SNP molecular marker combination for individual identification of blue pheasant and application thereof

Abstract

The application relates to a SNP molecular marker combination for individual identification of Gallicrex cinerea and application thereof, and relates to the technical field of biology. The SNP molecular marker combination comprises 59 SNP molecular markers. Based on the SNP molecular marker combination, target SNP site information of the Gallicrex cinerea can be quickly and accurately obtained, so that individual identification of the Gallicrex cinerea is carried out, and a foundation is laid for individual identification and genetic diversity research of the Gallicrex cinerea.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a SNP molecular marker combination for individual identification of Crossoptilon auritum and application thereof. BACKGROUND

[0002] Crossoptilon auritum, also known as Crossoptilon mantchuricum, Crossoptilon mantchuricum taiwanus, and Chinese Taiwan Crossoptilon mantchuricum, belongs to the family Phasianidae and the genus Crossoptilon. Crossoptilon auritum is endemic to China and only distributes in Taiwan, which is a typical island species. It has been listed as near threatened by the International Union for Conservation of Nature (IUCN) and as a first-class protected bird in the List of National Key Protected Wildlife in China. Crossoptilon auritum mainly inhabits in mountain forests below 2700 meters above sea level, and especially prefers dense primary broad-leaved forests and mature secondary broad-leaved forests, which has a unique position in the ecological system. Due to human activities such as forest cutting and land reclamation, the habitat of Crossoptilon auritum is continuously destroyed and fragmented. Not only does it compress the living space of Crossoptilon auritum, but also hinders the communication between its populations. Studies have found that the nucleotide polymorphism of the genome of Crossoptilon auritum is low, indicating that Crossoptilon auritum has a high degree of inbreeding. Inbreeding can easily lead to a decrease in genetic diversity, making the population less resistant to environmental changes, diseases, and other adverse factors. Therefore, developing a patent for identification technology of kinship and individual identification based on DNA molecular markers can accurately determine the kinship between individuals of Crossoptilon auritum, avoid inbreeding, and thus maintain the genetic diversity of the population of Crossoptilon auritum.

[0003] Single Nucleotide Polymorphisms (SNP) as the third generation of molecular marker technology, with unique characteristics such as wide distribution in the genome, high stability, and double alleles, and meets the requirements of high automation and high-throughput sequencing. SNP molecular markers have shown significant advantages in the field of individual identification and kinship identification of many rare species, providing a solid technical guarantee for in-depth genetic research, optimization of breeding strategies, and effective implementation of protection measures for Crossoptilon auritum. However, there is no research on individual identification of Crossoptilon auritum. Therefore, it is urgent to develop a SNP marker combination for individual identification of Crossoptilon auritum and corresponding primers and detection methods to meet the protection research needs of Crossoptilon auritum. SUMMARY

[0004] In view of the above problems, the present application provides a SNP molecular marker combination for individual identification of Crossoptilon auritum. Based on the SNP molecular marker combination, the target SNP site information of Crossoptilon auritum can be quickly and accurately obtained, thereby laying a foundation for individual identification and genetic diversity research of Crossoptilon auritum.

[0005] In order to achieve the above purpose, the present application provides a SNP molecular marker combination for individual identification of Crossoptilon auritum, which comprises the following 59 SNP molecular markers:

[0006] SNP site 1 is located at position 67974054 of chromosome 1, and the alleles are A / G;

[0007] SNP site 2 is located at position 125530636 of chromosome 1, and the alleles are C / T;

[0008] SNP site 3 is located at position 194739820 of chromosome 1, and the alleles are G / A;

[0009] SNP site 4 is located at position 97634433 of chromosome 2, and the alleles are T / C;

[0010] SNP site 5 is located at position 112377121 of chromosome 2, and the alleles are A / G;

[0011] SNP site 6 is located at position 31998099 of chromosome 3, and the alleles are T / C; SNP site 7 is located at position 60185546 of chromosome 3, and the alleles are T / C; SNP site 8 is located at position 76526823 of chromosome 3, and the alleles are T / C; SNP site 9 is located at position 99096322 of chromosome 3, and the alleles are T / C; SNP site 10 is located at position 5580707 of chromosome 4, and the alleles are T / C; SNP site 11 is located at position 61331953 of chromosome 4, and the alleles are G / A; SNP site 12 is located at position 72654237 of chromosome 4, and the alleles are T / C; SNP site 13 is located at position 4604233 of chromosome 5, and the alleles are C / T; SNP site 14 is located at position 30249593 of chromosome 5, and the alleles are C / T; SNP site 15 is located at position 50086450 of chromosome 5, and the alleles are A / G; SNP site 16 is located at position 18902332 of chromosome 6, and the alleles are G / A; SNP site 17 is located at position 29374172 of chromosome 6, and the alleles are G / T; SNP site 18 is located at position 41008232 of chromosome 6, and the alleles are A / G; SNP site 19 is located at position 53820188 of chromosome 6, and the alleles are C / T; SNP site 20 is located at position 239547 of chromosome 7, and the alleles are T / C;

[0012] SNP site 21 is located at position 35074517 of chromosome 7, the allelic gene is A / G; SNP site 22 is located at position 2606019 of chromosome 8, the allelic gene is T / C; SNP site 23 is located at position 26365282 of chromosome 8, the allelic gene is T / C; SNP site 24 is located at position 4482003 of chromosome 9, the allelic gene is C / T; SNP site 25 is located at position 28953883 of chromosome 9, the allelic gene is A / G; SNP site 26 is located at position 2763802 of chromosome 10, the allelic gene is C / T; SNP site 27 is located at position 25241662 of chromosome 10, the allelic gene is A / G; SNP site 28 is located at position 4905854 of chromosome 12, the allelic gene is A / G; SNP site 29 is located at position 20552335 of chromosome 12, the allelic gene is G / A; SNP site 30 is located at position 3719889 of chromosome 13, the allelic gene is G / A; SNP site 31 is located at position 21998632 of chromosome 13, the allelic gene is T / G; SNP site 32 is located at position 19292436 of chromosome 14, the allelic gene is C / T; SNP site 33 is located at position 18389002 of chromosome 15, the allelic gene is T / C; SNP site 34 is located at position 475472 of chromosome 16, the allelic gene is C / T; SNP site 35 is located at position 14465184 of chromosome 16, the allelic gene is C / T; SNP site 36 is located at position 4757476 of chromosome 17, the allelic gene is C / T; SNP site 37 is located at position 15979491 of chromosome 17, the allelic gene is C / T; SNP site 38 is located at position 3441805 of chromosome 18, the allelic gene is C / T; SNP site 39 is located at position 12982785 of chromosome 18, the allelic gene is A / G; SNP site 40 is located at position 5177 of chromosome 19, the allelic gene is C / T;

[0013] SNP site 41 is located at position 10836959 of chromosome 19, the allelic gene is C / T; SNP site 42 is located at position 20711 of chromosome 20, the allelic gene is T / C;

[0014] SNP site 43 is located at position 8462235 of chromosome 20, the allelic gene is G / A;

[0015] SNP site 44 is located at position 5667726 of chromosome 21, the allelic gene is A / G;

[0016] SNP site 45 is located at position 9106081 of chromosome 21, the allelic gene is A / G;

[0017] SNP site 46 is located at position 6590668 of chromosome 23, and the allelic bases are G / A;

[0018] SNP site 47 is located at position 4018058 of chromosome 25, and the allelic bases are T / C;

[0019] SNP site 48 is located at position 7757544 of chromosome 25, and the allelic bases are G / A;

[0020] SNP site 49 is located at position 1428759 of chromosome 26, and the allelic bases are T / C;

[0021] SNP site 50 is located at position 7526991 of chromosome 26, and the allelic bases are T / G;

[0022] SNP site 51 is located at position 15551 of chromosome 27, and the allelic bases are A / G;

[0023] SNP site 52 is located at position 4911414 of chromosome 28, and the allelic bases are T / C;

[0024] SNP site 53 is located at position 331268 of chromosome 29, and the allelic bases are A / G;

[0025] SNP site 54 is located at position 3839510 of chromosome 29, and the allelic bases are G / A;

[0026] SNP site 55 is located at position 29395 of chromosome 30, and the allelic bases are G / C;

[0027] SNP site 56 is located at position 2726139 of chromosome 30, and the allelic bases are C / A;

[0028] SNP site 57 is located at position 1154642 of chromosome 36, and the allelic bases are T / C;

[0029] SNP site 58 is located at position 890220 of chromosome 38, and the allelic bases are G / A;

[0030] SNP site 59 is located at position 1251302 of chromosome 39, and the allelic bases are C / T.

[0031] Although there are genetic marker studies on white pheasant, blue pheasant and white pheasant were differentiated into two different species 2.7 million years ago, there are certain differences between the genomes of the two species, and genome research shows that blue pheasant as a typical island species is only distributed in Taiwan, China, similar to the black pheasant which has one of the lowest genetic diversity in the world, the genome of blue pheasant is at a high inbreeding level, and a large number of SNP sites in the genome are in a homozygous state, and the percentage of long ROH (Runs of Homozygosity) of blue pheasant is close to 45%. White pheasant is widely distributed, and its genome has high genetic diversity, only lower than that of domestic chicken, and the long ROH of white pheasant accounts for only 0.1%. If the SNP site combination of white pheasant is used for individual identification of blue pheasant, firstly, SNP site amplification failure may occur due to differences in genome sequences; secondly, after obtaining SNP site typing, SNP site homozygosity or low allele frequency may occur, which greatly reduces the efficiency of individual identification. Therefore, for the highly inbred species of blue pheasant, it is necessary to screen SNP sites with high allele frequency for blue pheasant genome, so as to efficiently perform individual identification, and the genetic data and genetic markers of white pheasant cannot be simply used. Moreover, the white pheasant genome involved in the previous study is a scaffold, which does not reach the chromosome level, and the reference genome of blue pheasant at the high-quality chromosome level is assembled in the case, and the reference genome at the chromosome level can better select SNP markers representing the genome level for individual identification.

[0032] In one embodiment, the reference genome of the blue pheasant is GCA_030408155.1.

[0033] The application also provides a primer set for detecting the SNP molecular marker combination, which comprises the following primer pairs:

[0034] The primer pair 1 comprises SEQ ID No. 1 and SEQ ID No. 60;

[0035] The primer pair 2 comprises SEQ ID No. 2 and SEQ ID No. 61;

[0036] The primer pair 3 comprises SEQ ID No. 3 and SEQ ID No. 62;

[0037] Primer pair 4 comprises SEQ ID No. 4 and SEQ ID No. 63; primer pair 5 comprises SEQ ID No. 5 and SEQ ID No. 64; primer pair 6 comprises SEQ ID No. 6 and SEQ ID No. 65; primer pair 7 comprises SEQ ID No. 7 and SEQ ID No. 66; primer pair 8 comprises SEQ ID No. 8 and SEQ ID No. 67; primer pair 9 comprises SEQ ID No. 9 and SEQ ID No. 68; primer pair 10 comprises SEQ ID No. 10 and SEQ ID No. 69; primer pair 11 comprises SEQ ID No. 11 and SEQ ID No. 70; primer pair 12 comprises SEQ ID No. 12 and SEQ ID No. 71; primer pair 13 comprises SEQ ID No. 13 and SEQ ID No. 72; primer pair 14 comprises SEQ ID No. 14 and SEQ ID No. 73; primer pair 15 comprises SEQ ID No. 15 and SEQ ID No. 74; primer pair 16 comprises SEQ ID No. 16 and SEQ ID No. 75; primer pair 17 comprises SEQ ID No. 17 and SEQ ID No. 76; primer pair 18 comprises SEQ ID No. 18 and SEQ ID No. 77; primer pair 19 comprises SEQ ID No. 19 and SEQ ID No. 78; primer pair 20 comprises SEQ ID No. 20 and SEQ ID No. 79; primer pair 21 comprises SEQ ID No. 21 and SEQ ID No. 80; primer pair 22 comprises SEQ ID No. 22 and SEQ ID No. 81; primer pair 23 comprises SEQ ID No. 23 and SEQ ID No. 82; primer pair 24 comprises SEQ ID No. 24 and SEQ ID No. 83; primer pair 25 comprises SEQ ID No. 25 and SEQ ID No. 84; primer pair 26 comprises SEQ ID No. 26 and SEQ ID No. 85; primer pair 27 comprises SEQ ID No. 27 and SEQ ID No. 86; primer pair 28 comprises SEQ ID No. 28 and SEQ ID No. 87; primer pair 29 comprises SEQ ID No. 29 and SEQ ID No. 88; primer pair 30 comprises SEQ ID No. 30 and SEQ ID No. 89; primer pair 31 comprises SEQ ID No. 31 and SEQ ID No. 90; primer pair 32 comprises SEQ ID No. 32 and SEQ ID No. 91; primer pair 33 comprises SEQ ID No. 33 and SEQ ID No. 92; primer pair 34 comprises SEQ ID No. 34 and SEQ ID No.93; primer pair 35 comprises SEQ ID No. 35 and SEQ ID No. 94; primer pair 36 comprises SEQ ID No. 36 and SEQ ID No. 95; primer pair 37 comprises SEQ ID No. 37 and SEQ ID No. 96; primer pair 38 comprises SEQ ID No. 38 and SEQ ID No. 97; primer pair 39 comprises SEQ ID No. 39 and SEQ ID No. 98; primer pair 40 comprises SEQ ID No. 40 and SEQ ID No. 99;.

[0038] primer pair 41 comprises SEQ ID No. 41 and SEQ ID No. 100;

[0039] primer pair 42 comprises SEQ ID No. 42 and SEQ ID No. 101;

[0040] primer pair 43 comprises SEQ ID No. 43 and SEQ ID No. 102;

[0041] primer pair 44 comprises SEQ ID No. 44 and SEQ ID No. 103;

[0042] primer pair 45 comprises SEQ ID No. 45 and SEQ ID No. 104;

[0043] primer pair 46 comprises SEQ ID No. 46 and SEQ ID No. 105;

[0044] primer pair 47 comprises SEQ ID No. 47 and SEQ ID No. 106;

[0045] primer pair 48 comprises SEQ ID No. 48 and SEQ ID No. 107;

[0046] primer pair 49 comprises SEQ ID No. 49 and SEQ ID No. 108;

[0047] primer pair 50 comprises SEQ ID No. 50 and SEQ ID No. 109;

[0048] primer pair 51 comprises SEQ ID No. 51 and SEQ ID No. 110;

[0049] primer pair 52 comprises SEQ ID No. 52 and SEQ ID No. 111;

[0050] primer pair 53 comprises SEQ ID No. 53 and SEQ ID No. 112;

[0051] Primer pair 54 comprises SEQ ID No. 54 and SEQ ID No. 113;

[0052] Primer pair 55 comprises SEQ ID No. 55 and SEQ ID No. 114;

[0053] Primer pair 56 comprises SEQ ID No. 56 and SEQ ID No. 115;

[0054] Primer pair 57 comprises SEQ ID No. 57 and SEQ ID No. 116;

[0055] Primer pair 58 comprises SEQ ID No. 58 and SEQ ID No. 117;

[0056] Primer pair 59 comprises SEQ ID No. 59 and SEQ ID No. 118.

[0057] The present application also provides a detection system for individual identification of Gallus varius, comprising a multiplex PCR amplification system, wherein the multiplex PCR amplification system comprises the primer set.

[0058] In one embodiment, the reaction of the multiplex PCR amplification system comprises a first round of PCR and a second round of PCR.

[0059] The reaction conditions of the first round of PCR comprise: 95℃, 15min; 94℃, 30s, 60℃, 10min, 72℃, 30s, 4 cycles; 94℃, 30s, 60℃, 1min, 72℃, 30s, 20 cycles.

[0060] The reaction conditions of the second round of PCR comprise: 95℃, 15min; 94℃, 30s, 60℃, 4min, 72℃, 30s, 5 cycles; 94℃, 30s, 60℃, 1min, 72℃, 30s, 10 cycles.

[0061] The present application also provides a kit for individual identification of Gallus varius, wherein the kit comprises the primer set or the detection system.

[0062] The present application also provides the use of the SNP molecular marker combination, the primer set, the detection system or the kit in individual identification of Gallus varius.

[0063] In one embodiment, the individual identification comprises: genotyping of Gallus varius, analysis of the kinship of Gallus varius, and analysis of the genetic diversity of the population of Gallus varius.

[0064] The application further provides a method for individual identification of Phayre's pheasant, comprising the following steps: extracting DNA of a sample to be tested, performing multiplex PCR amplification by using the detection system or the kit, obtaining PCR amplification products, performing high-throughput sequencing, obtaining genotype data of the SNP molecular marker combination, and performing individual identification according to the genotype data.

[0065] Compared with the prior art, the application has the following beneficial effects:

[0066] The SNP molecular marker combination for individual identification of Phayre's pheasant and the application thereof can quickly and accurately obtain information of target SNP sites of Phayre's pheasant, so as to carry out individual identification of Phayre's pheasant and lay a foundation for individual identification and genetic diversity research of Phayre's pheasant. DETAILED DESCRIPTION

[0067] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant examples. However, the present application can be realized in many different forms and is not limited to the examples described herein. On the contrary, the purpose of providing these examples is to make the disclosure of the present application more thorough and comprehensive.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein only for the purpose of describing specific embodiments and is not intended to limit the present application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0069] Source:

[0070] The reagents, materials and equipment used in the examples are commercially available unless otherwise specified; the experimental methods are conventional experimental methods in the art unless otherwise specified.

[0071] Examples

[0072] 1. SNP site screening.

[0073] 1.1 SNP data set detection.

[0074] DNA was extracted from blood samples of 12 individuals of Meleagris gallopavo from Guangzhou Zoo. After constructing DNA library, whole genome re-sequencing data of at least 10 Gb per individual was obtained by Illumina NovaSeq 6000 platform. After quality control of the original reads data, the data was aligned to the chromosome-level reference genome of Meleagris gallopavo (NCBI accession number: GCA_030408155.1) to generate bam files, and duplicate reads were removed. Finally, SNP detection was performed using software, and quality control was performed with the conditions of DP < 59; DP > 534; QD < 2.0; FS > 60.0; MQ < 40.0; MQRankSum < -12.5; ReadPosRankSum < -8.0; SOR > 3.0, and finally a reliable SNP data set of Meleagris gallopavo was formed.

[0075] 1.2 SNP site screening.

[0076] The SNP data set of Meleagris gallopavo was further screened with the following criteria: (1) distributed in each autosome as much as possible; (2) the minimum allele frequency was greater than 0.4; (3) the physical distance between two SNP sites on the genome was greater than 5 Mb; (4) there was no insertion and deletion within 500 bp on both sides of the SNP site. Then, the 300 bp sequence containing the SNP site was blast aligned to the reference genome of Meleagris gallopavo, and only SNP sites with only one 95% or more homologous result were retained. Finally, primers were designed for the sequence containing the SNP site and evaluated, and 59 SNP sites were finally selected, located on 30 autosomes, as shown in Table 1:

[0077] Table 1 Information of 59 SNP sites of Meleagris gallopavo

[0078]

[0079]

[0080] II. SNP site amplification and genotyping.

[0081] 2.1 DNA extraction.

[0082] Twenty-two feather samples of 20 individuals of Meleagris gallopavo were randomly collected from Guangzhou Zoo, including two groups of two repeated samples each. All samples were extracted using HiPure Forensic DNA Kit.

[0083] 2.2 SNP site amplification.

[0084] Firstly, the 59 SNP loci of each individual were amplified by multiplex PCR, and the primer information is shown in Table 2. The multiplex PCR amplification system was 10 μL: 2 μL DNA, 1 μL Buffer (10×), 2 μL pre-mixed primer combination (50 nM), 0.8 μL dNTP (2.5 mM), 0.1 μL enzyme (5 U / μL), 1 μL Mg 2+ (100 mM), 3.2 μL ddH2O and 10 μL paraffin oil. The PCR program was as follows: 95℃, 15 min; 94℃, 30 s, 60℃, 10 min, 72℃, 30 s, 4 cycles; 94℃, 30 s, 60℃, 1 min, 72℃, 30 s, 20 cycles.

[0085] Then, the second round of PCR was performed, and a linker (Shanghai Yihao Biotechnology Co., Ltd.) was added to each sample to meet the subsequent high-throughput sequencing. 100 μL ddH2O was added to the multiplex PCR product of each sample in the first round, mixed evenly, and used as the DNA template for the second round of PCR, and the amplification reaction was performed, 20 μL system was 10 μL DNA template, 3.6 μL ddH2O, 2 μL Buffer (10×), 3.6 μL Barcode (2 μM), 0.8 μL dNTP (2.5 mM), 0.1 μL enzyme (5 U / μL), 1 μL Mg 2+ (100 mM) and 20 μL paraffin oil. The PCR program was as follows: 95℃, 15 min; 94℃, 30 s, 60℃, 4 min, 72℃, 30 s, 5 cycles; 94℃, 30 s, 60℃, 1 min, 72℃, 30 s, 10 cycles.

[0086] Table 2 Primer information for amplification of 59 SNP loci of blue pheasant

[0087]

[0088]

[0089]

[0090] 2.3 High-throughput sequencing.

[0091] 2.3.1 Library mixing.

[0092] 5 μL of the product was transferred to a U-shaped groove for mixing, then 200 μL of the mixed product in the U-shaped groove was transferred to a round-bottom centrifuge tube and vortexed for 30 seconds. Finally, the centrifuge tubes were fixed in parallel on a shaker, and the amplitude of the liquid in the tube was maximized. The mixed product was shaken overnight.

[0093] 2.3.2 Purification.

[0094] After electrophoresis, the target fragments were cut and placed in 4 1.5 ml low adsorption EP tubes. The magnetic bead method agarose gel DNA recovery kit (ENLIGHTEN, item number LD601-010) was used for purification according to the instructions. Finally, the four purified products were collected into one collection tube, shaken for 30 seconds and centrifuged, and stored at 4°C.

[0095] 2.3.3 High-throughput sequencing.

[0096] The purified product was accurately quantified and diluted to the required concentration for sequencing, and bridge PCR was performed. Then, the bridge PCR product was placed on the Illumina X-ten sequencing platform for sequencing, and the operation process was carried out according to the standard SOP.

[0097] 2.4 Genotyping.

[0098] After the data was downloaded, the index sequence was aligned based on the difference in the sample index sequence, and the original data of each sample was distinguished according to the difference in the index sequence, allowing at most one base mismatch when aligning the index sequence, and then removing the adapter sequence. The sequence alignment software was used to align the data after removing the adapter to the reference genome (NCBI accession number: GCA_030408155.1). Each sequencing read was subjected to SNP determination. For the SNP genotyping threshold, discard the amplicon with a sequencing depth <15x. Determine the genotype of the amplicon with sufficient sequencing depth: the heterozygote allele ratio is 20-80%, and the homozygote is outside this range, and count the number of reads and base sequencing quality (Q30). The average number of sequencing bases per individual is 1.03x10 8 The average sequencing depth of 59 SNP sites is 6686x, and the number of reads reaching Q30 is 95%, indicating that the sequencing data quality is high and can guarantee the reliability of SNP site genotyping. Through genotyping, 99.69% of the SNP sites were efficiently detected, and all 59 SNP sites were double alleles, as shown in Table 3. According to the genotyping result statistics, the average observed heterozygosity of the 59 SNP sites in this group is 0.378, the average expected heterozygosity is 0.459, and the average total polymorphism information is 0.345, as shown in Table 4. It is shown that the SNP site combination of the present application has good stability and polymorphism, which meets the requirements of individual identification.

[0099] Table 3 Genotyping of 59 SNP sites of blue pheasant

[0100]

[0101]

[0102]

[0103] Table 4 Genetic diversity statistics of 59 SNP loci of Phayre's pheasant

[0104]

[0105]

[0106] III. Individual identification

[0107] The genotype data of 59 SNP loci of 22 samples were analyzed for individual identification. The results showed that in the 22 samples, the genotypes of 59 SNP loci of two individuals in each pair of repeated samples were all the same, which were marked as "Exact match", i.e. determined as the same individual, indicating that the method is reliable and has good repeatability. The number of mismatched SNP loci between two samples of 20 different individuals ranged from 10 to 50, with an average of 36, and the identification results were all "Excluded", i.e. identified as different individuals, indicating that the use of the group of 59 loci can efficiently and accurately identify individuals.

[0108] According to the 59 SNP loci provided by the present application, since each SNP locus is a double allele, it can form three genotypes to form a 59-bit digital tag, which can theoretically give 3 59 Only the individual of Phayre's pheasant is provided with a molecular tag, which meets the individual identification needs of the Phayre's pheasant population. Therefore, through the group of 59 SNP loci, individual identification and genetic diversity evaluation of Phayre's pheasant can be performed.

[0109] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present application.

[0110] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of the patent of the present application should be subject to the appended claims.

Claims

1. Application of a SNP molecular marker combination in individual identification of Bubulcus coromandus, characterized in that, The reference genome of Phayre's palm is GCA_002973945.1, and the SNP molecular marker combination is composed of the following 59 SNP molecular markers: SNP site 1 is located at the position of 67974054 of chromosome 1, and the allele is A / G; SNP site 2 is located at the position of 125530636 of chromosome 1, and the allele is C / T; SNP site 3 is located at the position of 194739820 of chromosome 1, and the allele is G / A; SNP site 4 is located at the position of 97634433 of chromosome 2, and the allele is T / C; SNP site 5 is located at the position of 112377121 of chromosome 2, and the allele is A / G; SNP site 6 is located at the position of 31998099 of chromosome 3, and the allele is T / C; SNP site 7 is located at the position of 60185546 of chromosome 3, and the allele is T / C; SNP site 8 is located at the position of 76526823 of chromosome 3, and the allele is T / C; SNP site 9 is located at the position of 99096322 of chromosome 3, and the allele is T / C; SNP site 10 is located at the position of 5580707 of chromosome 4, and the allele is T / C; SNP site 11 is located at the position of 61331953 of chromosome 4, and the allele is G / A; SNP site 12 is located at the position of 72654237 of chromosome 4, and the allele is T / C; SNP site 13 is located at the position of 4604233 of chromosome 5, and the allele is C / T; SNP site 14 is located at the position of 30249593 of chromosome 5, and the allele is C / T; SNP site 15 is located at the position of 50086450 of chromosome 5, and the allele is A / G; SNP site 16 is located at the position of 18902332 of chromosome 6, and the allele is G / A; SNP site 17 is located at the position of 29374172 of chromosome 6, and the allele is G / T; SNP site 18 is located at the position of 41008232 of chromosome 6, and the allele is A / G; SNP site 19 is located at the position of 53820188 of chromosome 6, and the allele is C / T; SNP site 20 is located at the position of 239547 of chromosome 7, and the allele is T / C; SNP site 21 is located at the position of 35074517 of chromosome 7, and the allele is A / G; SNP site 22 is located at the position of 2606019 of chromosome 8, and the allele is T / C; SNP site 23 is located at the position of 26365282 of chromosome 8, and the allele is T / C; SNP site 24 is located at the position of 4482003 of chromosome 9, and the allele is C / T; SNP site 25 is located at the position of 28953883 of chromosome 9, and the allele is A / G; SNP site 26 is located at the position of 2763802 of chromosome 10, and the allele is C / T; SNP site 27 is located at the position of 25241662 of chromosome 10, and the allele is A / G; SNP site 28 is located at position 4905854 of chromosome 12, and the allelic bases are A / G; SNP site 29 is located at position 20552335 of chromosome 12, and the allelic bases are G / A; SNP site 30 is located at position 3719889 of chromosome 13, and the allelic bases are G / A; SNP site 31 is located at position 21998632 of chromosome 13, and the allelic bases are T / G; SNP site 32 is located at position 19292436 of chromosome 14, and the allelic bases are C / T; SNP site 33 is located at position 18389002 of chromosome 15, and the allelic bases are T / C; SNP site 34 is located at position 475472 of chromosome 16, and the allelic bases are C / T; SNP site 35 is located at position 14465184 of chromosome 16, and the allelic bases are C / T; SNP site 36 is located at position 4757476 of chromosome 17, and the allelic bases are C / T; SNP site 37 is located at position 15979491 of chromosome 17, and the allelic bases are C / T; SNP site 38 is located at position 3441805 of chromosome 18, and the allelic bases are C / T; SNP site 39 is located at position 12982785 of chromosome 18, and the allelic bases are A / G; SNP site 40 is located at position 5177 of chromosome 19, and the allelic bases are C / T; SNP site 41 is located at position 10836959 of chromosome 19, and the allelic bases are C / T; SNP site 42 is located at position 20711 of chromosome 20, and the allelic bases are T / C; SNP site 43 is located at position 8462235 of chromosome 20, and the allelic bases are G / A; SNP site 44 is located at position 5667726 of chromosome 21, and the allelic bases are A / G; SNP site 45 is located at position 9106081 of chromosome 21, and the allelic bases are A / G; SNP site 46 is located at position 6590668 of chromosome 23, and the allelic bases are G / A; SNP site 47 is located at position 4018058 of chromosome 25, and the allelic bases are T / C; SNP site 48 is located at position 7757544 of chromosome 25, and the allelic bases are G / A; SNP site 49 is located at position 1428759 of chromosome 26, and the allelic bases are T / C; SNP site 50 is located at position 7526991 of chromosome 26, and the allelic bases are T / G; SNP site 51 is located at position 15551 of chromosome 27, and the allelic bases are A / G; SNP site 52 is located at position 4911414 of chromosome 28, and the allelic bases are T / C; SNP site 53 is located at position 331268 of chromosome 29, and the allelic bases are A / G; SNP site 54 is located at position 3839510 of chromosome 29, and the allelic bases are G / A; SNP site 55 is located at position 29395 of chromosome 30, and the allelic bases are G / C; SNP site 56 is located at position 2726139 of chromosome 30, and the allelic bases are C / A; SNP site 57 is located at position 1154642 of chromosome 36, and the allelic bases are T / C; SNP site 58 is located at position 890220 of chromosome 38, and the allelic bases are G / A; SNP site 59 is located at position 1251302 of chromosome 39, and the allelic bases are C / T.

2. The primer set for detecting the SNP molecular marker combination in the application of claim 1 in the preparation of products for individual identification of Gallicrex cinerea, characterized in that, The primer set comprises the following primer pairs: Primer pair 1 comprises SEQ ID No. 1 and SEQ ID No. 60; Primer pair 2 comprises SEQ ID No. 2 and SEQ ID No. 61; Primer pair 3 comprises SEQ ID No. 3 and SEQ ID No. 62; Primer pair 4 comprises SEQ ID No. 4 and SEQ ID No. 63; Primer pair 5 comprises SEQ ID No. 5 and SEQ ID No. 64; Primer pair 6 comprises SEQ ID No. 6 and SEQ ID No. 65; Primer pair 7 comprises SEQ ID No. 7 and SEQ ID No. 66; Primer pair 8 comprises SEQ ID No. 8 and SEQ ID No. 67; Primer pair 9 comprises SEQ ID No. 9 and SEQ ID No. 68; Primer pair 10 comprises SEQ ID No. 10 and SEQ ID No. 69; Primer pair 11 comprises SEQ ID No. 11 and SEQ ID No. 70; Primer pair 12 comprises SEQ ID No. 12 and SEQ ID No. 71; Primer pair 13 comprises SEQ ID No. 13 and SEQ ID No. 72; Primer pair 14 comprises SEQ ID No. 14 and SEQ ID No. 73; Primer pair 15 comprises SEQ ID No. 15 and SEQ ID No. 74; Primer pair 16 comprises SEQ ID No. 16 and SEQ ID No. 75; Primer pair 17 comprises SEQ ID No. 17 and SEQ ID No. 76; Primer pair 18 comprises SEQ ID No. 18 and SEQ ID No. 77; Primer pair 19 comprises SEQ ID No. 19 and SEQ ID No. 78; Primer pair 20 comprises SEQ ID No. 20 and SEQ ID No. 79; Primer pair 21 comprises SEQ ID No. 21 and SEQ ID No. 80; Primer pair 22 comprises SEQ ID No. 22 and SEQ ID No. 81; Primer pair 23 comprises SEQ ID No. 23 and SEQ ID No. 82; Primer pair 24 comprises SEQ ID No. 24 and SEQ ID No. 83; Primer pair 25 comprises SEQ ID No. 25 and SEQ ID No. 84; Primer pair 26 comprises SEQ ID No. 26 and SEQ ID No. 85; Primer pair 27 comprises SEQ ID No. 27 and SEQ ID No. 86; Primer pair 28 comprises SEQ ID No. 28 and SEQ ID No. 87; Primer pair 29 comprises SEQ ID No. 29 and SEQ ID No. 88; Primer pair 30 comprises SEQ ID No. 30 and SEQ ID No. 89; Primer pair 31 comprises SEQ ID No. 31 and SEQ ID No. 90; Primer pair 32 comprises SEQ ID No. 32 and SEQ ID No. 91 ; Primer pair 33 comprises SEQ ID No. 33 and SEQ ID No. 92; Primer pair 34 comprises SEQ ID No. 34 and SEQ ID No. 93; Primer pair 35 comprises SEQ ID No. 35 and SEQ ID No. 94; Primer pair 36 comprises SEQ ID No. 36 and SEQ ID No. 95; Primer pair 37 comprises SEQ ID No. 37 and SEQ ID No. 96; Primer pair 38 comprises SEQ ID No. 38 and SEQ ID No. 97; Primer pair 39 comprises SEQ ID No. 39 and SEQ ID No. 98; Primer pair 40 comprises SEQ ID No. 40 and SEQ ID No. 99; Primer pair 41 comprises SEQ ID No. 41 and SEQ ID No. 100; Primer pair 42 comprises SEQ ID No. 42 and SEQ ID No. 101 ; Primer pair 43 comprises SEQ ID No. 43 and SEQ ID No. 102; Primer pair 44 comprises SEQ ID No. 44 and SEQ ID No. 103; Primer pair 45 comprises SEQ ID No. 45 and SEQ ID No. 104; Primer pair 46 comprises SEQ ID No. 46 and SEQ ID No. 105; Primer pair 47 comprises SEQ ID No. 47 and SEQ ID No. 106; Primer pair 48 comprises SEQ ID No. 48 and SEQ ID No. 107; Primer pair 49 comprises SEQ ID No. 49 and SEQ ID No. 108; Primer pair 50 comprises SEQ ID No. 50 and SEQ ID No. 109; Primer pair 51 comprises SEQ ID No. 51 and SEQ ID No. 110; Primer pair 52 comprises SEQ ID No. 52 and SEQ ID No. 111 ; Primer pair 53 comprises SEQ ID No. 53 and SEQ ID No. 112; Primer pair 54 comprises SEQ ID No. 54 and SEQ ID No. 113; Primer pair 55 comprises SEQ ID No. 55 and SEQ ID No. 114; The primer pair 56 comprises SEQ ID No. 56 and SEQ ID No.

115. The primer pair 57 comprises SEQ ID No. 57 and SEQ ID No.

116. The primer pair 58 comprises SEQ ID No. 58 and SEQ ID No.

117. The primer pair 59 comprises SEQ ID No. 59 and SEQ ID No.

118.

3. Use according to claim 2, characterized in that, The individual identification comprises genotyping of the blue pheasant, analysis of the genetic relationship of the blue pheasant, and analysis of the genetic diversity of the blue pheasant population.

4. The use of a detection system for detecting the SNP molecular marker combination in the application of claim 1 in the preparation of products for individual identification of Gallicrex cinerea. The detection system comprises a multiplex PCR amplification system comprising the primer set used in the application of claim 2.

5. Use according to claim 4, characterized in that, The individual identification comprises genotyping of the blue pheasant, analysis of the genetic relationship of the blue pheasant, and analysis of the genetic diversity of the blue pheasant population.

6. Use according to claim 4, characterized in that, The reaction of the multiplex PCR amplification system comprises a first round of PCR and a second round of PCR. The reaction conditions of the first round of PCR comprise 95℃, 15min; 94℃, 30s, 60℃, 10min, 72℃, 30s, 4 cycles; 94℃, 30s, 60℃, 1min, 72℃, 30s, 20 cycles. The reaction conditions of the second round of PCR comprise 95℃, 15min; 94℃, 30s, 60℃, 4min, 72℃, 30s, 5 cycles; 94℃, 30s, 60℃, 1min, 72℃, 30s, 10 cycles.

7. The use of a kit for detecting the SNP molecular marker combination in the application of claim 1 in the preparation of a product for individual identification of Gallicrex cinerea. The kit comprises the primer set used in the application of any one of claims 2-3 or the detection system used in the application of any one of claims 4-6.

8. Use according to claim 7, characterized in that, The individual identification comprises genotyping of the blue pheasant, analysis of the genetic relationship of the blue pheasant, and analysis of the genetic diversity of the blue pheasant population.

9. A method for individual identification of blue pheasants, characterized in that, The method comprises the following steps: DNA of the sample to be tested is extracted, and the detection system used in the application of any one of claims 4-6 or the kit used in the application of any one of claims 7-8 is used for multiplex PCR amplification to obtain a PCR amplification product, high-throughput sequencing is performed to obtain genotype data of the SNP molecular marker combination of claim 1, and individual identification is performed according to the genotype data.

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