CN106755328A - A kind of construction method of broad bean SSR finger-prints - Google Patents
A kind of construction method of broad bean SSR finger-prints Download PDFInfo
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- CN106755328A CN106755328A CN201611062135.6A CN201611062135A CN106755328A CN 106755328 A CN106755328 A CN 106755328A CN 201611062135 A CN201611062135 A CN 201611062135A CN 106755328 A CN106755328 A CN 106755328A
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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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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- 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 present invention provides a kind of construction method of broad bean SSR finger-prints, belongs to plant variety and differentiates and breeding technical field.The present invention provides 21 pairs of mark combinations of broad bean genome SSR primers composition, its nucleotide sequence is respectively as shown in SEQ ID NO.1 42, Vicia Faba is identified using PCR amplification techniques, native polyacrylamide gel electrophoresis or capillary electrophoresis detection technology, Broad Bean Seeds cultivar identification can be in a short time completed to be worked with Genetic diversity evaluation, with time saving, efficient, the advantages of quick, accurate, easy to operate, qualification result is difficult affected by environment.The method of the present invention can carry out effective monitoring to the Broad Bean Seeds true and false; hereditary variation and the affiliation of kind are disclosed from DNA level; protect crop varieties; prevent fake and forged kind from entering market; also for reasonable utilization of excellent germplasm in Vicia Faba Breeding Process provides technical support, have a good application prospect.
Description
Technical field
The present invention relates to molecular biology and plant variety authentication technique field, more particularly to a kind of broad bean SSR fingerprints
The finger-print of the construction method of collection of illustrative plates and the different cultivars broad bean obtained using structure is differentiated the method for Vicia Faba and answered
With.
Background technology
Broad bean (Vicia faba L.) as a kind of grain, vegetables and feed, green manure dual-purpose crop, in China crops
In occupy critical role.Broad bean has production and is distributed in each continent in the world, and the whole world has more than 43 country's production broad beans.Broad bean
In the cultivation history in China's existing more than 2000 years, in addition to Shandong, Hainan and three provinces in the northeast of China seldom plantation, remaining various regions has in the whole nation
Plantation.Nearly 10 years, the cultivated area of Chinese broad bean was up to more than 140 ten thousand hm2, the total cultivated area 50% or so in the world is accounted for, it is the world
One broad bean big producer.Broad bean can by with rhizobium symbiosis dross, the nitrogen in fixed air improving soil nutrient structure
In play an important role.Broad bean is nutritious, and protein average content is 27.6%, and vitamin content exceedes rice and small
Wheat.Therefore the effective examination for carrying out Vicia Faba is particularly important.
It is according to NY/T2345-2013 that current China carries out identification main method to Vicia Faba《New variety of plant is special
Property, uniformity and stability test guide broad bean》Field test is carried out to identify.The method is easy, economical, but due to many
Morphologic Characters qualification cycles is long, easily affected by environment, it is impossible to it is accurate, quickly for new varieties case of encroachment of right provide identify according to
According to while different testers also often occur has deviation to the understanding of some proterties, causing the problem that result of determination is inconsistent.
With the utilization centralization of breeding parent, traditional Identification of morphology and cytology method test character is more, workload big, it is difficult to
Accomplish accurately and really to identify the breed difference of broad bean, need to be analyzed identification on a molecular scale for this.
With the development of biotechnology, the DNA fingerprinting set up based on molecular marking technique
(fingerprint) played an important role in terms of kind, strain discriminating.The structure of kind DNA fingerprint database is in broad bean
Variety and authenticity identification, corresponding seed quality criteria, variety certification and property right registration protection, true and false kind is distinguished, kind is entangled
The aspect such as confused has significant application value, is probing into affiliation, Heterotic Groups division, the germplasm of China's resource of broad bean germplasm
The aspect such as germplasm innovation and breeding of new variety plays remarkable effect.It is currently used in the mark for building crop DNA fingerprinting
Note technology mainly has RFLP, RAPD, AFLP, EST, ISSR, SSR etc..Simple repeated sequence (Simple sequence
Repeat, SSR), it is the molecular marking technique of second generation PCR-based technology.Compared with other types of molecules are marked, SSR marker
Have the advantages that rich polymorphism, hereditary information are more, operation is convenient, codominance, high performance reproducibility, be widely used in planting
In the genetic research of goods and materials source and marker assisted selection practice.
DNA fingerprinting can identify interracial difference or genetic similarity from DNA level.Microsatellite (also known as
It is simple repeated sequence, SSR) section of DNA that repeatedly constitutes of the short sequence tandem sequence repeats that are made up of 1-6 nucleotides.By
The number of repetition of repeat unit is different in microsatellite, thus the length of the microsatellite sequence for amplifying is presented polymorphism.It is micro- to defend
Star sequence has polymorphism higher, repeatability and the features such as good stability, is highly effective molecular labeling, is widely used in
The fields such as cultivar identification, fingerprint map construction.In order to effectively protect the kind intellectual property of China's broad bean, it is necessary to opened in China
Exhibition broad bean exploitation microsatellite marker exploitation, and it is applied to the research of broad bean improved seeds " molecular identity card " structure.
The content of the invention
It is an object of the invention to provide a kind of construction method of broad bean SSR finger-prints, another object of the present invention exists
The method of a kind of efficiently, accurate, low cost utilization genome SSR finger-prints discriminating Vicia Faba in offer.It is of the invention
Purpose also resides in the application for providing the method.
To achieve the above object, the present invention with 48 parts of representative Vicia Faba genomic DNAs be template, by analyzing broad bean
Genome sequence and est sequence, synthesize core genome SSR and EST-SSR primer.By screening, there are 15 pairs of genomes respectively
The molecular labeling combination of SSR and 6 pair of EST-SSR primers composition can completely differentiate 48 parts of differences of Vicia Faba.Described 48 parts
Representative Vicia Faba is shown in Table 1.
1 48 parts of Vicia Fabas of table
By repeatedly screening, selection being uniformly distributed in linkage group, polymorphism is higher, PCR amplifications are relatively stablized, while full
Foot routine native gel electrophoresis detection and capillary fluorescence electrophoresis detect two kinds of technology platforms, finally filter out in Vicia Faba
Between polymorphism information content values highest, loci number are most, reproducible, amplification banding pattern definition is high, chromosome distribution is equal
The core primers that even SSR primers build as Vicia Faba fingerprint databases.
Based on above-mentioned screening technique, the molecular labeling combination the present invention is provided to differentiate Vicia Faba, it is characterised in that
Containing 15 SSR molecular markers and 6 EST-SSR molecular labelings, respectively SSR11908, EST181, SSR11771,
SSR12080、SSR13683、SSR12396、SSR14328、EST1672、EST1280、EST1741、SSR17991、
SSR10911、SSR13106、SSR760、EST1142、SSR11052、SSR13771、SSR11885、SSR12536、
SSR12868、EST1584；
The molecular labeling is obtained by following primer pair amplifies respectively：SEQ ID NO.1-2、SEQ ID NO.3-4、
SEQ ID NO.5-6、SEQ ID NO.7-8、SEQ ID NO.9-10、SEQ ID NO.11-12、SEQ ID NO.13-14、
SEQ ID NO.15-16、SEQ ID NO.17-18、SEQ ID NO.19-20、SEQ ID NO.21-22、SEQ ID NO.23-
24、SEQ ID NO.25-26、SEQ ID NO.27-28、SEQ ID NO.29-30、SEQ ID NO.31-32、SEQ ID
Shown in NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42
Primer.
The present invention provides a kind of primer combination, and containing following 21 pairs of specific primers pair, its nucleotide sequence is respectively such as SEQ
Shown in ID NO.1-42.
The present invention provides the kit containing the combination of above-mentioned primer.
The invention provides the application of the combination of above-mentioned molecular labeling or primer combination in Vicia Faba is identified.
The invention provides the application of the combination of above-mentioned molecular labeling or primer combination in resource of broad bean germplasm improvement.
The invention provides the application of the combination of above-mentioned molecular labeling or primer combination in broad bean SSR plant collection of illustrative plates is built.
The present invention also provides a kind of construction method of Vicia Faba SSR finger-prints, with of the present invention containing above-mentioned
Performing PCR amplification is entered in the primer combination of 21 pairs of primers to the DNA of different Vicia Fabas, and non denatured polyacrylamide is carried out to PCR primer
Amine gel electrophoresis is detected or PCR primer is carried out fluorescent capillary electrophoresis tube detection,
The allelic variation size data in homozygosis site is recorded as X/X, and wherein X is the size of the site allelic variation；
The allelic variation data of heterozygous sites are recorded as X/Y, and wherein X, Y is two different allelic variations on the site；
The size of invalid allelic variation is recorded as 0/0；
It is small fragment before above-mentioned "/", is large fragment after "/"；
By the Data Integration to different loci, the SSR finger-prints of different Vicia Fabas are formed.
Wherein, PCR amplification method is：
Response procedures are：95 DEG C of predegeneration 5min；95 DEG C of denaturation 35s, 60 DEG C of annealing 40s, 72 DEG C extend 45s, totally 32
Circulation；72 DEG C of extensions 5min, 10 DEG C of insulation 5min；
10 μ L amplification systems are：Upstream and downstream primer each 1 the μ L, DNA of 2 × Taq PCR Master Mix5 μ L, 2 μM/μ L
Template 1.5 μ L, ddH2O 2.5μL。
Wherein, it is using 8% non denatured polypropylene when electrophoresis detection is carried out using non-denaturing polyacrylamide gel
Acrylamide gel carries out electrophoresis, voltage 300V, electric current 280mA, power 260W.
It is that primer adds fluorescence labeling, details to be shown in Table 2 when being detected using fluorescent capillary electrophoresis tube.
The invention provides the Vicia Faba finger-print obtained using above-mentioned construction method structure.
The present invention builds the Vicia Faba finger-print for obtaining, and it is as shown in table 5- tables 16.
The present invention and then a kind of method for identifying Vicia Faba of offer, comprise the following steps：
(1) DNA of Vicia Faba to be checked is extracted, 21 pairs of primer pairs in being combined with the primer described in claim 2 are distinguished
DNA to examining Vicia Faba enters performing PCR amplification；
(2) amplified production is detected that silver staining develops the color, according to amplified production by native polyacrylamide gel electrophoresis
Relative position on running gel；Or
Fluorescent capillary electrophoresis tube detection is carried out to amplified production, according to the relative position of amplified production,
The allelic variation size data in homozygosis site is recorded as X/X, and wherein X is the size of the site allelic variation；
The allelic variation data of heterozygous sites are recorded as X/Y, and wherein X, Y is two different allelic variations on the site；
The size of invalid allelic variation is recorded as 0/0；It is small fragment before above-mentioned "/", is large fragment after "/"；
(3) the Vicia Faba finger-print of result table of comparisons 5- tables 16, Differences number of sites >=3 are then different product
Kind；Differences number of sites<3, then it is approximate kind.
The invention provides application of the above method in resource of broad bean germplasm improvement.
The construction method of broad bean DNA fingerprinting of the present invention, make use of SSR primers to combine to enter a certain Vicia Faba DNA
Row amplification, obtains one group of distinctive DNA fingerprint, and then the inventive method is entered using the method that primer combination differentiates to Vicia Faba
Row analysis and screening, make each kind have found its distinctive DNA fingerprint.The expansion of SSR and EST-SSR primers of the invention
Difference in size substantially, can be good at distinguishing different kinds between volume increase thing banding pattern.The amplification between same intravarietal individuality
Product, its banding pattern is consistent.Detection method of the invention can complete Broad Bean Seeds cultivar identification and genetic diversity in a short time
Property appraisal, with the advantage such as efficient, accurate, inexpensive, easy to operate.Detection method of the invention can be effective simultaneously
The Broad Bean Seeds true and false is monitored, hereditary variation and the affiliation of kind is disclosed from DNA level, protect crop varieties,
Prevent fake and forged kind from entering market, also for reasonable utilization of excellent germplasm in Vicia Faba Breeding Process provides technology ginseng
Examine.
Brief description of the drawings
Fig. 1 is 48 parts of representative Vicia Faba cluster analysis result figures.
Fig. 2 is 48 parts of PCR amplification electrophoretograms of representative Vicia Faba in embodiment 2.
Specific embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to the present invention
Scope.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment；
If not specializing, agents useful for same is commercially available in embodiment.
Kind decision principle of the present invention is carried out in accordance with following national standard, NY/T 2594-2014 plant variety identification of dna
Fingerprint method general provisions；NY/T 2345-2013《New variety of plant specificity, uniformity and stability test guide broad bean》；GB/T
19557.1 neies variety of plant specificity, uniformity and stability test guide general provisions.
Embodiment 1 is used for the screening of the SSR and EST-EST primers for identifying Vicia Faba
The present embodiment is with representative 48 broad bean (the being shown in Table 1) variety genome DNAs promoted in China's broad bean main producing region as mould
Plate, by analyzing broad bean genome sequence and est sequence, synthesizes core genome SSR and EST-SSR primer.By screening, point
The molecular labeling combination not being made up of 15 couples of genome SSR and the 6 pairs of EST-SSR primers can completely differentiate 48 parts of differences of Vicia Faba
It is different.
Final screening obtains 15 pairs of genome SSR molecular markers and 6 pairs of EST-SSR molecular labelings.
2 21 pairs of SSR core primers of table
The primer of above-mentioned table 1 corresponds to the SEQ ID NO.1-42 in sequence table respectively.
The structure of the Vicia Faba characteristic fingerprint pattern of embodiment 2 and the foundation of Vicia Faba authentication method
1st, Vicia Faba Genome DNA extraction
Experimental cultivar DNA is extracted using modified CTAB method：The fresh young leaflet tablet of 2g or so is taken, is pulverized in liquid nitrogen
Last shape is fitted into 2.0ml centrifuge tubes, is put into -80 DEG C of Refrigerator stores stand-by；65 DEG C of 2 × CTAB extract solutions will be preheating to, by accounting for
The ratio of the 1% of solution volume adds beta -mercaptoethanol, fully mixes；Ground leaf tissue is taken out, is added in every part of sample
Enter the CTAB extract solutions after 800 μ L preheatings, vortex 1-2min, 65 DEG C of water-bath 1h (turn upside down per 15min and gently turn over once, fully
Mix)；After heating water bath terminates, 800 μ L chloroforms/isoamyl alcohol (24 is added：1) solution mixes 15-20min in centrifuge tube,
The 15min of 10000rpm centrifugations afterwards；600 μ L or so supernatant is drawn to move to new centrifuge tube (amount can be reduced suitably, but can't be connect
Touch protein layer), 95% isometric ethanol is added afterwards, shake up and place 2h or 4 DEG C of refrigerator overnight after -20 DEG C of refrigerators, it is ensured that
Fully precipitate；12000rpm is centrifuged 10min, outwells supernatant, retains precipitation；White precipitate is cleaned with the μ L of 90% ethanol 500,
10000rpm is centrifuged 5min, carefully outwells supernatant；Room temperature is air-dried, and adds the 100 abundant dissolution precipitations of μ LddH2O.Utilize
Nanodrop2000/2000C instruments detect sample DNA concentration, and DNA concentration is diluted into 50ng/ μ L, and 4 DEG C save backup.
2nd, with 48 parts of Vicia Fabas described in embodiment 1 as material, using embodiment 1 screen obtain for expand 21 points
DNA of the primer of son mark respectively to 48 parts of representative Vicia Fabas described in embodiment 1 enters performing PCR amplification, and response procedures are：
95 DEG C of predegeneration 5min；95 DEG C of denaturation 35s, 60 DEG C of annealing 40s, 72 DEG C of extension 45s, totally 32 circulations；72 DEG C extend 5min, 10
DEG C insulation 5min；10 μ L amplification systems are：Each 1 μ L of upstream and downstream primer of 2 × Taq PCR Master Mix5 μ L, 2 μM/μ L,
DNA profiling 1.5 μ L, ddH2O 2.5μL。
3rd, pcr amplification product gel electrophoresis and silver staining develop the color：Amplified production is using 8% non-denaturing polyacrylamide gel electricity
Swimming detection, electrophoretic buffer is 0.5 × TBE, 200V voltage stabilizing electrophoresis 2-2.5h, electrophoresis when moving on to gel bottom to sample loading buffer
Terminate.Silver staining develops the color, film recording.
(1) glass plate cleaning clear water and detergent fully clean up flat, recessed glass plate, are then rushed with distilled water
Wash, be placed in glass plate rack and dry；To prevent thering is bubble to produce during encapsulating, glass plate is cleaned with alcohol before encapsulating, dried standby；Will
The glass plate level for assembling is put together, is fixed with clip.
(2) encapsulating adds the μ L of the TEMED 45 and μ LTEMED of 10% ammonium persulfate 450 in 45ml 8%PAGE glue
(usage amount of TEMED and ammonium persulfate should make corresponding adjustment according to environment temperature), encapsulating after rapid mixing.Treat that glue is full of
During glass plate interlayer, comb (comb insertion depth is voluntarily adjusted according to applied sample amount) is gently inserted on top, make its polymerization 25min
Left and right (specific polymerization time should make corresponding adjustment with varying environment temperature).During encapsulating, the generation of bubble should be prevented
If (there are minute bubbles, can be driven out of by plastic tab).
(3) pcr amplification product pretreatment adds 2.5 μ L6 × Loading buffer in 10 μ LPCR amplified productions,
Mix 30s stand-by.
(4) electrophoresis pipettor pressure-vaccum loading slot, removes impurity and bubble.1.5 μ L products are clicked and entered per well to be examined
Survey, electrophoresis 1-1.5 hours (voltage 300V, electric current 280mA, power 260W), electrophoresis time length can be according to target fragment size
Moderately adjusted with actual power.Treat that the index strip (dimethylbenzene is blue or green) on top reaches offset plate bottom, electrophoresis terminates, and carefully peels off poly- third
Acrylamide gel, it is to be dyed.
(5) silver staining rinsing：Using distilled water short rinse polyacrylamide gel 1 time, the time is no more than 10s.Silver staining：
Dyeed with gel is compared by following silver staining liquid, then parallel concussion dyeing 10min.
Table 3
Cleaning：After dyeing 10min, dyeing liquor is poured into waste liquid barrel, continuation is rinsed 2 times with distilled water, is no more than every time
15s.Colour developing：Matched by following nitrite ion, add appropriate nitrite ion in plastic casing, parallel concussion is placed in after gently mixing
Instrument, makes gel fully be acted on nitrite ion, until band is clear.
Table 4
Washing：Developer solution is outwelled and is cleaned 2-3 times with distilled water, every time no more than 15s.
Preserve：After cleaning up, by gel tiling on a glass, it is sealed against with preservative film, reads slice result simultaneously
Statistic record, then photograph is retained.
Result is recorded：The allelic variation size data in homozygosis site is recorded as X/X, and wherein X is the site allelic variation
Size；The allelic variation data of heterozygous sites are recorded as X/Y, and wherein X, Y is that two different allelic variations are (small on the site
Preceding, large fragment is rear for fragment)；The size of invalid allelic variation is recorded as 0/0.The polymorphism of primer is good as can be seen from Figure 2,
Can be good at differential variety.The finger-print of the 48 parts of broad beans for building is shown in Table 5- tables 16.
4 detection methods parallel with above-mentioned steps 3, can also use carries out fluorescent capillary electrophoresis tube inspection to pcr amplification product
Survey, it is specific as follows：
PCR primer preparation of samples and electrophoretic analysis result
The PCR primer of FAM (blueness) and HEX (green) fluorescence labeling is diluted 25 times with ultra-pure water, is taken respectively isometric
Above-mentioned 2 kinds of dilutions after solution be mixed to form mixed liquor, draw 1 μ L mixed liquors, 0.1 μ L LIZ500 molecular weight internal standards and 8.9 μ L
Deionized formamide is added separately in the special deep hole plate hole of DNA analysis instrument；Then it is denatured 5min for 95 DEG C in PCR instrument,
It is immediately placed on trash ice after taking-up, cooling 15min or so；Brief centrifugation 10s, carries out capillary electrophoresis detection, uses
GENEMAPPER carries out graphical analysis and data acquisition.It is DNA molecular amount standard, the equipotential in homozygosis site with DNA MarkerI
Variation size data is recorded as X/X, and wherein X is the size of the site allelic variation；The allelic variation data record of heterozygous sites
It is X/Y, wherein X, Y are two different allelic variations on the site；The size of invalid allelic variation is recorded as 0/0 (note：Small pieces
Duan Qian, large fragment is rear).By the Data Integration to multiple sites, the SSR finger-prints of different Vicia Fabas are ultimately formed
(table 5- tables 16).In addition to testing sample, each SSR site should also simultaneously include 3~5 amplified productions of Reference cultivars.
The most suitable extension rate of the amplified production of different fluorescence labelings determines preferably by trial test.
Testing result judges：Differences number of sites >=3, then be different cultivars；Differences number of sites<3, then for
Approximate kind；Compare 48 parts of finger print datas of kind, it is found that the interracial difference number of sites of any two is both greater than 3, illustrate this
The 21 pairs of core primers can be effective these Variety identifications are opened；Kind is carried out using Power Marker and MEGA software to gather
Class, analyzes interracial genetic affinity, it can be found that can be whole kinds when genetic similarity is less than 0.11 from figure
Distinguish (Fig. 1).Each Vicia Faba title in C1-C48 difference corresponding tables 1 in table 5- tables 16.
Table 5
Table 6
| SSR13771 | EST1142 | EST1584 | SSR11771 | SSR12868 | EST1672 | SSR13683 | |
| C1 | 163/163 | 213/219 | 183/183 | 149/149 | 144/146 | 181/187 | 147/150 |
| C2 | 157/161 | 206/217 | 184/187 | 136/142 | 143/146 | 190/190 | 145/145 |
| C3 | 158/158 | 230/236 | 185/185 | 139/146 | 146/146 | 181/184 | 146/150 |
| C4 | 164/164 | 206/206 | 185/185 | 149/149 | 144/146 | 187/193 | 144/144 |
| C5 | 163/163 | 206/217 | 184/184 | 143/143 | 143/143 | 186/186 | 149/149 |
| C6 | 162/162 | 208/218 | 187/187 | 146/146 | 144/147 | 181/181 | 144/144 |
| C7 | 162/162 | 217/221 | 188/188 | 146/146 | 144/144 | 190/190 | 150/150 |
| C8 | 159/159 | 206/206 | 185/185 | 142/142 | 143/143 | 187/187 | 150/150 |
| C9 | 163/167 | 206/206 | 184/184 | 142/142 | 143/143 | 181/193 | 144/144 |
| C10 | 160/164 | 206/208 | 188/188 | 141/146 | 144/144 | 181/181 | 145/145 |
| C11 | 159/159 | 219/219 | 187/187 | 142/146 | 143/143 | 186/186 | 150/150 |
| C12 | 159/159 | 219/237 | 182/182 | 146/149 | 144/147 | 181/181 | 144/147 |
Table 7
| SSR760 | SSR10911 | SSR13106 | SSR11052 | SSR14328 | SSR12536 | SSR17991 | |
| C1 | 168/168 | 166/169 | 167/167 | 187/187 | 177/177 | 192/198 | 181/181 |
| C2 | 165/168 | 163/169 | 167/177 | 184/184 | 177/179 | 202/202 | 158/181 |
| C3 | 165/174 | 160/176 | 167/167 | 184/184 | 177/177 | 195/195 | 158/158 |
| C4 | 165/174 | 166/180 | 167/171 | 184/193 | 177/177 | 198/198 | 159/181 |
| C5 | 171/171 | 166/166 | 167/167 | 193/193 | 171/178 | 198/198 | 158/158 |
| C6 | 168/168 | 158/158 | 167/169 | 193/193 | 177/177 | 198/198 | 159/159 |
| C7 | 168/171 | 157/166 | 169/169 | 184/184 | 171/171 | 198/198 | 158/158 |
| C8 | 168/168 | 163/163 | 167/167 | 193/193 | 178/178 | 198/198 | 159/159 |
| C9 | 168/167 | 166/166 | 167/167 | 184/193 | 177/182 | 198/198 | 159/180 |
| C10 | 159/159 | 163/176 | 169/169 | 193/193 | 177/177 | 201/201 | 158/158 |
| C11 | 168/168 | 166/166 | 167/167 | 184/184 | 170/170 | 201/201 | 159/159 |
| C12 | 158/168 | 166/169 | 164/164 | 184/187 | 177/177 | 192/198 | 158/158 |
Table 8
Table 9
| SSR13771 | EST1142 | EST1584 | SSR11771 | SSR12868 | EST1672 | SSR13683 | |
| C13 | 161/161 | 210/219 | 186/186 | 136/136 | 143/143 | 181/181 | 150/150 |
| C14 | 159/159 | 218/218 | 185/185 | 136/136 | 146/146 | 181/190 | 146/148 |
| C15 | 161/167 | 217/217 | 187/187 | 136/136 | 143/143 | 181/181 | 147/147 |
| C16 | 162/162 | 217/217 | 182/182 | 136/136 | 143/143 | 181/181 | 144/144 |
| C17 | 160/160 | 221/221 | 188/188 | 146/146 | 144/144 | 187/187 | 147/147 |
| C18 | 161/161 | 230/230 | 187/187 | 136/142 | 143/146 | 180/192 | 144/150 |
| C19 | 163/163 | 226/226 | 187/187 | 136/136 | 146/146 | 181/181 | 147/147 |
| C20 | 159/159 | 224/226 | 180/184 | 142/149 | 147/147 | 190/190 | 147/147 |
| C21 | 159/159 | 217/219 | 183/187 | 142/146 | 144/144 | 187/187 | 146/146 |
| C22 | 161/161 | 206/215 | 184/184 | 136/146 | 144/147 | 181/193 | 144/144 |
| C23 | 157/161 | 206/206 | 187/187 | 136/146 | 144/144 | 187/190 | 147/150 |
| C24 | 159/159 | 208/218 | 184/184 | 146/146 | 146/146 | 181/187 | 144/144 |
Table 10
| SSR760 | SSR10911 | SSR13106 | SSR11052 | SSR14328 | SSR12536 | SSR17991 | |
| C13 | 168/168 | 163/163 | 169/169 | 187/187 | 177/177 | 199/199 | 160/181 |
| C14 | 168/168 | 158/158 | 171/173 | 184/184 | 177/177 | 193/198 | 180/180 |
| C15 | 165/165 | 169/169 | 167/167 | 184/184 | 178/178 | 195/198 | 157/157 |
| C16 | 165/165 | 169/169 | 167/167 | 178/178 | 178/178 | 192/192 | 159/159 |
| C17 | 159/174 | 166/166 | 171/171 | 178/178 | 177/177 | 198/198 | 158/158 |
| C18 | 165/165 | 169/169 | 165/165 | 187/187 | 177/177 | 192/198 | 157/157 |
| C19 | 174/174 | 166/169 | 167/167 | 187/193 | 178/181 | 193/193 | 159/159 |
| C20 | 171/174 | 169/169 | 171/171 | 184/193 | 179/179 | 198/198 | 180/180 |
| C21 | 174/174 | 166/166 | 167/169 | 187/187 | 171/178 | 198/198 | 158/158 |
| C22 | 168/168 | 166/166 | 171/171 | 193/193 | 178/181 | 201/201 | 158/179 |
| C23 | 159/168 | 163/163 | 169/169 | 178/193 | 178/178 | 193/201 | 158/180 |
| C24 | 168/168 | 163/169 | 167/167 | 193/193 | 178/178 | 195/198 | 158/181 |
Table 11
Table 12
| SSR13771 | EST1142 | EST1584 | SSR11771 | SSR12868 | EST1672 | SSR13683 | |
| C25 | 157/157 | 217/221 | 185/188 | 146/146 | 144/150 | 181/181 | 146/150 |
| C26 | 159/159 | 206/206 | 187/187 | 136/136 | 146/146 | 181/181 | 145/145 |
| C27 | 159/159 | 206/206 | 187/187 | 146/146 | 143/143 | 193/193 | 144/144 |
| C28 | 158/167 | 213/217 | 187/187 | 146/146 | 146/146 | 181/193 | 145/147 |
| C29 | 160/160 | 223/225 | 188/188 | 136/142 | 144/147 | 181/184 | 145/145 |
| C30 | 159/159 | 212/227 | 187/187 | 136/146 | 146/146 | 181/181 | 146/146 |
| C31 | 161/161 | 221/221 | 187/187 | 146/146 | 144/144 | 187/187 | 145/145 |
| C32 | 159/159 | 217/226 | 182/182 | 136/149 | 144/147 | 187/193 | 145/145 |
| C33 | 161/163 | 216/216 | 187/187 | 146/146 | 144/147 | 187/187 | 145/145 |
| C34 | 163/163 | 218/218 | 187/187 | 146/146 | 144/144 | 187/187 | 145/145 |
| C35 | 159/159 | 206/206 | 188/188 | 146/146 | 144/144 | 187/187 | 145/145 |
| C36 | 159/159 | 221/221 | 188/188 | 143/143 | 143/143 | 187/187 | 144/144 |
Table 13
| SSR760 | SSR10911 | SSR13106 | SSR11052 | SSR14328 | SSR12536 | SSR17991 | |
| C25 | 159/168 | 166/166 | 165/165 | 184/196 | 171/178 | 195/195 | 160/181 |
| C26 | 168/168 | 163/163 | 167/167 | 193/193 | 181/181 | 202/202 | 158/158 |
| C27 | 168/168 | 158/166 | 171/171 | 193/193 | 178/178 | 198/198 | 158/158 |
| C28 | 171/171 | 166/169 | 167/171 | 184/184 | 177/177 | 198/198 | 180/180 |
| C29 | 158/167 | 168/171 | 171/177 | 184/184 | 178/178 | 198/198 | 158/158 |
| C30 | 168/171 | 166/169 | 165/165 | 178/184 | 177/177 | 198/198 | 157/180 |
| C31 | 168/168 | 163/163 | 171/171 | 136/136 | 178/178 | 198/198 | 158/158 |
| C32 | 174/174 | 163/169 | 167/167 | 187/187 | 171/181 | 198/201 | 180/180 |
| C33 | 168/168 | 166/166 | 171/171 | 193/193 | 177/177 | 198/198 | 158/158 |
| C34 | 174/174 | 163/163 | 169/171 | 193/239 | 177/177 | 198/198 | 181/181 |
| C35 | 174/174 | 166/166 | 169/169 | 194/194 | 177/177 | 199/199 | 158/158 |
| C36 | 168/168 | 163/163 | 171/171 | 236/236 | 178/178 | 198/198 | 158/158 |
Table 14
Table 15
| SSR13771 | EST1142 | EST1584 | SSR11771 | SSR12868 | EST1672 | SSR13683 | |
| C37 | 164/164 | 221/221 | 186/186 | 146/146 | 144/144 | 187/187 | 145/145 |
| C38 | 162/162 | 206/206 | 185/185 | 146/146 | 144/144 | 187/187 | 145/145 |
| C39 | 163/163 | 219/219 | 186/188 | 136/136 | 143/143 | 187/187 | 150/150 |
| C40 | 162/162 | 219/219 | 188/188 | 146/146 | 144/144 | 181/187 | 147/147 |
| C41 | 159/159 | 206/206 | 185/185 | 146/146 | 143/143 | 181/181 | 145/145 |
| C42 | 159/159 | 217/217 | 183/183 | 143/143 | 146/146 | 187/187 | 150/150 |
| C43 | 159/159 | 211/211 | 185/185 | 146/146 | 147/147 | 181/181 | 146/146 |
| C44 | 159/159 | 204/204 | 185/185 | 127/127 | 143/143 | 190/190 | 147/147 |
| C45 | 159/159 | 225/230 | 184/187 | 136/136 | 146/146 | 187/187 | 144/149 |
| C46 | 160/165 | 221/228 | 185/188 | 146/146 | 144/144 | 187/183 | 144/144 |
| C47 | 161/161 | 212/219 | 185/188 | 136/146 | 147/147 | 187/193 | 145/150 |
| C48 | 160/160 | 217/217 | 188/188 | 146/146 | 144/144 | 187/187 | 145/145 |
Table 16
| SSR760 | SSR10911 | SSR13106 | SSR11052 | SSR14328 | SSR12536 | SSR17991 | |
| C37 | 174/174 | 158/158 | 171/171 | 237/237 | 178/178 | 203/203 | 158/158 |
| C38 | 174/174 | 163/163 | 169/169 | 237/237 | 178/178 | 199/199 | 158/158 |
| C39 | 168/168 | 163/163 | 167/167 | 184/184 | 170/178 | 195/202 | 160/160 |
| C40 | 168/168 | 163/163 | 167/167 | 184/193 | 170/177 | 196/203 | 157/157 |
| C41 | 168/168 | 163/163 | 167/167 | 184/184 | 177/177 | 201/201 | 159/159 |
| C42 | 158/158 | 169/169 | 165/167 | 184/184 | 178/178 | 199/199 | 158/158 |
| C43 | 165/165 | 169/169 | 167/167 | 187/187 | 181/181 | 198/198 | 158/158 |
| C44 | 174/174 | 166/166 | 169/169 | 187/187 | 179/179 | 198/198 | 159/159 |
| C45 | 165/174 | 166/169 | 167/167 | 184/184 | 178/178 | 192/198 | 157/157 |
| C46 | 168/168 | 163/163 | 167/171 | 193/193 | 178/182 | 196/202 | 158/158 |
| C47 | 159/171 | 163/166 | 167/169 | 193/193 | 178/182 | 199/199 | 159/159 |
| C48 | 168/168 | 167/167 | 167/167 | 237/237 | 178/178 | 199/199 | 180/180 |
The application of the present invention identification Vicia Faba method of embodiment 3
6 leaf DNAs of broad bean sample of known variety name are extracted, the molecule for obtaining is screened with the embodiment of the present invention 1
21 pairs of primers in mark combination enter performing PCR amplification to the DNA of Vicia Faba to be checked respectively；With reference to the PCR of the embodiment of the present invention 2
Reaction system, PCR response procedures, polyacrylamide gel electrophoresis and variant sites recording method (method 1), while repeating reality
Test using fluorescent capillary electrophoresis tube method and variant sites recording method (method 2), the finger-print code that will be obtained is obtained with the present invention
The characteristic fingerprint pattern (table 5- tables 16) of Vicia Faba compare, to verify the authentication method that the embodiment of the present invention 2 is set up
The degree of accuracy.
Result finds：Performing PCR amplification, 6 silkworms that above two method is measured are entered in 21 pairs of primer combinations to 6 Vicia Fabas
Beans sample variation site is consistent, the results are shown in Table 17- tables 19, and the broad bean finger-print target variety set up with the present invention product
Interspecific difference number of sites ＜ 3, F1-F6 be 6 Vicia Fabas to be measured, through contrast identification obtain F1-F6 respectively correspond to face silkworm 3
Number, open beans 5, Qinghai 4, into No. 11 recklessly, wing silkworm No. two, kidney bean 83-63 is consistent with known variety name, the degree of accuracy
100%.Show that the embodiment of the present invention 1 is screened the 21 pairs of primers combination for obtaining and can preferably distinguish different Vicia Fabas.
Table 17
| DNA Numbering | EST181 | SSR11885 | SSR12396 | SSR11908 | SSR12080 | EST1280 | EST1741 |
| F1 | 200/202 | 167/167 | 164/164 | 163/169 | 211/214 | 184/184 | 181/181 |
| F2 | 208/208 | 167/167 | 171/171 | 166/166 | 207/207 | 186/186 | 186/186 |
| F3 | 206/206 | 170/170 | 170/170 | 163/163 | 210/210 | 184/184 | 189/189 |
| F4 | 208/208 | 168/171 | 164/164 | 160/163 | 211/211 | 184/186 | 181/181 |
| F5 | 200/207 | 167/167 | 170/170 | 161/161 | 204/211 | 183/183 | 181/181 |
| F6 | 208/208 | 167/167 | 170/170 | 166/166 | 204/204 | 198/198 | 180/180 |
Table 18
| DNA is numbered | SSR13771 | EST1142 | EST1584 | SSR11771 | SSR12868 | EST1672 | SSR13683 |
| F1 | 158/158 | 230/236 | 185/185 | 139/146 | 146/146 | 181/184 | 146/150 |
| F2 | 159/159 | 219/219 | 187/187 | 142/146 | 143/143 | 186/186 | 150/150 |
| F3 | 162/162 | 217/217 | 182/182 | 136/136 | 143/143 | 181/181 | 144/144 |
| F4 | 157/157 | 217/221 | 185/188 | 146/146 | 144/150 | 181/181 | 146/150 |
| F5 | 159/159 | 212/227 | 187/187 | 136/146 | 146/146 | 181/181 | 146/146 |
| F6 | 163/163 | 218/218 | 187/187 | 146/146 | 144/144 | 187/187 | 145/145 |
Table 19
| DNA is numbered | SSR760 | SSR10911 | SSR13106 | SSR11052 | SSR14328 | SSR12536 | SSR17991 |
| F1 | 165/174 | 160/176 | 167/167 | 184/184 | 177/177 | 195/195 | 158/158 |
| F2 | 168/168 | 166/166 | 167/167 | 184/184 | 170/170 | 201/201 | 159/159 |
| F3 | 165/165 | 169/169 | 167/167 | 178/178 | 178/178 | 192/192 | 159/159 |
| F4 | 159/168 | 166/166 | 165/165 | 184/196 | 171/178 | 195/195 | 160/181 |
| F5 | 168/171 | 166/169 | 165/165 | 178/184 | 177/177 | 198/198 | 157/180 |
| F6 | 174/174 | 163/163 | 169/171 | 193/239 | 177/177 | 198/198 | 181/181 |
Illustrate higher using the method accuracy of the discriminating Vicia Faba of the offer of the embodiment of the present invention 2, be adapted to promote the use of.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>A kind of construction method of broad bean SSR finger-prints
<130> KHP161117235.4
<160> 42
<170> PatentIn version 3.5
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tgcggaaaac agtaatattg aaa 23
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tgctggaaca acaacctgaa 20
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ctccgcgagc atcactttat 20
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Claims (10)
1. a kind of molecular labeling for differentiating Vicia Faba is combined, it is characterised in that containing 15 SSR molecular markers and 6
EST-SSR molecular labelings, respectively SSR11908, EST181, SSR11771, SSR12080, SSR13683, SSR12396,
SSR14328、EST1672、EST1280、EST1741、SSR17991、SSR10911、SSR13106、SSR760、EST1142、
SSR11052、SSR13771、SSR11885、SSR12536、SSR12868、EST1584；
The molecular labeling is obtained by following primer pair amplifies respectively：SEQ ID NO.1-2、SEQ ID NO.3-4、SEQ ID
NO.5-6、SEQ ID NO.7-8、SEQ ID NO.9-10、SEQ ID NO.11-12、SEQ ID NO.13-14、SEQ ID
NO.15-16、SEQ ID NO.17-18、SEQ ID NO.19-20、SEQ ID NO.21-22、SEQ ID NO.23-24、SEQ
ID NO.25-26、SEQ ID NO.27-28、SEQ ID NO.29-30、SEQ ID NO.31-32、SEQ ID NO.33-34、
Primer shown in SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42.
2. a kind of primer combination, it is characterised in that containing following 21 pairs of specific primers pair, its nucleotide sequence is respectively such as SEQ
Shown in ID NO.1-42.
3. the kit for being combined containing molecular labeling combination or primer described in claim 2 described in claim 1.
4. the molecular labeling combination described in claim 1 or the primer combination described in claim 2 are in Vicia Faba is identified
Using.
5. the molecular labeling combination described in claim 1 or the primer combination described in claim 2 are in resource of broad bean germplasm improvement
In application.
6. a kind of construction method of Vicia Faba SSR finger-prints, it is characterised in that combined with the primer described in claim 2
DNA to different Vicia Fabas enters performing PCR amplification, carries out native polyacrylamide gel electrophoresis to PCR primer and detects
Or carry out fluorescent capillary electrophoresis tube detection to PCR primer,
The allelic variation size data in homozygosis site is recorded as X/X, and wherein X is the size of the site allelic variation；
The allelic variation data of heterozygous sites are recorded as X/Y, and wherein X, Y is two different allelic variations on the site；
The size of invalid allelic variation is recorded as 0/0；
It is small fragment before above-mentioned "/", is large fragment after "/"；
By the Data Integration to different loci, the SSR finger-prints of different Vicia Fabas are formed.
7. construction method as claimed in claim 6, it is characterised in that PCR amplification method is：
Response procedures are：95 DEG C of predegeneration 5min；95 DEG C of denaturation 35s, 60 DEG C of annealing 40s, 72 DEG C of extension 45s, totally 32 circulations；
72 DEG C of extensions 5min, 10 DEG C of insulation 5min；
10 μ L amplification systems are：Each 1 μ L of upstream and downstream primer of 2 × Taq PCR Master Mix5 μ L, 2 μM/μ L, DNA profiling
1.5 μ L, ddH2O 2.5μL。
8. the construction method described in claim 6 or 7 builds the Vicia Faba finger-print for obtaining.
9. Vicia Faba finger-print as claimed in claim 8, it is 48 kinds of finger-prints of Vicia Faba, as follows respectively：
With
With
With
With
With
With
With
With
With
With
With
Above-mentioned C1-C48 refers to 48 kinds of Vicia Fabas respectively, the big white silkworm in Linxia is corresponded to respectively, is faced silkworm No. 2, is faced silkworm No. 3, opens beans one
Number, it is logical grind No. one, it is logical along No. three, open beans four, it is logical along No. two, open beans three, logical silkworm No. three, open beans 5, Qinghai three, blue or green
Sea No. five, Qinghai 1, Qinghai 2, Qinghai 4, Qinghai 6, Qinghai 8, Qinghai 9, Qinghai 10, No. Cheng Huyi, into recklessly
No. two, No. Cheng Hujiu, into recklessly No. 10, into recklessly No. 11, into recklessly No. 12, into recklessly No. 13, Chongli broad bean, wing silkworm No., wing silkworm No. two,
Kidney bean 83-324, kidney bean 83-315, kidney bean 83-190, kidney bean 83-63, kidney bean 81-37, Yundou324, kidney bean 690, phoenix beans 1,
Logical fresh No. 6 of silkworm, logical fresh No. 7 of silkworm open beans 2, Qinghai gingival cyst of mucous gland in the newborn, Qinghai 13, Qinghai 14, Qinghai 15, into No. 14 recklessly, into recklessly
No. 15, Yunnan 89147.
10. it is a kind of identify Vicia Faba method, it is characterised in that comprise the following steps：
(1) DNA of Vicia Faba to be checked is extracted, 21 pairs of primer pairs in being combined with the primer described in claim 2 are respectively to inspection
The DNA of Vicia Faba enters performing PCR amplification；
(2) amplified production is detected that silver staining develops the color, according to amplified production in electricity by native polyacrylamide gel electrophoresis
Relative position on swimming gel；Or
Fluorescent capillary electrophoresis tube detection is carried out to amplified production, according to the relative position of amplified production,
The allelic variation size data in homozygosis site is recorded as X/X, and wherein X is the size of the site allelic variation；
The allelic variation data of heterozygous sites are recorded as X/Y, and wherein X, Y is two different allelic variations on the site；
The size of invalid allelic variation is recorded as 0/0；It is small fragment before above-mentioned "/", is large fragment after "/"；
(3) result compares the Vicia Faba finger-print of claim 8 or 9, and Differences number of sites >=3 are then different product
Kind；Differences number of sites<3, then it is approximate kind.
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