BAS inbred line rat SNP locus typing and application

Abstract

The invention belongs to the technical field of experimental animal inbred line rat strain identification and genetic quality control, and particularly discloses 21 SNP markers in BAS inbred line rat strain identification, a preparation kit or a detection site sequence, a primer sequence, a nucleotide type and application of the 21 SNP markers in BAS inbred line rat strain identification and the 21 SNP markers in BAS inbred line rat strain identification and the 21 SNP markers in BAS inbred line rat strain identification and the 21 SNP markers in BAS inbred line rat strain identification. According to the method, 21 SNP markers are used for identifying a BAS inbred line rat strain, and each SNP marker comprises 21 SNP loci; the inbred line rats are inbred line rats of which the strain is BAS. The 21 SNP markers are used for identifying the BAS inbred line rats, the detection result shows that the 21 SNP loci of the BAS inbred line rats are all in a homozygous state, and the accuracy of inbred line rat line identification is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of inbred strain rat strain identification and genetic quality control of laboratory animals, and specifically discloses the application of 21 SNP markers in the identification of BAS inbred strain rats, the preparation of kits or detection site sequences, primer sequences, nucleotide types and their applications. Background Art

[0002] Laboratory animals refer to animals that are artificially bred, with their carried microorganisms controlled, clear genetic backgrounds or known origins, and are used for scientific research, teaching, production, verification, and other scientific experiments. The BAS inbred strain rats are a new inbred strain of rats cultivated by the Experimental Animal Center of Shaanxi Academy of Traditional Chinese Medicine. Inbred strain rats are one of the most widely used laboratory animals. Using inbred strain animals can reduce the number of laboratory animals and the number of experimental repetitions, and improve the comparability, repeatability, and accuracy of experimental results. During the cultivation process of inbred strain animals, in order to ensure the homozygosity of the genes of laboratory animals, it is very necessary to supplement genetic quality detection and timely discover and eliminate variant individuals. Common detections of inbred strain rats of laboratory animals include trait gene detection, skin transplantation experiments, biochemical marker detection, etc. With the development of molecular genetic detection technology, genetic markers have expanded from morphological genetic markers, cytogenetic markers, biochemical genetic markers, etc. to genetic markers at the genomic molecular level. Single nucleotide polymorphism markers (SNP) refer to DNA sequence polymorphisms caused by single nucleotide conversions, transversions, insertions, or deletions at the genomic level, and are the third-generation molecular markers for gene analysis. Common SNPs are mainly bi-allelic polymorphisms. Summary of the Invention

[0003] In order to be able to simultaneously identify the BAS inbred strain rat strain and improve the accuracy of the identification results of inbred strain rat strains, the present invention provides the application of 21 SNP markers in the identification of BAS inbred strain rats, the preparation of kits or detection site sequences, primer sequences, nucleotide types and their applications.

[0004] In the first aspect, the present invention provides an application of SNP markers in the identification of BAS inbred strain rats, adopting the following technical scheme:

[0005] 1. Use of SNP markers in the identification of BAS inbred rat strains, wherein the SNP markers include 21 SNP loci, namely: rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from BAS inbred rats.

[0006] Further, rs64750237 is located at the 255th position of the nucleotide sequence shown in SEQ ID NO: 1, and the base of the nucleotide molecule here is T or C;

[0007] rs106888236 is located at the 342nd position of the nucleotide sequence shown in SEQ ID NO: 2, and the base of the nucleotide molecule here is C or A;

[0008] rs106458396 is located at the 230th position of the nucleotide sequence shown in SEQ ID NO: 3, and the base of the nucleotide molecule here is A or C;

[0009] rs106672258 is located at the 197th position of the nucleotide sequence shown in SEQ ID NO: 4, and the base of the nucleotide molecule here is T or C;

[0010] rs65921305 is located at the 306th position of the nucleotide sequence shown in SEQ ID NO: 5, and the base of the nucleotide molecule here is T or G;

[0011] rs8152541 is located at the 378th position of the nucleotide sequence shown in SEQ ID NO: 6, and the base of the nucleotide molecule here is G or A;

[0012] rs106996961 is located at the 169th position of the nucleotide sequence shown in SEQ ID NO: 7, and the base of the nucleotide molecule here is G or T;

[0013] rs65970293 is located at the 424th position of the nucleotide sequence shown in SEQ ID NO: 8, and the base of the nucleotide molecule here is T or C;

[0014] The rs105187832 is located at the 359th position of the nucleotide sequence shown in SEQ ID NO: 9, where the base of the nucleotide molecule is C or A;

[0015] The rs13453252 is located at the 189th position of the nucleotide sequence shown in SEQ ID NO: 10, where the base of the nucleotide molecule is G or C;

[0016] The rs64102787 is located at the 292nd position of the nucleotide sequence shown in SEQ ID NO: 11, where the base of the nucleotide molecule is C or A;

[0017] The rs63860341 is located at the 201st position of the nucleotide sequence shown in SEQ ID NO: 12, where the base of the nucleotide molecule is C or T;

[0018] The rs107261116 is located at the 192nd position of the nucleotide sequence shown in SEQ ID NO: 13, where the base of the nucleotide molecule is G or C;

[0019] The rs106114574 is located at the 174th position of the nucleotide sequence shown in SEQ ID NO: 14, where the base of the nucleotide molecule is T or A;

[0020] The rs64682441 is located at the 321st position of the nucleotide sequence shown in SEQ ID NO: 15, where the base of the nucleotide molecule is T or C;

[0021] The rs65067166 is located at the 166th position of the nucleotide sequence shown in SEQ ID NO: 16, where the base of the nucleotide molecule is G or C;

[0022] The rs8167985 is located at the 221st position of the nucleotide sequence shown in SEQ ID NO: 17, where the base of the nucleotide molecule is T or G;

[0023] The rs106335590 is located at the 287th position of the nucleotide sequence shown in SEQ ID NO: 18, where the base of the nucleotide molecule is G or T;

[0024] The rs63814610 is located at the 241st position of the nucleotide sequence shown in SEQ ID NO: 19, where the base of the nucleotide molecule is C or T;

[0025] The rs105824121 is located at the 203rd position of the nucleotide sequence shown in SEQ ID NO: 20, where the base of the nucleotide molecule is A or G;

[0026] The rs65108991 is located at the 146th position of the nucleotide sequence shown in SEQ ID NO: 21, and the base of the nucleotide molecule here is A or T.

[0027] The present invention selects 21 SNP sites to form SNP sites for identifying the BAS inbred rat strain. These 21 SNP sites are located on 21 pairs of chromosomes of rats. By using the above 21 SNP markers to identify the BAS inbred rat strain, the detection results show that all 21 SNP sites of the BAS inbred rat are in the homozygous state, which improves the accuracy of identifying the inbred rat strain.

[0028] In a second aspect, the present invention provides an application of SNP markers in preparing a kit or detection, and the following technical solution is adopted:

[0029] An application of SNP markers in preparing a kit or detection, wherein the kit or detection is used for identifying the inbred rat strain; the SNP markers include 21 SNP sites, namely: rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from the BAS inbred rat.

[0030] By adopting the above solution, a kit for identifying the BAS inbred rat strain can be prepared by using the above SNP markers or the inbred rat strain can be identified by using the above SNP markers.

[0031] In a third aspect, the present invention further provides a primer sequence, and the following technical solution is adopted:

[0032] A primer sequence, which is used for the identification of inbred rat strains; the primer sequence is selected from primer sequence rs64750237, primer sequence rs106888236, primer sequence rs106458396, primer sequence rs106672258, primer sequence rs65921305, primer sequence rs8152541, primer sequence rs106996961, primer sequence rs65970293, primer sequence rs105187832, primer sequence rs13453252, primer sequence rs64102787, primer sequence in homozygous state rs63860341, primer sequence rs107261116, primer sequence rs106114574, primer sequence rs64682441, primer sequence rs65067166, rs8167985, primer sequence rs106335590, primer sequence rs63814610, primer sequence rs105824121, and primer sequence rs65108991; the inbred rat strain is selected from BAS inbred rats.

[0033] Preferably, in primer sequence rs64750237, primer sequence rs64750237-F is 5'-GCCAGCTAGCCAGGATACAC-3', and primer sequence rs64750237-R is 5'-CTGAAGCTGCAGAGTGGGTT-3';

[0034] And / or, in primer sequence rs106888236, primer sequence rs106888236-F is 5'-GCTGGTGGTTGTGTGAAGGT-3', and primer sequence rs106888236-R is 5'-TGGGAGCTAACATGTCCCCA-3';

[0035] And / or, in primer sequence rs106458396, primer sequence rs106458396-F is 5'-GATGGTCCCCAACCAAGAGC-3', and primer sequence rs106458396-R is 5'-TTACTCCACCCTCCCCCTAAG-3';

[0036] And / or, in primer sequence rs106672258, primer sequence rs106672258-F is 5'-GGCTGTGATCGGAACTAGCA-3', and primer sequence rs106672258-R is 5'-ACCTCCAAATAGCGCCACTC-3';

[0037] And / or, in the primer sequence rs65921305, the primer sequence rs65921305-F is 5'-GCTGTGCTTGGCATAATATC-3', and the primer sequence rs65921305-R is 5'-CCAGGTGATTATAGATTGTGTC-3';

[0038] And / or, in the primer sequence rs8152541, the primer sequence rs8152541-F is 5'-CAGGTGGTGATGGTGTAAGC-3', and the primer sequence rs8152541-R is 5'-CCTCATGCAGTAGCGAGTAAC-3';

[0039] And / or, in the primer sequence rs106996961, the primer sequence rs106996961-F is 5'-CTGCTGCCCTGATGAAGAAGT-3', and the primer sequence rs106996961-R is 5'-TTGGGAAGCTAGTCATGGGG-3';

[0040] And / or, in the primer sequence rs65970293, the primer sequence rs65970293-F is 5'-CTCTGGTTGGTCTGGTTGGTT-3', and the primer sequence rs65970293-R is 5'-AGATGCGGATGCCATTAAGTCT-3';

[0041] And / or, in the primer sequence rs105187832, the primer sequence rs105187832-F is 5'-GTAGGTCCACTGAGAGCTGA-3', and the primer sequence rs105187832-R is 5'-GTAATGGAGGCTCCTGTCCC-3';

[0042] And / or, in the primer sequence rs13453252, the primer sequence rs13453252-F is 5'-TGTGCAAGCTCTTCAGTCCT-3', and the primer sequence rs13453252-R is 5'-GCCAAGATGGCCCTGACTTA-3';

[0043] And / or, in the primer sequence rs64102787, the primer sequence rs64102787-F is 5'-GCTGGGCTGTGGTTAGACTT-3', and the primer sequence rs64102787-R is 5'-CCTGTTTGCAGGTTGAAGCC-3';

[0044] And / or, in the primer sequence rs63860341, the primer sequence rs63860341-F is 5'-ACCCAGTCATGCTTCATTTGTG-3', and the primer sequence rs63860341-R is 5'-CAAGGCTGAGGTGTGCAGTA-3';

[0045] And / or, in the primer sequence rs107261116, the primer sequence rs107261116-F is 5'-ACCTAGGTGATGACCTGCTC-3', and the primer sequence rs107261116-R is 5'-TCAGGCCACAAAACAGTGGA-3';

[0046] And / or, in the primer sequence rs106114574, the primer sequence rs106114574-F is 5'-CTCGTCATTTGGCCCCCTAA-3', and the primer sequence rs106114574-R is 5'-AGGCTTTTGCCTGCCTAGTT-3';

[0047] And / or, in the primer sequence rs64682441, the primer sequence rs64682441-F is 5'-TAGCCCTGCCTATGTGCATC-3', and the primer sequence rs64682441-R is 5'-ACAAGGCAGTACATCAAAACCA-3';

[0048] And / or, in the primer sequence rs65067166, the primer sequence rs65067166-F is 5'-CACTGTGGCCTCTCAGATGT-3', and the primer sequence rs65067166-R is 5'-AGTGGGTGCCTCCACTTTTC-3';

[0049] And / or, in the primer sequence rs8167985, the primer sequence rs8167985-F is 5'-ACCCACCTGGCTGCTAGTAT-3', and the primer sequence rs8167985-R is 5'-AGCCATGTGTTCGAGTGTCA-3';

[0050] And / or, in the primer sequence rs106335590, the primer sequence rs106335590-F is 5'-CCCTGTTACCGCTGATGAGA-3', and the primer sequence rs106335590-R is 5'-CATGGGGAGACTATGCCACA-3';

[0051] And / or, in the primer sequence rs63814610, the primer sequence rs63814610-F is 5'-GCTTCAGCTTCCCAGCTACT-3', and the primer sequence rs63814610-R is 5'-GTGTGGGGGCAAAGCCTAAAC-3';

[0052] And / or, in the primer sequence rs105824121, the primer sequence rs105824121-F is 5'-CCACTGGTGGTATGCTGTCT-3', and the primer sequence rs105824121-R is 5'-TAGTTCAGGGGCTGGTGACT-3';

[0053] And / or, in the primer sequence rs65108991, the primer sequence rs65108991-F is 5'-CACATCCCAGAAGCCCTGTT-3', and the primer sequence rs65108991-R is 5'-GTGCATTCTCCCAATTTCCACA-3'.

[0054] The present invention also provides the above 21 pairs of primer sequences, which are respectively used for amplifying the sequences corresponding to the above SNP sites.

[0055] In the fourth aspect, the present invention provides the application of the above primer sequences in the preparation of a kit or detection, and the kit or detection is used for the identification of inbred rat strains.

[0056] In the fifth aspect, the present invention provides the DNA sequences amplified by using the above primer sequences, and the above DNA sequences are selected from SEQ ID NO: 1-SEQ ID NO: 21.

[0057] The DNA sequence amplified by using the primer sequence rs64750237 is as shown in SEQ ID NO: 1.

[0058] The DNA sequence amplified by using the primer sequence rs106888236 is as shown in SEQ ID NO: 2.

[0059] The DNA sequence amplified by using the primer sequence rs106458396 is as shown in SEQ ID NO: 3.

[0060] The DNA sequence amplified by using the primer sequence rs106672258 is as shown in SEQ ID NO: 4.

[0061] The DNA sequence amplified by using the primer sequence rs65921305 is as shown in SEQ ID NO: 5.

[0062] The DNA sequence amplified using the primer sequence rs8152541 is as shown in SEQ ID NO: 6.

[0063] The DNA sequence amplified using the primer sequence rs106996961 is as shown in SEQ ID NO: 7.

[0064] The DNA sequence amplified using the primer sequence rs65970293 is as shown in SEQ ID NO: 8.

[0065] The DNA sequence amplified using the primer sequence rs105187832 is as shown in SEQ ID NO: 9.

[0066] The DNA sequence amplified using the primer sequence rs13453252 is as shown in SEQ ID NO: 10.

[0067] The DNA sequence amplified using the primer sequence rs64102787 is as shown in SEQ ID NO: 11.

[0068] The DNA sequence amplified using the primer sequence rs63860341 is as shown in SEQ ID NO: 12.

[0069] The DNA sequence amplified using the primer sequence rs107261116 is as shown in SEQ ID NO: 13.

[0070] The DNA sequence amplified using the primer sequence rs106114574 is as shown in SEQ ID NO: 14.

[0071] The DNA sequence amplified using the primer sequence rs64682441 is as shown in SEQ ID NO: 15.

[0072] The DNA sequence amplified using the primer sequence rs65067166 is as shown in SEQ ID NO: 16.

[0073] The DNA sequence amplified using the primer sequence rs8167985 is as shown in SEQ ID NO: 17.

[0074] The DNA sequence amplified using the primer sequence rs106335590 is as shown in SEQ ID NO: 18.

[0075] The DNA sequence amplified using the primer sequence rs63814610 is as shown in SEQ ID NO: 19.

[0076] The DNA sequence amplified using the primer sequence rs105824121 is as shown in SEQ ID NO: 20.

[0077] The DNA sequence amplified by using the primer sequence rs6510899 is shown in SEQ ID NO: 21.

[0078] In a sixth aspect, the present invention provides the application of the above DNA sequence in the identification of inbred rat strains.

[0079] (1) Identification of multiple inbred rat strains: Compare the base types of each SNP locus of the rat sample with the base types of each SNP locus of the database rats. If the base types are consistent with the base types of each SNP locus of the rat strain, it can be preliminarily determined that the rat sample comes from the corresponding rat strain.

[0080] (2) Genetic monitoring of inbred rats: Six rat individuals in the population can be selected as rat samples, and the genomes of each rat sample are obtained and the base types of 21 SNP loci are detected. If the base types of 21 SNP loci of all rat samples are consistent with the base types of each SNP locus of the rat, and are all monomorphic / homozygous, it is determined that the population is a qualified inbred rat strain; if the base types of 1 or more SNP loci of a rat individual are inconsistent with the base types of the corresponding SNP locus of the rat strain, or are polymorphic / heterozygous, the population is an unqualified inbred rat strain.

[0081] In summary, the present invention has the following beneficial effects:

[0082] 1. The present invention can identify multiple inbred rat strains by using the above 21 SNP loci.

[0083] 2. The SNP locus combination provided by the present invention is simple to operate and has low technical difficulties.

[0084] 3. The present invention also provides 21 pairs of primer sequences for amplifying the above SNP loci respectively, and the sequence amplified by each pair of primer sequences contains a specific SNP marker.

[0085] 4. The 21 SNP loci screened out by the present invention and the 21 pairs of primer sequences provided can not only be used for the identification of inbred rat strains, but also for the genetic monitoring of inbred rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 It is a sequencing result diagram of 21 SNP loci of BAS inbred rats in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0087] The present invention provides an application of SNP markers in the strain identification of inbred rats. The SNP markers include 21 SNP loci, namely rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from the BAS inbred rat strain.

[0088] rs64750237 is located on chromosome 4. The nucleotide sequence where rs64750237 is located is shown in SEQ ID NO: 1.

[0089] rs64750237 is located at the 255th position of the nucleotide sequence shown in SEQ ID NO: 1, and the base of the nucleotide molecule here is T or C. Among them, the base of the nucleotide molecule at the 357th position in SEQ ID NO: 1 of the BAS inbred rat is T.

[0090] rs106888236 is located on chromosome 16. The nucleotide sequence where rs106888236 is located is shown in SEQ ID NO: 2.

[0091] rs106888236 is located at the 342nd position of the nucleotide sequence shown in SEQ ID NO: 2, and the base of the nucleotide molecule here is C or A. Among them, the base of the nucleotide molecule at the 342nd position in SEQ ID NO: 2 of the BAS inbred rat is A.

[0092] rs106458396 is located on chromosome 15. The nucleotide sequence where rs106458396 is located is shown in SEQ ID NO: 3.

[0093] rs106458396 is located at the 230th position of the nucleotide sequence shown in SEQ ID NO: 3, and the base of the nucleotide molecule here is A or C. Among them, the base of the nucleotide molecule at the 230th position in SEQ ID NO: 3 of the BAS inbred rat is T.

[0094] rs106672258 is located on chromosome 12. The nucleotide sequence where rs106672258 is located is shown in SEQ ID NO: 4.

[0095] rs106672258 is located at position 197 of the nucleotide sequence shown in SEQ ID NO: 4, where the base of the nucleotide molecule is T or C. The base of the nucleotide molecule at position 197 in the SEQ ID NO: of BAS inbred rats is C.

[0096] rs65921305 is located on chromosome 13. The nucleotide sequence where rs65921305 is located is as shown in SEQ ID NO: 5.

[0097] rs65921305 is located at position 306 of the nucleotide sequence shown in SEQ ID NO: 5, where the base of the nucleotide molecule is T or G. The base of the nucleotide molecule at position 306 in the SEQ ID NO: 5 of BAS inbred rats is G.

[0098] rs8152541 is located on chromosome 18. The nucleotide sequence where rs105008665 is located is as shown in SEQ ID NO: 6.

[0099] rs8152541 is located at position 378 of the nucleotide sequence shown in SEQ ID NO: 6, where the base of the nucleotide molecule is G or A. The base of the nucleotide molecule at position 378 in the SEQ ID NO: 6 of BAS inbred rats is A.

[0100] rs106996961 is located on chromosome 2. The nucleotide sequence where rs106996961 is located is as shown in SEQ ID NO: 7.

[0101] rs106996961 is located at position 169 of the nucleotide sequence shown in SEQ ID NO: 7, where the base of the nucleotide molecule is G or T; the base of the nucleotide molecule at position 169 in the SEQ ID NO: 7 of BAS inbred rats is G.

[0102] rs65970293 is located on chromosome 1. The nucleotide sequence where rs65970293 is located is as shown in SEQ ID NO: 8.

[0103] rs65970293 is located at position 424 of the nucleotide sequence shown in SEQ ID NO: 8, where the base of the nucleotide molecule is T or C. The base of the nucleotide molecule at position 424 in the SEQ ID NO: 8 of BAS inbred rats is T.

[0104] rs105187832 is located on chromosome 7. The nucleotide sequence where rs105187832 is located is as shown in SEQ ID NO: 9.

[0105] rs105187832 is located at position 359 of the nucleotide sequence shown in SEQ ID NO: 9, where the base of the nucleotide molecule is C or A. The base of the nucleotide molecule at position 359 in the SEQ ID NO: 9 of BAS inbred rats is C.

[0106] rs13453252 is located on chromosome 20. The nucleotide sequence where rs13453252 is located is as shown in SEQ ID NO: 10.

[0107] rs13453252 is located at position 189 of the nucleotide sequence shown in SEQ ID NO: 10, where the base of the nucleotide molecule is G or C. The base of the nucleotide molecule at position 189 in the SEQ ID NO: 10 of BAS inbred rats is G.

[0108] rs64102787 is located on chromosome 6. The nucleotide sequence where rs64102787 is located is as shown in SEQ ID NO: 211.

[0109] rs64102787 is located at position 292 of the nucleotide sequence shown in SEQ ID NO: 11, where the base of the nucleotide molecule is C or A. The base of the nucleotide molecule at position 292 in the SEQ ID NO: 11 of BAS inbred rats is C.

[0110] rs63860341 is located on chromosome 3. The nucleotide sequence where rs63860341 is located is as shown in SEQ ID NO: 12.

[0111] rs63860341 is located at position 201 of the nucleotide sequence shown in SEQ ID NO: 12, where the base of the nucleotide molecule is C or T. The base of the nucleotide molecule at position 201 in the SEQ ID NO: 12 of BAS inbred rats is T.

[0112] rs107261116 is located on chromosome 3. The nucleotide sequence where rs107261116 is located is as shown in SEQ ID NO: 13.

[0113] rs107261116 is located at position 192 of the nucleotide sequence shown in SEQ ID NO: 13, where the base of the nucleotide molecule is G or C. The base of the nucleotide molecule at position 192 in the SEQ ID NO: 13 of BAS inbred rats is G.

[0114] rs106114574 is located on chromosome 8. The nucleotide sequence where rs106114574 is located is as shown in SEQ ID NO: 14.

[0115] rs106114574 is located at position 174 of the nucleotide sequence shown in SEQ ID NO: 14, where the base of the nucleotide molecule is T or A. The base of the nucleotide molecule at position 174 in SEQ ID NO: 14 of BAS inbred rats is A.

[0116] rs64682441 is located on chromosome 9. The nucleotide sequence where rs64682441 is located is as shown in SEQ ID NO: 15.

[0117] rs64682441 is located at position 321 of the nucleotide sequence shown in SEQ ID NO: 15, where the base of the nucleotide molecule is T or C. The base of the nucleotide molecule at position 321 in SEQ ID NO: 15 of BAS inbred rats is C.

[0118] rs65067166 is located on chromosome 10. The nucleotide sequence where rs65067166 is located is as shown in SEQ ID NO: 16.

[0119] rs65067166 is located at position 166 of the nucleotide sequence shown in SEQ ID NO: 16, where the base of the nucleotide molecule is G or C. The base of the nucleotide molecule at position 166 in SEQ ID NO: 16 of BAS inbred rats is G.

[0120] rs8167985 is located on chromosome 11. The nucleotide sequence where rs8167985 is located is as shown in SEQ ID NO: 17.

[0121] rs8167985 is located at position 221 of the nucleotide sequence shown in SEQ ID NO: 17, where the base of the nucleotide molecule is T or G. The base of the nucleotide molecule at position 221 in SEQ ID NO: 17 of BAS inbred rats is G.

[0122] rs106335590 is located on chromosome 14. The nucleotide sequence where rs106335590 is located is as shown in SEQ ID NO: 18.

[0123] rs106335590 is located at position 287 of the nucleotide sequence shown in SEQ ID NO: 18, where the base of the nucleotide molecule is G or T. The base of the nucleotide molecule at position 287 in SEQ ID NO: 18 of BAS inbred rats is G.

[0124] rs63814610 is located on chromosome 17. The nucleotide sequence where rs63814610 is located is as shown in SEQ ID NO: 19.

[0125] rs63814610 is located at position 241 of the nucleotide sequence shown in SEQ ID NO: 19, where the base of the nucleotide molecule is C or T. The base of the nucleotide molecule at position 241 in SEQ ID NO: 19 of BAS inbred rats is T.

[0126] rs105824121 is located on chromosome 19. The nucleotide sequence where rs105824121 is located is as shown in SEQ ID NO: 20.

[0127] rs105824121 is located at position 203 of the nucleotide sequence shown in SEQ ID NO: 20, where the base of the nucleotide molecule is A or G. The base of the nucleotide molecule at position 203 in SEQ ID NO: 20 of BAS inbred rats is A.

[0128] rs65108991 is located on the X chromosome. The nucleotide sequence where rs65108991 is located is as shown in SEQ ID NO: 21.

[0129] rs65108991 is located at position 146 of the nucleotide sequence shown in SEQ ID NO: 21, where the base of the nucleotide molecule is A or T. The base of the nucleotide molecule at position 146 in SEQ ID NO: 21 of BAS inbred rats is A.

[0130] The present invention also provides an application of an SNP marker in the preparation of a kit or detection, and the above-mentioned kit or detection is used for the identification of inbred rat strains. The SNP marker includes 21 SNP sites, namely rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from BAS inbred rats.

[0131] In addition, the present invention also provides a primer sequence, which is used for the identification of inbred rat strains. The primer sequence is selected from primer sequence rs64750237, primer sequence rs106888236, primer sequence rs106458396, primer sequence rs106672258, primer sequence rs65921305, primer sequence rs8152541, primer sequence rs106996961, primer sequence rs65970293, primer sequence rs105187832, primer sequence rs13453252, primer sequence rs64102787, primer sequence rs63860341, primer sequence rs107261116, primer sequence rs106114574, primer sequence rs64682441, primer sequence rs65067166, rs8167985, primer sequence rs106335590, primer sequence rs63814610, primer sequence rs105824121, and primer sequence rs65108991; the inbred rat strain is selected from BAS inbred rats.

[0132] Furthermore, the present invention also provides the application of the above primer sequence in the preparation of a kit or detection, and the kit or detection is used for the identification of inbred rat strains. Still further, the present invention also provides a DNA sequence obtained by amplifying the above primer sequence and the application of the above DNA sequence in the identification of inbred rat strains. The above DNA sequence is selected from SEQ ID NO: 1 - SEQ ID NO: 21.

[0133] The identification of inbred rat strains specifically includes the following steps:

[0134] 1. Extraction of genomic DNA

[0135] The method for extracting genomic DNA can adopt the phenol - chloroform extraction method or a genomic DNA extraction kit. Among them, the rat sample can be rat cells, rat tissues, or the tip of a rat tail. Among them, when the rat sample is rat cells or rat tissues, the sampling amount can be 0.1 g; when the rat sample is the tip of a rat tail, the sampling amount can be 2 mm.

[0136] Taking the extraction of genomic DNA using a genomic DNA extraction kit as an example, the method for extracting genomic DNA is specifically described below. Among them, the genomic DNA extraction kit can be a Blood / Cells / Tissues Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP304). It specifically includes the following steps:

[0137] (1) Put the rat sample into a 1.5 mL sterile centrifuge tube, add 200 μL of Buffer GA and 10 μL of proteinase K, and place it in a water bath at 56 °C for 12 h for lysis;

[0138] (2) Continuously add 200 μL of Buffer GB, invert and mix well, and place it in a water bath at 70 °C for 10 min;

[0139] (3) Continuously add 200 μL of absolute ethanol, shake and mix well, and centrifuge at 12000 rmp for 10 s to remove the liquid adhering to the wall;

[0140] (4) Transfer all the liquid in the sterile centrifuge tube after the previous step to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0141] (5) Add 500 μL of Buffer GD to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0142] (6) Continuously add 600 μL of Buffer PW to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0143] (7) Repeat step (6);

[0144] (8) Centrifuge the adsorption column at 12000 rmp for 2 min, discard the original 2 mL round-bottom centrifuge tube at the bottom, place the adsorption column in a new sterile EP tube, open the tube cap of the adsorption column, and let it stand at room temperature for 5 min;

[0145] (9) Continuously add 60 μL of Buffer TE to the adsorption column, let it stand at room temperature for 5 min, centrifuge at 12000 rmp for 30 s, and discard the waste liquid to obtain the genomic DNA of the rat sample. Store it at -20 °C for later use.

[0146] 2. Polymerase Chain Reaction (PCR) Amplification

[0147] Using the genomic DNA of the rat sample obtained in step 1 as a template, perform PCR amplification using the primer sequences of each SNP locus in the above-provided SNP markers.

[0148] PCR System: The total reaction volume is 20 μL, including 2×PCR Mix: 10 μL, upstream and downstream primers (100 pmol / μL) each 1 μL, genomic DNA: 1 μL, pure water (ddH2O): 7 μL.

[0149] PCR Reaction Program: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s; annealing temperature 60 °C for 30 s; extension at 72 °C for 45 s; 30 cycles; continue to extend at 72 °C for 7 min; store the amplified product at 4 °C.

[0150] 3. Detection of PCR Amplification Products

[0151] The PCR amplification products were detected by 1% agarose gel electrophoresis and photographed with a gel imaging system. The sample loading volume was 5 μL of PCR product per well, and the electrophoresis conditions were a constant voltage of 121 V for 15 min. After electrophoresis, the results were observed under ultraviolet transillumination.

[0152] 4. Sequencing and result judgment of PCR amplification products

[0153] The PCR amplification products were subjected to Sanger sequencing. The sequencing peak maps of each SNP locus were read and aligned using SnapGene software.

[0154] 5. Identification of inbred rat strains

[0155] The rat strain or genetic monitoring can be identified in at least the following 3 situations:

[0156] (1) Identification of BAS inbred rat strains: The base types of each SNP locus of the rat sample to be tested were compared with the base types of each SNP locus of the database rats. If the base types of 21 SNP loci of the rat sample were consistent with the base types of each SNP locus of this rat strain, it could be preliminarily determined that the rat sample came from the corresponding inbred rat strain.

[0157] (2) Genetic monitoring of inbred rats: Six rat individuals within the population could be selected as the rat sample, and the genomes of the inbred rat strain samples to be tested were obtained and the base types of 21 SNP loci were detected. If the base types of 21 SNP loci of all rat samples were consistent with the base types of each SNP locus of this rat strain and were all monomorphic / homozygous, it was determined that this population was a qualified inbred rat strain; if the base types of 1 or more SNP loci of the rat individuals were inconsistent with the base types of the corresponding SNP loci of this rat strain, or were polymorphic / heterozygous, this population was an unqualified inbred rat strain.

[0158] The present invention will be further described in detail below in combination with Preparation Examples 1-21 and Example 1.

[0159] Preparation Example

[0160] Preparation Examples 1-21

[0161] Preparation Examples 1-21 respectively provided a pair of primer sequences. The difference between each preparation example was that the SNP loci used for amplification were different. Specifically, it was shown in Table 1.

[0162] The design method of the primer sequences specifically included the following steps:

[0163] (1) Search for the nucleotide sequences upstream and downstream of the SNP loci on the Ensembl website (http: / / asia.ensembl.org / index.html), and use the online primer design website Primer blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) to design the primer sequences for PCR amplification of each SNP locus. The design results of the primer sequences are shown in Table 1. Among them, the rs (reference snp) number is a certain mutation that occurs at a certain locus of a gene, and these characteristic mutation loci were encoded in the Human Genome Project, which is the rs number.

[0164] (2) After the primer design is completed, entrust a third-party company to complete the synthesis of the primers.

[0165] Table 1 SNP loci corresponding to Preparation Examples 1-21 and the designed primer sequences

[0166]

[0167]

[0168] Examples

[0169] Example 1

[0170] This example provides a method for identifying an inbred rat strain. The types of 21 SNP loci of the rats identified in this example, which are BAS inbred rats and database rats, are shown in Table 2.

[0171] Table 2 Types of 21 SNP loci of BAS inbred rats

[0172]

[0173]

[0174] The method for identifying an inbred rat strain specifically includes the following steps:

[0175] 1. Use a genomic DNA extraction kit to extract genomic DNA. The genomic DNA extraction kit used is a Blood / Cell / Tissue Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP304). It specifically includes the following steps:

[0176] (1) Put 2 mm of the mouse tail tip into a 1.5 mL sterile centrifuge tube, add 200 μL of Buffer GA and 10 μL of proteinase K, and incubate at 56 °C in a water bath for 12 h for lysis;

[0177] (2) Continuously add 200 μL of Buffer GB, invert and mix well, and place it in a water bath at 70 °C for 10 min;

[0178] (3) Continuously add 200 μL of absolute ethanol, shake and mix well, and centrifuge at 12000 rmp for 10 s to remove the liquid adhering to the wall;

[0179] (4) Transfer all the liquid in the sterile centrifuge tube after the previous step to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0180] (5) Add 500 μL of Buffer GD to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0181] (6) Continuously add 600 μL of Buffer PW to the adsorption column, centrifuge at 12000 rmp for 30 s, and discard the waste liquid;

[0182] (7) Repeat step (6);

[0183] (8) Centrifuge the adsorption column at 12000 rmp for 2 min, discard the original 2 mL round-bottom centrifuge tube at the bottom, place the adsorption column in a new sterile EP tube, open the tube cap of the adsorption column, and let it stand at room temperature for 5 min;

[0184] (9) Continuously add 60 μL of Buffer TE to the adsorption column, let it stand at room temperature for 5 min, centrifuge at 12000 rmp for 30 s, discard the waste liquid, and obtain the genomic DNA of the rat sample. Store it at -20 °C for later use.

[0185] 2. PCR Amplification

[0186] Using the genomic DNA of the rat sample obtained in step 1 as a template, perform PCR amplification using the primer sequences of the 15 SNP markers provided in Preparation Examples 1-15.

[0187] PCR System: The total reaction volume is 12 μL, including 2×PCR Mix: 2 μL, upstream and downstream primers (100 pmol / μL) each 1 μL, genomic DNA: 1 μL, pure water (ddH2O): 7 μL.

[0188] PCR Reaction Program: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s; annealing temperature 60 °C for 30 s; extension at 72 °C for 45 s; 30 cycles; continue extension at 72 °C for 7 min; store the amplification product at 4 °C.

[0189] 3. Detection of PCR Amplification Products - 1% Agarose Gel Electrophoresis

[0190] The PCR amplification products were detected by 1% agarose gel electrophoresis and photographed with a gel imaging system. The sample loading volume was 5 μL of PCR product per well, and the electrophoresis conditions were a constant voltage of 121 V for 15 min. After electrophoresis, the results were observed under ultraviolet transillumination. All the target bands in the electrophoresis results were single and bright, indicating that the amplification products of the SNP sites were obtained.

[0191] 4. Sequencing and result judgment of PCR amplification products

[0192] The PCR amplification products obtained in step 3 were subjected to Sanger sequencing. The sequencing peak maps of the SNP sites were read and aligned with SnapGene software. The obtained identification results are as Figure 1 shown.

Claims

1. Application of 21 SNP markers in strain identification of BAS inbred rats, characterized in that, The SNP markers include 21 SNP sites, namely: rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from the BAS inbred rat strain.

2. Use of the SNP marker according to claim 1 in the strain identification of BAS inbred rats, characterized in that : The rs64750237 is located at the 255th position of the nucleotide sequence shown in SEQ ID NO: 1, and the base of the nucleotide molecule here is T or C; The rs106888236 is located at the 342nd position of the nucleotide sequence shown in SEQ ID NO: 2, and the base of the nucleotide molecule here is C or A; The rs106458396 is located at the 230th position of the nucleotide sequence shown in SEQ ID NO: 3, and the base of the nucleotide molecule here is A or C; The rs106672258 is located at the 197th position of the nucleotide sequence shown in SEQ ID NO: 4, and the base of the nucleotide molecule here is T or C; The rs65921305 is located at the 306th position of the nucleotide sequence shown in SEQ ID NO: 5, and the base of the nucleotide molecule here is T or G; The rs8152541 is located at the 378th position of the nucleotide sequence shown in SEQ ID NO: 6, and the base of the nucleotide molecule here is G or A; The rs106996961 is located at the 169th position of the nucleotide sequence shown in SEQ ID NO: 7, and the base of the nucleotide molecule here is G or T; The rs65970293 is located at the 424th position of the nucleotide sequence shown in SEQ ID NO: 8, and the base of the nucleotide molecule here is T or C; The rs105187832 is located at the 359th position of the nucleotide sequence shown in SEQ ID NO: 9, and the base of the nucleotide molecule here is C or A; The rs13453252 is located at the 189th position of the nucleotide sequence shown in SEQ ID NO: 10, and the base of the nucleotide molecule here is G or C; The rs64102787 is located at the 292nd position of the nucleotide sequence shown in SEQ ID NO: 11, and the base of the nucleotide molecule here is C or A; The rs63860341 is located at the 201st position of the nucleotide sequence shown in SEQ ID NO: 12, and the base of the nucleotide molecule here is C or T; The rs107261116 is located at the 192nd position of the nucleotide sequence shown in SEQ ID NO: 13, and the base of the nucleotide molecule here is G or C; The rs106114574 is located at the 174th position of the nucleotide sequence shown in SEQ ID NO: 14, and the base of the nucleotide molecule here is T or A; The rs64682441 is located at the 321st position of the nucleotide sequence shown in SEQ ID NO: 15, and the base of the nucleotide molecule here is T or C; The rs65067166 is located at the 166th position of the nucleotide sequence shown in SEQ ID NO: 16, and the base of the nucleotide molecule here is G or C; The rs8167985 is located at the 221st position of the nucleotide sequence shown in SEQ ID NO: 17, and the base of the nucleotide molecule here is T or G; The rs106335590 is located at the 287th position of the nucleotide sequence shown in SEQ ID NO: 18, and the base of the nucleotide molecule here is G or T; The rs63814610 is located at the 241st position of the nucleotide sequence shown in SEQ ID NO: 19, and the base of the nucleotide molecule here is C or T; The rs105824121 is located at the 203rd position of the nucleotide sequence shown in SEQ ID NO: 20, and the base of the nucleotide molecule here is A or G; The rs65108991 is located at the 146th position of the nucleotide sequence shown in SEQ ID NO: 21, and the base of the nucleotide molecule here is A or T.

3. Use of an SNP marker in preparing a kit or detection, characterized in that, The kit or detection is used for the identification of inbred large strains; the SNP markers include 21 SNP sites, namely: rs64750237, rs106888236, rs106458396, rs106672258, rs65921305, rs8152541, rs106996961, rs65970293, rs105187832, rs13453252, rs64102787, rs63860341, rs107261116, rs106114574, rs64682441, rs65067166, rs8167985, rs106335590, rs63814610, rs105824121 and rs65108991; the inbred rat strain is selected from the BAS inbred rat.

4. A primer sequence for amplifying the SNP marker described in any one of claims 1-2, characterized in that, The primer sequences are used for the strain identification of inbred rats; the primer sequences are selected from primer sequence rs64750237, primer sequence rs106888236, primer sequence rs106458396, primer sequence rs106672258, primer sequence rs65921305, primer sequence rs8152541, primer sequence rs106996961, primer sequence rs65970293, primer sequence rs105187832, primer sequence rs13453252, primer sequence rs64102787, primer sequence rs63860341, primer sequence rs107261116, primer sequence rs106114574, primer sequence rs64682441, primer sequence rs65067166, rs8167985, primer sequence rs106335590, primer sequence rs63814610, primer sequence rs105824121, and primer sequence rs65108991; the inbred rat strains are selected from BAS inbred rats.

5. The primer sequence according to claim 4, characterized in that : In the primer sequence rs64750237, primer sequence rs64750237-F is 5'-GCCAGCTAGCCAGGA TACAC-3', and primer sequence rs64750237-R is 5'-CTGAAGCTGCAGAGTGGGTT-3'; And / or, in the primer sequence rs106888236, primer sequence rs106888236-F is 5'-GCTGGTGGTTGTGTGAAGGT-3', and primer sequence rs106888236-R is 5'-TGGGAGCTAACATGTCCCCA-3'; And / or, in the primer sequence rs106458396, primer sequence rs106458396-F is 5'-GATGGTCCCCAACCAAGAGC-3', and primer sequence rs106458396-R is 5'-TTACTCCACCCTCCCCCTAAG-3'; And / or, in the primer sequence rs106672258, primer sequence rs106672258-F is 5'-GGCTGTGATCGGAACTAGCA-3', and primer sequence rs106672258-R is 5'-ACCTCCAAATAGCGCCACTC-3'; And / or, in the primer sequence rs65921305, primer sequence rs65921305-F is 5'-GCTGTGCTTGGCATAATATC-3', and primer sequence rs65921305-R is 5'-CCAGGTGATTATAGATTGTGTC-3'; And / or, in the primer sequence rs8152541, the primer sequence rs8152541-F is 5'-CAGGTGGTGATGGTGTAAGC-3', and the primer sequence rs8152541-R is 5'-CCTCATGCAGTAGCGAGTAAC-3'; And / or, in the primer sequence rs106996961, the primer sequence rs106996961-F is 5'-CTGCTGCCCTGATGAAGAAGT-3', and the primer sequence rs106996961-R is 5'-TTGGGAAGCTAGTCATGGGG-3'; And / or, in the primer sequence rs65970293, the primer sequence rs65970293-F is 5'-CTCTGGTTGGTCTGGTTGGTT-3', and the primer sequence rs65970293-R is 5'-AGATGCGGATGCCATTAAGTCT-3'; And / or, in the primer sequence rs105187832, the primer sequence rs105187832-F is 5'-GTAGGTCCACTGAGAGCTGA-3', and the primer sequence rs105187832-R is 5'-GTAATGGAGGCTCCTGTCCC-3'; And / or, in the primer sequence rs13453252, the primer sequence rs13453252-F is 5'-TGTGCAAGCTCTTCAGTCCT-3', and the primer sequence rs13453252-R is 5'-GCCAAGATGGCCCTGACTTA-3'; And / or, in the primer sequence rs64102787, the primer sequence rs64102787-F is 5'-GCTGGGCTGTGGTTAGACTT-3', and the primer sequence rs64102787-R is 5'-CCTGTTTGCAGGTTGAAGCC-3'; And / or, in the primer sequence rs63860341, the primer sequence rs63860341-F is 5'-ACCCAGTCATGCTTCATTTGTG-3', and the primer sequence rs63860341-R is 5'-CAAGGCTGAGGTGTGCAGTA-3'; And / or, in the primer sequence rs107261116, the primer sequence rs107261116-F is 5'-ACCTAGGTGATGACCTGCTC-3', and the primer sequence rs107261116-R is 5'-TCAGGCCACAAAACAGTGGA-3'; And / or, in the primer sequence rs106114574, the primer sequence rs106114574-F is 5'-CTCGTCATTTGGCCCCCTAA-3', and the primer sequence rs106114574-R is 5'-AGGCTTTTGCCTGCCTAGTT-3'; And / or, in the primer sequence rs64682441, the primer sequence rs64682441-F is 5'-TAGCCCTGCCTATGTGCATC-3', and the primer sequence rs64682441-R is 5'-ACAAGGCAGTACATCAAAACCA-3'; And / or, in the primer sequence rs65067166, the primer sequence rs65067166-F is 5'-CACTGTGGCCTCTCAGATGT-3', and the primer sequence rs65067166-R is 5'-AGTGGGTGCCTCCACTTTTC-3'; And / or, in the primer sequence rs8167985, the primer sequence rs8167985-F is 5'-ACCCACCTGGCTGCTAGTAT-3', and the primer sequence rs8167985-R is 5'-AGCCATGTGTTCGAGTGTCA-3'; And / or, in the primer sequence rs106335590, the primer sequence rs106335590-F is 5'-CCCTGTTACCGCTGATGAGA-3', and the primer sequence rs106335590-R is 5'-CATGGGGAGACTATGCCACA-3'; And / or, in the primer sequence rs63814610, the primer sequence rs63814610-F is 5'-GCTTCAGCTTCCCAGCTACT-3', and the primer sequence rs63814610-R is 5'-GTGTGGGGGCAAAGCCTAAAC-3'; And / or, in the primer sequence rs105824121, the primer sequence rs105824121-F is 5'-CCACTGGTGGTATGCTGTCT-3', and the primer sequence rs105824121-R is 5'-TAGTTCAGGGGCTGGTGACT-3'; And / or, in the primer sequence rs65108991, the primer sequence rs65108991-F is 5'-CACATCCCAGAAGCCCTGTT-3', and the primer sequence rs65108991-R is 5'-GTGCATTCTCCCAATTTCCACA-3'.

6. Use of the primer sequence according to any one of claims 4-5 in the preparation of a kit or detection, characterized in that, The kit or detection is used for the identification of inbred rat strains.

7. A DNA sequence amplified using the primer sequence according to any one of claims 4-5, characterized in that, The DNA sequence is selected from SEQ ID NO: 1 - SEQ ID NO:

21.

8. The application of the DNA sequence according to claim 7 in the identification of inbred rat strains.

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