CN111154888B - Microsatellite marker for detecting beagle closed group and combination thereof - Google Patents
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Abstract
The invention relates to a microsatellite marker for detecting beagle closed groups and a combination thereof. The microsatellite markers include at least one of FH2776, PEZ1, FH3972, C08.618, C14.866, REN06C11, REN85N14, CPH17, PEZ3, FH2626, REN87O21, REN239K24, FH3082, AF85862, and PEZ 2.
Description
Technical Field
The invention relates to a microsatellite marker for detecting beagle closed groups, in particular to a combination of the microsatellite markers for detecting the beagle closed groups.
Background
Beagle (Beagle), also known as miglu Beagle or Beagle dog, is the smaller of Beagle dogs in the uk as a native source and introduced into the united states in 1880, and is one of the international and universal breeds of dog. The dog bone carrier has the advantages of small size, mild and smooth character, uniform reaction, good repeatability, developed brain, strong adaptability and the like, is very suitable for life science and medical research, is an ideal experimental animal, and is an internationally recognized experimental dog. The application of beagle dogs in non-clinical researches of medicines such as acute toxicity tests is very wide, and the beagle dogs are also reported to be used in animal models for copying oral and urinary system diseases. In addition, beagle dogs are also used for research in various fields such as genetics, microbiology and biology. In 2015, the annual usage amount of the beagle dogs in China is 1.96 thousands of dogs, which accounts for 80% of the usage amount of experimental dogs in China. According to expert estimation, the dosage of beagle dogs in 2018 in China reaches more than 10 ten thousand dogs. Although beagle dogs have the characteristics of good uniformity, repeatability and comparability to experimental reactions, as beagle dogs are introduced in the early 1983, closed groups suitable for domestic feeding environments are basically formed through reproduction in nearly 30 years, and genetic information and biological characteristics of the beagle dogs are changed to a certain extent compared with foreign populations due to the influence of population, feeding environments and other facial factors. In the face of multi-region, multi-discipline and large-quantity market demands of beagle dogs, in order to ensure the quality of the beagle dogs, a beagle genetic quality detection method is established, and further establishment of a genetic quality standard of the beagle dogs becomes an urgent priority of a beagle production quality guarantee system.
Microsatellites (also known as Short Tandem Repeats (STRs)) refer to simple multiple tandem repeats consisting of a repeat unit of a few nucleotides (typically 2 to 6). In eukaryotes, microsatellites are widely present in almost all regions of the chromosome. Microsatellite markers can be classified into 1, 2, 3, 4, 5, 6 nucleotide microsatellites by the number of bases in their core unit. According to the structure of the microsatellite, Weber divides the microsatellite into complete, incomplete and composite microsatellites: the complete microsatellite is formed by connecting uninterrupted repeating units in series; incomplete microsatellites mean that less than 3 non-repetitive base intervals exist between repeat units, and the number of continuous repeats of the repeat units between two intervals is not less than 3; the compound microsatellite is composed of several types of tandem repeat units, the middle of the repeat units has 3 or less base intervals, and the number of the continuous repeat units is not less than 5. The microsatellite marker consists of a core sequence and two sides of conserved flanking sequences, wherein the conserved flanking sequences enable the microsatellite to be specifically positioned in a certain region of a chromosome, and the difference of the repetition number of the core sequence forms the high polymorphism of the microsatellite. The microsatellite DNA flanking sequence is used for designing a primer for PCR amplification, and due to different lengths of fragments formed by different repetition times, different electrophoresis band types are shown by imaging after electrophoretic separation.
The microsatellite DNA has the characteristics of abundant polymorphism, genetic linkage disequilibrium phenomenon, large quantity, uniform distribution, easy detection, good repeatability, time saving, suitability for automatic analysis, conservation among related species and the like, and is widely applied to the research of life science. The main manifestations are as follows: (1) the construction of the genetic map microsatellite is a codominant marker, the genetic analysis process is relatively simplified, the marker conversion among mapping groups is facilitated, and the transition from the genetic map to the physical map is also facilitated. (2) DNA fingerprinting was performed, and one of the first applications of microsatellites was to perform individual and breed (line) identification by making DNA fingerprinting. (3) Positioning functional genes and Quantitative Trait Loci (QTLs) using linkage between microsatellites and certain functional genes or QTLs, some functional genes or QTLs can be positioned on chromosomes or in linkage groups, which is a research that has been used in livestock (poultry). (4) The genetic identification and control of blood relationship are carried out, and pedigree records are very important in breeding and are firstly used for analyzing blood type and blood protein polymorphism, but the polymorphism is not abundant enough to be positioned in DNA fingerprint technology. (5) For population genetic diversity, genetic structure and origin studies, Nei et al (1994) concluded that microsatellites used to estimate genetic distances between closely related populations and to map phylogenetic trees are more accurate and efficient than other genetic marker techniques.
The International Society of Animal Genetics (ISAG) in 2001-2002 first verified the validity and reliability of different microsatellite DNA markers in canine paternity testing, and recommended 22 sites for reference. However, these microsatellite loci are completely unsuitable for the detection and monitoring of genetic quality of closed group beagle dogs.
Disclosure of Invention
One of the present invention provides a microsatellite marker comprising at least one of FH2776, PEZ1, FH3972, C08.618, C14.866, REN06C11, REN85N14, CPH17, PEZ3, FH2626, REN87O21, REN239K24, FH3082, AF85862 and PEZ 2; wherein the content of the first and second substances,
the FH2776 is obtained by taking the genome of beagle dogs as a template and taking SEQ ID No.1 and SEQ ID No.2 as primer pairs for PCR amplification;
the PEZ1 is obtained by carrying out PCR amplification by taking the genome of a beagle dog as a template and taking SEQ ID No.3 and SEQ ID No.4 as primer pairs;
the FH3972 is obtained by taking the genome of a beagle dog as a template and taking SEQ ID No.5 and SEQ ID No.6 as a primer pair for PCR amplification;
the C08.618 is obtained by taking the genome of the beagle dog as a template and taking SEQ ID No.7 and SEQ ID No.8 as primer pairs for PCR amplification;
the C14.866 is obtained by taking the genome of the beagle dog as a template and taking SEQ ID No.9 and SEQ ID No.10 as a primer pair for PCR amplification;
the REN06C11 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.11 and SEQ ID No.12 as a primer pair for PCR amplification;
the REN85N14 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.12 and SEQ ID No.14 as primer pairs for PCR amplification;
the CPH17 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.15 and SEQ ID No.16 as a primer pair for PCR amplification;
the PEZ3 is obtained by carrying out PCR amplification by taking the genome of a beagle dog as a template and taking SEQ ID No.17 and SEQ ID No.18 as primer pairs;
FH2626 through with the genome of beagle dog as the template, with SEQ ID No.19 and SEQ ID No.20 as the primer pair PCR amplification to get;
the REN87O21 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.21 and SEQ ID No.22 as primer pairs for PCR amplification;
the REN239K24 is obtained by performing PCR amplification by taking the genome of the beagle dog as a template and taking SEQ ID No.23 and SEQ ID No.24 as primer pairs;
the FH3082 is obtained by performing PCR amplification by taking the genome of the beagle dog as a template and taking SEQ ID No.25 and SEQ ID No.26 as primer pairs;
the AF85862 is obtained by performing PCR amplification by taking the genome of a beagle dog as a template and taking SEQ ID No.27 and SEQ ID No.28 as primer pairs;
the PEZ2 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID No.29 and SEQ ID No.30 as primer pairs.
The second invention provides a microsatellite marker combination, which comprises FH2776, PEZ1, FH3972, C08.618, C14.866, REN06C11, REN85N14, CPH17, PEZ3, FH2626, REN87O21, REN239K24, FH3082, AF85862 and PEZ 2.
In a specific embodiment, the combination further comprises at least one of FH2313, FH2263, FH2054, REN286P03, and PEZ 8; wherein the content of the first and second substances,
FH2313 is obtained by performing PCR amplification by using a beagle genome as a template and using SEQ ID No.31 and SEQ ID No.32 as primer pairs;
the FH2263 is obtained by PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID No.33 and SEQ ID No.34 as primer pairs;
the FH2054 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.35 and SEQ ID No.36 as primer pairs for PCR amplification;
the REN286P03 is obtained by taking the genome of beagle dog as a template and taking SEQ ID No.37 and SEQ ID No.38 as primer pairs for PCR amplification;
the PEZ8 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID No.39 and SEQ ID No.40 as primer pairs.
In a specific embodiment, said combination comprises said FH2313, said FH2263, said FH2054, said REN286P03 and said PEZ 8.
The third invention provides a primer pair, which comprises a primer pair consisting of SEQ ID No.1 and SEQ ID No.2, a primer pair consisting of SEQ ID No.3 and SEQ ID No.4, a primer pair consisting of SEQ ID No.5 and SEQ ID No.6, a primer pair consisting of SEQ ID No.7 and SEQ ID No.8, a primer pair consisting of SEQ ID No.9 and SEQ ID No.10, a primer pair consisting of SEQ ID No.11 and SEQ ID No.12, a primer pair consisting of SEQ ID No.12 and SEQ ID No.14, a primer pair consisting of SEQ ID No.15 and SEQ ID No.16, a primer pair consisting of SEQ ID No.17 and SEQ ID No.18, a primer pair consisting of SEQ ID No.19 and SEQ ID No.20, a primer pair consisting of SEQ ID No.21 and SEQ ID No.22, a primer pair consisting of SEQ ID No.23 and SEQ ID No.24, a primer pair consisting of SEQ ID No.25 and SEQ ID No.26, and a primer pair consisting of SEQ ID No.28, At least one of the primer pair consisting of SEQ ID No.29 and SEQ ID No. 30.
In a specific embodiment, the primer pair further comprises at least one of a primer pair consisting of SEQ ID No.31 and SEQ ID No.32, a primer pair consisting of SEQ ID No.33 and SEQ ID No.34, a primer pair consisting of SEQ ID No.35 and SEQ ID No.36, a primer pair consisting of SEQ ID No.37 and SEQ ID No.38, and a primer pair consisting of SEQ ID No.39 and SEQ ID No. 40.
The fourth aspect of the present invention provides a primer pair combination, which comprises a primer pair consisting of SEQ ID No.1 and SEQ ID No.2, a primer pair consisting of SEQ ID No.3 and SEQ ID No.4, a primer pair consisting of SEQ ID No.5 and SEQ ID No.6, a primer pair consisting of SEQ ID No.7 and SEQ ID No.8, a primer pair consisting of SEQ ID No.9 and SEQ ID No.10, a primer pair consisting of SEQ ID No.11 and SEQ ID No.12, a primer pair consisting of SEQ ID No.12 and SEQ ID No.14, a primer pair consisting of SEQ ID No.15 and SEQ ID No.16, a primer pair consisting of SEQ ID No.17 and SEQ ID No.18, a primer pair consisting of SEQ ID No.19 and SEQ ID No.20, a primer pair consisting of SEQ ID No.21 and SEQ ID No.22, a primer pair consisting of SEQ ID No.23 and SEQ ID No.24, a primer pair consisting of SEQ ID No.25 and SEQ ID No.26, and a primer pair consisting of SEQ ID No.28, A primer pair consisting of SEQ ID No.29 and SEQ ID No. 30.
In a specific embodiment, the combination further comprises a primer pair consisting of SEQ ID No.31 and SEQ ID No.32, a primer pair consisting of SEQ ID No.33 and SEQ ID No.34, a primer pair consisting of SEQ ID No.35 and SEQ ID No.36, a primer pair consisting of SEQ ID No.37 and SEQ ID No.38, and a primer pair consisting of SEQ ID No.39 and SEQ ID No. 40.
The fifth invention provides the application of the microsatellite marker in the first invention, the combination of the microsatellite markers in any one of the second invention, the primer pair in the third invention and the primer pair combination in the fourth invention in at least one of detection, identification and differentiation of beagle closed groups.
In a specific embodiment, the beagle dog is at least one of a small beagle dog, a medium beagle dog, and a large beagle dog.
The invention has the advantages of
The invention establishes a plurality of microsatellite loci for the first time, wherein the microsatellite loci can be used for beagle closed group detection. The method can be used for detecting the population genetic quality of the closed group beagle dog based on the combination of the microsatellite loci, so that a method capable of effectively detecting the genetic quality of the closed group beagle dog is established, and further the establishment of the genetic quality standard of the closed group beagle dog is facilitated, so that the production and the application of the beagle dog are standardized, the genetic condition of the closed group beagle dog is comprehensively, objectively and fairly evaluated, and a foundation is laid for realizing the effective preservation of the application and the resource of the closed group beagle dog in China and providing the genetic quality control standard and the corresponding technical specification of the closed group beagle dog.
Drawings
FIG. 1 shows the results of 0.8% agarose electrophoresis of a part of the genomic DNA of beagle dogs. Lanes M are 50bp marker, lanes 1 to 3 are genomic DNA from small beagle dogs, lanes 4 to 6 are genomic DNA from medium beagle dogs, and lanes 7 to 10 are genomic DNA from large beagle dogs.
FIG. 2 shows the results of 0.8% agarose electrophoresis of potential microsatellite loci from a portion of beagle dogs. Lane M is 50bpmarker, and lanes 1 to 10 are different potential microsatellite loci.
Figure 3 shows a small beagle colony clustering plot for 4 manufacturers based on genetic distance.
Detailed Description
Example 1
1.1.1 Experimental animals
A total of 30 non-littermates of unlimited sex purchased from Qingdao Bolong laboratory animals, Inc. were randomly selected. 5mL of blood was collected from each individual vein in each anticoagulated blood collection tube and stored in a refrigerator at 4 ℃ for use.
6 medium-size beagle dogs of different litters and different sexes and 6 large-size beagle dogs of different litters and different sexes purchased from Qingdao Bolong laboratory animals Co., Ltd are also randomly selected, 5mL of blood is extracted from each single vein into each anticoagulated blood collection tube, and the anticoagulated blood collection tubes are stored in a refrigerator at 4 ℃ for later use.
1.1.2 extraction and detection of genomic DNA from blood samples of beagle dogs
The genomic DNA was extracted from each of the beagle dogs' Blood Midi kit (Qiagen, Germany) using a Blood genomic DNA extraction kit according to the instructions. The concentration of the extracted genomic DNA was measured by a micro-UV spectrophotometer, and the A260/A280 values of all samples were between 1.8 and 2.0. The DNA stock was diluted to 80 ng/. mu.L. The diluted DNA was detected by 0.8% agarose electrophoresis, wherein the agarose gel electrophoresis pattern of a part of the beagle genomic DNA is shown in FIG. 1. The results show that the genomic DNA is relatively complete and can be used for the next PCR analysis.
1.1.3 Primary screening of potential microsatellite loci by amplification in common PCR reactions
6 DNA pools were prepared, each containing 1 of the above DNA samples from small, 1 medium and 1 large beagle dogs, and the DNA samples from each beagle dog between the different DNA pools were not repeated.
120 pairs of PCR primers for screening potential microsatellite loci were designed and synthesized.
A20. mu.L reaction was used: 80 ng/. mu.L of genomic DNA pool sample 2. mu.L, 1. mu.L each of upstream and downstream primers (10. mu. mol/. mu.L, respectively), 2. mu.L of 10 XPCR buffer, dNTP Mg2+plus (100. mu. mol/. mu.L) 4. mu.L, Taq enzyme (5U/. mu.L) 0.2. mu.L, supplemented with 9.8. mu.L of double distilled Water (ddH)2O). The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing for 1min (the annealing temperature at each site is determined by optimization between 53-60 ℃), and extension at 72 ℃ for 1 min; circulating for 35 times; continuing to extend for 10min at 72 ℃; the amplification product was stored at 4 ℃.
Agarose gel electrophoresis is used for primarily detecting PCR products, and a gel imaging system is used for image acquisition and analysis. The result showed that 101 of the 120 potential microsatellite loci were able to show an amplified band, indicating successful amplification, which was determined to be a positive band. Wherein the electrophoresis pattern of some microsatellite loci is shown in FIG. 2. Then, hybrid bands with high polymorphism (namely, electrophoretic detection bands are thicker or obvious main bands exist) and more concentrated product bands and no non-specificity in small, medium and large 3 populations are selected, and sites which are more uniformly covered on all 39 chromosome pairs of dogs (two sites are ensured on each chromosome pair) are selected, and finally 73 potential microsatellite sites are obtained, which is shown in table 1. And carrying out subsequent fluorescent primer marking grouping on the 73 sites according to the principle that the larger the size difference between target bands amplified by the same pair of primers is, the better the difference is and the closer the annealing temperature between different primer pairs is, the better the difference is.
1.1.4 STR scanning potential microsatellite loci amplified by fluorescent primers
Screening 73 potential microsatellite loci from the common primers to synthesize fluorescence labeled primers (synthesized by Shanghai Biotech Co., Ltd.), and respectively labeling the 5' ends of the upstream primers of the three loci with FAM, HEX and TAMRA when the same group comprises the three loci; when the same group comprises two sites, respectively marking FAM and TAMRA at the 5' ends of the upstream primers of the two sites; and labeling the 5' end of the upstream primer of the other site by using FAM. The site names, chromosomal location, fluorescent markers and annealing temperatures for the 73 microsatellite sites are shown in Table 1.
TABLE 1.73 beagle microsatellite locus names, chromosomal location, fluorescent markers and annealing temperatures
The PCR amplification of 30 DNA samples from small beagle dogs was performed using the same general primer protocol as in subsection 1.1.3 using fluorescent primers, wherein the annealing temperatures are shown in Table 1. STR scans were then performed to count the number of alleles and fragment size ranges for potential microsatellite loci and to genotype based on the scan results. The results show that 64 microsatellite loci can be successfully typed, the detection rate is high, and the results are detailed in table 2. It is demonstrated that these sites can be used for the analysis of the genetic structure of the beagle dog population, but the number of sites is large and the scanning pattern of some sites is not clear enough, such as REN107H05, so it is better to further optimize these sites.
TABLE 264 miniature beagle microsatellite locus names and the number and range of alleles detected in the Qingdao Borong closed group
1.1.5 statistical methods
Scanning results 30 samples were analyzed by genemarkerv2.2.0 software for amplified fragment size at 73 beagle microsatellite loci. The allele of each locus is recorded as the genotype of each sample a, b, c, d, e … … according to the sequence of the amplified fragment sizes from large to small, and can be recorded as aa, ab, ac, ad … …
1.1.6 analysis of Small beagle dog population genetic variation
And further selecting beagle microsatellite loci (49) with allele number more than or equal to 4 and good scanning results, and analyzing the genetic structure characteristics of the small beagle population by Popgene3.1 software (Table 3). The results showed that a total of 413 alleles were detected in small beagle dogs using the 49 microsatellite loci, with an average of 8.4 per microsatellite locus. Among them, the most abundant of alleles is FH3083 locus, there are 25 alleles; the minimum are FH2062, FH2890, FH2326, REN106I06, FH2885, REN164B05, FH2060 and FH2708, which have only 4 alleles respectively. Heterozygosity of the population is also calculated by Popgene3.1 software from allele frequencies of different microsatellite loci, and as shown in Table 3, heterozygosity of the population is different for different microsatellite loci. The average heterozygosity detected at this microsatellite locus was shown to be highest at FH3083 (0.9367), while the average heterozygosity detected at the FH2062 locus was shown to be lowest (0.3361). The overall average heterozygosity of the finally selected 49 microsatellite loci reaches 0.7375.
TABLE 3 allele number, average heterozygosity and polymorphism information content in the 49 microsatellite loci of Qingdao Bolong closed group of small beagle dogs
Polymorphism of sites is judged by Polymorphism Information Content (PIC), and accordingly, in 49 selected microsatellite sites in a small beagle dog population, the polymorphism information content is in the range of 0.3055-0.9333, so that the average polymorphism information content of the population reaches 0.7038 (obtained by calculation of Popgene software), and population high polymorphism is presented. Among them, 46 highly polymorphic sites (PIC >0.5), 3 moderately polymorphic sites (0.25 < PIC < 0.5) and 0 highly polymorphic sites (PIC < 0.25) were present (see Table 3 for details). Meanwhile, the average bloom index is also higher and reaches 1.6625, which indicates that the small beagle dog population is more balanced.
P-values from the Hardy-Weinberg genetic balance test for 49 microsatellite loci of small beagle dogs were calculated using popphen 1.32, see table 3. As a result, 12 microsatellite loci were very significantly off Hardy-Weinberg genetic equilibrium (P <0.01), 3 of them were significantly off Hardy-Weinberg genetic equilibrium (0.01< P <0.05), and the remaining 34 microsatellite loci were in Hardy-Weinberg genetic equilibrium (P >0.05) in the population genetics. Further, after considering several important indexes of allele number, chromosome position, shannon index and heterozygosity together, 20 microsatellite loci were selected from the several important indexes and combined together enough to be used for genetic testing analysis of beagle closed group (table 4).
Table 4 optimized 20 microsatellite locus names, chromosomal locations and number of alleles of beagle dogs in the celadon blond closed population.
Wherein, F is an upstream primer, and R is a downstream primer.
Example 2
30 small beagle closed groups from 3 domestic different manufacturers, namely Xian Dilep, Beijing Mas and Jiangsu megabiogenesis are purchased, and the genetic quality of the beagle closed groups is verified and evaluated by using 20 optimized microsatellite locus combinations suitable for genetic quality detection of the beagle closed groups.
Blood was collected in 5ml from each dog of each population. DNA extraction, detection, PCR, allele counts and allele range analysis were as in example 1. The specific sequence of the primer is detailed in Table 4, and the fluorescent label is detailed in Table 1. The results are shown in Table 5.
TABLE 520 allele counts and ranges for 3 populations of microsatellite loci
As can be seen from Table 5, the number of alleles in the Siann Dilep, Beijing Mas and Jiangsu megabiotic closed group reached 29, 14 and 14 at the maximum, respectively, and the minimum number of alleles was 2.
After the above-mentioned allelic typing was carried out on 3 closed groups of Cian Dilep, Beijing Ma Si and Jiangsu megabiosource using 20 loci, the 3 closed groups and each allelic type of the Qingdao clone closed group in example 1 were inputted into Popgene3.1 program for group genetic structure analysis, and the results are shown in Table 6. As can be seen from Table 6, the number of effective alleles for these closed groups was at least 5.45 and at most 9.95. The number of effective alleles was at least 3.3774 and at most 5.8897. The average heterozygosity is higher than 0.5, the bloom index is higher than 1.1, the polymorphic information content is good, and the high polymorphic level (PIC >0.5) is achieved. When these data were compared with data (table 3) obtained by analyzing the closed Qingdao Bolong population at 49 sites, it was found that the values obtained by detecting and calculating the genetic structure parameters of the population at 20 sites were equivalent to the results obtained by calculating 49 sites, regardless of the allele count, the effective allele count, the observed heterozygosity, the expected heterozygosity, the average heterozygosity, the Conlong index and the polymorphic information content. Therefore, the optimized detection effect of 20 sites is equal to 49 sites, but the reduction of the sites obviously improves the efficiency of detection work, reduces the workload and the cost, and is a relatively ideal combination.
TABLE 64 manufacturer's beagle population genetic Structure parameters
The closed groups of Xian Dilep, Beijing Ma Si and Jiangsu megabiogenesis and Qingdao Bolong were subjected to comparative analysis of Nei genetic similarity coefficient and genetic distance, and the results are shown in Table 7. The results show that the Nei genetic similarity coefficient of the Jiangsu megagenesis and the Beijing Mas population is the highest (0.6276), and the genetic distance is the closest (0.4659). The similarity coefficient of the Qingdao Bolong population and the similarity coefficient of the other three populations are smaller, and the genetic distance is longer.
Clustering analysis was performed on 4 closed clusters of Cian Dilep, Beijing Mas, Jiangsu megabiogenesis and Qingdao clone with Popgene3.1, and the results are shown in FIG. 3. As can be seen from fig. 3, the relation between the Qingdao Bolong closed group and the other three is far, and the relation between the Jiangsu megagen and the Beijing Ma is closer.
TABLE 74 comparison of Nei genetic similarity coefficients and genetic distances for the manufacturer beagle population
| Group ID | Sian Dilep | Beijing Maras | Jiangsu megagenesis | Qingdao Bolong |
| Sian Dilep | **** | 0.4648 | 0.5654 | 0.4764 |
| Beijing Maras | 0.7662 | **** | 0.6276 | 0.3746 |
| Jiangsu megagenesis | 0.5701 | 0.4659 | **** | 0.4576 |
| Qingdao Bolong | 0.7415 | 0.9818 | 0.7817 | **** |
Note: the genetic similarity coefficient is on the diagonal line, and the genetic distance is under the diagonal line
The analysis of the results is consistent with the actual source of the animals among the 4 closed groups. It can be seen that 20 microsatellite locus combinations can well evaluate the genetic characteristics of beagle closed groups.
The above results all show that the genetic detection of beagle closed groups can be effectively evaluated by repeatedly optimizing the determined 20 microsatellite locus combinations layer by layer.
Sequence listing
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<400> 7
caacccaggg tggaagc 17
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tagcaagaaa atgtgccca 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tgtcataata gttggaatga c 21
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ttagagctta ctcatgatat ctg 23
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgcagggcag aggctggagg 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gggggtgtcg gtggagttct 20
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaggcaggag gaggagcac 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tatggagatg gagggcacac 20
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gagaacaaaa gtcccatgca c 21
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gcattgatgc taatgcaaat g 21
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cactatgctg tacacctgaa tctaa 25
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cacttctcat acccagactc ct 22
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttctgccatt ctggaaaaca 20
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tccatggatc agcagcag 18
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tggctgtggc taaggctttg t 21
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ccccttccag cttcggtgta g 21
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ttcattggct ggtgactttg 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccccaggacc atttgttaga 20
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
agtaaagggt tctcaagtgt gc 22
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
catttgtcat tgtggaaaac c 21
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cattgatgct gaatttgact tc 22
<210> 28
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ttacacacac tattttgctg gtca 24
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcctctctaa ctgcctatgc 20
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gcccttgaat atgaacaatg acactgtatc 30
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agtagaagag gccacgcaaa 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cacgaagaaa gccatggttt 20
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
catgtagagt gattagttgg tctt 24
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ctgaatatcc tctgcccttc 20
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gccttattca ttgcagttag gg 22
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atgctgagtt ttgaactttc cc 22
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcacattcac aaagtggtgc 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gcaatggaaa gaggatggaa 20
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tgggaggcta aattggaggt gt 22
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ctgctcacca tggagtctac ttc 23
Claims (4)
1. A microsatellite marker combination comprising FH2776, PEZ1, FH3972, C08.618, C14.866, REN06C11, REN85N14, CPH17, PEZ3, FH2626, REN87O21, REN239K24, FH3082, AF85862, PEZ2, FH2313, FH2263, FH2054, REN286P03 and PEZ 8; wherein the content of the first and second substances,
the FH2776 is obtained by taking the genome of beagle dogs as a template and taking SEQ ID number 1 and SEQ ID number 2 as primer pairs for PCR amplification;
the PEZ1 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and SEQ ID number 3 and SEQ ID number 4 as primer pairs;
the FH3972 is obtained by taking the genome of beagle dogs as a template and taking SEQ ID number 5 and SEQ ID number 6 as primer pairs for PCR amplification;
the C08.618 is obtained by taking the genome of beagle dog as a template and SEQ ID number 7 and SEQ ID number 8 as primer pairs for PCR amplification;
the C14.866 is obtained by taking the genome of the beagle dog as a template and taking SEQ ID number 9 and SEQ ID number 10 as primer pairs for PCR amplification;
the REN06C11 is obtained by performing PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 11 and SEQ ID number 12 as primer pairs;
the REN85N14 is obtained by performing PCR amplification by taking the genome of beagle dogs as a template and taking SEQ ID number 13 and SEQ ID number 14 as primer pairs;
the CPH17 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and SEQ ID number 15 and SEQ ID number 16 as primer pairs;
the PEZ3 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 17 and SEQ ID number 18 as primer pairs;
the FH2626 is obtained by taking the genome of beagle dogs as a template and taking SEQ ID number 19 and SEQ ID number 20 as primer pairs for PCR amplification;
the REN87O21 is obtained by performing PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 21 and SEQ ID number 22 as primer pairs;
the REN239K24 is obtained by performing PCR amplification by taking the genome of beagle dogs as a template and taking SEQ ID number 23 and SEQ ID number 24 as primer pairs;
the FH3082 is obtained by performing PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 25 and SEQ ID number 26 as primer pairs;
the AF85862 is obtained by performing PCR amplification by taking the genome of a beagle dog as a template and taking SEQ ID number 27 and SEQ ID number 28 as primer pairs;
the PEZ2 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 29 and SEQ ID number 30 as primer pairs;
the FH2313 is obtained by performing PCR amplification by taking a beagle genome as a template and taking SEQ ID number 31 and SEQ ID number 32 as primer pairs;
the FH2263 is obtained by performing PCR amplification by taking the genome of beagle dogs as a template and taking SEQ ID number 33 and SEQ ID number 34 as primer pairs;
the FH2054 is obtained by performing PCR amplification by taking the genome of beagle dog as a template and taking SEQ ID number 35 and SEQ ID number 36 as primer pairs;
the REN286P03 is obtained by taking the genome of beagle dog as a template and taking SEQ ID number 37 and SEQ ID number 38 as primer pairs for PCR amplification;
the PEZ8 is obtained by carrying out PCR amplification by taking the genome of beagle dog as a template and SEQ ID number 39 and SEQ ID number 40 as primer pairs.
2. A primer pair combination comprising a primer pair consisting of SEQ ID number 1 and SEQ ID number 2, a primer pair consisting of SEQ ID number 3 and SEQ ID number 4, a primer pair consisting of SEQ ID number 5 and SEQ ID number 6, a primer pair consisting of SEQ ID number 7 and SEQ ID number 8, a primer pair consisting of SEQ ID number 9 and SEQ ID number 10, a primer pair consisting of SEQ ID number 11 and SEQ ID number 12, a primer pair consisting of SEQ ID number 13 and SEQ ID number 14, a primer pair consisting of SEQ ID number 15 and SEQ ID number 16, a primer pair consisting of SEQ ID number 17 and SEQ ID number 18, a primer pair consisting of SEQ ID number 19 and SEQ ID number 20, a primer pair consisting of SEQ ID number 21 and SEQ ID number 22, a primer pair consisting of SEQ ID number 2, a primer pair consisting of SEQ ID number 3 and SEQ ID number 4, The combination further comprises a primer pair consisting of SEQ ID number 23 and SEQ ID number 24, a primer pair consisting of SEQ ID number 25 and SEQ ID number 26, a primer pair consisting of SEQ ID number 27 and SEQ ID number 28, a primer pair consisting of SEQ ID number 29 and SEQ ID number 30, a primer pair consisting of SEQ ID number 31 and SEQ ID number 32, a primer pair consisting of SEQ ID number 33 and SEQ ID number 34, a primer pair consisting of SEQ ID number 35 and SEQ ID number 36, a primer pair consisting of SEQ ID number 37 and SEQ ID number 38, a primer pair consisting of SEQ ID number 39 and SEQ ID number 40.
3. Use of the microsatellite marker combination according to claim 1 and the primer pair combination according to claim 2 for at least one of detecting, identifying and differentiating beagle closed groups.
4. The use according to claim 3 wherein the beagle dog is at least one of a small beagle dog, a medium beagle dog and a large beagle dog.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321752A (en) * | 2010-08-19 | 2012-01-18 | 无锡中德美联生物技术有限公司 | Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2550219A1 (en) * | 2003-12-17 | 2005-06-30 | Fred Hutchinson Cancer Research Center | Methods and materials for canine breed identification |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321752A (en) * | 2010-08-19 | 2012-01-18 | 无锡中德美联生物技术有限公司 | Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Canine parentage testing based on microsatellite polymorphisms;Y Ichikawa等;《J Vet Med Sci.》;20011130;第63卷(第11期);1209-1213 * |
| 中国沙皮犬微卫星DNA分析的遗传多样性;刘清神 等;《动物学研究》;20080215;第29卷(第01期);33-38 * |
| 川东猎犬16个微卫星基因座多态性研究;杨前勇 等;《江西畜牧兽医杂志》;20151215(第06期);14-16 * |
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