CN103740840B - A kind of method identifying coffee germ plasm resource - Google Patents
A kind of method identifying coffee germ plasm resource Download PDFInfo
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- CN103740840B CN103740840B CN201410031317.1A CN201410031317A CN103740840B CN 103740840 B CN103740840 B CN 103740840B CN 201410031317 A CN201410031317 A CN 201410031317A CN 103740840 B CN103740840 B CN 103740840B
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Abstract
The present invention relates to technical field of biological information, disclose a kind of method identifying coffee germ plasm resource.Is the method by standard coffee kind DNA SEQ? ID? aligning primer shown in NO:1-10 carries out pcr amplification, there is site according to the Gel electrophoresis results of amplified production with coffee species numbering and band and be numbered horizontal stroke, ordinate zou, in the detected result of each horizontal stroke, ordinate zou interconnecting part record standard coffee species band, obtain the RAPD finger printing of standard coffee kind; Increased by the same primer PCR of coffee species DNA to be measured, amplified production builds the RAPD finger printing of coffee species to be measured as stated above, and with the RAPD finger printing contrast judgement of standard coffee kind.The present invention's application RAPD molecular marking technique has carried out precise Identification to coffee germ plasm resource from inheritance, solves the problem of coffee Idioplasm identification difficulty, and simply, easy to operate, detect rapidly.
Description
Technical field
The present invention relates to technical field of biological information, relate to a kind of method identifying coffee germ plasm resource specifically.
Background technology
Coffee and cocoa, the large beverage crops in the tea world three arranged side by side, its output, consumption, economic worth all account for first place, are after oil, the raw material type product of world rankings second.Domestic main producing region is in Yunnan and Hainan, and cultivated area has reached more than 50 ten thousand mu, and plan develops into 1,000,000 ~ 1,500,000 mu to " 12 " end.The classification of coffee in the past mainly relies on morphologic method, but affects greatly by factors such as environment due to most of morphological characters, is difficult to the requirement carrying out Idioplasm identification and fine-variety breeding.DNA molecular marker technology is a kind of genetic analysis method rapidly and efficiently, be widely used in the genetic research of plant, molecular mark is applied in the research of the Important Economic crops such as paddy rice, wheat and cotton with in producing, and obtains obvious economic benefit.
DNA molecular marker can reflect the DNA fragmentation of certain difference characteristic in biont or population genome, fundamentally can disclose the gene difference of plant inherence, has been widely applied to the research field such as Identifying Crop Cultivars and germ plasm resource classification in recent years.At present, applying more is SSR, RAPD, RFLP and AFLP equimolecular mark.
Wherein, one of important means that randomly amplified polymorphic DNA (RandomAmLplifiedPolymorphicDNA, RAPD) technology is studied as modern molecular biology, has the features such as amount of samples is few, detection efficiency is high, easy and simple to handle, expense is low.Now be widely used in the researchs such as germplasm identification, genetic diversity, molecular marker breeding.But build coffee DNA fingerprinting and utilize RAPD molecular marking technique to carry out the method for coffee germplasm identification there is not been reported.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method identifying coffee germ plasm resource, enable the qualification coffee germ plasm resource that the method utilizes RAPD molecular marking technique accurate, easy.
To achieve these goals, the invention provides following technical scheme:
Identify a method for coffee germ plasm resource, comprise the following steps:
Step 1, one or more in selection standard coffee species, carry out DNA extraction respectively, then the RAPD primer of more than or two in sequence shown in SEQIDNO:1-10 is adopted to carry out pcr amplification, gained amplified production carries out detected through gel electrophoresis, there is site according to gel electrophoresis figure result with standard coffee kind numbering and the band of all standard coffee kinds and be numbered horizontal stroke, ordinate zou, horizontal at each, the detected result of ordinate zou interconnecting part record standard coffee species band, band has been labeled as by what have an amplified band, be labeled as without band without amplified band, obtain the RAPD finger printing of standard coffee kind,
Step 2, extract the DNA of coffee species to be measured, the identical RAPD primer of step 1 is adopted to carry out pcr amplification, obtained amplified production is carried out detected through gel electrophoresis, there is site according to gel electrophoresis figure result with the band of all standard coffee kinds described in coffee species numbering to be measured and step 1 and be numbered horizontal stroke, ordinate zou, horizontal at each, the detected result of ordinate zou interconnecting part record coffee species band to be measured, band has been labeled as by what have an amplified band, be labeled as without band without amplified band, obtain the RAPD finger printing of coffee species to be measured, then compare with the RAPD finger printing of described standard coffee kind and judge.
Wherein, the present invention is optimized the reaction system of pcr amplification and amplification program, avoids the error that process of the test artificially produces, and makes result more stable and reliable.
Preferably, PCR amplification system of the present invention is: reaction cumulative volume is 20 μ L, wherein Mg
2+concentration is 1.5mmol/L, dNTP concentration is 0.3mmo/L, primer concentration is 0.4 μm of ol/L, the DNA consumption of coffee species is 20ng, Taq DNA polymerase consumption is 1.0U, 2 μ L10 × PCRbuffer.
Wherein, if more than one of primer, then the concentration of every bar primer is 0.4 μm of ol/L.
Preferably, described pcr amplification program is:
94 DEG C of denaturation 3min;
94 DEG C of sex change 1min, 38 DEG C of renaturation 1min45s, 72 DEG C of extension 2min, circulate 30 times;
72 DEG C extend 7min, 4 DEG C of preservations.
Gel of the present invention is preferably agarose gel electrophoresis, and described agarose gel electrophoresis preferably adopts gum concentration to be the sepharose of 1.5% further.
Preferably, described standard coffee kind be India's large seed, Shi Long slope, Charlie fruitlet, fruit in Charlie, the large fruit of Charlie, Ka Dimu CIFC7963, CATIMORP88, CCC24, Brazil, Ka Dimu, iron finish card, short card, Cameroon, Papua New Guinea's arabica, P3, No. 26, heat grinds No. 2, No. 27, No. 24, heat grinds No. 1, No. 24-10, emerging 28, emerging 29, emerging 30, emerging 31, emerging 32, emerging 33 and/or emerging 34.
In addition, the invention provides the method for a set of simple and effective extraction coffee high quality DNA preferably, as follows:
Steps A, get the fresh blade 0.15 ~ 0.3g of coffee half ripe, in liquid nitrogen, fine powder is ground to form after shredding, add successively immediately the mixing of 0.1gPVP powder, the mixing of 1ml chloroform, 2mlExtractionbuffer, 40 μ l final concentrations 2% beta-mercaptoethanol acutely shake, mixing;
Step B, by centrifugal in EP pipe for the mashed prod after mixing, only retain precipitation, often pipe throw out adds the beta-mercaptoethanol of 600 μ llysisbuffer, 12 μ l final concentrations 2%, incubation 0.5h at violent latter 65 DEG C of shake mixing;
Step C, Xiang Guanzhong add isopyknic volume ratio phenol: chloroform: primary isoamyl alcohol is the treatment solution of 25:24:l, abundant mixing, centrifugal after forming milk sap, upper water phase transition is in a new EP pipe, add the 3M sodium acetate of 1/10 aqueous phase volume and the cold ethanol of aqueous phase equal-volume, centrifugally abandon liquid phase, the washing with alcohol throw out with 70% 2 times, be dissolved in 100 μ lTE after drying;
Step D, add the RNase solution of 5 μ l concentration 1mg/ml, 37 DEG C of water-bath 30min clear up RNA, and obtain the DNA after extracting, 4 DEG C for subsequent use.
It is coffee Germplasms (see table 1) that the present invention chooses 28 kinds of standard coffee kinds, 100 RAPD primers are adopted to increase respectively the DNA of above-mentioned standard coffee germ plasm resource, easy differentiation degree according to the specificity of above-mentioned RAPD primer pcr amplification bands of a spectrum in 28 coffee kind matter, polymorphism and banding pattern is screened, and filters out that can to amplify rich polymorphism, banding pattern between different coffee kind matter clear and can stablize the RAPD primer of key band of repetition; Then, in these primers filtered out, select to amplify polymorphism information content between above-mentioned coffee kind matter the highest, accurately can distinguish the primer of all coffee kind matter, and obtain the RAPD primer of sequence as shown in SEQIDNO:1-10 through Optimizing Reconstruction, through RAPDistance software test without coincidence.
Due to practical situation restriction, the present invention exhaustive coffee species can not verify whether above-mentioned primer can distinguish all kinds, but any one in the RAPD primer of sequence shown in SEQIDNO:1-10 of the present invention all can be distinguished 28 coffee species of the present invention and the key band obtained is different after testing, even if selected coffee species is far beyond selected by the present invention 28 kinds and use a primer of the present invention to be not enough to distinguish, so along with using the increase of quantity of primer of the present invention can distinguish coffee species completely, therefore the method for the invention can select the number of primer according to practical situation.
Table 1 standard coffee germ plasm resource table
The primer of synthetic of the present invention is through the above-mentioned checking reaching the coffee species of 28 kinds, it is clear and can stablize the key band of repetition that it can amplify rich polymorphism between different coffee kind matter, banding pattern, show its RAPD-PCR amplification being suitable for all coffee species can ensure the accuracy of height.
In the fingerprint map construction process of the method for the invention, described by have amplified band be labeled as band and by the step be labeled as without band without amplified band, band and the conventional letter without band can be had by sets itself, as "+" and "-", " √ " and "×", " having " and "None" etc., and the present invention is preferred, the described band that has been labeled as is for being labeled as 1, describedly to be labeled as without band as being labeled as 0, so that with electronics coupling, improve the efficiency of band identification and process.In addition, coffee species numbering can be X-coordinate also can be ordinate zou, and accordingly, the site numbering that band exists can be ordinate zou can be also X-coordinate, and this belongs to the difference of representation, and the present invention does not do concrete restriction.Meanwhile, during the RAPD finger printing comparison of coffee species RAPD finger printing to be measured and standard coffee kind, need completely the samely could assert it is affiliated coffee species.
For the ease of understanding the present invention's design, " there is site according to gel electrophoresis figure result with standard coffee kind numbering and the band of all standard coffee kinds and be numbered horizontal stroke, ordinate zou " mentioned in the method for the invention is explained, as follows:
First determine that the kind that amplified band number is maximum (also can select other any one kinds, there is site in the band of just conveniently adding up selecting band number maximum), then with the band of this kind for reference, in conjunction with the pillar location of other kinds, by its oneself band be designated as band in the lump with the band that other kinds do not overlap and there is site, numbering, as ordinate zou, for following table, supposes that it is gel electrophoresis figure.
Coffee species D has 4 band, based on it, in conjunction with the band of other A, B, C tri-kinds, by all bands do not overlapped in the lump from the beginning numbering be designated as band and there is site numbering D1-D5, as long as so there is site at D2-D5 band to have band and there is site without the kind of band at D1 band and just can be accredited as coffee species D, by that analogy.
For verifying the accuracy of the method for the invention, the present invention with the standard coffee germ plasm resource in table 1 for material, build the RAPD finger printing of standard coffee kind, choose two kinds of the unknowns in addition, coffee species to be measured identifies by the method for the invention, send Ruili coffee Germplasm Resources to identify these two kinds of the unknowns, coffee species to be measured simultaneously.The result identified according to the method for the invention is: wherein a strain does not belong to any one (because standard coffee kind chooses the kind that several quantitative limitation fails to identify coffee to be measured) in the RAPD finger printing of above-mentioned standard coffee kind, and another strain is accredited as short card; Ruili coffee Germplasm Resources qualification result is: wherein a strain is S795 (C.arabicaL.), and another strain is accredited as short card, and this result is consistent with the method for the invention qualification result, shows the accuracy of the method for the invention height.
In addition, other coffee germ plasm resource outside table 1 standard coffee germ plasm resource can also be built the RAPD finger printing of standard coffee kind according to the method for the invention, or existing known all standard coffee germ plasm resources are built the RAPD finger printing of standard coffee kind, so just can identify more coffee species to be measured.
From above technical scheme; the present invention chooses the RAPD primer being suitable for coffee species; application RAPD molecular marking technique carries out precise Identification to coffee germ plasm resource from inheritance; solve the problem of coffee Idioplasm identification difficulty for a long time; and it is simple, easy to operate; detect rapidly, for the precise Identification of China's coffee germ plasm resource, breeding utilization and the protection of new variety power provide important evidence, for the structure in China's coffee germ plasm resource standard fingerprint storehouse is laid a good foundation.
Accompanying drawing explanation
Figure 1 shows that the agarose gel electrophoresis figure of the embodiment of the present invention 1 standard coffee kind;
Wherein, M is the label that DNAMarker, 1-28 represent coffee species in table 1 successively;
Figure 2 shows that the RAPD finger printing of the embodiment of the present invention 1 standard coffee kind;
Wherein, X-coordinate 1-28 represents the label of coffee species in table 1 successively, and ordinate zou 1-12 represents that band exists site numbering.
Embodiment
The embodiment of the invention discloses a kind of method identifying coffee germ plasm resource.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described and method are changed or suitably change with combination, realize and apply the technology of the present invention.
In order to understand the present invention further, a kind of identify that the method for coffee germ plasm resource is described in detail below in conjunction with embodiment to provided by the invention.
Embodiment 1: the RAPD finger printing building standard coffee kind
1, DNA extraction
With the fresh blade of half ripe of coffee germ plasm resource different in table 1 for material extraction plant genomic DNA.
Get the fresh blade 0.15 ~ 0.3g of half ripe, after shredding, in liquid nitrogen, grind to form fine powder.Add the mixing of 0.1gPVP powder immediately successively, 1ml chloroform mixes, 2mlExtractionbuffer (350mM sorbyl alcohol, 100mMTris-HClpH8.0,100mMEDTApH8.0,0.5% weight sodium sulfate), add 40 μ l beta-mercaptoethanols (final concentration 2%) and acutely shake, mixing.
Mashed prod after mixing is sub-packed in the EP pipe of 2 ~ 3 1.5ml, centrifugal 10min, 4 DEG C, 5000rpm, inhales and abandons upper phase and lower floor's chloroform.Often pipe throw out adds 600 μ llysisbuffer [0.2MTris-HCl (pH8.0), 0.05MEDTA, 2MNaCl, CTAB2%], add 12 μ l beta-mercaptoethanols (final concentration 2%), incubation 0.5h at violent latter 65 DEG C of shake mixing.
Xiang Guanzhong adds isopyknic phenol: chloroform: the treatment solution of primary isoamyl alcohol (25:24:l, v/v), tubule reversing about 10 times (fully mixing), centrifugal 15min after formation milk sap, 4 DEG C, 12000rpm; Repeat extracting once.
Upper water phase transition, to a new EP tubule, adds the ethanol that the 3M sodium acetate of 1/10 aqueous phase volume, equal-volume (600 μ l) are cold, centrifugal 10min, 4 DEG C, 6500rpm, abandons liquid phase, washing with alcohol throw out with 70% 2 times, is dissolved in after drying in 100 μ lTE.
Add 5 μ lRNase (1mg/ml) solution, 37 DEG C of water-bath 30min clear up RNA, and 4 DEG C for subsequent use.
2, RAPD-PCR amplification
Reaction cumulative volume is 20 μ L, wherein Mg
2+concentration for 1.5mmol/L, dNTP concentration be 0.3mmo/L, primer (shown in SEQIDNO:2 sequence) concentration is 0.4 μm of ol/L, the DNA consumption of coffee species is 20ng, Taq DNA polymerase consumption is 1.0U, 2 μ L10 × PCRbuffer.
Set up response procedures in PCR amplification instrument, response procedures comprises
94 DEG C of denaturation 3min;
94 DEG C of sex change 1min, 38 DEG C of renaturation 1min45s, 72 DEG C of extension 2min, circulate 30 times;
72 DEG C extend 7min, 4 DEG C of preservations.
3, RAPD-PCR amplification detects
Adopt agarose gel electrophoresis method, gum concentration is 1.5%, observes and records result, the results are shown in Figure 1 after DuGreen dyeing in sky energy 4200 ultraviolet gel imaging systems;
4, the RAPD fingerprint map construction of standard coffee kind
Electronics can not be relied on to arrange out RAPD finger printing according to gel figure result, see Fig. 2, the present invention also adopts other primers to carry out structure finger printing respectively in addition, all successfully can distinguish all 28 kinds of coffee species.
Qualification work for convenience, the present embodiment sets up finger printing according to the stripe information of gel imaging, employing fingerprint collection of illustrative plates automatic recognition system Gel2.0, Crosschecker, the DNA characteristics band of IMAGEpackage to described coffee germ plasm resource identifies and processes, there is site with the band of all standard coffee kinds and be numbered ordinate zou, X-coordinate is numbered with standard coffee kind, horizontal at each, the detected result of ordinate zou interconnecting part record standard coffee species band, amplification bands of a spectrum are had to be recorded as " 1 ", the bands of a spectrum that do not increase are recorded as " 0 ", construct the RAPD finger printing of standard coffee kind.
Build finger printing according to the present embodiment method, repeat 5 times altogether, each result is all consistent, in table 2.
The RAPD finger printing of table 2 standard coffee kind
The coffee species that note: 1-28 represents is in table 1.
Embodiment 2: the qualification of coffee species to be measured
Choosing two kinds of unknown coffee species is detected materials, carries out DNA extraction, RAPD-PCR amplification, the detection of RAPD-PCR amplification according to the method in embodiment 1;
Stripe information according to gel imaging sets up finger printing, employing fingerprint collection of illustrative plates automatic recognition system Gel2.0, Crosschecker, the DNA characteristics band of IMAGEpackage to described coffee germ plasm resource identifies and processes, there is site with the band of standard coffee kinds all in embodiment 1 and be numbered X-coordinate, ordinate zou is numbered with coffee species to be measured, horizontal at each, the detected result of ordinate zou interconnecting part record coffee species band to be measured, amplification bands of a spectrum are had to be recorded as " 1 ", the bands of a spectrum that do not increase are recorded as " 0 ", construct the RAPD finger printing of coffee species to be measured, in table 3.
The RAPD finger printing of table 3 coffee species to be measured
Table 3 and table 2 are compared, unknown coffee species 1 is different from the RAPD finger printing of set up standard coffee kind, proves that it does not belong to 28 kinds of coffee species in table 1 any one; And unknown coffee species 2 is completely the same with the finger printing of the standard coffee kind numbering 12 in the RAPD finger printing of the standard coffee kind set up, be accredited as short card.
Send Ruili coffee Germplasm Resources to identify these two kinds of the unknowns, coffee species to be measured simultaneously.The result that peace is identified according to the method for the invention is: wherein a strain does not belong to any one (because standard coffee kind chooses the kind that several quantitative limitation fails to identify coffee to be measured) in the RAPD finger printing of above-mentioned standard coffee kind, and another strain is accredited as short card; Ruili coffee Germplasm Resources qualification result is: wherein a strain is S795 (C.arabicaL.), and another strain is accredited as short card, and this result is consistent with the method for the invention qualification result, shows the accuracy of the method for the invention height.
In order to prove the accuracy of the method for the invention further, according to embodiment 1 method build standard S795 (C.arabicaL.) coffee species RAPD finger printing (still with the band of all standard coffee kinds exist site numbering and standard coffee kind be numbered horizontal stroke, ordinate zou), in result and table 3, the RAPD finger printing of unknown coffee species 1 is completely the same, shows that the method for the invention has high degree of accuracy.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (4)
1. identify a method for coffee germ plasm resource, it is characterized in that, comprise the following steps:
Step 1, selection standard coffee species, carry out DNA extraction respectively, then the RAPD primer of sequence shown in SEQIDNO:2 is adopted to carry out pcr amplification, gained amplified production carries out detected through gel electrophoresis, there is site according to gel electrophoresis figure result with standard coffee kind numbering and the band of all standard coffee kinds and be numbered horizontal stroke, ordinate zou, in the detected result of each horizontal stroke, ordinate zou interconnecting part record standard coffee species band, band has been labeled as by what have an amplified band, be labeled as without band without amplified band, obtain the RAPD finger printing of standard coffee kind; Described standard coffee kind is fruit in India's large seed, Shi Long slope, Charlie fruitlet, Charlie, the large fruit of Charlie, Ka Dimu CIFC7963, CATIMORP88, CCC24, Brazil, Ka Dimu, the complete card of iron, short card, Cameroon and Papua New Guinea's arabica;
Described PCR amplification system is:
Reaction cumulative volume is 20 μ L, wherein Mg
2+concentration is 1.5mmol/L, dNTP concentration is 0.3mmo/L, primer concentration is 0.4 μm of ol/L, the DNA consumption of coffee species is 20ng, Taq DNA polymerase consumption is 1.0U, 2 μ L10 × PCRbuffer;
Described pcr amplification program is:
94 DEG C of denaturation 3min;
94 DEG C of sex change 1min, 38 DEG C of renaturation 1min45s, 72 DEG C of extension 2min, circulate 30 times;
72 DEG C extend 7min, 4 DEG C of preservations;
Step 2, extract the DNA of coffee species to be measured, the identical RAPD primer of step 1 is adopted to carry out pcr amplification, obtained amplified production is carried out detected through gel electrophoresis, there is site according to gel electrophoresis figure result with the band of all standard coffee kinds described in coffee species numbering to be measured and step 1 and be numbered horizontal stroke, ordinate zou, horizontal at each, the detected result of ordinate zou interconnecting part record coffee species band to be measured, band has been labeled as by what have an amplified band, be labeled as without band without amplified band, obtain the RAPD finger printing of coffee species to be measured, then compare with the RAPD finger printing of described standard coffee kind and judge,
Described gel electrophoresis is agarose gel electrophoresis, and described agarose gel electrophoresis adopts gum concentration to be 1.5% sepharose.
2. method according to claim 1, is characterized in that, described in be labeled as band for being labeled as 1.
3. method according to claim 1, is characterized in that, described in be labeled as without band as being labeled as 0.
4. method according to claim 1, it is characterized in that, described DNA extraction method is as follows:
Steps A, get the fresh blade 0.15 ~ 0.3g of coffee half ripe, in liquid nitrogen, fine powder is ground to form after shredding, add successively immediately the mixing of 0.1gPVP powder, the mixing of 1ml chloroform, 2mlExtractionbuffer, 40 μ l final concentrations 2% beta-mercaptoethanol acutely shake, mixing;
Step B, by centrifugal in EP pipe for the mashed prod after mixing, only retain precipitation, often pipe throw out adds the beta-mercaptoethanol of 600 μ llysisbuffer, 12 μ l final concentrations 2%, incubation 0.5h at violent latter 65 DEG C of shake mixing;
Step C, Xiang Guanzhong add isopyknic volume ratio phenol: chloroform: primary isoamyl alcohol is the treatment solution of 25:24:l, abundant mixing, centrifugal after forming milk sap, upper water phase transition is in a new EP pipe, add the 3M sodium acetate of 1/10 aqueous phase volume and the cold ethanol of aqueous phase equal-volume, centrifugally abandon liquid phase, the washing with alcohol throw out with 70% 2 times, be dissolved in 100 μ lTE after drying;
Step D, add the RNase solution of 5 μ l concentration 1mg/ml, 37 DEG C of water-bath 30min clear up RNA, and obtain the DNA after extracting, 4 DEG C for subsequent use.
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