CN107794306B - Method for identifying authenticity of green sword bean variety - Google Patents
Method for identifying authenticity of green sword bean variety Download PDFInfo
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- CN107794306B CN107794306B CN201610711969.9A CN201610711969A CN107794306B CN 107794306 B CN107794306 B CN 107794306B CN 201610711969 A CN201610711969 A CN 201610711969A CN 107794306 B CN107794306 B CN 107794306B
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Abstract
The invention relates to a method for identifying the authenticity of a green sword bean variety, belonging to the field of biology. The identification of variety genuineness is increasingly important to maintaining the interests of producers and breeders. The green sword beans are strict self-pollinated crops, the varieties are mainly conventional varieties, the conventional varieties have no parents, and the varieties can be reserved automatically, so that the new variety protection of the green sword beans is more difficult. The method for identifying the authenticity of the green sword bean variety by using the morphological indexes consumes long time and has poor reliability, so that the invention of the method for identifying the authenticity of the green sword bean variety by using the molecular marker becomes urgent. The technical scheme is that 6 pairs of SSR primers (SEQ ID NO.1-SEQ ID NO.12) and 1 RAPD primer (SEQ ID NO.13) are utilized to carry out PCR amplification on three varieties and a control, 12 generated differential sites are assigned with values of '1' and '0', and a variety fingerprint is constructed for authenticity identification. The method carries out more accurate and more effective identification and protection on the green sword bean varieties 'Yonglv No. 1', 'Yonglv No. 3' and 'Yonglv No. 4' by a more sensitive identification means and lower time and labor cost.
Description
One, the technical field
The invention belongs to the field of biology, and particularly relates to an identification method for authenticity of green sword bean varieties 'Yonglv No. 1', 'Yonglv No. 3' and 'Yonglv No. 4'.
Second, background Art
The green sword beans (Phaseolus vulgaris), namely the kidney beans, kidney beans and small sword beans, are annual winding herbaceous plants, and the bean pods are rich in nutrition and protein, can be directly sold on the market for fresh eating, can also be prepared into quick-frozen vegetables for exporting earning, and have high economic benefit. The green sword beans are strict self-pollinated crops, the varieties are mainly conventional varieties, and the conventional varieties have no parents and can be reserved by themselves, so that the problems of unclear sources, synonyms, foreign matters and the like of the green sword beans circulating on the market generally occur, and the difficulty is increased for protecting new varieties of the green sword beans. The conventional identification method for the authenticity of the green sword bean variety mainly utilizes morphological indexes of grains, a seedling stage or a plant forming stage for identification, the phenotypic indexes are easily influenced by the plant development period and the growth environment, the identification period is long, and the identification result is low in accuracy. Therefore, establishing a method for identifying the authenticity of a green sword bean variety is extremely interesting for maintaining producers and breeders.
The Yonglv No.1 ', Yonglv No.3 ' and Yonglv No.4 ' are new green sword bean varieties which are independently bred by agricultural science research institute in Ningbo city, have the characteristics of heat resistance, high yield, fresh green bean pods and the like, and are popular with farmers after popularization. At present, only field morphology identification is carried out on Yonglv No.1, Yonglv No.3 and Yonglv No.4, a convenient and effective variety authenticity identification method is lacked, and variety protection is far from being lacked.
Third, the invention
Aiming at the problems, the invention provides a method for identifying the authenticity of the varieties of green sword bean, namely Yonglv No.1, Yonglv No.3 and Yonglv No.4 by using a DNA fingerprint.
In order to achieve the purpose of the invention, the inventor provides the following technical scheme: extracting young leaf DNA of 'Yonglv No. 1', 'Yonglv No. 3' and 'Yonglv No. 4' and comparison varieties thereof by adopting a CTAB method; performing PCR amplification by using 6 pairs of SSR primers and 1 RAPD primer (the corresponding relation between the primer name and the sequence number in the sequence table is shown in table 1, and the specific sequence of the primer is shown in the sequence table finally attached to the specification); and (3) carrying out agarose electrophoresis separation and ethidium bromide staining on the amplification product, and detecting polymorphism on an ultraviolet fluoroscope. The polymorphism of the amplification product reflects the polymorphism of the genome. The difference allelic sites, the amplified band is marked as '1', and the band at the same position is not marked as '0'; these differentially amplified fragments were converted into a string consisting of 1 and 0, i.e., a DNA fingerprint was constructed to distinguish 3 varieties of green sword beans and their controls.
The invention has the advantages that: compared with conventional morphological identification, the method has the advantages of accurate and reliable identification result, short time consumption, no environmental influence, simple operation, capability of identifying a plurality of varieties simultaneously, convenience and rapidness.
Description of the drawings
FIG. 1 shows the amplification of SSR primers SD43/44, SD63/64, SD103/104, SD111/112, SD113/114, SD151/152 and RAPD primer P158 in ` Yonglv No.1 `, ` Yonglv No.3 ` and ` Yonglv No.4 ` and their control materials ` Xianglv No.1 ` and ` south Taihu ` in the examples of the present invention.
In FIG. 1, the three left pictures are, from top to bottom, the amplifications of SSR primers SD43/44, SD63/64 and SD103/104 in Yonglv No.1 ', ' Yonglv No.3 ' and ' Yonglv No.4 ' and their control materials ' Bright Green No.1 ' and ' south Taihu ', respectively; the four images on the right side are the amplifications of SSR primers SD111/112, SD113/114, SD151/152 and RAPD primer P158 in Yonglv No.1 ', ' Yonglv No.3 ' and ' Yonglv No.4 ' and their control materials ' Bright Green No.1 ' and ' south Taihu ' respectively from top to bottom. The two groups of picture samples are sequentially arranged from left to right by DNAmker, Yonglv No.1, Yonglv No.3, Yonglv No.4, fresh green No.1 and south Taihu. The left side of the picture is marked with DNAmarker standard molecular weight, and the right side is marked with the name of the primer and the size of the segment of the differential site.
Fifth, detailed description of the invention
1. Sampling: sowing Yonglv No.1, Yonglv No.3 and Yonglv No.4, and collecting 1g of tender leaves when the plants grow to 5-6 true leaves, and storing at-80 deg.C.
2. DNA extraction: fresh leaf total genomic DNA was extracted by the CTAB method (see: Murray HG, Thompson WF. Rapid isolation of high her weight DNA. nucleic acids Res,1980,8: 4321).
3. And (3) PCR amplification: the mix system for SSR and RAPD was 15. mu.l (Fazio G, Staub JE, Stevens MR. genetic mapping and QTL analysis of Horticultural tracks in cuumber (Cucumis sativus L.) using recombinant in branched lines. TheorAppl Genet,2003,107: 864. 874) including 1.5. mu.l dNTP (1.25mM), 1.5. mu.l 10 XBuffer, 1.5. mu.l MgCl2(25mM), 2.0ul of primers (5 mM; 1.0ul of each primer of SSR), 5ul of DNA (10ng/ul), 0.05ul of Taq (1U), and 3.45ul of ultrapure water. The PCR amplification procedure for SSR was: pre-denaturation at 94 ℃ for 1 min; denaturation at 93 deg.C for 1min, annealing at 40 deg.C for 1min, and extension at 72 deg.C for 2min (40 cycles); extension at 72 ℃ for 1 min. The PCR amplification procedure for RAPD was: pre-denaturation at 94 ℃ for 4 min; denaturation at 93 ℃ for 15sec, annealing at 37 ℃ for 1min for 30sec, and elongation at 72 ℃ for 2min (40 cycles); extension was carried out at 72 ℃ for 6 min.
4. And (3) detecting a PCR product: SSR used 2.5% high resolution standard gel agarose (DNA fragment separation range 40-1Kbp), RAPD amplification product used 2% common standard gel agarose (DNA fragment separation range 500-20 Kbp). Add 20. mu.l EB (ethidium bromide ) for color development. Add 2. mu.l of Loading Dye to the PCR product, electrophorese for 2-3 hours, voltage 200-. After the electrophoresis was finished, the results were observed by using a gel imaging system.
5. Data collection and analysis: evaluation is carried out on the amplification results of 6 pairs of SSR primers SD43/44, SD63/64, SD103/104, SD111/112, SD113/114, SD151/152 and 1 RAPD primer P158 in 'Yonglv No. 1', 'Yonglv No. 3' and 'Yonglv No. 4' and control materials thereof 'Xionglv No. 1' and 'Nantaihu', a strip is marked as '1' when a certain allele is amplified, and a strip without amplification at the same site is marked as '0'. These differentially amplified fragments were converted into a string consisting of 1 and 0, constituting a variety fingerprint (Table 1). 12 polymorphic sites generated by 7 molecular markers are utilized to construct fingerprint spectrums of Yonglv No.1 ' of 1-0-0-1-0-1, fingerprint spectrums of ' Yonglv No.3 ' of 0-1-0-1-0-1-0-1-1-1, fingerprint spectrums of ' Yonglv No.4 ' of 0-1-0-0-0-1-1-1, fingerprint spectrums of ' fresh green No.1 ' of 1-0-0-1-0-1-0, and fingerprint spectrums of ' south Taihu ' of 0-1-1-1 -0-1-1-1-1. The fingerprint maps can effectively distinguish the three varieties, and can also distinguish the three varieties from similar varieties with easily confused forms, thereby playing roles in variety identification and protection.
TABLE 1 Green sword bean variety to be tested and reference number, name and SSR fingerprint
Claims (1)
1. A method for identifying the authenticity of a green sword bean variety comprises the following steps:
1. sampling: sowing Yonglv No.1, Yonglv No.3 and Yonglv No.4, and collecting 1g of tender leaves when the plants grow to 5-6 true leaves, and storing at-80 deg.C;
2. DNA extraction: extracting fresh leaf total genome DNA by referring to a CTAB method;
3. and (3) PCR amplification: SSR and RAPD, namely a mixed sample system of 6 pairs of SSR primers SD43/44, SD63/64, SD103/104, SD111/112, SD113/114, SD151/152 and 1 RAPD primer P158 is 15 ul, and comprises 1.25mM dNTP 1.5ul, 1.5ul 10 XBuffer, 25mM MgCl21.5 ul, and 1.0ul of 2.0ul primer/5 mM RAPD primer P1582.0 ul, 10ng/ul DNA 5ul, 1U Taq 0.05ul and 3.45ul ultrapure water which are respectively added into two primers of 5mM SSR; the PCR amplification procedure for SSR was: pre-denaturation at 94 ℃ for 1 min; denaturation at 93 deg.C for 1min, annealing at 40 deg.C for 1min, and extension at 72 deg.C for 2min for 40 cycles; extending at 72 ℃ for 1 min; the PCR amplification procedure for RAPD was: pre-denaturation at 94 ℃ for 4 min; denaturation at 93 ℃ for 15sec, annealing at 37 ℃ for 1min for 30sec, and extension at 72 ℃ for 2min for 40 cycles; extending at 72 ℃ for 6 min;
4. and (3) detecting a PCR product: SSR uses 2.5% high resolution standard gel agarose, in which the DNA fragments are separated in the range of 40-1Kbp, and RAPD amplification products use 2% common standard gel agarose, in which the DNA fragments are separated in the range of 500-20 Kbp. Adding 20 μ l EB, ethidium bromide, for color development; adding 2 μ l Loadingdye into PCR product, electrophoresis for 2-3 hours, voltage 200-; after electrophoresis is finished, observing the result by using a gel imaging system;
5. data collection and analysis: assigning values to amplification results of 6 pairs of SSR primers SD43/44, SD63/64, SD103/104, SD111/112, SD113/114, SD151/152 and 1 RAPD primer P158 in 'Yonglv No. 1', 'Yonglv No. 3' and 'Yonglv No. 4' and control materials thereof 'Xianchu No. 1' and 'Nantaihu', wherein a band is marked as '1' when a certain allele is amplified, and a band which is not amplified at the same site is marked as '0'; converting the differential amplification fragments into character strings consisting of 1 and 0, namely forming a variety fingerprint;
wherein, the sequence of SD43/44 in the 6 pairs of SSR primers is shown as SEQ ID NO.1 and SEQ ID NO.2, the sequence of SD63/64 is shown as SEQ ID NO.3 and SEQ ID NO.4, the sequence of SD103/104 is shown as SEQ ID NO.5 and SEQ ID NO.6, the sequence of SD111/112 is shown as SEQ ID NO.7 and SEQ ID NO.8, the sequence of SD113/114 is shown as SEQ ID NO.9 and SEQ ID NO.10, the sequence of SD151/152 is shown as SEQ ID NO.11 and SEQ ID NO.12, and the sequence of 1 RAPD primer P158 is shown as SEQ ID NO. 13.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105420368A (en) * | 2015-12-17 | 2016-03-23 | 黑龙江八一农垦大学 | Method for constructing fingerprint of phaseolus vulgaris according to SSR (simple sequence repeat) molecular marker and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420368A (en) * | 2015-12-17 | 2016-03-23 | 黑龙江八一农垦大学 | Method for constructing fingerprint of phaseolus vulgaris according to SSR (simple sequence repeat) molecular marker and application |
Non-Patent Citations (2)
| Title |
|---|
| Genetic diversity in local and commercial dry bean(Phaseolus vulgaris)accessions based on microsatellite markers;P.D.S.Cabral et al.;《Genetics and Molecular Research》;20110201;第10卷(第1期);全文 * |
| Genetic diversity of common bean(Phaseolus vulgaris L.)landraces grown in northeast anatolia of Turkey assessed with simple sequence repeat markers;Maya Izar KHAIDIZAR et al.;《Turkish Journal of Field Crops》;20121231;第17卷(第2期);全文 * |
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