CN105063223A - Method for identifying coffee germplasm resource by using ISSR (inter-simple sequence repeat) fingerprint chromatography - Google Patents
Method for identifying coffee germplasm resource by using ISSR (inter-simple sequence repeat) fingerprint chromatography Download PDFInfo
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- CN105063223A CN105063223A CN201510556607.2A CN201510556607A CN105063223A CN 105063223 A CN105063223 A CN 105063223A CN 201510556607 A CN201510556607 A CN 201510556607A CN 105063223 A CN105063223 A CN 105063223A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to the technical field of biological information, and discloses a method for identifying a coffee germplasm resource by using ISSR (inter-simple sequence repeat) fingerprint chromatography. The method comprises the following steps: carrying out PCR (polymerase chain reaction) amplification on primer/primers disclosed as SEQ ID NO:1 and/or 2 by using standard coffee species DNA (deoxyribonucleic acid), establishing horizontal and vertical coordinates by using coffee species number and strip existence site number according to the gel electrophoresis result of the amplification product, and recording the detection result of the standard coffee species strip at each horizontal/vertical coordinates cross site to obtain an ISSR fingerprint chromatogram of the standard coffee species; and carrying out PCR amplification on the to-be-detected coffee species DNA by using the same primer, constructing the ISSR fingerprint chromatogram of the to-be-detected coffee species of the amplification product by the process above, and carrying out comparison judgment with the ISSR fingerprint chromatogram of the standard coffee species. The method performs accurate identification on the coffee germplasm resource from the essence of inheritance by using the ISSR molecular marker technique, solves the problem of difficulty in coffee germplasm identification, and is simple and easy to operate and quick for detection.
Description
Technical field
The present invention relates to technical field of biological information, relate to a kind of method of ISSR finger print identification 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 a kind of tropical cash crop of high added value.Domestic main producing region is in Yunnan and Hainan, and cultivated area has reached more than 180 ten thousand mu, and output more than 11 ten thousand tons, the output value reaches 1,600,000,000 yuan.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, ISSR, RAPD and AFLP equimolecular mark.Wherein, ISSR (Inter-simplesequencerepeat), i.e. simple sequence repeats interval amplification, it is a kind of molecular marking technique being equaled establishment in 1994 by Montreal, CAN university Zietkiewicz, its principle holds grappling 1-4 base as primer at 3 ＇ of SSR or 5 ＇, one section of sequence both sides to reversed arrangement SSR increases, instead of amplification SSR itself.Anchored base avoids the slip of SSR on genome, thus substantially increases stability and the repeatability of pcr amplification.
ISSR molecular marking technique has the advantage of SSR, RAPD, RFLP, AFLP equimolecular mark concurrently, and compared with SSR, ISSR does not need know sequence information in advance and greatly reduce costs, and polymorphism is abundanter; Compared with RAPD, ISSR annealing temperature, at about 55 DEG C, ensure that stability and the repeatability of pcr amplification; Compared with RFLP, AFLP, ISSR utilizes the normal simple repeated sequence design primer occurred in Plant Genome, and without the need to Cloning and sequencing in advance, technical requirements is not high, speed, with low cost.At present, ISSR labeling technique is widely applied in the researchs such as the precise Identification of germ plasm resource, genetic diversity, the assignment of genes gene mapping, genetic map construction and molecular marker assisted selection.But build coffee DNA fingerprinting and utilize ISSR molecular marking technique to carry out the method for coffee germplasm identification there is not been reported, and the ISSR finger print identification of the germ plasm resource of other species often needs nearly several ISSR primers to carry out the specificity of each kind DNA bands of a spectrum of pcr amplification, this is very inconvenient in the qualification process of reality.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method utilizing ISSR finger print identification coffee germ plasm resource, enable the method adopt the less primer pair coffee germ plasm resource of one to two by accurate, the easy qualification of ISSR molecular marking technique.
To achieve these goals, the invention provides following technical scheme:
Utilize a method for ISSR finger print identification 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 ISSR primer in sequence shown in SEQIDNO:1 and/or 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, 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 ISSR finger printing of standard coffee kind,
Step 2, extract the DNA of coffee species to be measured, the identical ISSR 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 ISSR finger printing of coffee species to be measured, then compare with the ISSR 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 4min;
94 DEG C of sex change 15s, 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C or 55 DEG C of renaturation 1min, 72 DEG C of extension 78s, circulate 40 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 1.3% further.
In the specific embodiment of the present invention, it is that coffee Germplasms (see the table 1) accuracy to the method for the invention is verified that the present invention have chosen 30 kinds of standard coffee kinds.Due to practical situation restriction, the present invention exhaustive coffee species can not verify whether above-mentioned primer can distinguish all kinds in this area, but the present invention only needs the ISSR primer of sequence shown in SEQIDNO:1 can distinguish 28 kinds of 30 coffee species of the present invention, only has aligning primer shown in 2 kinds of needs SEQIDNO:2 jointly to identify.
In addition, other coffee germ plasm resource outside table 1 standard coffee germ plasm resource can also be built the ISSR finger printing of standard coffee kind according to the method for the invention, or existing known standard coffee germ plasm resource is built the ISSR finger printing of standard coffee kind, so just can identify more coffee species to be measured.
Table 1 standard coffee germ plasm resource table
| Numbering | Coffee species | Numbering | Chinese name | Numbering | Chinese name |
| 1 | No. 27 | 11 | Emerging 30 | 21 | Card Supreme Being is not (Brazil) |
| 2 | No. 24 | 12 | Emerging 29 | 22 | CTM1 |
| 3 | Heat grinds No. 1 | 13 | Emerging 28 | 23 | CTM2 |
| 4 | 24-2 | 14 | No. 26 | 24 | CTM4 |
| 5 | No. 24-10 | 15 | Heat grinds No. 2 | 25 | CTM19 |
| 6 | No. 24-11 | 16 | S288 | 26 | Huang Guo |
| 7 | Emerging 34 | 17 | Cameroon | 27 | Purple leaf |
| 8 | Emerging 33 | 18 | Meng Duonuowo | 28 | S28 |
| 9 | Emerging 32 | 19 | Ka Dula (height) | 29 | Card finished by iron |
| 10 | Emerging 31 | 20 | No. 2, Malaysia | 30 | Ripple nation |
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, described in be labeled as strip markers be 1, described in be labeled as without strip markers be 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 ISSR finger printing comparison of coffee species ISSR 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 ISSR 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, these two kinds of the unknowns, coffee species to be measured and Institute of Spices and Beverage's coffee Germplasm Resources live body conserving species matter are carried out contrast simultaneously and identify.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 ISSR finger printing of above-mentioned standard coffee kind, and another strain is accredited as emerging 28; Institute of Spices and Beverage's coffee Germplasm Resources qualification result is: wherein a strain is emerging 1, and another strain is accredited as emerging 28, 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 ISSR 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 ISSR 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 ISSR primer being suitable for coffee species; application ISSR 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 embodiment of the present invention 1 is with the agarose gel electrophoresis figure after SEQIDNO:1 primer amplification standard coffee kind;
Wherein, M is the label that DNAMarker, 1-30 represent coffee species in table 1 successively;
Figure 2 shows that the embodiment of the present invention 1 is with the agarose gel electrophoresis figure of SEQIDNO:2 primer amplification standard coffee kind;
Wherein, M is the label that DNAMarker, 1-30 represent coffee species in table 1 successively.
Embodiment
The embodiment of the invention discloses a kind of method utilizing ISSR finger print identification 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.
The present invention can adopt this area common method to extract DNA in the process extracting DNA, and commercial DNA extraction kit also can be adopted to extract.In specific implementation process of the present invention, sky root plant genes group DNA extraction kit is adopted to extract plant genomic DNA, utilize agarose gel electrophoresis and total spectrophotometer to detect DNA quality simultaneously, make the STb gene integrity of extraction good, and A260/A280 ratio is between 1.6-2.0, be diluted to 10ng/ μ L ,-80 DEG C save backup.
In order to understand the present invention further, be described in detail below in conjunction with the method for embodiment to a kind of ISSR of utilization finger print identification coffee germ plasm resource provided by the invention.
Embodiment 1: the ISSR 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, plant genomic DNA is extracted by sky root plant genes group DNA extraction kit, agarose gel electrophoresis and total spectrophotometer is utilized to detect DNA quality, make the STb gene integrity of extraction good, and A260/A280 ratio is between 1.6-2.0, be diluted to 10ng/ μ L ,-80 DEG C save backup;
2, ISSR-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:1-2 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 4min;
94 DEG C of sex change 15s, 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C or 55 DEG C of renaturation 1min, 72 DEG C of extension 78s, circulate 40 times;
72 DEG C extend 7min, 4 DEG C of preservations.
3, ISSR-PCR amplification detects
Adopt agarose gel electrophoresis method, gum concentration is 1.3%, observes and records result, the results are shown in Figure 1 and Fig. 2 after DuGreen dyeing in sky energy 4200 ultraviolet gel imaging systems;
4, the ISSR fingerprint map construction of standard coffee kind
For convenience of later qualification work, 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 ISSR finger printing of standard coffee kind.
Build finger printing according to the present embodiment method, in table 2, table 3, gel figure is shown in Fig. 1 and Fig. 2.
ISSR (SEQIDNO:1 primer) finger printing of table 2 standard coffee kind
ISSR (SEQIDNO:2 primer) finger printing of table 3 standard coffee kind
The coffee species that note: 1-30 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, ISSR-PCR amplification, the detection of ISSR-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 ISSR finger printing of coffee species to be measured, in table 4 and table 5.
ISSR (SEQIDNO:1 primer) finger printing of table 4 coffee species to be measured
ISSR (SEQIDNO:2 primer) finger printing of table 5 coffee species to be measured
Table 4 and table 2, table 5 and table 3 are compared respectively, unknown coffee species 1 is different from the ISSR finger printing of set up standard coffee kind, proves that it does not belong to 30 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 13 in the ISSR finger printing of the standard coffee kind set up, be accredited as emerging 28.
These two kinds of the unknowns, coffee species to be measured and Institute of Spices and Beverage's coffee Germplasm Resources live body conserving species matter are contrasted simultaneously, qualification result is: wherein a strain is emerging 1, and another strain is accredited as emerging 28, 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, the ISSR finger printing (still there is site numbering with the band of all standard coffee kinds and standard coffee kind is numbered horizontal stroke, ordinate zou) of standard emerging 1 coffee species is built according to the method for embodiment 1, in result and table 4, table 5, the ISSR 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 (8)
1. utilize a method for ISSR finger print identification coffee germ plasm resource, it is characterized in that, comprise the following steps:
Step 1, one or more in selection standard coffee species, carry out DNA extraction respectively, then the ISSR primer in sequence shown in SEQIDNO:1 and/or 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, 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 ISSR finger printing of standard coffee kind,
Step 2, extract the DNA of coffee species to be measured, the identical ISSR 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 ISSR finger printing of coffee species to be measured, then compare with the ISSR finger printing of described standard coffee kind and judge.
2. method according to claim 1, it is characterized in that, described standard coffee kind is:
No. 27, No. 24, heat grind No. 1,24-2, No. 24-10,24-11, emerging 34, emerging 33, emerging 32, emerging 31, emerging 30, emerging 29, emerging 28, No. 26, heat grind No. 2, S288, Cameroon, Meng Duonuowo, Ka Dula, No. 2, Malaysia, Ka Dimu (Brazil), CTM1, CTM2, CTM4, CTM19, Huang Guo, purple leaf, S28, iron finish block, ripple nation.
3. method according to claim 1, it is characterized in that, 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.
4. method according to claim 1, it is characterized in that, described pcr amplification program is:
94 DEG C of denaturation 5min;
94 DEG C of sex change 30s, 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C or 55 DEG C of renaturation 1min, 72 DEG C of extension 1.5min, circulate 38 times;
72 DEG C extend 10min, 4 DEG C of preservations.
5. method according to claim 1, is characterized in that, described in be labeled as band for being labeled as 1.
6. method according to claim 1, is characterized in that, described in be labeled as without band as being labeled as 0.
7. method according to claim 1, it is characterized in that, described gel electrophoresis is agarose gel electrophoresis.
8. method according to claim 7, is characterized in that, described agarose gel electrophoresis adopts gum concentration to be 1.3% sepharose.
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