CN104017859A - Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique - Google Patents
Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique Download PDFInfo
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- CN104017859A CN104017859A CN201410090829.5A CN201410090829A CN104017859A CN 104017859 A CN104017859 A CN 104017859A CN 201410090829 A CN201410090829 A CN 201410090829A CN 104017859 A CN104017859 A CN 104017859A
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses a method for identifying sugarcane germplasm resources. According to the method, SSR (Simple Sequence Repeats) molecular marker and CE (capillary electrophoresis) technique are combined, and fingerprint spectrums of 52 sugarcane varieties are established by utilizing 12 pairs of primers and are applied to identification of sugarcane germplasm, variety identification and protection. The specific method comprises the following steps: extracting genomic DNAs of different varieties of sugarcane, carrying out PCR (polymerase chain reaction) amplification, detecting amplification results by utilizing CE, establishing fingerprint spectrums of different sugarcane germplasms according different sizes of DNA strips and utilizing the fingerprint spectrums in the identification of germplasm resources. Compared with the conventional germplasm resource identification and biochemical detection, the method for identifying the sugarcane germplasm resources are not influenced by growth environment of plants and seasons and has the advantages that the resolution is high, the result is exact and reliable, the operation is simple, quick and direct, and the method is especially suitable for mass multiple-batch experimental data integration.
Description
Technical field
The invention belongs to biological technical field, relate to the high-flux detection method of a kind of SSR of utilization and capillary electrophoresis technique qualification sugarcane germplasm.
Background technology
China is in the world one of ancient cane planting country, and successive dynasties cultivation of sugar cane variety type is a lot.In recent years, China introduce again, seed selection be adapted to the sugar cane breed promoted in various ecotype sugarcanes district more than 120.But the hereditary basis of current cultivation of sugar cane kind is narrow, the biological characteristics such as form, the economical character difference of kind is little, how to identify that cultivation of sugar cane kind and filial generation thereof have become one of heavy difficult point of current sugarcane scientific effort.
Before the widespread use of DNA molecular technological method, people are mainly by experimental techniques such as form and economical character, nucleus type analysis, isoenzyme mark, chromosome analysis technology, cultivation of sugar cane kind is identified, but these methods are restricted by the factors such as environment, limited marker number, can not extensively be promoted.DNA molecular marker technology is the direct reflection of genetic diversity on DNA level, have quantity many, stablize, without phenotypic effect, the advantage such as not affected by environment.
DNA fingerprint technology refers to and can reflect between biont or population a kind of Protocols in Molecular Biology of DNA Site discrepancy in genome.DNA molecular marker has been widely used in the aspects such as cultivation of sugar cane kind fingerprint map construction, genetic analysis, the assignment of genes gene mapping and molecular marker assisted selection at present.Compared with other molecule marker, SSR molecular marking technique has the advantages such as rich polymorphism, codominant inheritance mode, analytical procedure be simple, reproducible.At present, the PCR product detection method of SSR labeling technique is mainly contained to the polyacrylamide gel electrophoresis method of fluorescent dye primer sequencing, isotopic labeling primer, and silver dyes polyacrylamide PAGE(polyacrylamide gel electrophoresis) method.PAGE is current the most frequently used detection method, but it dyes because of needs silver, makes testing process become complicated, and can not accurately read clip size, can only "ball-park" estimate.
Capillary electrophoresis (capillary electrophoresis, CE) be also HPCE (HPCE).It is a kind of separate analytical technique emerging rapidly in the world in the later stage eighties 20th century.Capillary gel electrophoresis is compared with common electrophoresis, has the advantages such as highly sensitive, high resolving power, high-speed, sample is few; Therefore, CE is progressively applied to the pcr amplification product of detection molecules labeling technique.
Summary of the invention
The object of the invention is to solve sugarcane germplasm and identify difficult problem, a kind of high-throughput, simple to operate, the result authentication method of sugarcane seed resource are accurately and reliably provided.And capillary electrophoresis technique is combined with SSR molecular marking technique, can provide strong support for the qualification of sugarcane kinship, cultivar identification, seed authenticity qualification and Purity Identification.
Sugarcane germplasm authentication method provided by the invention, the steps include: to extract the fresh tender leaf DNA of sugarcane, utilize 12 pairs of SSR fluorescent primers to combine with capillary electrophoresis technique and build 52 at the extensively sugar cane breed DNA fingerprint database of plantation of China, kind to be measured or strain DNA fingerprint data are analyzed with the data in the sugarcane DNA fingerprint database building, judge the kind title of kind to be measured or strain.
As further restriction of the present invention, described 12 pairs of SSR fluorescent primers are the core primers that obtains of screening, and its primer information is as following table:
As further restriction of the present invention, described DNA extraction comprises some steps: by powdered in liquid nitrogen grinding sugarcane tender leaf, add the SDS extracting solution of 65 DEG C of preheatings of 1.2 ml, shake up, 65 DEG C of water-bath 45 min; Add 200 μ l KAc (5 mol/L, pH4.8), after mixing, ice bath 15 min; 4 DEG C, centrifugal 15 min of 10000 rpm, get 1 ml supernatant liquor, add equal-volume chloroform to mix; 4 DEG C, centrifugal 7 min of 12000 rpm, get supernatant liquor, add the NaAc (3 mol/L, pH5.2) of 1/10 volume and the precooling dehydrated alcohol of 2 times of volumes, ice bath 20 min; 4 DEG C, centrifugal 10 min of 10000 rpm, abandon supernatant, with 70% alcohol washing 2 times, dry precipitation; Add the 200 sterilized TE solution of μ l (pH8.0), dissolution precipitation, obtains DNA solution, is placed in-20 DEG C and saves backup;
As further restriction of the present invention, the amplification program of described pcr amplification is: first 95 DEG C of denaturation 5 min; 30 circulations are subsequently: 94 DEG C of sex change 30 sec, the actual temp table 1 of annealing 30 sec(different primers), 72 DEG C are extended 30 sec; Last 72 DEG C of downward-extension 2 min.
As further restriction of the present invention, described capillary electrophoretic analysis method is for after diluting 100 times by obtained PCR product, on BECKMAN COULTER CEQ 8000 genetic analysis systems instrument, utilize capillary electrophoresis to detect, in each CE sample, contain 1 μ l PCR product, 0.2 μ l Genescan-400 molecular weight standard product and 20 μ l SLS solution.In capillary gel electrophoresis process, the fluorescent signal of PCR product and Genescan-400 molecular weight standard product is preserved automatically by genetic analysis instrument.
As further restriction of the present invention, the SSR labeled fragment that polymorphism is higher is only added up in being configured to of described finger printing in the amplified production of all primers, participate in the experiment in material, be designated as " A " if contain these these special SSR labeled fragment, if be designated as " C " without expanding.For a certain cultivation of sugar cane kind, the i.e. fingerprint of cultivation of sugar cane kind for this reason of the combination that has or not these special SSR marks, the SSR labeled fragment fingerprint of different sugarcane germplasms has formed fingerprints database.
This research and utilization SSR fluorescent mark combines with capillary gel electrophoresis technology, easier with respect to traditional gel analysis, the unified of the complete paired data of computer software that the technology utilization of SSR-capillary gel electrophoresis and genetic system match processed, without the preparation of traditional gel, silver dye, the step such as rinsing, not only avoid the basis of experiment operator and objectionable impurities, and greatly dwindle workload, easy and simple to handle, realize molecule marking research and combination efficient, automatic technology.Capillary gel electrophoresis can accurately be read the size of amplification allelotrope fragment simultaneously, is conducive to extensive, multiple batches of data gathering and analysis, has reduced personal errors, makes experimental result more accurate.
Embodiment
Embodiment 1: utilize 12 pairs of SSR fluorescent primers to set up the plant collection of illustrative plates of 52 cultivation of sugar cane kinds, selected 52 cultivation of sugar cane kinds see the following form.
1, the extraction of genomic dna
(1) get sugarcane tender leaf in mortar, add liquid nitrogen grinding powdered, pack in 2 ml centrifuge tubes;
(2) to the SDS extracting solution (Tris-HCl 200 mmol/L, the pH8.0 that add 65 DEG C of preheatings of 1.2 ml in centrifuge tube; EDTA 50 mmol/L pH8.0; NaCl 500 mmol/L; 3% SDS), sample powder and extracting solution are shaken up rapidly and make it not agglomerating, 65 DEG C of water-bath 45 min, therebetween jogs 4-6 time;
(3) add 200 μ l KAc (5 mol/L, pH 4.8), after mixing, ice bath 15 min;
(4) in 4 DEG C of refrigerated centrifuges, centrifugal 15 min of 10000 rpm;
(5) get 800 μ l supernatant liquors, add 800 μ l chloroforms to mix, in 4 DEG C, centrifugal 7 min of 12000 rpm;
(6) get 400 μ l supernatant liquors, add 40 μ l NaAc (3 mol/L, pH 5.2) and 800 μ l precooling dehydrated alcohols, ice bath 20 min;
(7) in 4 DEG C, centrifugal 10 min of 10000 rpm, abandon supernatant liquor, by 2 precipitations of 70% washing with alcohol, dry precipitation;
(8) add the 200 sterilized TE solution of μ l (pH8.0), dissolution precipitation, obtains DNA solution, is placed in-20 DEG C and saves backup;
2, pcr amplification
PCR reaction system (20 μ l): 1 μ l 25ng/ μ l DNA profiling, 2 μ l 10 × Buffer(are containing 20 mM Mg
2+), 0.5 μ l 10 mM dNTP, 0.8 μ l mol/L SSR primer, (5 U/ μ l), add ddH to 0.2 μ l DNA pfu enzyme
2o to 20 μ l(reagent is purchased from Sheng Gong biotechnology limited-liability company).
Pcr amplification program: first 95 DEG C of denaturation 5 min; 30 circulations subsequently: 94 DEG C of sex change 30 sec, the concrete annealing temperature of annealing 30 sec(different primers is in table 1), 72 DEG C are extended 30 sec; Last 72 DEG C of downward-extension 2 min.
3, capillary gel electrophoresis detects
Upper model: by 100 times of obtained PCR product dilutions, add respectively the PCR product after 20 μ l methane amide 0.2 μ l Genescan-400 molecular weight standard product and 1 μ l dilution in each hole of 96 orifice plates.
Dash plate: add parting liquid in each hole of 96 orifice plates, be approximately 2/3 of every hole.
Gentle upper model punching is put in BECKMAN COULTER CEQ 8000 genetic analysis systems instrument (U.S., BECKMAN company produces) to 90 DEG C of sex change 2 min; Sample introduction voltage 2 kV, times 30 s; Separation voltage 7.5 kV, times 40 min.In capillary gel electrophoresis process, the fluorescent signal of PCR product and Genescan-400 molecular weight standard product is preserved automatically by genetic analysis instrument.
4, data analysis
The data analysis of BECKMAN COULTER CEQ 8000 genetic analysis systems instrument being collected with GeneMapper-V3.0 software, draw the amplified fragments of each SSR mark for examination material by manual analysis and software statistics, software system, by comparing according to the Genescan-400 molecular weight standard product in the position of amplified fragments target peak and same swimming lane, obtain fragment length size.And data are carried out to 2-D data statistical study.
5, the structure of finger printing
In amplified production in 12 pairs of primers, only add up 141 SSR labeled fragments that polymorphism is higher, participate in the experiment in material, be designated as " A " if contain these 141 special SSR labeled fragments, if be designated as " C " without expanding.For a certain cultivation of sugar cane kind, the i.e. fingerprint of cultivation of sugar cane kind for this reason of the combination that has or not these 141 special SSR marks, the SSR labeled fragment fingerprint of different sugarcane germplasms has formed fingerprints database, can be used for identifying the true and false of germ plasm resource and the qualification of purity of hybrid, also can be used for analyzing genetic distance and sibship between germ plasm resource.
6, interpretation of result
Table 3 is SSR mark fingerprints of No. 19, cultivation of sugar cane kind Guilin bamboo cane and osmanthus sugar, both are except the SSR labeled fragment of primer SMC486CG amplification is in full accord, other primer amplification not consistent, shows that this method can form the fingerprints database of cultivation of sugar cane kind.
Embodiment 2: the qualification of sugar cane breed to be measured
1, choosing two kinds of unknown sugar cane breeds is detected materials, carries out DNA extraction, SSR-PCR amplification, capillary gel electrophoresis detection and result data analysis according to the method in embodiment 1;
2, according to read stripe size, according in embodiment 1 141 special SSR labeled fragments, be designated as " A " if contain these 141 special SSR labeled fragments, if be designated as " C " without expanding, construct the SSR finger printing of sugar cane breed to be measured.And the standard sugarcane finger printing of setting up in result and embodiment 1 is compared, identifying sugar cane breed is No. 19, osmanthus sugar.
Claims (6)
1. a high-throughput SSR sugarcane germplasm authentication method, after it is characterized in that extracting the fresh tender leaf DNA of sugarcane, utilize 12 pairs of SSR fluorescent primers to carry out pcr amplification, then by PCR product process capillary electrophoresis analysis, construct 52 at the extensively sugar cane breed DNA fingerprint database of plantation of China, kind to be measured or strain DNA fingerprint data are analyzed with the data in the sugarcane DNA fingerprint database building, judge the kind title of kind to be measured or strain.
2. high-throughput SSR sugarcane germplasm authentication method method according to claim 1, it is characterized in that, described 12 pairs of SSR fluorescent primers are the core primers that screening obtains, and wherein forward primer 5' holds with CY5 phosphorus fluorochrome label, synthetic fluorescent primer, and keep in Dark Place; Primer information is as follows:
Primer title: SMC7CUQ, stripe size: 158-171Bp, primer annealing temperature: 60 DEG C, primer sequence:
Forward: 5 '-GCCAAAGCAAGGGTC ACT AGA-3 '
Reverse: 5 '-AGCTCTATCAGTTGAAACCGA-3 ';
Primer title: SMC18SA, stripe size: 130-165Bp, primer annealing temperature: 62 DEG C, primer sequence:
Forward: 5 '-ATTCGGCTCGAC CTC GGG AT-3 '
Reverse: 5 '-AGTCGAAAGGTAGCGTGGTGTTAC-3 ';
Primer title: SMC22DUQ, stripe size: 140-163Bp, primer annealing temperature: 62 DEG C, primer sequence:
Forward: 5 '-CCA TTC GAC GAA AGC GTC CT-3 '
Reverse: 5 '-CAA GCG TTG TGC TGC CGA GT-3 ';
Primer title: SMC24DUQ, stripe size: 121-142Bp, primer annealing temperature: 64 DEG C, primer sequence:
Forward: 5 '-CGCAACGACATATACACTTCGG-3 '
Reverse: 5 '-CGACATCACGGAGCA ATC AGT-3 ';
Primer title: SMC31CUQ, stripe size: 138-185Bp, primer annealing temperature: 62 DEG C, primer sequence:
Forward: 5 '-CATGCCAACTTCCAATACAGACT-3 '
Reverse: 5 '-AGTGCCAATCCA TCT CAG AGA-3 ';
Primer title: SMC36BUQ, stripe size: 104-254Bp, primer annealing temperature: 64 DEG C, primer sequence:
Forward: 5 '-GGGTTTCAT CTC TAG CCT ACC-3 '
Reverse: 5 '-TCAGTAGCAGAGTCAGACGCTT-3 ';
Primer title: SMC119CG, stripe size: 105-131Bp, primer annealing temperature: 58 DEG C, primer sequence:
Forward: 5 '-TTCATCTCT AGC CTA CCC CAA-3 '
Reverse: 5 '-AGC AGC CAT TTA CCC AGG A-3 ';
Primer title: SMC278CS, stripe size: 140-182Bp, primer annealing temperature: 64 DEG C, primer sequence:
Forward: 5 '-TTCTAGTGCCAATCCATCTCAGA-3 '
Reverse: 5 '-CATGCCAACTTCCAAACAGACT-3 ';
Primer title: SMC334BS, stripe size: 132-165Bp, primer annealing temperature: 60 DEG C, primer sequence:
Forward: 5 '-CAATTCTGACCG TGC AAA GAT-3 '
Reverse: 5 '-CGATGAGCTTGA TTG CGA ATG-3 ';
Primer title: SMC336BS, stripe size: 141-185Bp, primer annealing temperature: 62 DEG C, primer sequence:
Forward: 5 '-ATTCTAGTGCCAATCCATCTC A-3 '
Reverse: 5 '-CATGCCAACTTC CAA ACA GAC-3 ';
Primer title: SMC486CG, stripe size: 224-245Bp, primer annealing temperature: 58 DEG C, primer sequence:
Forward: 5 '-GAA ATT GCC TCC CAG GAT TA-3 '
Reverse: 5 '-CCAACTTGAGAATTGAGATTCG-3 ';
Primer title: SMC597CS, stripe size: 144-179Bp, primer annealing temperature: 64 DEG C, primer sequence:
Forward: 5 '-GCACACCACTCGAATAACGGAT-3 '
Reverse: 5 '-AGTATATCGTCCCTGGCATTCA-3 '.
3. high-throughput SSR sugarcane germplasm authentication method method according to claim 1, it is characterized in that, described DNA extraction comprises the following steps: by powdered in liquid nitrogen grinding sugarcane tender leaf, add the SDS extracting solution of 65 DEG C of preheatings of 1.2 ml, shake up 65 DEG C of water-bath 45 min; Add 200 μ l KAc, after mixing, ice bath 15 min; 4 DEG C, centrifugal 15 min of 10000 rpm, get 1 ml supernatant liquor, add equal-volume chloroform to mix; 4 DEG C, centrifugal 7 min of 12000 rpm, get supernatant liquor, add the NaAc of 1/10 volume and the precooling dehydrated alcohol of 2 times of volumes, ice bath 20 min; 4 DEG C, centrifugal 10 min of 10000 rpm, abandon supernatant, with 70% alcohol washing 2 times, dry precipitation; Add the sterilized TE solution of 200 μ l, dissolution precipitation, obtains DNA solution, is placed in-20 DEG C and saves backup.
4. high-throughput SSR sugarcane germplasm authentication method method according to claim 1, is characterized in that, described pcr amplification program is: first 95 DEG C of denaturation 5 min; 30 circulations are subsequently: 94 DEG C of sex change 30 sec, and annealing 30 sec, 72 DEG C are extended 30 sec; Last 72 DEG C of downward-extension 2 min.
5. high-throughput SSR sugarcane germplasm authentication method according to claim 1, it is characterized in that: described capillary electrophoretic analysis method is for after diluting 100 times by obtained PCR product, on genetic analysis systems instrument, utilize capillary electrophoresis to detect, in each CE sample, contain 1 μ l PCR product, 0.2 μ l Genescan-400 molecular weight standard product and 20 μ l SLS solution; In capillary gel electrophoresis process, the fluorescent signal of PCR product and Genescan-400 molecular weight standard product is preserved automatically by genetic analysis instrument.
6. high-throughput SSR sugarcane germplasm authentication method method according to claim 1, it is characterized in that: the SSR labeled fragment that polymorphism is higher is only added up in being configured to of described fingerprint database in the amplified production of all primers, participate in the experiment in material, be designated as " A " if contain these these special SSR labeled fragment, if be designated as " C " without expanding; For a certain cultivation of sugar cane kind, the i.e. fingerprint of cultivation of sugar cane kind for this reason of the combination that has or not these special SSR marks, the SSR labeled fragment of different sugarcane germplasms has formed fingerprints database.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106561445A (en) * | 2016-10-20 | 2017-04-19 | 华南农业大学 | Breeding method of new fruit sugarcane variety |
| CN107058508A (en) * | 2017-02-27 | 2017-08-18 | 浙江理工大学 | A kind of Salvia miltiorrhiza Bge authentication method |
| CN107151696A (en) * | 2017-02-27 | 2017-09-12 | 浙江理工大学 | A kind of germplasm identification method of fringe chinaroot greenbrier |
| CN108504771A (en) * | 2018-06-22 | 2018-09-07 | 福建农林大学 | A method of exploitation sugarcane SSR marker and identification Sugarcane Breeding affiliation |
| CN108841983A (en) * | 2018-06-22 | 2018-11-20 | 福建农林大学 | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development |
| CN109521066A (en) * | 2018-10-29 | 2019-03-26 | 浙江树人学院 | A kind of construction method of yellow rice wine electrochemistry finger-print and application |
| CN110205329A (en) * | 2019-06-19 | 2019-09-06 | 福建农林大学 | Saccharum S. spontaneum distinguished sequence and its identification method |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106561445A (en) * | 2016-10-20 | 2017-04-19 | 华南农业大学 | Breeding method of new fruit sugarcane variety |
| CN107058508A (en) * | 2017-02-27 | 2017-08-18 | 浙江理工大学 | A kind of Salvia miltiorrhiza Bge authentication method |
| CN107151696A (en) * | 2017-02-27 | 2017-09-12 | 浙江理工大学 | A kind of germplasm identification method of fringe chinaroot greenbrier |
| CN107058508B (en) * | 2017-02-27 | 2020-04-17 | 浙江理工大学 | Salvia miltiorrhiza germplasm resource identification method |
| CN107151696B (en) * | 2017-02-27 | 2020-06-05 | 浙江理工大学 | Method for identifying germplasm resource of smilax china |
| CN108504771A (en) * | 2018-06-22 | 2018-09-07 | 福建农林大学 | A method of exploitation sugarcane SSR marker and identification Sugarcane Breeding affiliation |
| CN108841983A (en) * | 2018-06-22 | 2018-11-20 | 福建农林大学 | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development |
| CN109521066A (en) * | 2018-10-29 | 2019-03-26 | 浙江树人学院 | A kind of construction method of yellow rice wine electrochemistry finger-print and application |
| CN109521066B (en) * | 2018-10-29 | 2021-06-08 | 浙江树人学院 | Construction method and application of electrochemical fingerprint spectrum of yellow wine |
| CN110205329A (en) * | 2019-06-19 | 2019-09-06 | 福建农林大学 | Saccharum S. spontaneum distinguished sequence and its identification method |
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Application publication date: 20140903 |