CN111690768A - Construction method of excellent variety DNA fingerprint spectrum database of oil peony and special primer pair combination thereof - Google Patents

Abstract

The invention discloses a construction method of a DNA fingerprint database of excellent oil peony varieties and a special primer pair combination thereof, belonging to the technical field of biology. The method comprises the following steps: extracting peony genome DNA for oil; performing PCR amplification reaction of SSR primers and performing capillary electrophoresis on amplification products; reading band information to establish an oil peony DNA digital identity card; and converting the digital code into a two-dimensional code picture to establish the peony electronic identity card for oil. The method for constructing the DNA fingerprint of the peony for oil, provided by the invention, is simple and rapid to operate, and reliable, stable and accurate in result; according to the fingerprint spectrum database of the 87 parts of excellent oil peony varieties, DNA fingerprint information of each oil peony variety has uniqueness, so that a technical basis can be provided for identifying the authenticity of the excellent oil peony varieties, supervision and inspection on oil peony seedling production, identification of new varieties and protection of intellectual property rights of breeding workers are of great significance.

Description

Construction method of excellent variety DNA fingerprint spectrum database of oil peony and special primer pair combination thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method of a peony fine variety DNA fingerprint spectrum library for oil and a special primer pair combination thereof.

Background

Peony is a generic name of the peony (Paeonia) group (sect. moutan DC) plant, and is a perennial deciduous shrub. The oil peony is a peony type which can be used for producing edible peony oil processed by seeds, or a peony variety with the seed oil yield of more than or equal to 22%. The content of unsaturated fatty acid in the peony seed oil is up to 92.26%, wherein the content of alpha-linolenic acid is 43.18% (6 times of soybean oil), the content of oleic acid is 21.93%, and the content of linoleic acid is 27.15%. Meanwhile, the peony seed oil also contains nutrient substances such as paeonol, paeoniflorin, peony polysaccharide, fucosterol, squalene and the like, and multiple indexes of the nutrient substances exceed those of olive oil, so that the peony seed oil is high-quality edible oil. The national ministry of health has approved peony seed oil as a new resource food in 2011. In 2014, the State Council released implementation opinions on accelerating the development of the woody oil industry, and clearly listed peony as the third largest woody oil crop. The oil peony is used as a new woody oil crop which is fully popularized in China, the planting area and range are rapidly increased, and the most popular oil peony varieties at present comprise paeonia ostii (P.ostii) and paeonia rockii (P.rockii). In the vigorous development process of the oil peony industry, real and reliable excellent varieties are the basis and key for ensuring the continuous and healthy development of the oil peony industry.

The DNA fingerprint is a DNA electrophoresis pattern which is established by utilizing molecular markers and can identify differences among biological individuals, can quickly and accurately identify the authenticity and purity of plant varieties, has the characteristics of high individual specificity, good environmental stability and the like, and plays an important role in the aspects of plant genetic breeding and variety protection. SSR (simple sequence repeat) is a repetitive sequence universally existing in eukaryotic genomes and consists of tandem repetitive sequences with the length of 1-6 bp. The SSR marker is a sequence with the highest mutation rate in a genome, has high specificity and polymorphism, and is the most suitable molecular marker for constructing a DNA fingerprint. The fingerprint of the excellent variety of the oil peony constructed by utilizing the SSR molecular marker technology not only can provide a technical basis for identifying the authenticity of the excellent variety of the oil peony, but also has important significance for supervising and checking the production of the seedling of the oil peony, identifying new varieties and protecting the intellectual property rights of breeding workers.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a special primer pair combination for constructing a peony DNA fingerprint spectrum for oil. The invention aims to solve another technical problem of providing a construction method of a DNA fingerprint database of an excellent variety of oil peonies.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a special primer pair combination for constructing a DNA fingerprint of oil peony is disclosed, and the sequences of the special primer pairs are as follows:

the special primer pair combination for constructing the peony for oil DNA fingerprint is applied to constructing the peony for oil DNA fingerprint.

Further, PCR amplification is respectively carried out on the genome DNA of the peony for oil by utilizing the 1 st to 10 th primer pairs, and an amplification band type is obtained by carrying out capillary electrophoresis on an amplification product, namely the DNA fingerprint of the peony for oil.

Further, the reaction system for PCR amplification comprises: template DNA25ng, 20ng each of the upstream and downstream primers labeled with fluorophore FAM at the 5' end, 200. mu.M dNTP, 0.5U Taq polymerase, 1.5. mu.L 10 XPCR buffer, and sterilized deionized water to make up the reaction volume to 15. mu.L.

Further, the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94 ℃ for 2 min; then denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min.

A method for constructing a DNA fingerprint database of oil peonies comprises the following steps: the application is utilized to obtain the DNA fingerprint of the peony for oil, the amplification band type of the DNA fingerprint of the peony for oil is subjected to coding treatment to obtain the peony DNA fingerprint code represented by a character string, and the character string is further converted into a two-dimensional code graph by utilizing two-dimensional code conversion software. The method specifically comprises the following steps:

(1) extracting genome DNA of a peony sample for certain oil;

(2) carrying out PCR amplification on the peony DNA samples for oil by using the 1 st to 10 th pairs of primers respectively to obtain amplification products;

(3) detecting the amplification product by using capillary electrophoresis, and reading the amplification band types of the 1 st to 10 th pairs of primers according to the direction from small to large of the fragments;

(4) recording the size of the amplified bands of the 1 st to 10 th pairs of primers, omitting units, and connecting by using a negative line if more than 2 bands are amplified by one pair of primers simultaneously; if no band is amplified, the DNA fingerprint code of the peony sample for oil is obtained by indicating 0, adding letters indicating the name of the primer in front of a numeric string to obtain the 1 st to 10 th serial numbers of the peony DNA for oil corresponding to the 1 st to 10 th primers, and connecting the 1 st to 10 th serial numbers in series to form a character string consisting of letters, Arabic numbers and hyphen;

(5) and generating a fingerprint spectrum two-dimensional code, processing the DNA fingerprint code of the oil peony sample by using a two-dimensional code technology, converting the DNA fingerprint code into a two-dimensional code graph, and using the two-dimensional code graph as an electronic identity card of the oil peony.

The excellent variety DNA fingerprint database of the peony for oil obtained by the construction method of the DNA fingerprint database of the peony for oil is shown in the following table:

the special primer pair combination for constructing the DNA fingerprint of the peony for oil, or the application of the special primer pair combination for constructing the DNA fingerprint of the peony for oil in the construction of the DNA fingerprint of the peony for oil, or the construction method of the DNA fingerprint database of the peony for oil, or the application of the DNA fingerprint database of the excellent variety of the peony for oil in the identification of the germplasm resources of the peony for oil.

Compared with the prior art, the invention has the beneficial effects that:

(1) a set of special primer pair combination for constructing the DNA fingerprint map of the peony for oil is developed based on SSR markers, amplification bands of the primer pair combination are intensively distributed between 100 and 400bp, the detection is sensitive and easy to interpret, and the primer pair combination has the advantages of good specificity, high polymorphism, good repeatability and the like;

(2) the construction of the fingerprint by using the genome DNA is not limited by seasons and environmental conditions, the operation method is simple and quick, and the detection result is reliable, stable and accurate;

(3) the primer combination and the method are utilized to obtain 87 parts of DNA fingerprint spectrum databases (digital codes and two-dimensional code graphs) of excellent oil peony varieties, the DNA fingerprint information of each variety is accurate, reliable and unique, and can be directly used for identifying the excellent oil peony varieties and provide a basis for identifying the genetic authenticity of the oil peony;

(4) the fingerprint code information is converted into the two-dimensional code graph by the two-dimensional code generator, so that the method is convenient for market circulation and cultivation management, has good practicability and wide application prospect, and can produce better economic benefits and social benefits.

Drawings

FIG. 1 is a capillary electrophoresis chart of primer A after amplification of Paeonia ostii-Shandong lotus-3;

FIG. 2 is a capillary electrophoresis chart of primer B pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 3 is a capillary electrophoresis pattern of primer C after amplification of Paeonia ostii-Shandong lotus-3;

FIG. 4 is a capillary electrophoresis pattern of primer D pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 5 is a capillary electrophoresis pattern of primer E pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 6 is a capillary electrophoresis pattern of the primer F pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 7 is a capillary electrophoresis pattern of the primer G pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 8 is a capillary electrophoresis pattern of primer H after amplification of Paeonia ostii-Shandong lotus-3;

FIG. 9 is a capillary electrophoresis pattern of primer I pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 10 is a capillary electrophoresis pattern of primer J pair "Paeonia ostii-Shandong lotus-3" after amplification;

FIG. 11 is a two-dimensional code picture of DNA fingerprint of Paeonia ostii-Shandong Neze-3.

Detailed Description

The invention is further described with reference to specific examples.

Example 1

The construction and application of the DNA fingerprint of the peony for oil will be specifically described below.

1. A large number of microsatellite sequences obtained based on a peony whole genome sequence are used for developing SSR primers, Primer 3.0 software is used for designing the primers in batches, the length of the primers is 18-26bp, the GC content is 40-60%, the Tm value is 50-65 ℃, the product prediction length is 100-400bp, and no dimer and hairpin structure appear in the primers. 150 pairs of microsatellite primers were selected and synthesized by Nanjing Kinshire Biotechnology Inc. The synthesized primers are used for carrying out PCR amplification on the peony DNA for oil, the 150 pairs of SSR primers are preliminarily screened by using 1.5 percent agarose gel electrophoresis and 8 percent polyacrylamide gel electrophoresis, and a primer combination with clear bands, easy interpretation and high polymorphism is selected. The screened SSR primers are specifically shown in Table 1.

TABLE 1 SSR primer sequence detailed Table

2. Collecting excellent variety of oil peony

87 parts of excellent oil peony varieties collected from Shandong, Hunan, Henan, Shaanxi, Anhui, Hubei, Gansu, Sichuan and other places are subjected to genotype detection by using the 1 st to 10 th pairs of primers in the table 1. The specific method comprises the following steps:

genomic DNA was extracted from young leaves of oil peony, according to the method of "Rapid isolation of high molecular weight plant DNA" (Murray and Thomson (1980)). Then using the extracted DNA sample as a template, and respectively using 1 st to 10 th pairs of primers in ABI-Veri^TMPCR amplification was performed on a thermal cycler. The total reaction system of PCR is 15 μ L, and the reaction system comprises: template DNA25ng, 20ng each of the upstream and downstream primers (downstream of each pair of primers)The 5' end of the primer has a fluorescence group FAM mark), 200 mu M dNTP, 0.5U Taq polymerase, 1.5 mu L10 × PCRbuffer, sterile deionized water is added to supplement the reaction volume to 15 mu L, the PCR reaction condition is that pre-denaturation is carried out for 2min at 94 ℃, then denaturation is carried out for 30s at 94 ℃, annealing is carried out for 30s at 50 ℃, extension is carried out for 1min at 72 ℃, after the circulation is finished, extension is carried out for 5min at 72 ℃, the amplification product is subjected to capillary electrophoresis in an ABI-3730XL sequencer, the obtained electrophoresis band is analyzed by using GeneMapper genotype analysis software, the number and the size of the amplification band of the 1-10 primer corresponding to each variety of peony are obtained, the capillary electrophoresis after Fengdan-Shangze-3 amplification shown in figures 1-10 is carried out, the coding treatment is carried out on the amplification band type, the size of the amplification band of 1-10 primer pair is recorded, the unit is omitted, if a pair of primers are amplified simultaneously, the bands above 2 are connected with a fingerprint, the number of the serial number of the peony-10, the number of the serial number of the peony number 1-10 is obtained by the number of the serial.

TABLE 2 DNA fingerprint code of 87 parts of improved oil peony species obtained in this example

After the DNA fingerprint expressed as a string as shown in table 2 is obtained, the string is converted into a two-dimensional code pattern by a two-dimensional code generator (in this embodiment, a forage two-dimensional code generator is used), for example, fig. 11 shows the two-dimensional code pattern of the "paeonia ostii-shandong spearmint-3" DNA fingerprint, i.e., the electronic identification card of the sample. Each oil peony variety has a unique electronic identity card, so that the oil peony is convenient to further popularize and utilize in the fields of market circulation, cultivation management and the like of the oil peony, and can also be applied to the construction and management of a seedling tracing system.

Example 2:

the SSR primers in the embodiment 1 and the constructed DNA fingerprint database of the peony for oil are used for identifying the excellent variety of the peony for oil and further expanding the data of the fingerprint database, and the process is as follows: extracting genome DNA of a peony sample to be detected as oil, using the extracted genome DNA as a template of PCR reaction, and performing PCR amplification by using the 1 st-10 th primer pair special primers in the table 1 in the example 1 respectively, wherein the amplification reaction system and the amplification conditions are the same as those in the example 1. Performing capillary electrophoresis on the amplification product, reading the band information of the sample to be detected, including the number and the size of bands, and further obtaining the DNA fingerprint code of the sample to be detected according to the method in the embodiment 1. Comparing the digital identity card of the sample to be detected with the 87 parts of the peony fine variety DNA fingerprint map database constructed in the embodiment 1, and determining the genetic authenticity and variety information of the sample to be detected if the digital identity information of the sample to be detected is completely consistent with the comparison of the DNA fingerprint codes of any variety in the database; if the comparison result of the DNA fingerprint codes of the sample to be detected and any variety in the database is different, the DNA fingerprint codes of the sample, the source, the type and other information are recorded into the DNA fingerprint database of the peony for oil together, so that the database information is further enriched, and information is provided for future detection of more unknown samples. Experiments prove that the method is accurate and reliable.

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Claims (7)

1. A special primer pair combination for constructing a DNA fingerprint of oil peony is characterized in that the sequences of the special primer pairs are as follows:

2. the application of the special primer pair combination for constructing the DNA fingerprint of the peony for oil in claim 1 in constructing the DNA fingerprint of the peony for oil.

3. Use according to claim 2, characterized in that: PCR amplification is respectively carried out on oil peony genome DNA by utilizing the special primer pair combination, and an amplification product is subjected to capillary electrophoresis to obtain an amplification band type, namely the DNA fingerprint of the oil peony.

4. The use according to claim 3, wherein the reaction system for PCR amplification comprises: template DNA25ng, 20ng each of the upstream and downstream primers labeled with fluorophore FAM at the 5' end, 200. mu.M dNTP, 0.5U Taq polymerase, 1.5. mu.L 10 XPCR buffer, and sterilized deionized water to make up the reaction volume to 15. mu.L.

5. The use according to claim 4, wherein the PCR amplification is performed under the following conditions: pre-denaturation at 94 ℃ for 2 min; then denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min.

6. A method for constructing a DNA fingerprint database of oil peonies is characterized by comprising the following steps:

(1) extracting genome DNA of a peony sample for certain oil;

(2) carrying out PCR amplification on the oil peony DNA samples by respectively using the 1 st to 10 th pairs of primers of claim 1 to obtain amplification products;

(3) detecting the amplification product by using capillary electrophoresis, and reading the amplification band types of the 1 st to 10 th pairs of primers according to the direction from small to large of the fragments;

(4) recording the size of the amplified bands of the 1 st to 10 th pairs of primers, omitting units, and connecting by using a negative line if more than 2 bands are amplified by one pair of primers simultaneously; if no band is amplified, the DNA fingerprint code of the peony sample for oil is obtained by indicating 0, adding letters indicating the name of the primer in front of a numeric string to obtain the 1 st to 10 th serial numbers of the peony DNA for oil corresponding to the 1 st to 10 th primers, and connecting the 1 st to 10 th serial numbers in series to form a character string consisting of letters, Arabic numbers and hyphen;

(5) and generating a fingerprint spectrum two-dimensional code, processing the DNA fingerprint code of the oil peony sample by using a two-dimensional code technology, converting the DNA fingerprint code into a two-dimensional code graph, and using the two-dimensional code graph as an electronic identity card of the oil peony.

7. The method for constructing the peony DNA fingerprint database for oil according to claim 6 is used for obtaining the peony fine variety DNA fingerprint database for oil, and the method is as follows:

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