CN102965431B - Molecular identification method for distant hybridization progeny of Sesame indicum L. and Sesame radiatum - Google Patents
Molecular identification method for distant hybridization progeny of Sesame indicum L. and Sesame radiatum Download PDFInfo
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- CN102965431B CN102965431B CN201210318496.8A CN201210318496A CN102965431B CN 102965431 B CN102965431 B CN 102965431B CN 201210318496 A CN201210318496 A CN 201210318496A CN 102965431 B CN102965431 B CN 102965431B
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Abstract
Belonging to the field of biotechnologies, the invention discloses a molecular identification method for a distant hybridization progeny of Sesamum indicum L. and Sesame radiatum. The method includes: extracting the genomic DNA of Sesame indicum L., 2n=26, the Sesamum radiatum, 2n=64 and the hybridization progeny F1, conducting PCR amplification and gel electrophoresis detection, and determining whether the distant hybridization progeny F1 is true or false according to whether parental specific bands can be amplified simultaneously. The molecular identification method for a sesame distant hybridization progeny established in the invention solves the problems of long distant hybrid identification cycle, low accuracy and the like in the process of sesame distant hybridization breeding study, and fills the vacancy of sesame distant hybridization breeding technology in China. By means of the method, the sesame distant hybridization progeny F1 can be identified efficiently and rapidly, thus laying the material and technology bases for breeding of new disease-resistant sesame varieties.
Description
Technical field
The present invention relates to a kind of plant hybridization offspring's molecular assay method, particularly relate to the molecular assay method of a kind of sesame cultivar (2n=26) and wild species (Sesamum radiatum, 2n=64) distant hybrid progeny, belong to biological technical field.
Background technology
Sesame (Sesamum indicum L., 2n=26) belongs to pedaliaceae flax and belongs to, and is in the world one of the most ancient characteristic oil crops.Sesame, at China's cultivation history of existing more than 3,000 year, because of best in quality, occupies critical role in international production and trade.But, because the disease-resistant waterlogging tolerance of sesame is poor, being subject to the impact of envrionment conditions, the yield stability of output is not good.Recent two decades comes, and carries out the key task that the disease-resistant degeneration-resistant breeding of new variety of stable high yield is China's sesame genetic breeding always.
Sesame wild species (Sesamum radiatum, 2n=64) have good disease-resistant adverse-resistant characteristic, and the disease-resistant adverse-resistant characteristic of wild species is proceeded to cultivar, are the important technology approach that improves sesame variety resistance feature.But because sesame distant hybirdization does not have affinity, offspring almost can not self-fertility, can only adopt at present that embryo rescue is cultivated, tissue cultured seedling transplanting method obtains distant hybrid progeny.Aspect distant hybrid progeny qualification, limited by investigative technique, only adopt the authentication method such as mode of appearance, biology to judge the true and false of sesame distant hybirdization F1 generation at present.Research discovery, different from seedling, between sesame distant hybrid progeny tissue cultured seedling, aspect mode of appearance, difference is not obvious, but all very remarkable with parent's difference.Therefore select the method such as mode of appearance, biology to identify distant hybrid progeny, result reliability is poor, and qualification cycle is longer.Therefore, be at present badly in need of setting up the authentication method of the efficient sesame distant hybrid progeny of a kind of fast and stable, for improving sesame distant hybridization breeding technology, accelerate sesame distant hybridization breeding paces technical support is provided.
DNA is biological genetic material, and the polymorphism of DNA is the essential content of species diversity.At present, along with development and the widespread use of molecular marking technique, the various molecular marking techniques of increasing application in offspring's qualification of plant hybridization breeding both at home and abroad, as RAPD, ISSR, AFLP, SSR equimolecular mark.Compared with conventional breeding, molecule marker can be directly with the form performance of DNA, all can detect in each tissue, each etap of organism, be not subject to season, environmental restraint, there is not the expression problem such as whether yet.Wherein, SSR mark because its polymorphism is high, codominance and good stability, chromosome position is clear is widely applied, closely linked SSR mark can be used for marker assisted selection and union breeding, and in application molecule marker carries out the qualification process of filial generation authenticity, specific primer is key point.
Summary of the invention
The object of this invention is to provide the molecular assay method of a kind of sesame cultivar and Sesamum radiatum wild species distant hybrid progeny.
In order to realize above object, the technical solution adopted in the present invention is:
The SSR primer that detects sesame distant hybrid progeny, comprising:
Primer pair HS209
Forward primer sequence: 5 '-GCAAGAAGCCGACATGTTTA-3 ' (Tm=53.35 DEG C)
Reverse primer sequence: 5 '-ACTCCAATTCCTCCACCAAG-3 ' (Tm=55.4 DEG C).
Above-mentioned primer is for the molecular assay method of sesame cultivar and Sesamum radiatum wild species distant hybrid progeny, and concrete steps are as follows:
(1) select sesame cultivar and the Sesamum radiatum wild species seed of full health, June after planting in late July plant enter the florescence, carry out artificial pollination hybridization, adopt tissue culture method to cultivate filial generation rataria, obtain distant hybirdization F
1offspring;
(2) sesame cultivar (Sesamum indicum L., 2n=26), wild species (Sesamum radiatum, 2n=64) and filial generation F in extraction step (1)
1genomic dna, for subsequent use as template;
(3) DNA profiling step (2) being extracted is applied to PCR reaction system, and operation pcr amplification program, under the guiding of primer pair HS209, obtains PCR reaction product;
(4) the PCR reaction product of step (3) is carried out to detected through gel electrophoresis, it is true hybrid that detected result has cultivar specific band 210bp and wild species specific band 200bp, 230bp's; All the other are pseudostationary.
Described sesame parent and the extracting method of filial generation genomic dna: take CTAB extraction method, get plant leaf, add the Extraction buffer of precooling, grind, 4 DEG C centrifugal, abandons supernatant, the lysis buffer that adds preheating in precipitation, 65 DEG C of water-baths, add chloroform/primary isoamyl alcohol mixed solution, mix, 15 DEG C centrifugal, gets supernatant, adds the Virahol of precooling, mix, leave standstill, centrifugal, abandon supernatant, in precipitation, add washing with alcohol, centrifugal, outwell after supernatant dryly, add TE damping fluid, more than 4 DEG C of dissolving DNA 30min, save backup in 20 DEG C.
Described Extraction buffer comprises: 0.35M Glucose, and 0.1M Tris-HCl, 5mM Na-EDTA, 2%PVP, 1% (V/V) β-Me, surplus is aseptic ultrapure water, 4 DEG C of preservations.
Described lysis buffer comprises: 1.4M NaCl, and 0.1M Tris-HCl, 20mM Na-EDTA, 2%CTAB, 2%PVP, 1% (V/V) β-Me, surplus is aseptic ultrapure water, room temperature preservation.
Described TE damping fluid comprises: 10mM Tris-HCl (PH8.0), and 1mM EDTA (PH8.0), surplus is aseptic ultrapure water.
Described PCR reaction system is: DNA masterplate 50ng, and 10 × Buffer1 μ L, 10mmolL-1dNTPs0.2 μ L, 10 μ gL-1 primer pair HS2091 μ L, 2.5U μ L-1Taq DNA polymerase0.5 μ L, adds aseptic ultrapure water to final volume 10 μ L.
Described pcr amplification program is: 94 DEG C of sex change 3min, and 94 DEG C of sex change l min, 58 DEG C of annealing 45s, 72 DEG C are extended 60s, 30 circulations, 72 DEG C of total elongation 10min; 4 DEG C of preservations.
Described detected through gel electrophoresis is: adopt native polyacrylamide gel electrophoresis, and gel strength 10%, electrophoretic buffer is 1 × TBE, 150V constant voltage electrophoresis 2 hours, after electrophoresis finishes, solidify fixed first glue, then silver dyes, rinsing, colour developing, records reading of data after rinsing gel.
Beneficial effect of the present invention:
(1) HS209SSR is labeled as specific marker (GenBank, JP643611) between the kind of independent development, and result is reliable and stable.
(2) test is carried out and is not subject to sesame production season limit, can be taking Characteristics of Sesame blade as material, and few with material, the cycle is short, the pre-treatment of material and test operation step are simple and easy to do.
(3) adopt SSR labeling technique to identify the distant hybrid progeny of wild species and cultivar sesame, filled up the blank of sesame distant hybrid progeny molecular biology identification technique.Compared with flow cytometer cytology detection method, the method does not need genome with reference to material, and data are more reliable and more stable; In addition, the technology of the present invention main points all belong to conventional biology techniques method, and cost is low, can complete at short notice the qualification of large quantities of test materialss.Can be widely used in the research such as Sesame germplasm innovation, distant hybridization breeding from now on.
Brief description of the drawings
Fig. 1 distant hybrid progeny tissue cultured seedling F
1field growing way;
The SSR Marker Identification electrophorogram of Fig. 2 parent and sesame distant hybrid progeny;
Note: 1:Marker DL2000; 2: Henan sesame 11; 3: wild sesame No. 1; 4: true hybrid F1No.IH07; 5: true hybrid F1No.IH25; 6: pseudostationary F1No.IH01; 7: pseudostationary F1No.IH13.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated, but do not form any limitation of the invention.
Embodiment
The present embodiment is for sesame Henan sesame No. 11 (Sesamum indicum L., 2n=26) and No. 1 (Sesamum radiatum, 2n=64) hybridization F of wild sesame
1the qualification in generation, concrete operation step is as follows:
(1) distant hybirdization parent and rear culture: No. 11 (the Sesamum indicum L. of sesame Henan sesame that select full health, 2n=26) with No. 1 (Sesamum radiatum of wild sesame, 2n=64) seed, June after planting in late July plant enter the florescence, carry out artificial pollination hybridization.Adopt tissue culture method to cultivate filial generation rataria, obtain distant hybrid progeny F
1totally 4 strains (Fig. 1);
(2) extraction sesame Henan sesame No. 11, wild sesame No. 1 and filial generation F
1genomic dna, for subsequent use as template;
CTAB extraction method is in a small amount taked in the extraction of genomic dna:
A. get parent, the each 2-3 sheet of remote hybrid F1 offspring tender leaf, add the freshly prepared Extraction buffer of 600 μ l precoolings, electricity turns grinding, then in 5000rpm4 DEG C of centrifugal 20min, abandons supernatant;
B. in precipitation, add the lysis buffer of 600 μ l65 DEG C preheatings, 65 DEG C of water-bath 30min, upset during this time mixes 2-3 time;
C. add 800 μ l chloroform/primary isoamyl alcohol (chloroform: primary isoamyl alcohol is 24:1) mixed solution, overturn more than 50 times, 9000rpm15 DEG C of centrifugal 20min, supernatant is proceeded in the centrifuge tube of 1.5ml, add the Virahol of the precooling of 0.6 volume, slowly overturn 30 times, mix, leave standstill 10min, the centrifugal 10min(RT of 9000rpm), abandon supernatant, in precipitation, add the washing with alcohol of 1ml70%, the centrifugal 2min of 1000rpm, outwells supernatant liquor, air seasoning 20min;
D. add 30 μ l TE damping fluids, more than 4 DEG C of dissolving DNA 30min ,-20 DEG C save backup.
Extraction buffer comprises: 0.35M Glucose, and 0.1M Tris-HCl, 5mM Na-EDTA, 2%PVP, 1% (V/V) β-Me, surplus is aseptic ultrapure water, 4 DEG C of preservations.
Lysis buffer comprises: 1.4M NaCl, and 0.1M Tris-HCl, 20mM Na-EDTA, 2%CTAB, 2%PVP, 1% (V/V) β-Me, surplus is aseptic ultrapure water, room temperature preservation.
TE damping fluid comprises: 10mM Tris-HCl (PH8.0), and 1mM EDTA (PH8.0), surplus is aseptic ultrapure water.
(3) DNA profiling step (2) being extracted is applied to PCR reaction system, and operation pcr amplification program, under the guiding of primer pair HS209, obtains PCR reaction product;
PCR reaction system is: DNA masterplate 50ng, 10 × Buffer1 μ L, 10mmolL
-1dNTPs0.2 μ L, 10 μ gL
-1primer pair HS2091 μ L, 2.5U μ L
-1taq DNA polymerase0.5 μ L, adds aseptic ultrapure water to final volume 10 μ L).
Pcr amplification program is: 94 DEG C of sex change 3min, and 94 DEG C of sex change l min, 58 DEG C of annealing 45s, 72 DEG C are extended 60s, 30 circulations, 72 DEG C of total elongation 10min; 4 DEG C of preservations.
(4) the PCR reaction product of step (3) is carried out to detected through gel electrophoresis.
Detected through gel electrophoresis is: adopt native polyacrylamide gel electrophoresis, and gel strength 10%, gel size 180mm × 120mm × 2mm, electrophoretic buffer is 1 × TBE, 150V constant voltage electrophoresis 2 hours.After electrophoresis finishes, gel is first fixed to (10% ethanol, 0.5% glacial acetic acid) 6 minutes; The silver of 0.2% silver nitrate aqueous solution infiltration afterwards dyes 12 minutes; After 2 minutes twice of distilled water rinsing, appropriateness colour developing in 1.5% sodium hydroxide and 0.4% formaldehyde mixing solutions; Last clear water rinsing gel also records reading of data.
Qualification result
Utilize HS209 primer to amplify 1 special band (210bp) male parent Henan sesame 11 parents, on No. 1 parent of the wild sesame of female parent, amplify 2 special bands (being respectively 200bp and 230bpp).According to pinciple of gene reconfiguration, real distant hybrid progeny F1 should have two parent's specific bands simultaneously.Analyze above-mentioned PCR result known, No.IH07,25 has the specific band of Henan sesame No. 11 and wild sesame No. 1 simultaneously.Therefore, judge that No.IH07,25 is No. 1 real distant hybrid progeny of Henan sesame No. 11 and wild sesame.No.IH01,13, with maternal identical, is false offspring (Fig. 2).
For verifying the reliability of above-mentioned technical evaluation result, simultaneously to distant hybrid progeny F
1carry out the qualification that biological method and fluidic cell DNA content are measured.
1. biological method
Distant hybrid progeny F
1biological character between group training transplanting plant is comparatively similar, and be not easily distinguishable seedling stage, but F
1is entirely almost sterile, and biological character obvious difference between parent.Therefore, by judging plant F
1fertility feature identify, can educate for pseudostationary, can not educate is true hybrid.
2. fluidic cell DNA content is measured
In fluidic cell DNA content detects, select soybean (Williams82, Genome Size 950Mb) as DNA content standard control.The Genome Size that Henan sesame 11 and wild sesame are No. 1 is respectively 383.60Mb, 636.50Mb, and therefore whether significant difference can be genuine distant hybrid progeny according to the Genome Size preliminary judgement of material.Use BD FACSCaibur
tmthe cell DNA content that flow cytometer carries out test material detects.Utilize DNA-ACQ software to obtain scatter plot of data and histogram, use ModFit LT software analysis to determine sample G
0/ G
1the peak variation coefficient (CV%), then, according to the 2C value (pg) of (2003) method calculation sample core DNA such as Seker, result is as table 1.
The fertility feature of table 1 sesame distant hybrid progeny and cell DNA content detect
According to table 1 plant fertility and fluidic cell DNA content qualification result, think No.IH07,25 hybrid F
1for the real distant hybrid progeny of Henan sesame No. 11 and wild sesame No. 1, No.IH01,13 hybrid F
1for pseudostationary.Two kinds of methods are all in full accord with Molecular Identification result, therefore, can think that the molecular assay method that utilizes the SSR mark of having developed to set up is applicable to the distant hybrid progeny qualification of sesame cultivar and Sesamum radiatum wild species.
Claims (6)
1. the molecular assay method of sesame cultivar and Sesamum radiatum wild species distant hybrid progeny, is characterized in that, concrete steps are as follows:
(1) select sesame cultivar and the Sesamum radiatum wild species seed of full health, June after planting in late July plant enter the florescence, carry out artificial pollination hybridization, adopt tissue culture method to cultivate filial generation rataria, obtain distant hybirdization F
1offspring;
(2) sesame cultivar, Sesamum radiatum wild species and filial generation F in extraction step (1)
1genomic dna, for subsequent use as template;
(3) DNA profiling step (2) being extracted is applied to PCR reaction system, and operation pcr amplification program, under the guiding of primer pair HS209, obtains PCR reaction product;
(4) the PCR reaction product of step (3) is carried out to detected through gel electrophoresis, what detected result had cultivar specific band 210bp and wild species specific band 200bp and 230bp is true hybrid; All the other are pseudostationary;
Described primer pair HS209 is:
Forward primer sequence: 5 '-GCAAGAAGCCGACATGTTTA-3 ',
Reverse primer sequence: 5 '-ACTCCAATTCCTCCACCAAG-3 ';
Described PCR reaction system is: DNA masterplate 50ng, 10 × Buffer1 μ L, 10mmolL
-1dNTPs0.2 μ L, 10 μ gL
-1primer pair HS2091 μ L, 2.5U μ L
-1taq archaeal dna polymerase 0.5 μ L, adds aseptic ultrapure water to final volume 10 μ L;
Described pcr amplification program is: 94 DEG C of sex change 3min, and 94 DEG C of sex change 1min, 58 DEG C of annealing 45s, 72 DEG C are extended 60s, 30 circulations, 72 DEG C of total elongation 10min; 4 DEG C of preservations.
2. molecular assay method according to claim 1, it is characterized in that, the extracting method of described genomic dna: get plant leaf, add the Extraction buffer of precooling, grind, 4 DEG C centrifugal, abandon supernatant, in precipitation, add the lysis buffer of preheating, 65 DEG C of water-baths, add chloroform/primary isoamyl alcohol mixed solution, mix, 15 DEG C centrifugal, get supernatant, add the Virahol of precooling, mix, leave standstill, centrifugal, abandon supernatant, in precipitation, add washing with alcohol, centrifugal, after outwelling supernatant, be dried, add TE damping fluid, more than 4 DEG C of dissolving DNA 30min, save backup in 20 DEG C.
3. molecular assay method according to claim 2, is characterized in that, described Extraction buffer comprises: 0.35M glucose, and 0.1M Tris-HCl, 5mM Na-EDTA, 2%PVP, 1% β-dredge base ethanol, surplus is aseptic ultrapure water.
4. molecular assay method according to claim 2, is characterized in that, described lysis buffer comprises: 1.4M NaCl, and 0.1M Tris-HCl, 20mM Na-EDTA, 2%CTAB, 2%PVP, 1% β-dredge base ethanol, surplus is aseptic ultrapure water.
5. molecular assay method according to claim 2, is characterized in that, described TE damping fluid comprises: 10mM Tris-HCl, and 1mM EDTA, surplus is aseptic ultrapure water; The pH value that the pH value of described Tris-HCl is 8.0, EDTA is 8.0.
6. molecular assay method according to claim 1, it is characterized in that, detected through gel electrophoresis is: polyacrylamide concentration is 10%, and electrophoretic buffer is 1 × TBE, 150V constant voltage electrophoresis 2 hours, after electrophoresis finishes, solidify fixed first glue, then silver dyes, rinsing, colour developing, records reading of data after rinsing gel.
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| CN113584018A (en) * | 2021-08-24 | 2021-11-02 | 海南师范大学 | Improved CTAB method for extracting nucleic acid from mallotus japonicus |
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|---|---|---|---|---|
| CN102154459A (en) * | 2011-01-07 | 2011-08-17 | 哈尔滨师范大学 | Inter-simple sequence repeat ISSR-SCAR marker specific to E-group chromosomes of agropyron elongatum |
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| CN102154459A (en) * | 2011-01-07 | 2011-08-17 | 哈尔滨师范大学 | Inter-simple sequence repeat ISSR-SCAR marker specific to E-group chromosomes of agropyron elongatum |
Non-Patent Citations (4)
| Title |
|---|
| Development and validation of genic-SSR markers in sesame by RNA-seq;Zhang et al.;《BMC Genomics》;20120716;第13卷(第316期);第3页及附件1的表格S1 * |
| Zhang et al..Development and validation of genic-SSR markers in sesame by RNA-seq.《BMC Genomics》.2012,第13卷(第316期),第3页及附件1的表格S1. |
| 张平湖.花生杂交Fl代真假杂种SSR标记鉴定体系的建立.《广东农业科学》.2009,(第10期),49-50. |
| 花生杂交Fl代真假杂种SSR标记鉴定体系的建立;张平湖;《广东农业科学》;20091231(第10期);49-50 * |
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