CN103233065A - Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method - Google Patents
Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method Download PDFInfo
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Abstract
The present invention provides molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera. The sequences of the primers are: an upstream primer: 5'-CCTATGGTGCCTCTTTGTTTGATGT-3'; and downstream primer: 5'-GTGACAAGTTGAACTCTAGCACAAA-3'. By employing the molecular specific marker primers of the present invention, quick identification of the No. 4 and No.32 of the improved variety Changlin of Camellia oleifera in early stage can be achieved; the method is simple, fast and accurate, so it is a molecular method that can not be substituted by using apparent characteristics to distinguish improved varieties of Camellia oleifera.
Description
(1) technical field
The present invention relates to the molecular specificity labeled primers of the long woods of oil tea breeding No. 4 and long woods No. 32, and utilize this primer the long woods of oil tea breeding No. 4 and long woods to be carried out the method for Rapid identification for No. 32.
(2) background technology
The woody oil tree species that oil tea is China's planting area maximum, distribution range is wider.Greatly develop camellia oleiferaindustry, to ensureing grain and oil safety, promote increasing peasant income, advance mountain area comprehensive exploitation and building a New Socialist Countryside, all have very important meaning.China's camellia oleiferaindustry is in the ascendant, and development potentiality is huge.At present, China's camellia oleifera lam total area reaches more than 4,500 ten thousand mu, but output is very low, and every per mu yield tea oil is only about 4 kilograms.Its basic reason is, most camellia oleifera lam variety and quality differences or serious degradation, and a large amount of countries and provincial (recognizing) the fixed improved seeds of examining are not applied.Simultaneously, some local seedling weak quality consciousness, operation control is extensive.Be to ensure the good and fast development of camellia oleiferaindustry, fully realize the oil tea breeding to the importance of development camellia oleiferaindustry, must accelerate the oil tea good seed and develop and strengthen quality control.
China mainly relies on every system that management layer is formulated, and does not obtain key breakthrough as yet at technological layer at present to the supervision and management of oil tea seedling quality, particularly lacks the early stage quick identification technology system of oil tea breeding.9 long woods kind series by national forest breeding authorization are respectively No. 3, No. 4, No. 18, No. 21, No. 23, No. 27, No. 40, No. 53, No. 55 at present.These oil tea breedings have characteristics such as real high yield, stable yields, seed-producing rate height, oleaginousness height, strong resistance, wide adaptability early.To put forth effort to solve the trouble waters of oil tea seedling in the present production to the supervision and management of oil tea seedling quality, and at first from technological layer, long woods oil tea breeding be differentiated protection.
The discriminating of oil tea kind mainly according to appearance features, but because the cycle that many morphological characterss are identified is long, affected by environment big, and kind quantity is on the increase, is made more and more difficulty of cultivar identification.Since this century, the molecular marking technique of some PCR-based such as RAPD(Random Amplified Polymorphic DNA, randomly amplified polymorphic DNA), ISSR(Inter-Simple Sequence Repea, amplification polymorphism between the simple sequence iteron) and SRAP(sequence-related amplified polymorphism, SRAP) is used for the germ plasm resource genetic diversity of oil tea in succession, genetic distance research, but chain for molecule marker and oil tea proterties, the interracial molecule Study on Identification of oil tea seldom, it is that moreover these molecule markers adopt or be general random primer or be the combination at random of team design primer, its pcr amplification collection of illustrative plates is not only complicated, poor repeatability, therefore and be not suitable for cultivar identification and specificity is not high.SCAR(Sequence Characterized Amplified Region, characteristic fragment amplification zone) mark is to be proposed on the RAPD basis by Paran and Michelmore in 1993, it is based on the order-checking to special RAPD fragment, primer according to a pair of 18～24 bases of two ends sequences Design, carry out under higher annealing temperature that specific amplified realizes, the competition between the random primer binding site has been got rid of in the employing of its specificity primer, thereby it is a kind of very stable molecule marker, have rapidly in application, easy, characteristics cheaply, up to now, the research report that does not have the SCAR tag application to differentiate in the oil tea kind as yet.
(3) summary of the invention
The object of the invention provides the molecular specificity labeled primers of the long woods of oil tea breeding No. 4 and long woods No. 32, and a kind of method that can carry out Rapid identification to the long woods of oil tea breeding No. 4 and long woods for No. 32.
The technical solution used in the present invention is:
The molecular specificity labeled primers that the long woods of oil tea breeding No. 4 and long woods are No. 32, described primer sequence is:
Upstream primer: 5 '-CCTATGGTGCCTCTTTGTTTGATGT-3 ';
Upstream primer: 5 '-GTGACAAGTTGAACTCTAGCACAAA-3 '.
This primer is to being the employing round pcr, through a large amount of shaker tests, adopt ISSR to be labeled as the DNA fragment specific that primer obtains the long woods of oil tea breeding No. 4 and long woods No. 32, with this fragment cloning order-checking, based on the dna sequence dna that obtains, designed Auele Specific Primer carries out pcr amplification No. 32 with this primer to the long woods of oil tea breeding No. 4 and long woods, can obtain the specific fragment of 350bp size.Need to prove that molecular specificity labeled primers of the present invention only limits to the evaluation (identifying whether it is long woods No. 4 or No. 32) of oil tea breeding, namely testing sample only limits to oil tea.
The invention still further relates to a kind of described molecular specificity labeled primers grows woods No. 4 and grows the method that woods carries out Rapid identification for No. 32 the oil tea breeding, described method is: extract the genomic dna of oil tea kind blade to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 350bp appears in electrophoresis result, then oil tea kind to be measured is grown woods No. 4 or is grown woods No. 32 for the oil tea breeding; Otherwise then deny;
Described molecular specificity labeled primers sequence is:
Upstream primer 5 '-CCTATGGTGCCTCTTTGTTTGATGT-3 ';
Upstream primer 5 '-GTGACAAGTTGAACTCTAGCACAAA-3 '.
The inventive method key is the selection of amplimer, and DNA extraction, PCR reaction system and reaction conditions are determined, and electrophoresis detection, all can carry out according to this area ordinary method.
Preferably, PCR reaction system of the present invention is composed as follows:
The PCR reaction conditions is as follows:
Behind 95 ℃ of pre-sex change 4min; 95 ℃ of sex change 1min, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
PCR Buffer final concentration is 1 *, refer to that the concentration of each component in reaction system is identical with 1 * PCR Buffer among the PCR Buffer, selecting volume usually for use is 10 * PCRBuffer of reaction system volume 1/10.100mM Tris-HCl(pH8.5), 500mM KCl, 25mM MgCl 10 * PCR Buffer composition is:
2And 1.0%Triton-X-100, solvent is ddH
2O.
Concrete, the method for the invention is as follows:
(1) gets oil tea blade 0.01g to be measured, add liquid nitrogen and thoroughly grind, extract the genomic dna of oil tea blade to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 25 μ l are composed as follows for the PCR reaction system:
The PCR reaction conditions is as follows:
Behind 95 ℃ of pre-sex change 4min; 95 ℃ of sex change 1min, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ l, with 1 μ l0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, electrophoresis finishes back EB dyeing (dyeing is 30 minutes in the aqueous solution that contains 0.5 μ g/m1EB), take a picture on automatic gel images analyser, if the DNA band of 350bp appears in electrophoresis result, then oil tea to be measured grows woods No. 4 or grows woods No. 32 for the oil tea breeding; Otherwise then deny.
Beneficial effect of the present invention is mainly reflected in: molecular specificity labeled primers of the present invention can be differentiated for No. 32 fast in early days to the long woods of oil tea breeding No. 4 or long woods, method is simple, quick, accurate, is that appearance features is distinguished the irreplaceable molecule means of oil tea breeding.
(4) description of drawings
Fig. 1 is for carrying out the result of pcr amplification to the oil tea breeding; M is dna molecular amount standard; The arrow indication is for being the special DNA band of 350bp from being numbered the long woods of 2 oil tea breeding No. 4 and being numbered the molecular weight that 6 the long woods of oil tea breeding amplifies for No. 32; All the other are numbered other oil tea breeding.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) extraction of oil tea fine breed gene group DNA:
Get oil tea breeding young leaflet tablet 0.01g to be measured, add liquid nitrogen and thoroughly grind, the SDS-CTAB method is adopted in the extraction of genomic dna, through repeatedly extracting, extracts the genomic dna crude extract that obtains the oil tea breeding.The DNA crude extract carries out purifying (rich day Bioer through Magabio nucleic acid purification test kit again, Hangzhou, China) after, the agarose gel electrophoresis by 1.5% and DNA/RNA ultraviolet spectrophotometer (GeneQuant Pro, GE Healthcare) detect integrity, purity and concentration.OD
260/ OD
2801.8 DNA sample is used for follow-up pcr amplification.The DNA extraction thing is standby in-20 ℃ of refrigerator storages.
(2) design specific PCR amplimer, the right sequence of primer is:
Upstream primer 5 '-CCTATGGTGCCTCTTTGTTTGATGT-3 ' and downstream primer 5 '-GTGACAAGTTGAACTCTAGCACAAA-3 ', synthetic by Shanghai biotechnology company limited.
(4) pcr amplification of SCAR molecule marker:
The PCR reaction solution is formed: 10 * PCR Buffer2.5 μ l, 10mmol/L dNTPs2.5 μ l, 25mmol/L MgCl
22.5 μ l, 5U/ μ l Taq DNA enzyme 0.2 μ l, 10mM special primer be to each 1 μ l, 20ng/ μ l template DNA 3 μ l, ddH
2O complements to 25 μ l.
Amplified reaction carries out at TC-XP type amplification instrument.Amplification condition: behind 95 ℃ of pre-sex change 4min; 95 ℃ of sex change 1min, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
(4) electrophoresis detection: get step (3) pcr amplification product 5ul, with 1ul0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, in 1 * TAB damping fluid, electrophoresis under the 5V/cm voltage, after electrophoresis finished, dyeing was 30 minutes in the aqueous solution that contains 0.5 μ g/m1EB, took a picture at the automatic gel images analyser of the clear JS-380A of training then.
According to the method described above, (numbering 1～10 represents the oil tea breeding and is followed successively by: 1: long woods No. 3 to a plurality of oil tea breedings respectively; 2: long woods No. 4; 3: long woods No. 18; 4: long woods No. 21; 5: long woods No. 27; 6: long woods No. 32; 7: long woods No. 40; 8: long woods No. 53; 9: long woods No. 55; 10: long woods No. 66; ) detect, electrophoresis result is seen Fig. 1.
Wherein be numbered the long woods of 2 oil tea breeding No. 4 and be numbered 6 the long woods of oil tea breeding No. 32, having amplified molecular weight is the special DNA band of 350bp, and all the other are numbered other oil tea breedings, and not seeing has the special DNA band of 350bp to produce.
Claims (4)
1. the molecular specificity labeled primers of the long woods of oil tea breeding No. 4 and No. 32, described primer sequence is as follows:
Upstream primer: 5 '-CCTATGGTGCCTCTTTGTTTGATGT-3 ';
Upstream primer: 5 '-GTGACAAGTTGAACTCTAGCACAAA-3 '.
2. one kind is utilized the described molecular specificity labeled primers of claim 1 that the oil tea breeding is grown the method that woods carries out Rapid identification for No. 4 and No. 32, described method is: extract the genomic dna of oil tea kind blade to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 350bp appears in electrophoresis result, then oil tea kind to be measured is grown woods No. 4 or No. 32 for the oil tea breeding; Described molecular specificity labeled primers sequence is:
Upstream primer: 5 '-CCTATGGTGCCTCTTTGTTTGATGT-3 ';
Upstream primer: 5 '-GTGACAAGTTGAACTCTAGCACAAA-3 '.
3. method as claimed in claim 2 is characterized in that described pcr amplification condition is as follows: 95 ℃ of pre-sex change 4min; 95 ℃ of sex change 1min, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
4. method as claimed in claim 2 is characterized in that described method is as follows:
(1) gets oil tea blade to be measured, add liquid nitrogen and grind, extract the genomic dna of oil tea blade to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 25 μ l are composed as follows for the PCR reaction system:
The PCR reaction conditions is as follows:
95 ℃ of pre-sex change 4min; 95 ℃ of sex change 1min, 60 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ l, with 1 μ l0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, electrophoresis finishes back EB dyeing, take a picture on automatic gel images analyser, if the DNA band of 350bp appears in electrophoresis result, then oil tea to be measured is long woods No. 4 or No. 32.
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