 1 is got the Purity Identification field from vegetables institute base, its individual plant is numbered, extract individual plant DNA to carry out detecting (see Fig. 3), detected result is all true hybrid, seed purity is 100%, consistent with Fields detection result, accuracy rate is 100%.
Above embodiment shows, exquisite joint melon cenospecies and its female parent (No. 4, More female lines) seed can effectively be distinguished by method of the present invention, detect seed purity fast, accurately.
The present invention will be described more than to enumerate specific embodiment.It is pointed out that above embodiment is only for the invention will be further described, do not represent protection scope of the present invention, the nonessential amendment that other people prompting according to the present invention is made and adjustment, still belong to protection scope of the present invention.
Claims (5)
1. the SSR primer for exquisite joint melon hybrid seed purity qualification, it is characterized in that: described primer comprises upstream primer ChQSSR304-F and the downstream primer ChQSSR304-R of primer ChQSSR304, the nucleotide sequence of described upstream primer ChQSSR304-F is as shown in SEQIDNO.1, and the nucleotide sequence of described downstream primer ChQSSR304-R is as shown in SEQIDNO.2.
2. an authentication method for exquisite joint melon hybrid seed purity, is characterized in that: containing following steps:
(1) genomic dna of joint melon seedling is extracted;
(2) to save melon genomic dna for template, the primer ChQSSR304 in claim 1 is used to carry out pcr amplification;
(3) gel electrophoresis is carried out to amplified production;
(4) electrophoresis result is analyzed, only have the individual plant simultaneously with parent's specific band to be just real cross-fertilize seed, any band lacked wherein is designated as pseudostationary, calculates seed purity, wherein, primer ChQSSR304 can produce the male parent specific marker ChQSSR304 of 185bp
185and the maternal specific marker ChQSSR304 of 156bp
156.
3. the authentication method of exquisite joint melon hybrid seed purity according to claim 2, is characterized in that: the reaction system adopted during pcr amplification in step (2) is: genomic dna 10ng, containing Mg
2+10 × PCRbuffer2 μ L, 2.5MmdNTP:1.5 μ L, SSR primer ChQSSR3040.25mmolL
-1, Taq enzyme 1U, ddH
2o complements to 20 μ L.
4. the authentication method of exquisite joint melon hybrid seed purity according to claim 2, it is characterized in that: in step (2), during pcr amplification, amplification program is: after 95 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, after 35 circulations, 72 DEG C of ends extend 7min, are placed in 4 DEG C of preservations.
5. the authentication method of exquisite joint melon hybrid seed purity according to claim 2, is characterized in that: adopt 8% native polyacrylamide gel electrophoresis during gel electrophoresis in step (3).
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| CN110029184B (en) * | 2019-03-05 | 2020-11-03 | 广西大学 | SSR molecular marker primer for identifying purity of hybrid seeds of white gourd and application thereof |
| CN111286554B (en) * | 2020-03-18 | 2021-03-02 | 广东省农业科学院蔬菜研究所 | SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer |
| CN117385086B (en) * | 2023-11-27 | 2024-04-16 | 广东省农业科学院蔬菜研究所 | SSR (simple sequence repeat) marker primer for melon germplasm resources and construction method of fingerprint |
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