CN115011715B - Finger-print of small bean constructed based on SSR molecular marker - Google Patents
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Abstract
The invention discloses a finger print of small bean constructed based on SSR molecular markers, and relates to the technical field of plant variety identification. The molecular markers include VaSSR01-VaSSR13, which are expressed by SEQ ID NO: 1-26. The invention also discloses a polypeptide shown as SEQ ID NO:1-SEQ ID NO:26, the kit comprising the primer combination and the construction method of the red bean fingerprint map construct the red bean fingerprint map by the constructed DNA fingerprint map, the fingerprint map can accurately identify different varieties of red beans by 100 percent, and the kit has the advantages of good detection effect, time saving, cost saving and the like, and can provide technical support for the DNA fingerprint identification of the red bean varieties.
Description
Technical Field
The invention relates to the field of plant variety identification, in particular to a finger print for constructing small beans based on SSR molecular markers.
Background
Vigna angularis (Vigna angularis) originated in china and belongs to one of the cowpea cultivars (Vigna). The planting of the small beans in China has at least more than 2000 years of history. The small bean belongs to coarse cereals, is rich in nutrients such as protein, vitamins, mineral elements and the like, has the function of inducing diuresis to reduce edema when being eaten properly, and is a popular crop for both medical and food. The red bean germplasm resources of China are rich, and as long as 2008, the preservation quantity of the national germplasm bank is nearly 5000 parts, and because the varieties are frequently exchanged among different regions, the types and varieties of the red bean are various, the sources of the red bean are complex, and a large number of 'same-name heterogeneous' germplasms and 'one-product multiple-name' germplasms exist. The red bean varieties are disordered and hard to distinguish, a plurality of producers cannot distinguish the locally planted varieties, and the quality of the produced high-quality and high-yield red beans cannot be guaranteed, so that serious loss is caused. For many years, variety identification was based on morphological markers. Although the method is simple and economical, the method has long period, high cost and season limitation, and the expression of a plurality of characters is influenced by cultivation measures and environmental factors, thereby restricting the accuracy of identification. The DNA molecular marker technology has the advantages of high efficiency, accuracy, no influence of environmental conditions, simple experiment operation and the like, and is widely applied to the authenticity identification and purity detection research of plant varieties. SSR markers are second-generation molecular markers established on the basis of PCR and developed in recent years. The SSR molecular marker has the characteristics of abundant quantity, high polymorphism, genetic codominance, stable band amplification, high precision, short detection time, mature technology and the like, and is applied to crop genetics research. At present, the small bean is also researched by using a molecular marker technology, but an SSR molecular marker which is developed only aiming at small bean varieties and a technical system constructed by using the SSR molecular marker are absent.
Disclosure of Invention
The invention aims to provide a fingerprint of small bean constructed based on SSR molecular marker, which can be used for solving the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker combination for identifying small beans, wherein the molecular markers comprise VaSSR01, vaSSR02, vaSSR03, vaSSR04, vaSSR05, vaSSR06, vaSSR07, vaSSR08, vaSSR09, vaSSR10, vaSSR11, vaSSR12 and VaSSR13; the VaSSR01 is expressed by the nucleotide sequence shown as SEQ ID NO:1-2, and the VaSSR02 is obtained by amplifying a primer pair shown in SEQ ID NO:3-4, and the VaSSR03 is obtained by amplifying a primer pair shown as SEQ ID NO:5-6, wherein the VaSSR04 is obtained by amplifying a primer pair shown as SEQ ID NO:7-8, wherein the VaSSR05 is obtained by amplifying a primer pair shown as SEQ ID NO:9-10, wherein the VaSSR06 is obtained by amplifying a primer pair shown as SEQ ID NO:11-12, and the VaSSR07 is obtained by amplifying a primer pair shown in SEQ ID NO:13-14, and the VaSSR08 is obtained by amplifying a primer pair shown as SEQ ID NO:15-16, and the VaSSR09 is obtained by amplifying a primer pair shown in SEQ ID NO:17-18, and the VaSSR10 is obtained by amplifying a primer pair shown in SEQ ID NO:19-20, wherein the VaSSR11 is obtained by amplifying a primer pair shown as SEQ ID NO:21-22, and the VaSSR12 is obtained by amplifying a primer pair shown as SEQ ID NO:23-24, and the VaSSR13 is obtained by amplifying a primer pair shown as SEQ ID NO: 25-26.
The invention also provides a primer combination for identifying the small beans, which comprises 13 pairs of specific primer pairs, wherein the nucleotide sequences of the primer pairs are respectively shown as SEQ ID NO:1-SEQ ID NO: shown at 26.
The invention also provides a kit for identifying the small beans, which comprises the primer combination.
The invention also provides a construction method of the small bean fingerprint spectrum, which comprises the following steps: the small beans with different growth habits are collected and numbered, the primer combination is utilized to carry out PCR amplification on DNA of the small beans with different varieties, then electrophoresis detection is carried out on the amplified products, and the small bean fingerprint spectrum is constructed according to the electrophoresis detection result.
Furthermore, adzuki bean varieties include north 23 with resource pool number XD017, north 26 with resource pool number XD021, shandong jujube village farmhouse with resource pool number XD035, baoqing red with resource pool number XD036, LZX031 with resource pool number XD045, LZX051 with resource pool number XD050, red adzuki bean with resource pool number XD053, LZX063 with resource pool number XD054, red pearl with resource pool number XD075, HD2 with resource pool number XD084, FBX01 with resource pool number XD099, FJMS01 with resource pool number XD113, FLD01 with resource pool number XD116, tianjin red with resource pool number XD120, JI red with resource pool number XD128, A485 with resource pool number XD130, miscellaneous beans with resource pool number XD182, XD 1-XD 182, XD 1-199 with resource pool number XD199, XD H199-18, and XD H199-18-199.
Further, the reaction system for PCR amplification is: DNA template 0.8. Mu.L, L0pmo1/L forward primer 0.2. Mu.L, 10pmo1/L reverse primer 0.2. Mu.L, 10 XPCR buffer 2. Mu.L, 25mmol/LMg 2+ 1.2. Mu.L, 10mmol/L dNTP 0.5. Mu.L, 5U/. Mu.L Taq enzyme 0.2. Mu.L, ddH 2 O14.4 μ L; the PCRbuffer does not contain Mg 2+ 。
Further, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30 s; extension at 72 ℃ for 5min and storage at 4 ℃.
The invention also provides application of the molecular marker combination, the primer combination or the kit in acquisition of the small bean fingerprint.
The invention also provides application of the molecular marker combination, the primer combination, the kit or the construction method in identifying the red bean varieties.
The invention discloses the following technical effects:
the invention discloses a molecular marker combination for identifying small beans, which can be amplified by only using 13 pairs of small bean primer pairs, so that small bean varieties can be effectively identified, and the identification accuracy rate is up to 100%. Therefore, the molecular marker combination and the primer combination disclosed by the invention have great potential in the aspect of further identifying a large number of small bean varieties, have the advantages of better effect, time saving, cost saving and the like compared with the conventional morphological identification, and can provide technical support for DNA fingerprint identification of the small bean varieties.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram of the electrophoretic band pattern of 25 portions of red bean material with M being Marker and 01-25 being used for detecting and recording DNA fingerprint.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 construction method of finger-print of adzuki bean
1. Experimental Material
The method is characterized in that 25 small bean varieties are used as sample materials, and fingerprint data of the small bean varieties are constructed according to the developed SSR molecular markers so as to achieve the purpose of variety identification.
The information of 25 kinds of adzuki beans is shown in Table 1. In the following examples, the species names and resource pool numbers of the small beans are from the germplasm resource pool of the research center of the minor cereal engineering technology.
TABLE 1 variety information of 25 kinds of small beans
2. Construction of standard DNA fingerprint spectrum library of small beans of different varieties
2.1 Total DNA extraction of various varieties of Small beans
Planting red bean in soil, culturing at room temperature, taking young leaf of red bean after growing for 10-15d, freezing in liquid nitrogen, and storing at-80 deg.C for use. Taking 0.2g of young and tender leaves of each variety of small beans, putting the young and tender leaves into a centrifugal tube with the diameter of 1.5m, adding liquid nitrogen, and grinding the leaves into powder. DNA is extracted by a kit method, and the operation experiment steps are carried out according to the kit instruction. The total DNA was analyzed for mass by agarose gel electrophoresis and spectrophotometer, and then diluted to 100 ng/. Mu.L and stored at-20 ℃ until use.
2.2 screening of primers
SSR marker synthesis: an SSR molecular marker amplification primer is synthesized by a biological company, the purity requirement of the primer synthesis is PAGE purification, the primer synthesis is divided into 10D/tube, and the concentrations of the upstream primer and the downstream primer are both diluted to 10pmol/L.
SSR molecular marker primer combination screening method
Step one, screening a primer: screening SSR primary screening primers which have polymorphism and clear band patterns among different small bean varieties and can be stably repeated.
The following primers were obtained by screening, and their labels, primer names, and positions are shown in Table 2.
TABLE 2 primer tag names
Screening of SSR-labeled amplification primer combinations
Taking small bean varieties as materials, respectively carrying out PCR amplification on DNA of each small bean variety by using an amplification primer (shown in table 3) of a synthesized SSR molecular marker, wherein a PCR reaction system adopted in a PCR reaction test is a 20-mu-L system: 0.8. Mu.L of DNA template, 0.2. Mu.L of F-primer (10 pmol/L), 0.2. Mu.L of R-primer (10 pmol/L), 10 XPCR buffer (Mg-free) 2+ )2μL，Mg 2+ (25 mmo 1/L) 1.2. Mu.L, dNTP (10 mmol/L) 0.5. Mu.L, taq enzyme (5U/. Mu.L) 0.2. Mu.L, ddH 2 O14.4. Mu.L, 20.0. Mu.L in total.
The PCR reaction program is: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30 s; extension at 72 ℃ for 5min and storage at 4 ℃.
d. Gel electrophoresis and Gel-red color development
The amplification products were detected by 10% native polyacrylamide gel electrophoresis in a Sequi-Gen GT nucleic acid electrophoresis system (Bio-Rad, USA).
Color development: fixing for 30min by using Gelred, placing the gel into gel imaging (Bio-red) for photographic recording, recording an electrophoresis result according to the standard DNA molecular weight and the mobility of primers, recording a strip with the maximum mobility of each pair of primers as 1, recording a strip with the second mobility as 2, repeating the steps until no strip is recorded as 0, establishing SSR genotype information data, and coding according to the sequence of the primers to obtain a fingerprint code which is the characteristic fingerprint of the variety; the recording mode is shown in fig. 1.
3.3 construction of DNA characteristic fingerprint of small bean
TABLE 3 molecular marker fingerprint code of 25 parts of red bean material
| Serial number | Name(s) | Resource pool number | Fingerprint code |
| 1 | North 26 | XD021 | 1111211212121 |
| 2 | North 23 | XD017 | 1121111211111 |
| 3 | LZX031 | XD045 | 1111221112111 |
| 4 | LZX051 | XD050 | 2111121122221 |
| 5 | Small red bean (Zhangdi Yingzi village) | XD053 | 2111122111121 |
| 6 | Shandong jujube village farmhouse | XD035 | 1121111111112 |
| 7 | Baoqinghong wine | XD036 | 2122121222211 |
| 8 | Small red bean 1-1 | XD132 | 2121112122111 |
| 9 | LZX063 | XD054 | 2111222111221 |
| 10 | Red pearl | XD075 | 2111221121211 |
| 11 | HD2 | XD084 | 2112221121121 |
| 12 | FBX01 | XD099 | 1211111121212 |
| 13 | FJMS01 | XD113 | 1211121121211 |
| 14 | FLD01 | XD116 | 1111222111122 |
| 15 | Tianjin Red | XD120 | 1211212211212 |
| 16 | A485 | XD128 | 1211222111211 |
| 17 | Mixed beans | XD130 | 1112212131111 |
| 18 | Small red bean | XD166 | 1212121111111 |
| 19 | H05 | XD178 | 1111222112211 |
| 20 | H06 | XD180 | 1111111112112 |
| 21 | H07 | XD182 | 2211222112211 |
| 22 | Jihong 9218 | XD184 | 1212222112211 |
| 23 | H09-1 | XD188 | 1112222122211 |
| 24 | H14-1 | XD199 | 1212211212222 |
| 25 | Guayule 876-16 | XD143 | 1121222111112 |
As can be concluded from Table 3, the DNA molecular marker fingerprints of any two varieties are different, and it can be determined that 25 small bean varieties to be tested are different, and the corresponding DNA molecular fingerprint codes are the unique fingerprints of the varieties.
The result shows that the 13 pairs of primer combination can be used for identifying the genetic characteristics of the small bean varieties and protecting the rights of the varieties from being invaded.
The invention constructs 25 small bean DNA finger prints by utilizing the technical system and the screened 13 pairs of small bean SSR polymorphic primers, can distinguish 25 small bean varieties and provides technical support for identification and variety right protection of the small beans.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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Claims (8)
1. A molecular marker combination for identifying adzuki beans, wherein the molecular markers include VaSSR01, vaSSR02, vaSSR03, vaSSR04, vaSSR05, vaSSR06, vaSSR07, vaSSR08, vaSSR09, vaSSR10, vaSSR11, vaSSR12, and VaSSR13; the VaSSR01 is expressed by a nucleotide sequence as shown in SEQ ID NO:1-2, and the VaSSR02 is obtained by amplifying a primer pair shown as SEQ ID NO:3-4, and the VaSSR03 is obtained by amplifying a primer pair shown as SEQ ID NO:5-6, wherein the VaSSR04 is obtained by amplifying a primer pair shown in SEQ ID NO:7-8, wherein the VaSSR05 is obtained by amplifying a primer pair shown as SEQ ID NO:9-10, wherein the VaSSR06 is obtained by amplifying a primer pair shown as SEQ ID NO:11-12, and the VaSSR07 is obtained by amplifying a primer pair shown as SEQ ID NO:13-14, and the VaSSR08 is obtained by amplifying a primer pair shown as SEQ ID NO:15-16, and the VaSSR09 is obtained by amplifying a primer pair shown as SEQ ID NO:17-18, wherein the VaSSR10 is obtained by amplifying a primer pair shown in SEQ ID NO:19-20, wherein the VaSSR11 is obtained by amplifying a primer pair shown as SEQ ID NO:21-22, and the VaSSR12 is obtained by amplifying a primer pair shown as SEQ ID NO:23-24, and the VaSSR13 is obtained by amplifying a primer pair shown as SEQ ID NO: 25-26.
2. A primer combination for identifying small beans is characterized by comprising 13 pairs of specific primer pairs, wherein nucleotide sequences of the primer pairs are respectively shown as SEQ ID NO:1-SEQ ID NO: shown at 26.
3. A kit for identifying adzuki beans, comprising the primer combination according to claim 2.
4. A construction method of small bean fingerprint spectrum is characterized by comprising the following steps: collecting and numbering adzuki beans with different growth habits, carrying out PCR amplification on DNA of adzuki beans with different varieties by using the primer combination according to claim 2, carrying out electrophoresis detection on an amplification product, recording an electrophoresis result according to standard DNA molecular weight and the mobility of primers, recording a strip with the maximum mobility of each pair of primers as 1, recording a strip with the second mobility as 2, repeating the steps until no strip is recorded as 0, establishing SSR genotype information data, and coding according to the sequence of the primers to construct the adzuki bean fingerprint;
the varieties of the small bean comprise northern 23 with resource library number XD017, northern 26 with resource library number XD021, shandong jujube farmer's race with resource library number XD035, baoqing red with resource library number XD036, LZX031 with resource library number XD045, LZX051 with resource library number XD050, small red bean with resource library number XD053, LZX063 with resource library number XD054, red pearl with resource library number XD075, HD2 with resource library number XD084, FBX01 with resource library number XD099, FJMS01 with resource library number XD113, FLD01 with resource library number XD116, tianjin red with resource library number XD120, A485 with resource library number XD128, XD130, miscellaneous red bean with resource library number XD182, XD 1-XD 188, XD H199-XD H199, XD H18-XD H06, XD H199-XD H94, XD H199-XD H94, and XD H188;
the resource libraries are germplasm resource libraries of the national coarse cereal engineering technical research center.
5. The construction method according to claim 4, wherein the reaction system of the PCR amplification is: DNA template 0.8. Mu.L, L0pmo1/L forward primer 0.2. Mu.L, 10pmo1/L reverse primer 0.2. Mu.L, 10 XPCR buffer 2. Mu.L, 25mmol/LMg 2+ 1.2. Mu.L, 10 mmol/. Mu.L dNTP 0.5. Mu.L, 5U/. Mu.L of LTaq enzyme, ddH 2 O14.4 μ L; the PCRbuffer does not contain Mg 2+ 。
6. The construction method according to claim 4, wherein the reaction procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 3min; 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30 s; extension at 72 ℃ for 5min and storage at 4 ℃.
7. The application of the molecular marker combination according to claim 1, the primer combination according to claim 2 or the kit according to claim 3 in obtaining the fingerprint of adzuki bean, wherein the adzuki bean comprises north 23 with resource library number XD017, north 26 with resource library number XD021, shandong juzhuang nong's breed with resource library number XD035, baoqing red with resource library number XD036, LZX031 with resource library number XD045, LZX051 with resource library number XD050, adzuki bean with resource library number XD053, LZX063 with resource library number XD054, adzuki pearl with resource library number XD075, HD2 with resource library number XD084, x01 with resource library number XD099, FJMS01 with resource library number 113, FLD01 with resource library number XD116, FLD 120 with resource library number XD120, XD with resource library number XD # 102, XD 1-102 with resource library number XD, XD18 XD H-102 with resource library number XD # 102, XD # 14, and 14-94 with resource library number XD # 14, and 14-188, and 132 with resource library number 14 and 132;
the resource libraries are germplasm resource libraries of the national coarse cereal engineering technology research center.
8. Use of the molecular marker combination according to claim 1, the primer combination according to claim 2, the kit according to claim 3, or the method according to any one of claims 4 to 6 for identifying adzuki bean varieties; it is characterized in that the varieties of the small bean include north 23 with resource library number XD017, north 26 with resource library number XD021, shandong jujube village farmyard with resource library number XD035, baoqing red with resource library number XD036, LZX031 with resource library number XD045, LZX051 with resource library number XD050, small red bean with resource library number XD053, LZX063 with resource library number XD054, red pearl with resource library number XD075, HD2 with resource library number XD084, FBX01 with resource library number XD099, FJMS01 with resource library number XD113, FLD01 with resource library number XD116, tianhong with resource library number XD120, A485 with resource library number 128, XD130, miscellaneous beans with resource library number XD182, XD 1-XD 182, XD H199-18 with resource library number XD 199-18, XD H199-18 with resource library number XD 199-199, XD-18, and XD H09;
the resource libraries are germplasm resource libraries of the national coarse cereal engineering technology research center.
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