CN113832236B - Primer group, kit and application for identifying sika deer, red deer and hybridized deer - Google Patents
Primer group, kit and application for identifying sika deer, red deer and hybridized deer Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and application for identifying sika deer, red deer and hybridized deer, and belongs to the technical field of biology. The primer group comprises SEQ ID NO:1 to SEQ ID NO: 400. An application of the MNP marker loci, the application comprising: 200 MNP marker loci as shown in Table 1 of the specification were used for genetic differential analysis of sika deer, red deer and hybrid deer and pedigree analysis of sika deer, red deer and hybrid deer. The primer set provided by the embodiment of the invention has good differentiation, reproducibility and accuracy, and the kit using the primer set can have the advantages at the same time, and can effectively ensure the detection accuracy.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a kit and application for identifying sika deer, red deer and hybridized deer.
Background
The pilose antler is a traditional and rare Chinese medicinal material in China, has remarkable health care curative effects of enhancing organism functions and eliminating fatigue, and is widely applied to clinical medicine. Definition in the pharmacopoeia of the people's republic of China (2015 edition): the cornu Cervi Pantotrichum is young horn of deer (Cervus Nippon Temminck) or Cervus elaphus Linnaeus). At present, researches show that the contents of amino acid, nucleoside component, phospholipid component, cholesterol and polyamine in sika deer antler are different from those in sika deer antler, wherein the content of nucleoside component in sika deer antler is obviously higher than that of sika deer antler, which may be the reason for the difference of medicinal value of the two deer antler. In addition, the quality of the spotted deer in northeast is generally considered to be optimal, the quality of the spotted deer in northeast is considered to be better, and the quality of the spotted deer in northwest is considered to be lower, namely, the spotted deer in northwest is considered to be 'western Ma Rong'. Therefore, the deer antler is more valuable in the market, and the price of the deer antler is generally about 3 times higher than that of the deer antler. Because the sika deer antler is expensive, the phenomenon that the sika deer antler is replaced by the sika deer antler or the hybridization deer antler bred by the sika deer is frequent in China, the standardization of the traditional Chinese medicine market and the development of industry are seriously affected, and the legal rights and interests of consumers are simultaneously infringed. Therefore, the establishment of an accurate and efficient deer antler identification method has important significance.
At present, PCR (Polymerase Chain Reaction ) technology is mostly adopted to detect COI genes of mitochondria or SRY genes on Y chromosome in the method for identifying the authenticity of the pilose antler and the mixed and imitative products thereof, and the amplified COI genes of the mitochondria are amplified and then subjected to electrophoresis to obtain amplified products, and the size and the shape of the amplified products are identified to distinguish the source of the pilose antler. Since the mitochondria are from the female parent, a series of identification methods according to the COI gene can only be used as the basis for identifying the female parent, and for the hybridized deer, the female parent can be sika deer, and the male parent can be red deer or deer of other varieties, so that the method can not accurately identify the hybridized deer antler of which the male parent is non-sika deer.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a primer group, a kit and application for identifying sika deer, red deer and hybridized deer. The technical scheme is as follows:
in one aspect, the invention provides a primer set for identifying sika deer, red deer and hybridized deer, wherein the primer set comprises a sequence shown in SEQ ID NO:1 to SEQ ID NO: 400.
In yet another aspect, embodiments of the present invention provide a kit for identifying sika deer, red deer and hybrid deer, the kit comprising the primer set of claim 1.
In yet another aspect, embodiments of the present invention provide an application of MNP marker loci, the application comprising: 200 MNP marker loci as shown in Table 1 of the specification were used for genetic differential analysis of sika deer, red deer and hybrid deer and pedigree analysis of sika deer, red deer and hybrid deer.
The technical scheme provided by the embodiment of the invention has the beneficial effects that: the primer group provided by the embodiment of the invention is used for identifying sika deer, red deer and hybridized deer, and utilizes multiple PCR amplification and fusion of a second generation sequencing platform to sequence amplification products, so that the advantages of multiple targets, high flux, high efficiency and high accuracy of deer antler detection are realized, the primer group has very high individual distinction degree, higher reproducibility and accuracy, multiple targets can be detected once by utilizing multiple PCR, thus the problems of high false negative and low sensitivity caused by single target amplification failure are effectively avoided, meanwhile, the primer group is utilized to detect the accuracy, hundreds of times of sequencing are performed on amplification products by matching with a second generation high flux sequencer, and the output result is a base sequence, so that no parallel experiment is needed, random comparison can be performed on data, and the data sharing performance is strong. The kit using the primer group can have the advantages at the same time, and can effectively ensure the reproducibility and accuracy of detection. The MNP marker loci are used for genetic difference analysis of sika deer, red deer and hybrid deer and blood system analysis of sika deer, red deer and hybrid deer, alleles of the MNP marker loci are rich, polymorphism is high, 2n types of alleles are higher than SSR and SNP on a single MNP marker locus, and the MNP marker loci are utilized to distinguish species.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a cluster tree diagram according to an embodiment of the present invention;
FIG. 2 is a cluster distribution diagram according to a second embodiment of the present invention;
fig. 3 is a diagram for analyzing the blood system proportion of sika deer according to the third embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example 1
The embodiment of the invention provides a primer group for identifying sika deer, red deer and hybridized deer, which comprises the following components: the upstream primer and the downstream primer, in particular, the primer group comprises the nucleotide sequence shown as SEQ ID NO:1 to SEQ ID NO: 400.
100 sika deer and 68 sika deer individual genome sequencing data (receiving number: PRJNA 355630) obtained from the SRA database of NCBI are combined by using the published sika genome sequence GCA_002197005.1 as a reference genome, and Samtools (Version 1.2) and BCFtools (Version 1.2) are used for identifying SNP sites on the genome, and compared with the NT library of NCBI for analysis, and screening of MNP markers is performed according to the following principle:
(1) The marker sequence is common to sika deer and red deer, but is not present in other species;
(2) Differences in sequence among multiple discrete SNPs;
(3) The length of the marker sequence is between 200 and 300 bp.
Further screening MNP marker loci by comparing the sequencing data of the known sika deer and red deer, and finally screening 200 MNP marker loci.
In use, the sequence of SEQ ID NO:1 to SEQ ID NO: 400. Specifically, SEQ ID NO:1 to SEQ ID NO:400 were mixed at 5. Mu.L each.
In yet another aspect, an embodiment of the present invention provides a kit for identifying sika deer, red deer and hybrid deer, the kit comprising the above primer set.
The performance of the primer set was evaluated as follows:
the primer group provided by the embodiment of the invention is used for respectively testing MNP (MNP) marking sites of 4 sika deer DNA samples and 4 sika deer antler DNA samples provided by the Chinese inspection and quarantine scientific institute, and the DNA of 4 sika deer antler and the DNA source of 4 sika deer antler provided by the embodiment of the invention are both presented by the Chinese inspection and quarantine scientific institute. Specifically, the primer set provided by the embodiment of the invention is adopted to carry out multiplex PCR amplification on 8 DNA samples, the amplification cycle number is designed to be 20, amplification products are obtained, a high-throughput sequencing library is constructed by adopting the amplification products, the Miseq platform of illuminea is adopted to carry out second generation sequencing on the high-throughput sequencing library, a sequencing fragment set is obtained, and analysis and statistics results are respectively carried out according to 200 MNP (MNP) marker loci shown in table 1.
Table 1 shows 200 MNP labeling sites and corresponding primer sequence listing
To verify the accuracy of the results, each DNA sample was divided into two parts before detection, 16 libraries were constructed at two different times (7 days between two times) and subjected to second generation sequencing to obtain 16 sequencing fragment groups, and the typing results of the 16 sequencing fragment groups were counted according to the 200 MNP marker locus analyses shown in table 1, and the specific results are shown in table 2.
Table 2 is a table of evaluation information for 200 MNP marker loci
As can be seen from the combination table 2, the DNA sample of sika deer and the DNA sample of red deer detect 194 MNP marking sites at least, the site detection rate exceeds 97%, the average detection rate of the obtained 16 high-throughput sequencing libraries reaches 98.13%, which indicates that the primer group can detect the screened MNP marking sites efficiently, the primer specificity is strong, and the primer amplification efficiency is high; comparing the typing results of the MNP marker loci detected in the high-throughput sequencing library repeatedly constructed for each sample, the repeated typing results of 3140 MNP marker loci are identical, and the repeatability is 100% and the accuracy is 100%. Comparing the typing results of MNP marking sites among samples, judging that the MNP marking sites are different according to the principle that at least 1 SNP is different among the samples, wherein at least 94.33% of MNP marking sites are different among DNA samples of the sika deer antler and DNA samples of the sika deer antler, and at least 10.25% of MNP marking sites are different among sika deer individuals, and the clustering analysis result is shown in figure 1. The result shows that the 200 MNP marking sites provided by the embodiment of the invention have high detection rate, high typing accuracy and high variety discrimination, and can be used for obviously distinguishing deer from deer antler of sika deer origin. Meanwhile, MNP marker loci have rich alleles and high polymorphism, 2n types of alleles are higher than SSR and SNP on a single MNP marker locus, and the MNP marker loci have strong capability of distinguishing species. Moreover, the MNP marking site detection efficiency is high, and hundreds of thousands of MNP marking sites of hundreds of samples can be typed at one time by adopting the ultra-multiplex PCR combined with the second-generation high-throughput sequencing technology to detect the MNP marking sites. In addition, the primer set provided by the embodiment of the invention has high site amplification specificity and high detection rate, multiple targets can be detected at one time by utilizing multiple PCR, so that the problems of high false negative and low sensitivity caused by single target amplification failure are effectively avoided, meanwhile, the primer set is high in detection accuracy, the amplification products are sequenced hundreds of times by matching with a second-generation high-throughput sequencer, and the output result is a base sequence, so that no parallel experiment is needed, random comparison can be performed on the data, and the data sharing performance is strong.
Example two
The embodiment of the invention provides an application of MNP marking sites, which comprises the following steps: the application comprises the following steps: the sequence shown in SEQ ID NO:401 to SEQ ID NO:600 is used for genetic difference analysis of sika deer, red deer and hybridized deer and blood system analysis of sika deer, red deer and hybridized deer.
The 200 MNP marking sites and the kit provided by the embodiment of the invention are utilized to carry out MNP marking site difference analysis on 29 sika deer, 32 sika deer and 7 hybridized deer F1 generations given by the Chinese inspection and quarantine scientific research institute, 68 deer individual DNAs in total, and all samples can be divided into three major categories of sika deer, sika deer and hybridized deer through cluster analysis, and the result is shown in figure 2. The 14 sika deer, 2 sika deer samples and the hybridized deer are gathered into one type, and the result is supposed to be consistent with a PCoA analysis result of a main component by using MNP marking difference among samples, which is presumably caused by sample source error in sample collection, so that the MNP marking site can be used for genetic difference analysis of sika deer, sika deer and hybridized deer. Sika deer, red deer and hybrid deer are each gathered into a class, and individuals between groups and within groups can be distinguished remarkably.
Example 3 sika deer, red deer, and analysis of blood System of sika deer in individuals of hybridized deer
The sample sequencing data of which the sample names are inconsistent with the classification in the example 2 are removed, MNP marking typing data of the rest 26 sika deer and 25 sika deer are analyzed, specific MNP alleles only existing in the sika deer are screened, 78 MNP marking loci are obtained in total, the sika deer specific allele numbers (T) of the samples at the 78 loci are analyzed, the specific allele proportion of the sika deer in each sample is calculated, and a sika deer blood system calculation method is established, namely, the sika deer blood system proportion (M) =sika deer specific allele number (T)/78 MNP marking allele total number (N), and the higher the M value is indicated to be closer to the sika deer blood relationship. According to the method, sika deer blood system analysis is carried out on 65 deer individuals in the embodiment 2, the analysis result is shown in fig. 3, and as can be known from fig. 3, the sika deer individual sample M average value is 1 (namely, all sika deer blood systems are obtained), the sika deer M average value is 0 (namely, sika deer blood systems are not contained), the M average value of the hybridized deer F1 is 0.46 (namely, sika deer blood systems are approximately half contained), and the result shows that the sika deer blood system is consistent with the actual situation and high in accuracy when the sika deer blood system is judged by the sika deer blood system calculation method established by the embodiment of the invention. In addition, the genetic difference cluster in example 2 shows that the M average value of 13 red deer samples of the hybridized deer is 0.71, which indicates that the red deer samples do contain red deer blood rims, belong to the hybridized deer type, and are consistent with the clustering result.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (3)
1. A primer set for identifying sika deer, red deer and hybridized deer, characterized in that the primer set comprises a primer set sequence as shown in SEQ ID NO:1 to SEQ ID NO: 400.
2. A kit for identifying sika deer, red deer and hybrid deer, comprising the primer set of claim 1.
3. An application of MNP marker loci, the application comprising: 200 MNP marker loci as shown in Table 1 of the specification were used for genetic differential analysis of sika deer, red deer and hybrid deer and pedigree analysis of sika deer, red deer and hybrid deer.
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| CN114672575B (en) * | 2022-04-19 | 2024-04-12 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybridized pilose antler based on RAA amplification |
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| CN111793700A (en) * | 2020-08-21 | 2020-10-20 | 长春科技学院 | SNP (Single nucleotide polymorphism) marker for yield traits of pilose antler, detection primer pair, kit and application |
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| 基于COI与SRY序列建立梅花鹿、马鹿及其杂交鹿茸的分子鉴别方法;魏艺聪等;中国中药杂志;42(23);4588-4592 * |
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