Nucleic acid molecule, primer group, kit and method for identifying dama dama

Abstract

The invention discloses a primer group, kit and method for identifying dama dama, the primer group comprises a primer pair composed of an upstream primer H1F and a downstream primer H1R, the upstream primer H1F has a nucleotide sequence as shown in SEQ ID NO: 2, and the downstream primer H1R has a nucleotide sequence as shown in SEQ ID NO: 3. In the technical scheme provided by the invention, the genome sequence of the dama dama is taken as a reference sequence, the SNP of the dama dama is annotated, a candidate gene sequence of an enriched SNP region is excavated, and the nucleic acid molecule, the primer group, the kit and the method for identifying the dama dama are developed, so that a basic basis is provided for identifying products such as fur, antler and meat of the dama dama; the DNA amplification product is identified through electrophoresis, species identification of the dama dama can be carried out, compared with the conventional common mitochondrial sequence design primer, the identification process is greatly simplified, the identification work is more efficient and convenient, and the problem of rapid identification of the dama dama product is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a primer set, a kit and a method for identifying fallow deer. Background Art

[0002] China is a country extremely rich in deer resources, accounting for 41% of the world's deer resources. The fallow deer is native to Europe. Since it was introduced into China last century, artificial breeding and related utilization work have been widely carried out across the country as an ornamental animal. At the same time, many other deer species in China, such as elk, sika deer, and white-lipped deer, are included in the list of national key wild animal protection. When carrying out artificial breeding of deer and business activities related to their products, accurately distinguishing fallow deer from individuals and products of these related species is extremely crucial for promoting the work of wildlife protection and utilization scientifically and reasonably. This not only helps to standardize legal business utilization behaviors and avoid illegal infringement of protected species, but also provides accurate basis for protection decision-making and realizes sustainable management of deer resources.

[0003] Currently, species identification methods at home and abroad mainly include traditional morphological techniques, biochemical identification techniques based on protein immunomics, and modern molecular detection techniques based on molecular biology. Among them, molecular detection techniques, with various means such as fluorescence quantitative PCR, fragment length polymorphism detection, sequencing techniques, and specific locus detection techniques, show the advantages of good stability, high repeatability, high efficiency, specificity, sensitivity and rapidity, and are widely used in the field of species identification. With the rapid development of high-throughput resequencing technology, research on the systematic differentiation and classification of deer using sequencing technology has gradually been carried out, accumulating basic data for solving the problem of deer species identification at the molecular level.

[0004] Although molecular detection techniques have many advantages in species identification, there are still difficulties in the effective identification of fallow deer. Currently, specific loci clearly used for the effective identification of fallow deer have not been determined, and there is also a lack of simple and easy detection methods. In the past, mitochondrial sequences were commonly used to design primers in species identification. However, the amplification products need to be detected by sequencing or SNP detection kits, and the whole process is time-consuming and laborious, making it difficult to meet the requirements of rapid and efficient identification of fallow deer in actual work. Therefore, developing a primer set, a kit and a convenient method for fallow deer identification to overcome the deficiencies of the existing technology has become an urgent problem to be solved. Summary of the Invention

[0005] The main object of the present invention is to propose a primer set, a kit and a method for identifying fallow deer, aiming to be able to quickly and effectively identify fallow deer samples and accurately distinguish individuals and products of fallow deer and its related species.

[0006] To achieve the above object, the present invention provides a primer set for identifying fallow deer, which comprises a primer pair consisting of an upstream primer H1F and a downstream primer H1R, wherein the upstream primer H1F has the nucleotide sequence shown in SEQ ID NO: 2, and the downstream primer H1R has the nucleotide sequence shown in SEQ ID NO: 3.

[0007] Preferably, the primer set further comprises a primer pair consisting of an upstream primer H1F and a downstream primer H2R, wherein the upstream primer H1F has the nucleotide sequence shown in SEQ ID NO: 2, and the downstream primer H2R has the nucleotide sequence shown in SEQ ID NO: 4.

[0008] The present invention also provides a kit for identifying fallow deer, which contains the above-mentioned primer set.

[0009] Preferably, the kit further comprises amplification reaction reagents.

[0010] The present invention also provides an application of the primer set for identifying fallow deer as described above in the preparation of fallow deer identification products.

[0011] Preferably, the product is a kit, and the kit comprises the above-mentioned primer pair.

[0012] Preferably, the primer pair comprises an upstream primer H1F and a downstream primer H1R, or comprises an upstream primer H1F and a downstream primer H2R.

[0013] Preferably, the test sample includes tissue or blood.

[0014] The present invention also provides a method for identifying fallow deer, which comprises the following steps:

[0015] (1) Amplifying a test sample with the primer set for identifying fallow deer as described above;

[0016] Or (2) Amplifying a test sample with the above-mentioned kit.

[0017] Compared with the prior art, the beneficial effect of the present invention is that:

[0018] (1) Based on the genome re-sequencing of fallow deer, sika deer, and elk, this invention uses the genome information of fallow deer as the reference sequence. By screening the genome, gene sequences enriched in SNP hotspots of three deer species are selected as characteristic sequences. A total of 200 sequences are screened, primers are designed for them and screened, and a total of 2 pairs of effective species identification primers are obtained. Using these 2 pairs of primers, amplification is carried out in 60 individuals of 6 deer species (elk, sika deer, fallow deer, white-lipped deer, albino red deer, and red deer) and related wild horse species. The results show that the specific primer H1 is effectively amplified in the genomes of 6 deer species, but not effectively amplified in the genomes of wild horses, roe deer, and cattle; the length of the amplified band C of fallow deer is significantly greater than that of the other 5 deer species. This invention can achieve the species identification of fallow deer by electrophoretically identifying the DNA amplification products, solving the problem of rapid identification of products of the Cervinae subfamily and fallow deer products.

[0019] (2) In the technical solution provided by this invention, using the genome sequence of fallow deer as the reference sequence, SNPs are annotated, and candidate gene sequences in the SNP-enriched regions are mined to develop nucleic acid molecules, primer sets, kits, and methods for identifying fallow deer, providing a basic basis for the identification of products such as the fur, antlers, and meat of fallow deer. By electrophoretically identifying the DNA amplification products, the species identification of fallow deer can be achieved. Compared with the conventional method of designing primers using mitochondrial sequences, the identification process is greatly simplified, making the identification work more efficient and convenient, and solving the problem of rapid identification of fallow deer products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the technical solutions in the embodiments of this invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following described drawings are only some embodiments of this invention. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0021] Figure 1 The specific amplification results of primer H1 in DNA samples of 6 deer species (elk, sika deer, fallow deer, white-lipped deer, albino red deer, red deer) and related species wild horses, roe deer, and cattle. Among them, lane 1 is elk, lane 2 is sika deer, lane 3 is fallow deer, lane 4 is white-lipped deer, lane 5 is albino red deer, lane 6 is red deer, lanes 7-9 are DNA samples of related species wild horses, roe deer, and cattle, and M is 2K DNA Marker;

[0022] Figure 2Specific amplification results of primer H2 in DNA samples of 6 Cervidae animals (Père David's deer, Sika deer, Fallow deer, White-lipped deer, Albino red deer, Red deer) and related species Wild horse, Roe deer and Cattle. Among them, lane 1 is Père David's deer, lane 2 is Sika deer, lane 3 is Fallow deer, lane 4 is White-lipped deer, lane 5 is Albino red deer, lane 6 is Red deer, lanes 7-9 are DNA samples of related species Wild horse, Roe deer and Cattle, and M is 2K DNA Marker.

[0023] The realization, functional features and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Detailed implementation manners

[0024] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those skilled in the art can implement it. When the combination of technical solutions conflicts or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0025] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0026] Example 1 Genome resequencing of 3 Cervidae animals

[0027] 1. Sample collection and resequencing

[0028] A total of 20 individuals' blood or muscle tissues from 3 species (Père David's deer, Sika deer and Fallow deer) were collected for sequencing analysis. The reference genome of Fallow deer, Dama dama, is about 3.1G (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_033118175.2 / ), containing 284 scaffolds, and the GC content is 42.5%.

[0029] 2. Data filtering

[0030] The raw data obtained from the sequencer was filtered by SOAPnuke (Chen et al., 2017) to obtain clean data. Reads with 50% or more of the bases matching the adapter sequence were removed; reads with 50% or more of the bases having a quality score below 12 were removed; reads with 10% or more of the bases being 'N' were removed. After filtering, the clean data for each individual was 98.64 - 99.96%.

[0031] 3. Data Alignment

[0032] The filtered clean reads were aligned to the reference genome using the Burrows - Wheeler Aligner (BWA) (Li, 2013) software. Then, the SAMTools was used to sort the alignment results, and Picard was used to mark duplicate reads. The results showed that the average genome coverage rate was 99.07% and the average sequencing depth was 19.85X.

[0033] 4. SNP Detection and Annotation

[0034] The Genome Analysis Toolkit (GATK) (McKenna et al., 2010) was used to detect and filter single nucleotide polymorphisms (SNPs) in the resulting BAM file. To ensure the accuracy of SNPs, the following parameters were used for filtering: ① QD, the ratio of variant confidence to depth ≥ 2; ② FS, the p - value of strand bias obtained by Fisher's exact test to detect strand bias ≤ 60, a too high value indicates that the variant may be a false positive; ③ RMSMappingQuality, the root mean square value of the alignment quality of all samples ≥ 40; ④ MQRankSum, which evaluates the confidence based on the alignment quality of the reads of REF and ALT, and requires the value ≥ - 12.5; ⑤ ReadPosRankSum, the position evaluation of the variant on the read ≥ - 8.0; comprehensively evaluate the possibility of strand bias StrandOddsRatio (SOR) > 3.0.

[0035] After the final filtering and analysis, the number of SNP sites for each individual was 2,208,115 - 71,618,582, with an average of 55,365,871.2 SNP sites per individual.

[0036] Example 2 Design of Specific Marking Primers for 26 Cervidae Animals

[0037] From 2,208,115 SNP sites, using the reference genome of fallow deer as the reference sequence, 30 samples of 6 Cervidae animals (Père David's deer, sika deer, fallow deer, white-lipped deer, albino red deer, and red deer) were screened for inter-regional specific gene loci. Adopting the principle of giving priority to the hotspots of SNP enrichment in 6 species and the annotated sequences of functional genes, the SNP site hotspots in this region of each Cervidae sample were searched, and sequences of 250 bp on both sides of the variant sites in this region of the reference sequence were intercepted. 200 hot sequences of SNP enrichment were initially screened out. Through gene alignment, these 200 sequences were mainly immune genes and ncRNAs with rich genetic diversity.

[0038] 50 of these sequences were selected for primer design and screening, and PCR amplification and electrophoresis detection were carried out on 6 Cervidae animals (Père David's deer, sika deer, fallow deer, white-lipped deer, albino red deer, and red deer) and their related species wild horses. Finally, it was identified that the specific primer H1 was effectively amplified in 6 Cervidae animals, but not in other related species outside the Cervidae family. The specific amplification sequences and the primer H1 sequence are shown in Table 1 and Table 2. The PCR product of fallow deer was selected for sequencing to obtain its corresponding specific sequence. Through comparative analysis, it was found that this section of the sequence of fallow deer had an inserted fragment with a length of 25 bp compared with the other 5 deer species, resulting in a difference in the length of the PCR product.

[0039] The downstream primer design site was changed, and the primer sequence was set in the indel sequence of fallow deer and red deer. However, this region was conserved in Père David's deer, sika deer, white-lipped deer, and albino red deer. The H2 primer was designed in this region, and its primer sequence is shown in Table 2.

[0040] Table 1 Specific Sequences of Fallow Deer

[0041]

[0042] Table 2 Primer Sequences

[0043]

[0044] Example 3 Identification of Fallow Deer Samples

[0045] 1. Sample Collection

[0046] A total of 35 blood and tissue samples were selected from representative Cervidae species such as Père David's deer, sika deer, fallow deer, white-lipped deer, albino red deer, red deer and their related species wild horses. Among them, there were 5 samples of Père David's deer, 5 samples of sika deer, 5 samples of fallow deer, 5 samples of white-lipped deer, 5 samples of albino red deer, 5 samples of red deer, and 5 samples of their related species wild horses.

[0047] 2. Genomic DNA Extraction

[0048] The method for extracting genomic DNA from blood is as follows: Collect a blood sample and place it in a collection tube containing an appropriate amount of anticoagulant. Invert the collection tube repeatedly to thoroughly mix the anticoagulant with the blood. Centrifuge at 2000 - 3000 rpm for 10 min (centrifuge within 1 h after blood sample collection at room temperature; if the blood sample is placed on ice after collection, it can be processed within 4 h). The middle layer is white blood cells; store at -80 °C in a refrigerator. Vortex and mix the white blood cell sample, lysis buffer, and proteinase K, and let it lysate at room temperature; use a blood extraction kit, isopropanol, and magnetic beads, and invert and mix; perform magnetic separation, discard the supernatant, wash the magnetic beads 3 times, and dry them with the lid open at room temperature; add elution buffer, mix, and let it elute at room temperature; perform magnetic separation, transfer the supernatant to a new centrifuge tube for downstream experiments.

[0049] The method for extracting genomic DNA from tissues is as follows: Homogenize or grind the tissue sample, then add lysis buffer and proteinase K, and vortex and mix; use a tissue extraction kit, isopropanol, and magnetic beads, and invert and mix; perform magnetic separation, discard the supernatant, wash the magnetic beads 3 times, and dry them with the lid open at room temperature; add elution buffer, mix, and let it elute at room temperature; perform magnetic separation, transfer the supernatant to a new centrifuge tube for downstream experiments.

[0050] 3. Primer Sequences

[0051] The primer sequences are shown in Table 2.

[0052] 4. Amplification System and Amplification Conditions

[0053] The PCR amplification system for H1 and H2 is shown in Table 3 below.

[0054] Table 3 PCR Amplification System

[0055]

[0056] H1 and H2 use a touchdown PCR amplification program (Table 4).

[0057] Table 4 Touchdown PCR Amplification Program for H1

[0058]

[0059] 5. Amplification Results are as Figure 1 and Figure 2 shown.

[0060] In Figure 1Among them, the PCR product gel electrophoresis imaging of elk, sika deer, white-lipped deer, albino red deer and wapiti samples showed a single bright band of approximately 450 bp; the PCR product gel electrophoresis imaging of fallow deer samples showed a single bright band of approximately 490 bp; while there were no bands in the PCR product gel imaging of Przewalski's horse, roe deer and cattle; Primer H1 can effectively identify fallow deer DNA samples. Figure 1 The results showed that primer H1 was effectively amplified in all 6 deer species, but not in related species. Using primer H1 to amplify blood and tissue samples of 30 individuals of 6 deer species, including elk, sika deer, fallow deer, white-lipped deer, albino red deer and wapiti, amplification products could be obtained, while amplification products could not be obtained from samples such as tissues of related species Przewalski's horse, indicating that primer H1 can be used as a specific detection primer for deer origin. At the same time, the length of the amplification product of fallow deer was significantly higher than that of the other 5 deer species, so it can be used as a specific detection primer for fallow deer.

[0061] In Figure 2 Among them, the PCR product gel electrophoresis imaging of elk, sika deer, white-lipped deer, and albino red deer samples showed a single bright band of approximately 445 bp; there were no bands in the PCR product gel electrophoresis imaging of fallow deer and wapiti samples; while there were no bands in the PCR product gel imaging of Przewalski's horse, roe deer and cattle. Figure 2 The results showed that primer H2 was effectively amplified in 4 deer species, but not in fallow deer and wapiti. Using primer H2 to amplify blood and tissue samples of 30 individuals of 6 deer species, including elk, sika deer, fallow deer, white-lipped deer, albino red deer and wapiti, amplification products could be obtained from elk, sika deer, white-lipped deer and albino red deer, while amplification products could not be obtained from samples such as tissues of fallow deer, wapiti and related species Przewalski's horse, indicating that primer H2 combined with primer H1 can be used as a specific detection primer for fallow deer and wapiti. Generally speaking, in Figure 1 Among them, fallow deer samples can be effectively distinguished by the difference in the length of PCR products; while in Figure 2 Among them, there was no effective amplification in fallow deer and wapiti; The application of primers H2 and H1 can effectively identify fallow deer and wapiti DNA samples.

[0062] In summary, it is shown that the above primers H1 and H2 can be used for species identification and differentiation of fallow deer and wapiti.

[0063] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A primer set for identifying fallow deer, characterized in that: The primer pair comprises an upstream primer H1F and a downstream primer H1R, wherein the upstream primer H1F has a nucleotide sequence as shown in SEQ ID NO:2, and the downstream primer H1R has a nucleotide sequence as shown in SEQ ID NO:

3.

2. The primer set according to claim 1, characterized in that The primer set further includes a primer pair consisting of an upstream primer H1F and a downstream primer H2R, wherein the upstream primer H1F has a nucleotide sequence as shown in SEQ ID NO:2, and the downstream primer H2R has a nucleotide sequence as shown in SEQ ID NO:

4.

3. A kit for identifying fallow deer, characterized in that: The kit contains the primer set according to claim 1 or 2.

4. The kit for identifying fallow deer according to claim 3, characterized in that: The kit also includes amplification reaction reagents.

5. Use of the primer set for identifying fallow deer as claimed in claim 1 or 2 in preparing a fallow deer identification product.

6. The use according to claim 5, characterized in that: The product is a kit, which includes the primer pair as described in claim 1 or 2.

7. The use according to claim 6, characterized in that: The primer pair includes an upstream primer H1F and a downstream primer H1R, or includes an upstream primer H1F and a downstream primer H2R.

8. The use according to claims 5-7, characterized in that: Test samples include tissue or blood.

9. A method for identifying fallow deer, characterized in that: The following steps are involved: (1) amplifying a test sample using the primer set for identifying fallow deer as described in claim 1 or 2; or (2) amplifying the test sample using the kit as described in claim 3 or 4.

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