CN103540678A - Method for constructing sugarcane capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and authenticating sugarcane variety resource - Google Patents
Method for constructing sugarcane capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and authenticating sugarcane variety resource Download PDFInfo
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Abstract
The invention discloses a method for constructing a sugarcane capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and authenticating a sugarcane variety resource. The method for constructing the sugarcane capillary electrophoresis DNA fingerprint spectrum and authenticating the sugarcane variety resource comprises the following steps of 1) extracting the total DNA of a sugarcane lamina; 2) carrying out PCR (polymerase chain reaction) under the effect of an SSR (simple sequence repeat) primer; 3) carrying out capillary electrophoresis analysis on PCR amplification products; and 4) establishing a sugarcane sample DNA fingerprint spectrum by utilizing the position, the height and the area parameters of an electrophoresis peak generated by capillary electrophoresis. According to the invention, the rich SSR genetic diversity in the sugarcane DNA is fully utilized, and the DNA fingerprint spectrum is high in information amount, strong in practicability, correct in result, stable, good in repeatability and high in sensitivity. The method has an extremely wide application prospect.
Description
Technical field
The present invention relates to Sugarcane Variety Resource authenticate technology, a kind of method that specifically structure of sugarcane capillary electrophoresis DNA finger printing and Sugarcane Variety Resource are identified.
Background technology
Sugarcane is the important sugar crop in China and the world.Sugarcane belongs to Gramineae saccharum (Saccharum) in classification.At present, the discriminating of Sugarcane Variety Resource is mainly according to appearance character.Appearance character is differentiated experience and the sensation that too relies on discriminating personnel, and subjectivity is strong, and confidence level is lower.Appearance character is affected by environment large simultaneously, also can cause differentiating mistake, and the similarity of sugar cane breed is more and more higher in addition, causes differentiating more and more difficult.Due to DNA genetic material affected by environment little, polymorphism is high, DNA fingerprint authenticate technology has become the effective means of Identifying Crop Cultivars, progressively applies also starting aspect the discriminating of Sugarcane Variety Resource at present.
DNA fingerprint authenticate technology is to utilize in organism carrier of genetic information DNA to the living species method for distinguishing that reflects, and has been widely used in recent years the evaluation of plant variety resource.The molecule marking method that is applied at present constructed dna finger printing mainly contains: restriction fragment length polymorphism (Restriction Fragment Length Polymorphisms, RFLP) analytical technology, random amplification DNA polymorphism (Random Amplified Polymorphic DNA, RAPD) analytical technology, amplified fragment length polymorphism (Amplified Fragment Length Polymorphisms, AFLP) analytical technology, simple repeated sequence (Simple sequence repeat, SSR) analytical technology, single nucleotide polymorphism (Single nucleotide polymorphism, SNP) analytical technology, in RAPD and RFLP technical foundation, set up sequence-specific amplification region (Sequence characterized amplified regions, SCAR), enzyme is cut the polymorphic sequence of amplification (Cleaved amplified polymorphic sequence, CAPS) and DNA cloning fingerprint (DNA amplified fingerprints, the analytical technology such as DAF).Wherein Moore equals in conjunction with round pcr, to have founded SSR labeling technique in 1991, SSR also claims microsatellite DNA, it is the DNA sequence dna that a class is connected and repeated to form by the motif of several (mostly being 1～6) based composition, wherein modal is that dinucleotide repeats, the core sequence structure of each microsatellite DNA is identical, 10～60 of repeating unit's numbers, it is different that its height polymorphism is mainly derived from serial number object.Different genetic stocks multiplicity are different, caused the variability of SSR length, this variability basis that SSR mark produces just.Compare with other molecular marking techniques, SSR tool has the following advantages:
1.SSR primer is according to the specific short sequences Design at micro-satellite repetitive sequence two ends, has very strong specificity;
2.SSR sequence conventionally has very much higher state property in colony, and is generally codominance;
3.SSR repeatability and good stability.
Capillary electrophoresis (Capillary Electrophoresis, CE) has quick, sensitive, resolving power advantages of higher.CE can meet ssr analysis to the high-resolution requirement of amplified production, and resolution 1bp accurately represents the polymorphism of amplification; CE analyzes the position that the electrophoresis peak obtaining can be located pcr amplification product migration accurately, the i.e. size of pcr amplification product; CE analyzes the peak height at the electrophoresis peak obtaining and the amount that peak area can react pcr amplification product accurately.
In recent years, Cordeiro, G.M., Pan, Y.B., Henry, R.J. (2003) .Sugarcane microsatellites for the assessment of genetic diversity in sugarcane germplasm.Plant Science, 165 (1), 181-189, Liang Jun, PAN Yong-Bao, Li Yangrui, Fang Fengxue. (2010). the genetic analysis that utilizes SSR mark and capillary electrophoresis to carry out saccharum. GUIHAIA, 30 (1): 106-111, Qi, Y.W., Pan, Y.B., Lao, F.Y., Zhang, C.M., Fan, L.N., He, H.Y., Liu, R., Wang, Q.N., Liu, S.M., Liu, F.Y., Li, Q.W., Deng, H.H. (2012) .Genetic Structure and Diversity of Parental Cultivars Involved in China Mainland Sugarcane Breeding Programs as Inferred from DNA Microsatellites.Journal of Integrative Agriculture, 11 (11), 1794-1803, You, Q., Xu, L., Zheng, Y., Que, Y. (2013) .Genetic Diversity Analysis of Sugarcane Parents in Chinese Breeding Programmes Using gSSR Markers.The Scientific World Journal, 2013. grades utilize SSR molecule marker in conjunction with capillary electrophoresis technique, to carry out the research of the aspects such as sugarcane germplasm genetic diversity.Simultaneously, Australia Piperidis aspect the research of sugarcane DNA fingerprinting, G., Rattey, A.R., Taylor, G.O., Cox, M.C., (2004) .DNA markers:A tool for identifying sugarcane varieties.Austral Sugarcane, 8 (suppl): 1-8; U.S. Pan, Y.B. (2010) .Databasing molecular identities of sugarcane (Saccharum spp.) clones constructed with microsatellite (SSR) DNA markers.American Journal of Plant Sciences, 1 (2), the investigators such as 87-94. use SSR molecule marker to set up sugarcane molecular identity card database separately in conjunction with capillary electrophoresis technique.But, no matter be, utilize capillary electrophoresis to carry out the research that sugarcane germplasm is lost research or DNA fingerprinting aspect, all these research is all that to utilize having or not of capillary electrophoresis peak be 1 after digitizing, 0 data analysis research, all do not relate to the utilization of capillary electrophoresis peak characteristic, the present invention is the application of the characteristic based on capillary electrophoresis peak.
Summary of the invention
The object of this invention is to provide a kind of DNA fingerprinting construction process and authentication method based on capillary electrophoresis technique that Sugarcane Variety Resource is differentiated that can be used for.
The present invention achieves the above object by the following technical programs: a kind of construction process of sugarcane capillary electrophoresis DNA finger printing, it is characterized in that, and comprise the steps:
1) sugarcane DNA extraction: the methods such as CTAB, SDS or DNA extraction test kit of pressing are extracted sugarcane DNA;
2) pcr amplification of sugarcane DNA: the sugarcane DNA extracting of take carries out pcr amplification as template, and the primer of employing is SSR primer;
3) analysis of pcr amplification product: adopt capillary electrophoresis analysis pcr amplification product, record capillary electrophoresis collection of illustrative plates;
4) capillary electrophoresis collection of illustrative plates validity feature data gathering, comprises that the position at big or small electrophoresis peak of corresponding pcr amplification product is, the area at the height at electrophoresis peak, electrophoresis peak;
5) stdn of capillary electrophoresis collection of illustrative plates validity feature data, the characteristic at the electrophoresis peak of electrophoresis peak heights is the highest or electrophoresis peak area maximum is standardized as 1, and all the other electrophoresis peaks carry out stdn by its electrophoresis peak heights and the ratio at high voltage electrophoresis peak or the ratio at electrophoresis peak area and maximum electrophoresis peak;
6) utilize the position at big or small electrophoresis peak of corresponding pcr amplification product and the electrophoresis peak characteristic standardized value of correspondence thereof to build sugarcane DNA fingerprinting.
An authentication method for sugarcane capillary electrophoresis DNA fingerprint, comprises the steps:
1) sugarcane sample DNA extracts: the methods such as CTAB, SDS or DNA extraction test kit of pressing are extracted sugarcane DNA;
2) pcr amplification of sugarcane sample DNA: the sugarcane DNA extracting of take carries out pcr amplification as template, and the primer of employing is SSR primer;
3) analysis of pcr amplification product: adopt capillary electrophoresis analysis pcr amplification product, record capillary electrophoresis collection of illustrative plates;
4) foundation of sugarcane sample capillary electrophoresis DNA finger printing: the DNA fingerprinting of setting up sugarcane sample with the construction process of above-mentioned structure sugarcane capillary electrophoresis DNA finger printing;
5) sugarcane sample survey: utilize the dependency of DNA fingerprinting in correlation analysis algorithm comparative sample DNA fingerprinting and sugarcane DNA fingerprinting database, according to relation conefficient, sugarcane sample DNA is identified.
Described sugarcane DNA fingerprinting is saccharum sugarcane Hybrid or germ plasm resource DNA fingerprinting.
The primer that the pcr amplification of described sugarcane DNA adopts is SSR(simple sequence repeat, and simple sequence repeats, and also claims micro-satellite) primer.
Outstanding advantages of the present invention is:
Utilize SSR molecule marker to build sugarcane DNA fingerprinting in conjunction with capillary electrophoresis technique, the shortcoming such as overcome traditional sepharose or polyacrylamide gel electrophoresis resolving power is low, sensitivity is low, tolerance range is low, interpretation of result is difficult;
Utilize the characteristic at capillary electrophoresis peak to carry out constructed dna finger printing, than routine application electrophoretic band, have or not data such as (being digitized as 1,0 data) with more quantity of information; Application correlation analysis algorithm is carried out identification and analysis to sample to be identified, fully reflects the feature of DNA fingerprinting, and result is objective, accurate.
Accompanying drawing explanation
Accompanying drawing 1 is sugarcane SSR molecule marker capillary electrophoresis collection of illustrative plates and electrophoresis peak eigenwert.
The size that in figure, X-coordinate is pcr amplification product, ordinate zou is electrophoresis peak heights; Electrophoresis summit end shows numerical value, is above electrophoresis peak heights value, and lower is electrophoresis peak area.
Accompanying drawing 2 is continuous sugarcane SSR molecule marker capillary electrophoresis collection of illustrative plates and electrophoresis peak eigenwert.
The size that in figure, X-coordinate is pcr amplification product, ordinate zou is electrophoresis peak heights; Electrophoresis summit end shows numerical value, is above electrophoresis peak heights value, and lower is electrophoresis peak area.
Embodiment
Below in conjunction with case study on implementation, technical scheme of the present invention is described further.
Embodiment 1
The construction process of sugarcane capillary electrophoresis DNA finger printing of the present invention, comprises the steps:
1. sugarcane DNA extraction
After Sugarcane Leaves is clayed into power in liquid nitrogen, the Caulis Sacchari sinensis leaf powder transfer of 0.1g, in 2mL centrifuge tube, is added to 2 * CTAB damping fluid of 500 μ L65 ℃ preheatings, then in 65 ℃ of incubation 30min, centre is shaken 4-5 time, after taking-up centrifuge tube is cooling, add 500 μ L chloroforms: primary isoamyl alcohol mixes by 24:1, then at 12000rpm, room temperature, centrifugal 10min, shift supernatant liquor to new centrifuge tube, the Virahol that adds 2/3 volume, mixes, and-20 ℃ standing 1 hour; At 4 ℃, the centrifugal 5min of 10000rpm, removes supernatant; Add 500 μ L76% washing with alcohol precipitation 2 times, room temperature is dried, and adds 100 μ L TE buffer dissolving DNAs ,-20 ℃ of preservations.
2.PCR amplification
The sugarcane DNA of take carries out pcr amplification as template, and the SSR primer using in the implementation case is SMC1604SA, and fluorescent mark is FAM, and primer sequence is:
SMC1604SA-F（5'-3'）：FAM-AGG?GAA?AAG?GTA?GCC?TTG?G
SMC1604SA-R（5'-3'）：TTC?CAA?CAG?ACT?TGG?GTG?G
PCR reaction system is 20 μ L, 50ng DNA profiling 1 μ L wherein, 1 * PCR Buffer2 μ L, 3mmol Mg2+0.6 μ L, 2mmol/each dNTP2 μ L, each 0.5 μ L of 10 μ mol SMC1604SA-F and SMC1604SA-R primer, 5U/ μ L Taq enzyme 0.2 μ L, ddH2O13.2 μ L.
PCR response procedures is: 95 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 59 ℃ of annealing 60s, 72 ℃ are extended 60s, 35 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.
3.PCR product capillary electrophoresis analysis
The capillary electrophoresis instrument WeiABI PRISM3730 of company sequenator, sample loading is processed with reference to instrument explanation requirement, and capillary electrophoresis sample introduction voltage be-15kV, and sample injection time is 40s, and separation voltage is-15kV that temperature control is 25 ℃.
4. the foundation of sugarcane capillary electrophoresis DNA finger printing
The capillary electrophoresis collection of illustrative plates obtaining according to step 3, select the electrophoresis peak that meets SSR design of primers target product size and repeating unit's feature, carry out the data gathering of capillary electrophoresis collection of illustrative plates validity feature, comprise that the position at big or small electrophoresis peak of corresponding pcr amplification product is, the area at the height at electrophoresis peak, electrophoresis peak; Then carry out the stdn of capillary electrophoresis collection of illustrative plates validity feature data, the characteristic at the electrophoresis peak of electrophoresis peak heights is the highest or electrophoresis peak area maximum is standardized as 1, and all the other electrophoresis peaks carry out stdn by its electrophoresis peak heights and the ratio at high voltage electrophoresis peak or the ratio at electrophoresis peak area and maximum electrophoresis peak; Finally utilize the position at big or small electrophoresis peak and the electrophoresis peak characteristic of correspondence thereof of corresponding pcr amplification product to build sugarcane DNA fingerprinting.As Fig. 1, Fig. 2, table 1, shown in table 2.
Embodiment 2
Evaluation by sugarcane capillary electrophoresis DNA finger printing of the present invention for certain sugarcane sample
After Sugarcane Variety Resource capillary electrophoresis DNA fingerprints database is set up, in the time of will identifying certain sample, first extract its DNA, and carry out pcr amplification as DNA profiling, the SSR primer using is consistent with the primer of setting up Sugarcane Variety Resource capillary electrophoresis DNA fingerprints database, pcr amplification product carries out capillary electrophoresis analysis, capillary electrophoresis peak characteristic is carried out to stdn, application correlation analysis method compares the data in testing sample and database, according to relation conefficient, testing sample is identified.
Qualification result: if in testing sample and middle database certain Sugarcane Variety Resource Xiang Guan Xi Shuo≤99%, can determine this testing sample identity; If in testing sample and database all Sugarcane Variety Resources relation conefficient be all less than 95%, can determine that this testing sample is a novel material, can be appended in database; If in testing sample and database all Sugarcane Variety Resources relation conefficient between 95-99%, need to judge by human assistance.
If having or not by electrophoresis peak between Sugarcane smut osmanthus sugar 97-40 and POJ2931 in case study on implementation 1,1,0 data come more result for both are 100% consistent, if judged but apply the relation conefficient of authentication method of the present invention by two kinds of electrophoresis peak standardized feature values, find that relation conefficient is between the two r=0.8767, according to standard of perfection, both can be made a distinction.
Table 1 is sugarcane capillary electrophoresis DNA finger printing capillary electrophoresis peak feature raw data.
Table 2 is sugarcane capillary electrophoresis DNA finger printing capillary electrophoresis peak characteristic standard data.
Claims (3)
1. a construction process for sugarcane capillary electrophoresis DNA finger printing, is characterized in that, comprises the steps:
1) sugarcane DNA extraction: the method for pressing CTAB, SDS or DNA extraction test kit is extracted sugarcane DNA;
2) pcr amplification of sugarcane DNA: the sugarcane DNA extracting of take carries out pcr amplification as template, and the primer of employing is SSR primer;
3) analysis of pcr amplification product: adopt capillary electrophoresis analysis pcr amplification product, record capillary electrophoresis collection of illustrative plates;
4) capillary electrophoresis collection of illustrative plates validity feature data gathering, comprises that the position at big or small electrophoresis peak of corresponding pcr amplification product is, the area at the height at electrophoresis peak, electrophoresis peak;
5) stdn of capillary electrophoresis collection of illustrative plates validity feature data, the characteristic at the electrophoresis peak of electrophoresis peak heights is the highest or electrophoresis peak area maximum is standardized as 1, and all the other electrophoresis peaks carry out stdn by its electrophoresis peak heights and the ratio at high voltage electrophoresis peak or the ratio at electrophoresis peak area and maximum electrophoresis peak;
6) utilize the position at big or small electrophoresis peak of corresponding pcr amplification product and the electrophoresis peak characteristic standardized value of correspondence thereof to build sugarcane DNA fingerprinting.
2. an authentication method for sugarcane capillary electrophoresis DNA fingerprint, is characterized in that, comprises the steps:
1) sugarcane sample DNA extracts: the method for pressing CTAB, SDS or DNA extraction test kit is extracted sugarcane DNA;
2) pcr amplification of sugarcane sample DNA: the sugarcane DNA extracting of take carries out pcr amplification as template, and the primer of employing is SSR primer;
3) analysis of pcr amplification product: adopt capillary electrophoresis analysis pcr amplification product, record capillary electrophoresis collection of illustrative plates;
4) foundation of sugarcane sample capillary electrophoresis DNA finger printing: the DNA fingerprinting of setting up sugarcane sample with the construction process of described structure sugarcane capillary electrophoresis DNA finger printing;
5) sugarcane sample survey: utilize the dependency of DNA fingerprinting in correlation analysis algorithm comparative sample DNA fingerprinting and sugarcane DNA fingerprinting database, according to relation conefficient, sugarcane sample DNA is identified.
3. the construction process of sugarcane capillary electrophoresis DNA finger printing as claimed in claim 1, is characterized in that, described sugarcane DNA fingerprinting is saccharum sugarcane Hybrid or germ plasm resource DNA fingerprinting.
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| CN103911442A (en) * | 2014-03-13 | 2014-07-09 | 南宁益谱检测技术有限公司 | Rice fingerprint constructing method based on SSR marking and capillary electrophoresis technology |
| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN106916904A (en) * | 2017-05-11 | 2017-07-04 | 广西壮族自治区农业科学院甘蔗研究所 | A kind of preparation method and application of sugarcane EST SSR markers |
| 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 |
| CN114034753A (en) * | 2021-11-18 | 2022-02-11 | 辽宁省农业科学院 | Method for identifying fruiting phenotype of oudemansiella radicata based on efficient capillary electrophoresis |
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| CN103911442A (en) * | 2014-03-13 | 2014-07-09 | 南宁益谱检测技术有限公司 | Rice fingerprint constructing method based on SSR marking and capillary electrophoresis technology |
| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN106916904A (en) * | 2017-05-11 | 2017-07-04 | 广西壮族自治区农业科学院甘蔗研究所 | A kind of preparation method and application of sugarcane EST SSR markers |
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| 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 |
| CN114034753A (en) * | 2021-11-18 | 2022-02-11 | 辽宁省农业科学院 | Method for identifying fruiting phenotype of oudemansiella radicata based on efficient capillary electrophoresis |
| CN114034753B (en) * | 2021-11-18 | 2024-04-09 | 辽宁省农业科学院 | Method for identifying fruiting phenotype of agrocybe cylindracea based on efficient capillary electrophoresis |
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Application publication date: 20140129 |