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CN118109638B - MNP (MNP) marking site for chrysanthemum variety identification, primer composition, kit and application - Google Patents

MNP (MNP) marking site for chrysanthemum variety identification, primer composition, kit and application Download PDF

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CN118109638B
CN118109638B CN202410522297.1A CN202410522297A CN118109638B CN 118109638 B CN118109638 B CN 118109638B CN 202410522297 A CN202410522297 A CN 202410522297A CN 118109638 B CN118109638 B CN 118109638B
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chrysanthemum
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赵秦
刘艳芳
杨晓洪
毛进
彭海
李甜甜
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INSTITUTE OF QUALITY STANDARD AND DETECTION TECHNOLOGY YUNNAN ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The invention relates to the technical field of molecular biological identification, in particular to MNP (MNP) marking sites for chrysanthemum variety identification, a primer composition, a kit and application, wherein the MNP marking sites are genome regions which are screened on chrysanthemum genome and have a plurality of nucleotide polymorphisms in chrysanthemum population, and the primer is shown as SEQ ID No. 1-SEQ ID No. 974. The MNP has the advantages of more marker loci, high polymorphism and strong variety distinguishing capability, and meets the requirements of identifying the authenticity of the chrysanthemum variety and identifying the substantial derivative variety; the primers are not interfered with each other, the identification accuracy is high, the result reproducibility is strong, and the requirement of constructing a DNA fingerprint database is met; the method can be applied to large-scale chrysanthemum variety identification, and has great application value in the aspects of chrysanthemum variety market supervision, intellectual property protection, new variety cultivation and the like.

Description

MNP (MNP) marking site for chrysanthemum variety identification, primer composition, kit and application
Technical Field
The invention relates to the technical field of molecular biological identification, in particular to MNP (MNP) marking sites for chrysanthemum variety identification, a primer composition, a kit and application.
Background
The chrysanthemum (Chrysanthemum multiplied by moriforlium Ramat.) belongs to the chrysanthemum genus (Chrysanthemum L.) of the Compositae (Compositae), is known as one of the two flowers in the world as a special breeding aspect in the flower world, is one of ten flowers in China, one of four jun fruits (Meilanzhu chrysanthemum) in flowers, and one of four cut flowers in the world (chrysanthemum, china rose, carnation and gladiolus) in the flower world, and the yield of the cut flowers is the first place each year. The chrysanthemum originates in China and is widely planted worldwide, is mainly distributed in mountain areas, hills and plain areas of Asian temperate zones and subtropical areas, and has long planting and cultivation history in China. The chrysanthemum has various postures, extremely high ornamental value, is popular with the people, and can be used for carrying out chrysanthemum exhibition once every year in many places of the whole country so as to drive local travel and economic chrysanthemum culture. Besides ornamental, the chrysanthemum also has economic values such as eating, medical use and the like.
However, with the vigorous development of the chrysanthemum industry, the chrysanthemum is introduced and cultivated for a long time, and the chrysanthemum is added with complicated genetic background and chaotic genetic relationship, so that great difficulty and obstacle are brought to the chrysanthemum variety identification. The phenotype-based variety testing technology can not scientifically and effectively finish variety discrimination and identification before huge number of chrysanthemum varieties, and the problems of variety counterfeit infringement, homonymous and heteronymous varieties in the market are prominent. Meanwhile, as the genome of the chrysanthemum has complexity and diversity, the cultivated chrysanthemum is an isohexaploid hybrid complex, has high heterozygosity, and is more difficult to identify by utilizing a molecular marker technology compared with diploid crops, a method capable of accurately and efficiently identifying varieties is urgently needed in chrysanthemum variety management so as to protect legal rights of variety owners and promote healthy development of chrysanthemum industry.
The polynucleotide polymorphism (Multiple Nucleotide Polymorphism, MNP) marking method is another national standard for plant variety identification in 2020 after secondary molecular markers SSR (Simple Sequence Repeats, SSR) and third-generation molecular markers SNP (Single Nucleotide Polymorphism, SNP), and has been used in variety identification of 16 crops such as rice, corn, cotton and the like. The MNP labeling method is to select a sequence containing 2-10 SNP as MNP label, the SNP has only 4 genotypes, if there are n SNP in MNP sequence, the genotype number can reach 4 n . The MNP labeling method overcomes the defects of the SSR labeling technology and the SNP labeling technology, combines the advantages of the SSR labeling technology and the SNP labeling technology, and has the outstanding advantages of high precision, high flux, simplicity, convenience, rapidness, cheapness and the like.
Based on the advantages, MNP marking technology is widely applied to crops such as rice, corn, tomatoes, kiwi fruits and the like, no research report on MNP marking is available in chrysanthemum variety identification at present, and corresponding technology is lacking. Therefore, the method develops an MNP mark in the chrysanthemum, can be used for accurate authorization, false making and right maintenance of plant species rights, and provides a reliable standard means for the implementation of seed law.
Disclosure of Invention
The invention aims to provide MNP (MNP) marking sites, primer compositions, kits and applications thereof for identifying chrysanthemum varieties, which not only can identify the authenticity of the varieties and the substantive derivative varieties of the chrysanthemum varieties, but also can carry out genetic analysis on the chrysanthemum varieties, and have the effects of strong differentiation, high flux and high accuracy.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the first object of the invention is to provide an MNP marker locus for chrysanthemum variety identification, wherein the MNP marker locus is a genome region which is screened on a chrysanthemum genome and has a plurality of nucleotide polymorphisms in the chrysanthemum population, and the MNP marker locus comprises marker loci of MNP-1-MNP-487 on the chrysanthemum genome.
The second object of the present invention is to provide a multiplex PCR primer composition for detecting the MNP labeling site, wherein the multiplex PCR primer composition comprises 487 pairs of primers, and the nucleotide sequences of the 487 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 974.
It is a third object of the present invention to provide a kit for detecting the MNP marker loci, the kit comprising the primer composition.
Further, the kit also comprises a multiplex PCR premix.
The fourth object of the invention is to provide the application of the MNP marking locus or the primer composition or the kit in the identification of the authenticity of the chrysanthemum varieties.
The fifth object of the invention is to provide the application of the MNP marking locus or the primer composition or the kit in the identification of the chrysanthemum substantive derivative variety.
The sixth object of the invention is to provide the application of the MNP marking locus or the primer composition or the kit in the genetic diversity analysis of chrysanthemum germplasm resources.
The seventh object of the invention is to provide the application of the MNP marking locus or the primer composition or the kit in constructing a DNA fingerprint database of chrysanthemum varieties.
In the above application, the specific application steps are as follows:
Firstly, obtaining total DNA of a variety to be detected; the kit is utilized to carry out a first round of multiplex PCR amplification on the DNA of the sample to be detected, and the number of cycles is 15; purifying the amplified product, and adding a sample tag and a second generation sequencing joint based on the second PCR amplification; quantifying after purifying the second round of amplification products;
When a plurality of chrysanthemum variety samples are detected, the amplification products of the second round are mixed in equal quantity and then subjected to high-throughput sequencing;
And performing data quality control and data analysis on sequencing data of a sample to be tested, and comparing a sequencing result to the chrysanthemum reference sequence to obtain the number of detected loci of the sample to be tested on the MNP loci, the number of sequencing sequences covering each MNP locus and the MNP locus genotype data.
When used for variety identification and substantial derivative variety identification, the kit and the detection method are formulated, and the accuracy and the degree of distinction of the kit and the detection method are evaluated;
When used for genetic analysis of chrysanthemum individuals, genetic difference analysis between populations and inside populations is included. Genotype data of individuals to be compared at 487 MNP sites are obtained by using the kit and the method. And analyzing the co-detected MNP loci of individuals to be compared on the 487 MNP loci through genotype comparison, wherein whether the main genotypes of each locus are different or not, and counting the difference proportion. And judging the genetic difference distribution situation among individuals through the difference proportion.
When the kit and the method are used for constructing a chrysanthemum variety DNA fingerprint database, genotype data of the chrysanthemum variety to be detected at 487 MNP sites are obtained by using the kit and the method.
The beneficial technical effects of the invention are as follows: the invention provides MNP marking loci, a primer composition and a kit for chrysanthemum variety identification and application thereof, wherein 487 MNP loci of chrysanthemum and the primer composition thereof can be subjected to multiplex PCR amplification, and the amplification products are sequenced by combining a second generation sequencing platform; therefore, the 487 MNP marking loci and primer combination provided by the invention can be applied to chrysanthemum variety authenticity identification, substantial derivative identification, germplasm resource genetic diversity analysis and other related applications, provides powerful technical support for chrysanthemum variety molecular breeding, variety right management, market supervision, intellectual property protection and other aspects in China, ensures grain safety, and promotes the healthy development of industry.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a statistical chart showing the number of MNP-labeling sites on chrysanthemum chromosomes in example 1 of the present invention.
FIG. 2 is a plot of data and marker detection rate statistics for 30 samples of chrysanthemum in example 2 of the present invention.
FIG. 3 shows the distribution of MNP-marked contrast between samples of chrysanthemum according to example 2 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, 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 embodiments of the invention belong. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
screening MNP markers suitable for detecting the population organisms as detection targets; MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over others:
(1) The alleles are rich, the polymorphism is high, and 2n types of alleles are higher than SSR and SNP markers on a single MNP locus;
(2) The variety distinguishing capability is strong, and the variety identification can be realized by only a small amount of MNP marks due to the fact that the MNP marks have more allelic genotypes, so that the detection error rate is reduced;
(3) The efficiency is high, and a plurality of sites can be amplified simultaneously in a single-tube PCR reaction, for example, 317-1042 MNP marks can be amplified simultaneously in the traditional national standard GB/T38551; in combination with high throughput sequencing, hundreds or thousands of samples can be detected simultaneously.
(4) The accuracy is high, the amplification mark product is sequenced hundreds of times by the second-generation high-throughput sequencer, the output result is a base sequence, no parallel experiment is needed, the data can be arbitrarily compared, and the data sharing performance is strong.
Based on the advantages and characteristics, MNP marking technology is widely applied to crops such as mango, potato, rice, corn, melon, mushroom, medlar, osmanthus fragrans, kiwi fruit, arundo donax and the like, no report about MNP marking in chrysanthemum exists at present, and corresponding technology is lacking.
Therefore, the invention screens a set of chrysanthemum MNP marker loci 487 with high polymorphism through the developed marker screening rules (see in particular example 1) based on chrysanthemum reference genome and combining sequencing data of main chrysanthemum varieties, wherein the loci are located in genome regions screened on chrysanthemum genome and provided with a plurality of nucleotide polymorphisms in chrysanthemum population.
Next, the present invention contemplates multiplex PCR primer compositions for amplifying these MNP marker loci, comprising 487 pairs of primers, the nucleotide sequences of which 487 pairs of primers are shown in the appendix (SEQ ID No. 1-SEQ ID No. 974). The primers do not conflict with each other, efficient amplification can be performed through multiple PCR, the identification accuracy is high, the result reproducibility is strong, and the requirement of DNA fingerprint database construction is met.
The multiplex PCR primer composition can be used in a detection kit for detecting MNP labeling sites.
The primer and the detection kit can be applied to the identification of the authenticity of the chrysanthemum varieties, the substantial derivative identification, the genetic diversity analysis of germplasm resources and other related fields.
The MNP labeling sites, primer compositions and kits for chrysanthemum variety identification according to the present application and their applications will be described in detail with reference to examples, comparative examples and experimental data.
Examples
MNP (MNP) marker locus screening and multiplex PCR (polymerase chain reaction) amplification primer design for chrysanthemum variety identification
S1, screening MNP (MNP) marker loci for chrysanthemum variety identification
The genome sequencing is simplified by selecting 30 varieties of the main varieties from the Netherlands and China (Henan, jiangsu, jiangxi, anhui, hebei, beijing, hubei and Yunnan), which are various in character expression types and representative 5-class chrysanthemum groups (edible chrysanthemum, potted and ground chrysanthemum, traditional chrysanthemum, cut flower small chrysanthemum and cut flower large chrysanthemum) as experimental materials for the research. The sequencing data of 30 chrysanthemums were aligned to the reference genome Chrysanthemum nankingense to screen for MNP sites, respectively. Firstly, carrying out sequence analysis by Samtools (v 1.2) and BCFtools (v 1.2) to obtain SNP loci on chrysanthemum genome, comparing and analyzing with NT library of NCBI, and screening MNP markers according to the following principle:
(1) The marker sequence is unique only to chrysanthemum and is not present in other species;
(2) A single copy of the sequence in the genome;
(3) The difference of at least three discontinuous SNP on the marker sequence; the length of the marker sequence is less than 250bp;
And further comparing the discrimination of the screened candidate MNP markers in the obtained chrysanthemum sample sequencing data, and finally screening 487 MNP marker loci.
After site development, the more complete data publication of the chrysanthemum reference genome (https:// doi. Org/10.6084/m9.Figshare.21655364. V2) compares 487 MNP markers with the new genome, where 14 MNP markers were aligned to fragments without chromosomal annotation or not aligned and the remaining MNP markers were evenly distributed over 27 chromosomes, as shown in FIG. 1.
S2, design of multiplex PCR amplification primer
The multiplex PCR amplification primers of MNP loci are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be amplified normally in one amplification reaction, and the amplified primer sequences are shown in a sequence table.
Examples
MNP (MNP) mark for chrysanthemum variety identification, primer composition and evaluation of kit of MNP mark
After 487 primer synthesis, 5. Mu.L of each primer was mixed in equal amounts to form a primer mix of 1:1 equal amounts of F and R terminal primers. And (3) testing the detection rate, accuracy and distinction of MNP marking sites by utilizing the evaluation of MNP marking, primers and kits developed by 30 chrysanthemum varieties collected by a unit.
(1) Multiplex PCR library construction system
Multiplex PCR system and procedure: using a 0.2mLPCR tube/96 well PCR plate, the reaction was configured as follows: MNP labeled primer mix 4. Mu.L, template DNA amount (20-200 ng) X. Mu.L, high-fidelity DNA polymerase reaction solution 10. Mu.L and water (16-X). Mu.L (total 30. Mu.L).
After the system configuration is completed, shaking, mixing and centrifuging are carried out, and the mixture is placed in a PCR instrument, wherein the PCR reaction program is 95 ℃ for 15min; then denaturation is carried out at 95 ℃ for 30 s, annealing and extension are carried out at 60 ℃ for 4min, and the total cycle is 15 times; extending at 70deg.C for 4min.
And then purifying the amplified product by using a DNA Beads system, wherein the purified product is used for connecting a corresponding connector and a sample barcode by using an Illumina or Huada sequencer.
Taking an Illumina sequencer system as an example, a sequencing library is established: purified product was added to the system as follows, and different samples were selected for different barcode primers. After the PCR amplified product is purified, the PCR amplified product is directly used for high-throughput sequencing by a sequencer.
The system comprises: high-fidelity DNA polymerase reaction solution 10 μ L, P5barcode (5 μM) 2 μ L, P7barcode (5 μM) 2 μL and water 16 μL (total 30 μL). PCR reaction procedure: 3 min at 95 ℃; subsequently denatured at 95℃for 15s, annealed at 58℃for 15s, extended at 70℃for 30s, and cycled 8 times in total; finally, the mixture was stretched at 72℃for 5min and finally cooled to 4 ℃.
(2) MNP marker detection rate
The kit is used for carrying out multiplex PCR amplification and construction of a sequencing library, and carrying out multiplex amplification, second-generation high-throughput sequencing and data analysis on the 30 chrysanthemum DNA samples, so that detection of 30 x 487=14610 markers in one experiment is realized, the average coverage multiple of sequencing of each sample is more than 700 times, and the high efficiency of MNP marker detection is shown.
The distribution diagram of the number of the sites for detecting the MNP marks of the chrysanthemum is shown in figure 2, the detection number of the MNP marks is counted in the sequencing data of the 30 samples, 465 marks can be detected on average for each sample, the average detection rate is 95.46%, and the requirement of the MNP marking method on the detection rate of the variety identification marks in the national standard is met.
(3) Chrysanthemum MNP labeling method accuracy analysis
In order to test the accuracy of the chrysanthemum MNP marker, a reproducibility experiment (two independent experiments performed by different personnel, different batches of reagents and different instruments) is performed on 8 chrysanthemum germplasm, and a comparison analysis is performed on the marker sites which are common among 2 batches of data of each sample, so that the typing accuracy rate is calculated, and the accuracy rate=1- (1-accuracy)/2. Wherein, the accuracy refers to the proportion of the marking sites which are consistent with the parting results of the two experiments to all marking sites. The reproducibility experiment simulates the identification of different batches, and the high accuracy rate means that the identification results of different laboratories can be accurately compared with each other. The statistical results are shown in Table 1, and the results show that the total of 3710 MNP markers are compared, the proportion of sites which are not reproduced is 0.27%, and the typing accuracy is 99.87%. The result shows that the retained 487 chrysanthemum MNP markers and the detection method thereof have the characteristic of high accuracy.
Table 1 MNP marker locus accuracy evaluation information table for chrysanthemum
(4) Chrysanthemum MNP (MNP) marked variety discrimination degree
In 30 chrysanthemum varieties, the number of the detected alleles of each MNP marking site is between 1 and 133, and 13.85+/-12.39 alleles are detected averagely, wherein 438 loci contain more than 5 alleles. The genotypes of the 30 chrysanthemum samples at the 487 MNP marking loci are counted, the samples are compared pairwise, and based on the principle that at least 1 SNP is judged to be different on the allele type of the same MNP marking locus in different varieties, the number of different MNP marking between every two comparisons of the 30 samples is counted, and the result is shown in figure 3. And (3) carrying out pairwise comparison on MNP (MNP) marker fingerprints of 30 chrysanthemum samples, and analyzing the difference of MNP marker loci among the samples to obtain 435 comparison results. The ratio of MNP markers of each pair of differences between varieties is referred to as the inter-variety distance, and the inter-variety distance directly shows the discrimination capability of the MNP markers for the varieties. The result shows that at least 312 marks are different among any chrysanthemum varieties, the average distance among varieties reaches 85.15%, and the set of MNP marks has strong variety distinguishing capability.
Examples
Identification of authenticity and determination of substantial derivative variety of chrysanthemum variety
Taking the analysis of table 1 in example 2 as an example, we have seen in reproducibility experiments that the marker typing accuracy (reproducibility) for each variety is at least 99.87%. Thus, if natural genetic variation of a variety during planting is excluded, the threshold for judging that two varieties are identical may be as strict as 99.87%. However, after the cultivar is bred, there is always genetic variation during propagation and production, and some degree of genetic variation is tolerated. Therefore, the judgment threshold for variety authenticity should be below 99.87% in view of the allowable genetic variation.
The judging threshold value of the variety authenticity of the existing variety industry standard and national standard is about 96%. In order to solve the contradiction between DNA identification and trait DUS testing, when a pair of samples is judged to be "similar variety" or "very similar variety or the same variety", further field planting identification can be performed according to GB/T19557.19-2018.
The quality of 5 chrysanthemum germplasm varieties is identified by the threshold value, and the results are shown in Table 2. The results show that 1 group of germplasm (same variety) with the genetic similarity being more than 96 percent and the rest less than 96 percent are different varieties. Considering the characteristic of asexual propagation of chrysanthemum, the genetic variation is smaller, and the result meets the expectations.
TABLE 2 authentication results between chrysanthemum varieties
Sample 1 Sample 2 Number of co-detection sites Number of typing non-differential sites Genetic similarity coefficient (GS) Identification conclusion of variety authenticity
JL-1 JZZ 447 91 20.36% Different varieties
JL-1 CS 442 56 12.67% Different varieties
JL-1 YY 441 61 13.83% Different varieties
JL-1 JL-2 459 456 99.35% Very similar or identical varieties
JZZ CS 434 58 13.36% Different varieties
JZZ YY 435 58 13.33% Different varieties
JZZ JL-2 440 89 20.23% Different varieties
CS YY 434 130 29.95% Different varieties
CS JL-2 438 58 13.24% Different varieties
YY JL-2 439 59 13.44% Different varieties
The variety DNA fingerprint data obtained by adopting MNP marking technology is a base sequence, can be stored for a long time and has strong data traceability, so that the sequencing data of each variety can be stored to form a DNA fingerprint database. Furthermore, the allele type obtained by the variety to be detected can be compared with data in the DNA fingerprint database in a large quantity, so that the application of accurate screening of similar varieties, accurate determination of the infringement objects of the varieties, accurate identification of substantial derivative varieties and the like are greatly facilitated. The application needs to compare the parting result of a variety with the parting result of thousands of varieties, thousands of experiments are needed to be carried out on the assumption that the parallel experiments adopting the traditional identification method are not feasible, only one experiment is needed through the marking, the primer and the kit thereof, and then the comparison work is handed to a computer for processing, meanwhile, the number of MNP marks in the invention is 487, and the number of identified marking sites is more, so that the application of accurate screening of approximate varieties, accurate identification of the infringement objects of the varieties, accurate identification of the substantial derivative varieties and the like becomes possible.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.

Claims (7)

1. The MNP marking site multiplex PCR primer composition for chrysanthemum variety identification is characterized by comprising 487 pairs of primers, wherein the nucleotide sequence of the 487 pairs of primers is shown as SEQ ID No. 1-SEQ ID No. 974.
2. A MNP marker locus detection kit for chrysanthemum variety identification, characterized in that the kit comprises the primer composition of claim 1.
3. The kit of claim 2, further comprising a multiplex PCR premix.
4. Use of the primer composition of claim 1 or the kit of any one of claims 2-3 in the identification of the authenticity of a chrysanthemum variety.
5. Use of a primer composition according to claim 1 or a kit according to any one of claims 2 to 3 for the identification of a substantial derivative of chrysanthemum.
6. Use of the primer composition of claim 1 or the kit of any one of claims 2-3 in genetic diversity analysis of germplasm resources of chrysanthemum.
7. Use of the primer composition of claim 1 or the kit of any one of claims 2-3 for constructing a database of DNA fingerprints of chrysanthemum varieties.
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CN117089644A (en) * 2023-08-02 2023-11-21 武汉兰多生物科技有限公司 MNP (MNP) marking site for identification of arundo donax varieties, primer composition, kit and application of MNP marking site
CN117210597A (en) * 2023-08-08 2023-12-12 南京农业大学 Chrysanthemum DNA fingerprint based on SNP markers and construction method and application thereof

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