Method for identifying tan sheep and non-tan sheep based on genotyping

Abstract

The application discloses a method for identifying Tan sheep and non-Tan sheep based on genotyping. The method comprises the following steps: detecting nucleotides at each SNP site in a specific SNP site combination in the genome of a to-be-detected sheep, and determining the genotype of the to-be-detected sheep corresponding to the specific SNP site combination; and then determining whether the to-be-detected sheep is a Tan sheep according to the following: if the genotype of the to-be-detected sheep corresponding to the specific SNP site combination meets a standard genotype of Tan sheep, the to-be-detected sheep is or is a candidate for a Tan sheep; and if the genotype of the to-be-detected sheep corresponding to the specific SNP site combination meets a standard genotype of non-Tan sheep, the to-be-detected sheep is or is a candidate for a non-Tan sheep. The application successfully obtains a SNP site combination for identifying Tan sheep and non-Tan sheep, and provides support for the identification, breeding and genetic breeding of Tan sheep in the future.

Description

[0001] This application is a divisional application of application No. 201810409670.7, with the application date of May 2, 2018, and the invention name of “a method for identifying Tan sheep and non-Tan sheep based on genotyping” TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to a method for identifying Tan sheep and non-Tan sheep based on genotyping. BACKGROUND

[0003] Tan sheep is an important livestock resource in Ningxia, with an annual output of 6 million, and is highly respected by the market due to its unique flavor and meat quality, and is an important advantage brand in Ningxia. However, the recent appearance of hybrid offspring of Tan sheep and other sheep breeds has caused great damage to the Tan sheep brand. In order to stabilize the quality of Tan sheep and restore the brand image of Tan sheep, we will make Tan sheep a nationally famous brand. We identify Tan sheep and non-Tan sheep to lay a foundation for the identification and protection of Tan sheep breeds and genetic breeding in the future.

[0004] In recent years, with the development of molecular biology, gene detection technology has made great progress in biological species identification. Gene detection technology is widely used in animal and plant species identification because it can identify almost all biological materials. Gene detection technology for identifying animals and plants is the fastest developing and most accurate technology in species identification.

[0005] Whole genome resequencing is the sequencing of the whole genome of an individual whose genome sequence is known. With the decrease in sequencing cost and the increase in known genome sequences of species, resequencing has become one of the most effective methods in animal and plant breeding research. Sequenom SNP genotyping technology combines sensitive PCR amplification and reliable MALDI-TOF mass spectrometry through primer extension reaction to realize genotyping detection. This technology is particularly suitable for verifying the results of whole genome research. SUMMARY

[0006] The purpose of the present application is to provide a method for identifying Tan sheep and non-Tan sheep based on genotyping.

[0007] In the first aspect, the present application claims a method for identifying Tan sheep and non-Tan sheep based on genotyping.

[0008] The method for identifying Tan sheep and non-Tan sheep based on genotyping provided by the present application can be any of the following:

[0009] The first method is based on 11 SNP site combinations. The method can specifically include the following steps: detecting the nucleotides at each SNP site in the 11 SNP site combinations in the genome of the sheep to be tested, determining the 11 genotypes of the sheep to be tested corresponding to the 11 SNP site combinations; then determining whether the sheep to be tested is a Tan sheep according to the following: if the 11 genotypes of the sheep to be tested corresponding to the 11 SNP site combinations all meet the standard genotypes of Tan sheep of the corresponding SNP site combinations, the sheep to be tested is or is a candidate for Tan sheep; if the 11 genotypes of the sheep to be tested corresponding to the 11 SNP site combinations all meet the standard genotypes of non-Tan sheep of the corresponding SNP site combinations, the sheep to be tested is or is a candidate for non-Tan sheep.

[0010] The 11 SNP site combinations are (a1)-(a11); the standard genotypes of non-Tan sheep and Tan sheep corresponding to the 11 SNP site combinations shown in (a1)-(a11) are (b1)-(b11) respectively. That is, the standard genotypes of non-Tan sheep and Tan sheep corresponding to the SNP site combination shown in (a1) are (b1); the standard genotypes of non-Tan sheep and Tan sheep corresponding to the SNP site combination shown in (a2) are (b2); and so on, the standard genotypes of non-Tan sheep and Tan sheep corresponding to the SNP site combination shown in (a11) are (b11).

[0011] (a1) contains 5 SNP sites, CM001598.2:69009397, CM001602.2:49927473, CM001603.2:38707088, CM001603.2:46619514 and CM001606.2:34435705;

[0012] (a2) contains 5 SNP sites, CM001582.2:86006851, CM001598.2:69009397, CM001600.2:45516740, CM001601.2:47737780 and CM001603.2:46619514;

[0013] (a3) contains 5 SNP sites, CM001582.2:86006851, CM001583.2:42344562, CM001584.2:11650210, CM001584.2:12200323 and CM001588.2:9423474;

[0014] (a4) contains 5 SNP sites CM001592.2:57058734, CM001595.2:48968164, CM001598.2:69009397, CM001601.2:47737780 and CM001603.2:38707088, respectively;

[0015] (a5) contains 5 SNP sites CM001585.2:30868868, CM001589.2:75561002, CM001590.2:56924859, CM001598.2:69009397 and CM001606.2:34435705, respectively;

[0016] (a6) contains 5 SNP sites CM001585.2:30868868, CM001588.2:84941340, CM001598.2:45312379, CM001598.2:69009397 and CM001603.2:46619514, respectively;

[0017] (a7) contains 5 SNP sites CM001585.2:30868868, CM001588.2:84941340, CM001594.2:45477903, CM001594.2:80961940 and CM001603.2:38707088, respectively;

[0018] (a8) contains 5 SNP sites CM001585.2:30868868, CM001588.2:84941340, CM001593.2:1397868, CM001595.2:14087769 and CM001602.2:49927473, respectively;

[0019] (a9) contains 5 SNP sites CM001585.2:30868868, CM001588.2:84941340, CM001592.2:34421625, CM001592.2:53664048 and CM001595.2:48968164, respectively;

[0020] (a10) contains 5 SNP sites CM001584.2:79082958, CM001584.2:157466635, CM001601.2:47737780, CM001602.2:49927473 and CM001603.2:38707088, respectively;

[0021] (a11) contains 5 SNP sites, CM001584.2:79082958, CM001585.2:5250467, CM001588.2:84941340, CM001595.2:43390933 and CM001595.2:48968164 respectively;

[0022] The physical position of each SNP site in the 11 groups of SNP site combinations is determined based on the alignment of the whole genome standard sequence of Tan sheep, the version number of which is Ovis. Aries V4.0; a total of 29 SNP sites, as follows:

[0023] CM001582.2:86006851 is located at position 86006751 on chromosome 1, and its deoxynucleotide is G or T;

[0024] CM001583.2:42344562 is located at position 42344462 on chromosome 2, and its deoxynucleotide is T or C;

[0025] CM001584.2:11650210 is located at position 11650110 on chromosome 3, and its deoxynucleotide is T or C;

[0026] CM001584.2:12200323 is located at position 12200223 on chromosome 3, and its deoxynucleotide is C or G;

[0027] CM001584.2:79082958 is located at position 79082958 on chromosome 3, and its deoxynucleotide is A or G;

[0028] CM001584.2:157466635 is located at position 157466635 on chromosome 3, and its deoxynucleotide is C or T;

[0029] CM001585.2:5250467 is located at position 5250467 on chromosome 4, and its deoxynucleotide is C or G;

[0030] CM001585.2:30868868 is located at position 30868868 on chromosome 4, and its deoxynucleotide is T or C;

[0031] CM001588.2:9423474 is located at position 9423374 on chromosome 7, and its deoxynucleotide is A or C;

[0032] CM001588.2:84941340 is located at position 84941340 on chromosome 7, and its deoxynucleotide is A or C;

[0033] CM001589.2:75561002 is located at position 75561002 on chromosome 8, and the deoxynucleotide is C or G;

[0034] CM001590.2:56924859 is located at position 56924859 on chromosome 9, and the deoxynucleotide is A or G;

[0035] CM001592.2:34421625 is located at position 34421625 on chromosome 11, and the deoxynucleotide is T or C;

[0036] CM001592.2:53664048 is located at position 53664048 on chromosome 11, and the deoxynucleotide is A or G;

[0037] CM001592.2:57058734 is located at position 57058734 on chromosome 11, and the deoxynucleotide is C or G;

[0038] CM001593.2:1397868 is located at position 1397868 on chromosome 12, and the deoxynucleotide is A or G;

[0039] CM001594.2:45477903 is located at position 45477903 on chromosome 13, and the deoxynucleotide is A or G;

[0040] CM001594.2:80961940 is located at position 80961940 on chromosome 13, and the deoxynucleotide is A or C;

[0041] CM001595.2:14087769 is located at position 14087769 on chromosome 14, and the deoxynucleotide is A or T;

[0042] CM001595.2:43390933 is located at position 43390933 on chromosome 14, and the deoxynucleotide is A or G;

[0043] CM001595.2:48968164 is located at position 48968164 on chromosome 14, and the deoxynucleotide is T or G;

[0044] CM001598.2:45312379 is located at position 45312379 on chromosome 17, and the deoxynucleotide is T or C;

[0045] CM001598.2:69009397 is located at position 69009397 on chromosome 17, and the deoxynucleotide is A or C;

[0046] CM001600.2: 45516740 located at 45516740 on chromosome 19, its deoxynucleotide is A or C;

[0047] CM001601.2: 47737780 located at 47737780 on chromosome 20, its deoxynucleotide is A or G;

[0048] CM001602.2: 49927473 located at 49927473 on chromosome 21, its deoxynucleotide is T or C;

[0049] CM001603.2: 38707088 located at 38707088 on chromosome 22, its deoxynucleotide is T or C;

[0050] CM001603.2: 46619514 located at 46619514 on chromosome 22, its deoxynucleotide is A or G;

[0051] CM001606.2: 34435705 located at 34435705 on chromosome 25, its deoxynucleotide is C or G;

[0052] Of course, the version number of the said Tan sheep whole genome standard sequence adopted in the actual operation process is not limited to Ovis. Aries V4.0. No matter which version number of the said Tan sheep whole genome standard sequence is adopted, as long as the SNP site involved is the same as the above-mentioned 29 SNP sites of the present application, it falls within the protection scope of the present application.

[0053] (b1) Non-Tan sheep standard genotype is any one of the following: CC_TT_CC_AA_CG; CC_TC_CC_AA_GG; CC_TT_CC_AA_GG; CC_TC_TC_AA_GG; AC_TT_CC_AA_GG; AC_TT_CC_AA_CG; CC_CC_CC_AA_CG; AC_CC_TC_AA_GG; AC_CC_CC_AA_CG; CC_CC_CC_AA_GG; CC_TC_TC_AA_CG; CC_TT_TC_AA_GG; CC_TC_CC_AA_CG; CC_TT_TC_AA_CG; Tan sheep standard genotype is any one of the following: CC_CC_TT_AA_CG; AA_CC_CC_AA_CG; CC_TC_TT_AA_CG; CC_CC_CC_GA_CC; AC_TC_TC_AA_CG; AC_CC_TC_AA_CG; CC_CC_TC_AA_CC; AC_TC_CC_AA_GG; AA_CC_TC_GA_CG; CC_CC_CC_GG_CG; AA_CC_CC_GG_CC; AC_CC_TC_GA_GG; AA_CC_TC_AA_CC; CC_CC_TT_GA_GG; AC_CC_CC_AA_CC; AC_CC_TC_GA_CC.

[0054] (b2) Non-Tan sheep standard genotype is any one of the following: TT_CC_CC_GG_AA; TT_AC_CC_GG_AA; TT_CC_CC_AG_AA; TT_AC_AC_AG_AA; TT_CC_AC_AG_AA; TT_CC_AC_GG_AA; TT_CC_CC_AA_AA; Tan sheep standard genotype is any one of the following: TT_AA_AC_AG_GA; TT_CC_AA_AG_AA; GT_AC_CC_AG_AA; TT_AC_AC_GG_AA; GG_AC_AC_GG_GA; TT_AC_CC_AG_AA; GT_AC_AC_AG_AA; GG_CC_CC_AG_GG; GG_AC_AC_AA_GA; TT_CC_AC_GG_GA; GT_AA_AC_AG_AA; GG_CC_AA_AA_GA; GT_AC_AC_AG_GA; TT_AC_AC_AA_GA; TT_CC_AC_AG_GA.

[0055] (b3) non-Tan sheep standard genotype is as follows any one of: TT_TC_TT_CG_AC; TT_TT_TT_GG_AC; TT_TC_TT_GG_CC; TT_CC_CT_GG_CC; TT_TT_CT_GG_CC; TT_TT_TT_GG_CC; TT_CC_TT_GG_CC; TT_TT_TT_CG_CC; TT_TC_CT_GG_CC; TT_TC_TT_CG_CC; TT_TC_TT_GG_AC; GT_TT_TT_GG_CC; Tan sheep standard genotype is as follows any one of: TT_CC_CT_CG_CC; TT_TC_CC_GG_CC; TT_TT_CT_CC_CC; GT_CC_CT_CG_AC; GG_CC_TT_GG_AC; GT_CC_TT_GG_CC; TT_CC_TT_CG_AC; GG_TT_CC_CC_AA; TT_CC_CT_GG_AC; TT_CC_CC_CG_AA; GT_CC_CT_CG_CC.

[0056] (b4) the non-Tan sheep standard genotype is any one of: CC_GT_CC_GG_CC; GC_TT_CC_GG_CC; GG_TT_CC_AG_TC; GG_GT_AC_GG_CC; GC_GT_CC_GG_CC; GC_TT_CC_AG_TC; CC_GT_AC_AG_CC; GC_TT_CC_GG_TC; GG_TT_AC_GG_TC; GG_TT_CC_GG_CC; CC_TT_CC_AG_CC; CC_TT_AC_GG_CC; GC_TT_CC_AG_CC; CC_TT_CC_GG_CC; CC_TT_CC_GG_TC; GC_GT_CC_AA_TC; GG_TT_CC_AG_CC; GG_GT_CC_GG_CC; GC_GT_CC_GG_TC; the Tan sheep standard genotype is any one of: CC_GT_AA_AG_TC; GC_GT_AC_GG_CC; GC_GT_AC_AG_TC; CC_GT_AA_AA_CC; GC_GG_AA_AG_TC; CC_GT_AC_AG_TT; CC_GG_CC_AA_CC; GC_GG_AC_GG_TC; CC_TT_AC_GG_TT; CC_GT_AC_AG_TC; CC_GG_CC_AG_TC; CC_GG_AC_AG_TC; CC_TT_CC_AG_TT; CC_GT_CC_AG_CC; CC_GG_AA_AG_CC; CC_TT_AC_AA_TC; CC_GG_AA_AG_TC; CC_GT_CC_AA_TT; CC_GG_AC_AG_TT; CC_GT_AC_AA_CC; CC_GT_AC_AA_TC; CC_TT_AC_AG_CC.

[0057] (b5) the non-Tan sheep standard genotype is any one of the following: CC_GG_AA_CC_CG; CC_GG_GA_AC_GG; CC_GG_GA_CC_GG; CC_CC_GA_CC_CG; CC_CC_AA_CC_GG; TC_GG_GG_AC_GG; CC_CG_GG_CC_CG; CC_CG_GA_CC_GG; TC_CG_GA_AC_CG; CC_CG_GA_CC_CG; TC_CG_GA_AC_GG; CC_CG_GG_CC_GG; TC_CG_AA_CC_GG; CC_CG_AA_CC_GG; CC_GG_GG_CC_GG; CC_GG_GA_AC_CG; TC_GG_GG_CC_GG; TC_GG_GG_CC_CG; TC_GG_GA_CC_CG; TC_CG_GG_CC_CG; TC_GG_GA_CC_GG; the Tan sheep standard genotype is any one of the following: TC_CG_GA_AC_CC; TC_CC_GA_CC_CG; TT_CG_AA_AA_CG; CC_CC_AA_CC_CC; TT_CG_GA_AC_CG; TC_CC_GA_AC_CG; TT_CC_AA_CC_CC; TC_CG_AA_CC_CG; TC_CG_GA_CC_CG; TC_GG_GA_AC_GG; CC_CC_AA_CC_CG; TT_CG_GA_AA_CC; CC_CC_AA_AA_GG; TT_CC_GA_AC_CG; CC_CC_GA_AC_CC.

[0058] (b6) Non-Tan sheep standard genotype is any one of the following: CC_CA_CC_CC_AA; TC_AA_TC_CC_AA; CC_AA_CC_AC_AA; CC_CA_TC_CC_AA; CC_CA_TT_CC_AA; TC_CA_CC_AC_AA; CC_AA_CC_CC_AA; CC_AA_TC_CC_AA; TC_AA_CC_CC_AA; TC_CC_TC_CC_AA; CC_AA_TT_CC_AA; CC_CC_TC_CC_AA; TC_CA_CC_CC_AA; Tan sheep standard genotype is any one of the following: TC_CA_TC_AA_GA; TC_AA_TT_CC_AA; TT_CC_TC_AA_AA; TT_AA_TT_AC_GA; TC_CA_TC_CC_AA; TC_AA_TC_AC_GA; TC_CC_TC_AC_AA; TC_AA_TT_AC_AA; TT_CC_CC_CC_AA; TC_AA_TC_AC_AA; TC_CA_TT_AC_GA; CC_CA_TT_CC_GA; TT_CC_TT_AA_AA; TC_CC_TC_CC_GA; TT_CC_TC_AC_GA; CC_CA_TT_AC_AA.

[0059] (b7) Non-Tan sheep standard genotype is any one of the following: CC_CA_GG_AA_CC; TC_AA_GG_CA_CC; CC_CA_GG_AA_TC; TC_CA_GG_AA_CC; CC_AA_GG_CA_CC; TC_CA_GG_CA_CC; TC_AA_GG_CA_TC; CC_AA_GG_CA_TC; CC_CA_GG_CC_CC; CC_AA_GG_AA_CC; CC_CA_AG_AA_TC; TC_CC_GG_CA_CC; TC_AA_GG_AA_TC; TC_CA_AG_AA_CC; CC_CC_GG_AA_CC; TC_AA_AG_AA_CC; Tan sheep standard genotype is any one of the following: TC_CA_AG_AA_TC; TC_AA_AG_AA_TC; TT_AA_GG_AA_TC; TT_CC_AG_AA_CC; TT_CC_GG_AA_TC; TT_AA_AG_AA_TC; TC_CA_AA_AA_TT; TC_CA_AA_AA_CC; TC_CC_AG_AA_TC; TC_CA_GG_AA_TC; TC_CA_GG_AA_TT; TT_CC_AG_AA_TC; TC_CC_AG_AA_CC; CC_CA_AG_AA_CC.

[0060] (b8) the non-Tan sheep standard genotype is any one of the following: CC_CA_GG_AA_TT; CC_AA_GG_AA_TC; CC_CA_AG_AA_TT; CC_CA_GG_TT_TC; CC_CA_AG_AA_TC; TC_CA_AG_TA_TT; CC_AA_AG_TT_CC; CC_AA_AG_TA_TC; CC_AA_AA_TA_CC; TC_AA_AG_TT_CC; CC_AA_GG_TT_TC; TC_AA_GG_AA_TT; CC_AA_GG_TT_CC; CC_AA_AG_TA_CC; TC_AA_AG_TT_TC; CC_CA_AG_TT_CC; CC_AA_AG_TT_TC; CC_AA_AG_AA_CC; CC_AA_GG_AA_CC; CC_CA_GG_TA_TC; TC_CC_GG_AA_TT; TC_AA_GG_TA_CC; TC_AA_AG_AA_TC; CC_AA_GG_TT_TT; CC_AA_GG_TA_TT; CC_AA_AA_TA_TC; CC_CA_GG_TT_TT; CC_CC_GG_TA_CC; TC_AA_GG_TA_TC; TC_CA_GG_TT_TC; CC_AA_GG_TA_TC; TC_AA_AG_AA_TT; the Tan sheep standard genotype is any one of the following: TC_CA_AA_AA_TC; TC_CA_AG_AA_CC; TC_AA_AG_TA_CC; TT_CC_AA_AA_TC; TT_AA_AA_AA_CC; TC_AA_AG_TA_TC; TC_CC_AG_AA_CC; TC_CA_GG_AA_CC; TC_CA_AG_AA_TC; TC_AA_AG_AA_CC; TT_CC_GG_AA_CC; TC_CC_AA_AA_CC; CC_CA_AA_AA_TC; TT_CC_AA_AA_CC; TT_AA_GG_AA_CC; CC_CA_GG_AA_CC.

[0061] (b9) the non-Tan sheep standard genotype is any one of: CC_CA_TT_GG_GT; TC_AA_TT_GG_GT; CC_AA_TT_AG_GT; TC_CA_TT_GG_GT; CC_AA_TT_GG_GT; CC_AA_CT_GG_TT; CC_AA_TT_GG_TT; TC_AA_TT_AG_GT; TC_AA_TT_GG_TT; CC_CA_TT_GG_TT; CC_CA_TT_AG_TT; TC_CC_TT_GG_TT; CC_CC_TT_AG_GT; TC_CA_TT_AG_TT; the Tan sheep standard genotype is any one of: TC_CA_TT_AG_GT; TT_AA_CT_AG_GT; TT_CC_TT_AG_GG; TC_AA_TT_AG_TT; TC_AA_TT_GG_GG; TT_CC_CT_AG_GG; TC_CA_TT_GG_TT; TC_CA_CT_GG_GT; CC_CA_TT_AG_GG; TT_CC_CT_AA_GG; TC_CC_CT_AG_GG; TC_CC_CT_AG_GT; TT_AA_TT_AA_TT; TT_CC_TT_AA_GT; CC_CA_CT_AG_TT.

[0062] (b10) the non-Tan sheep standard genotype is any one of the following: AA_CC_GG_TT_CC; AA_CT_GG_TT_CC; GA_CT_GG_TT_CC; GA_CC_AG_TC_TC; GA_CC_AG_CC_CC; GA_CT_GG_TC_TC; GA_CC_GG_TC_CC; AA_CT_GG_CC_CC; GA_CT_AG_CC_CC; AA_CT_AG_CC_CC; GA_TT_GG_CC_CC; AA_CC_AG_CC_CC; AA_CT_AG_TC_TC; AA_CT_GG_TC_TC; GG_TT_GG_TT_CC; GA_TT_AA_TT_TC; GG_CC_AG_TC_CC; GA_CC_AG_TT_CC; GA_CT_AG_TC_CC; AA_CC_GG_CC_CC; GA_TT_GG_TC_CC; AA_CC_GG_TC_CC; GA_CT_GG_TC_CC; AA_CC_GG_TT_TC; the Tan sheep standard genotype is any one of the following: GA_CT_AG_TC_TC; AA_TT_GG_CC_CC; GA_CT_AG_CC_TT; GG_TT_AG_TC_TT; GG_TT_GG_TC_TT; GG_CT_GG_TC_CC; GG_CT_AA_CC_CC; GG_CT_GG_TC_TC; GG_CT_GG_TC_TT; AA_TT_AG_CC_TC; GA_CT_GG_CC_TC; GA_TT_AG_CC_TC; AA_CT_AA_CC_TC; AA_CT_AG_TC_CC; GG_TT_AG_CC_TC; GG_TT_AG_CC_CC; GG_CT_AG_CC_CC; GG_CT_AA_CC_TC; GG_TT_GG_TC_TC; GG_TT_AA_CC_CC; GA_TT_AA_CC_TC; GA_TT_AG_CC_CC.

[0063] (b11) the non-Tan sheep standard genotype is any one of: AA_GC_CA_AG_GT; GA_GC_AA_AG_GT; GG_GC_AA_GG_GT; AA_CC_CA_AG_TT; GA_GC_CA_AG_TT; AA_CC_CA_GG_GT; GA_GC_AA_GG_GT; GA_CC_AA_AA_GT; GA_CC_AA_AG_TT; AA_GG_AA_AG_TT; GA_CC_AA_GG_TT; AA_GG_AA_GG_TT; AA_CC_AA_GG_TT; GA_GC_AA_GG_TT; AA_GC_AA_GG_TT; AA_GC_CA_AG_TT; AA_CC_AA_AG_TT; GA_GC_AA_AG_TT; AA_GC_AA_AG_TT; AA_CC_CA_GG_TT; GA_CC_CC_AG_TT; AA_CC_AA_AA_TT; GG_CC_AA_GG_TT; GA_CC_CA_AG_GT; AA_CC_CC_AG_GT; GA_CC_AA_AA_TT; AA_CC_AA_GG_GT; and the Tan sheep standard genotype is any one of: AA_GC_AA_GG_GT; AA_GC_CC_AG_GT; GA_GC_CA_AA_GT; GA_GG_CA_AG_TT; GA_GG_AA_AG_GG; AA_GG_CC_AG_GG; GG_GC_CC_AA_GT; GG_GC_CA_AG_GT; GG_GC_CC_AA_GG; GG_GC_CA_AA_TT; GA_GC_CA_AG_GG; GG_GC_CC_AG_GG; GG_CC_CC_AA_GG; GG_GC_CC_AG_GT; GA_GC_CA_AG_GT; GA_GC_CC_AG_GT; AA_GG_CC_GG_GT.

[0064] The genotype shown in (b1)-(b11) above is represented as X1X2_X3X4_X5X6_X7X8_X9X 10 , wherein X1 to X 10 all represent A or C or T or G, X1X2 represents the nucleotide at the first SNP site in the SNP site combination (for example, AA represents a homozygous type in which the nucleotide at the SNP site is A; for example, AT represents a heterozygous type in which the nucleotide at the SNP site is A and T), X3X4 represents the nucleotide at the second SNP site in the SNP site combination, …, X9X 10 represents the nucleotide at the fifth SNP site in the SNP site combination.

[0065] The second method is based on a combination of 1 SNP site group. The method can be any one of the following:

[0066] (1) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a1) in the genome of the sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep as follows: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b1), the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b1), the sheep to be tested is or is a candidate for non-Tan sheep.

[0067] (2) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a2) in the genome of the sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep as follows: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b2), the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b2), the sheep to be tested is or is a candidate for non-Tan sheep.

[0068] (3) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a3) in the genome of the sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep as follows: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b3), the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b3), the sheep to be tested is or is a candidate for non-Tan sheep.

[0069] (4) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a4) in the genome of the sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep as follows: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b4), the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b4), the sheep to be tested is or is a candidate for non-Tan sheep.

[0070] (5) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a5) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b5) above, the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b5) above, the sheep to be tested is or is a candidate for non-Tan sheep.

[0071] (6) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a6) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b6) above, the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b6) above, the sheep to be tested is or is a candidate for non-Tan sheep.

[0072] (7) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a7) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b7) above, the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b7) above, the sheep to be tested is or is a candidate for non-Tan sheep.

[0073] (8) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a8) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b8) above, the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b8) above, the sheep to be tested is or is a candidate for non-Tan sheep.

[0074] (9) comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a9) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b9) above, the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b9) above, the sheep to be tested is or is a candidate for non-Tan sheep.

[0075] (10) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a10) described above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b10) described above, the sheep to be tested is or is a candidate for a Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b10) described above, the sheep to be tested is or is a candidate for a non-Tan sheep.

[0076] (11) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a11) described above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; determining whether the sheep to be tested is a Tan sheep according to the following: if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b11) described above, the sheep to be tested is or is a candidate for a Tan sheep; if the genotype of the sheep to be tested meets the standard genotype of non-Tan sheep in (b11) described above, the sheep to be tested is or is a candidate for a non-Tan sheep.

[0077] In a second aspect, the present application claims a method for identifying Tan sheep based on genotyping.

[0078] The method for identifying Tan sheep based on genotyping provided by the present application can be any one of the following:

[0079] The first one is a method for identifying based on 11 SNP site combinations. The method can comprise the steps of: detecting the nucleotides at each SNP site in the 11 SNP site combinations in the genome of a sheep to be tested, determining the 11 genotypes of the sheep to be tested corresponding to the 11 SNP site combinations; if the 11 genotypes of the sheep to be tested corresponding to the 11 SNP site combinations all meet the standard genotype of Tan sheep of the corresponding SNP site combination, the sheep to be tested is or is a candidate for a Tan sheep.

[0080] The 11 SNP site combinations are (a1)-(a11) described above; the standard genotype of Tan sheep corresponding to the 11 SNP site combinations shown in (a1)-(a11) is the standard genotype of Tan sheep in (b1)-(b11) described above, respectively.

[0081] The second one is a method for identifying based on 1 SNP site combination. The method can be any one of the following:

[0082] (1) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a1) described above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b1) described above, the sheep to be tested is or is a candidate for a Tan sheep.

[0083] (2) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a2) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b2) above, the sheep to be tested is or is a candidate for Tan sheep.

[0084] (3) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a3) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b3) above, the sheep to be tested is or is a candidate for Tan sheep.

[0085] (4) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a4) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b4) above, the sheep to be tested is or is a candidate for Tan sheep.

[0086] (5) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a5) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b5) above, the sheep to be tested is or is a candidate for Tan sheep.

[0087] (6) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a6) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b6) above, the sheep to be tested is or is a candidate for Tan sheep.

[0088] (7) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a7) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b7) above, the sheep to be tested is or is a candidate for Tan sheep.

[0089] (8) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination shown in (a8) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep in (b8) above, the sheep to be tested is or is a candidate for Tan sheep.

[0090] (9) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination as described in (a9) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep as described in (b9) above, the sheep to be tested is or is a candidate for Tan sheep.

[0091] (10) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination as described in (a10) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep as described in (b10) above, the sheep to be tested is or is a candidate for Tan sheep.

[0092] (11) comprising the steps of: detecting the nucleotides at each SNP site in the SNP site combination as described in (a11) above in the genome of a sheep to be tested, determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested meets the standard genotype of Tan sheep as described in (b11) above, the sheep to be tested is or is a candidate for Tan sheep.

[0093] In the methods of the first and second aspects, the sheep to be tested can be specifically Tan sheep or non-Tan sheep (such as Mongolian sheep).

[0094] In the methods of the first and second aspects, the specific method of detecting the nucleotides at the SNP site in the genome of the sheep to be tested can be whole genome resequencing. Further, the DNA sample used for performing the whole genome resequencing is taken from the blood or ear tissue of the sheep to be tested.

[0095] In a third aspect, the present application also claims the following applications.

[0096] The first is the application of a substance for detecting the nucleotides at each SNP site in the 11 SNP site combinations as described above in the genome of a sheep (or a substance for detecting the nucleotides at the 29 SNP sites as described above in the genome of a sheep) in identifying Tan sheep and / or non-Tan sheep, or in the preparation of a kit for identifying Tan sheep and / or non-Tan sheep.

[0097] The second is the application of a substance for detecting the nucleotides at each SNP site in any one of the 11 SNP site combinations as described above in the genome of a sheep in identifying Tan sheep and / or non-Tan sheep, or in the preparation of a kit for identifying Tan sheep and / or non-Tan sheep.

[0098] In the above two applications, the substance can be specifically a PCR primer and / or a sequencing primer designed for the SNP site to be detected.

[0099] Further, the primers for detecting the CM001582.2:86006851 are two single-stranded DNAs shown in SEQ ID No. 1 and SEQ ID No. 2; the primers for detecting the CM001583.2:42344562 are two single-stranded DNAs shown in SEQ ID No. 4 and SEQ ID No. 5; the primers for detecting the CM001584.2:11650210 are two single-stranded DNAs shown in SEQ ID No. 7 and SEQ ID No. 8; the primers for detecting the CM001584.2:12200323 are two single-stranded DNAs shown in SEQ ID No. 10 and SEQ ID No. 11; the primers for detecting the CM001584.2:79082958 are two single-stranded DNAs shown in SEQ ID No. 13 and SEQ ID No. 14; the primers for detecting the CM001584.2:157466635 are two single-stranded DNAs shown in SEQ ID No. 16 and SEQ ID No. 17; the primers for detecting the CM001585.2:5250467 are two single-stranded DNAs shown in SEQ ID No. 19 and SEQ ID No. 20; the primers for detecting the CM001585.2:30868868 are two single-stranded DNAs shown in SEQ ID No. 22 and SEQ ID No. 23; the primers for detecting the CM001588.2:9423474 are two single-stranded DNAs shown in SEQ ID No. 25 and SEQ ID No. 26; the primers for detecting the CM001588.2:84941340 are two single-stranded DNAs shown in SEQ ID No. 28 and SEQ ID No. 29; the primers for detecting the CM001589.2:75561002 are two single-stranded DNAs shown in SEQ ID No. 31 and SEQ ID No. 32; the primers for detecting the CM001590.2:56924859 are two single-stranded DNAs shown in SEQ ID No. 34 and SEQ ID No. 35; the primers for detecting the CM001592.2:34421625 are two single-stranded DNAs shown in SEQ ID No. 37 and SEQ ID No. 38; the primers for detecting the CM001592.2:53664048 are two single-stranded DNAs shown in SEQ ID No. 40 and SEQ ID No. 41; the primers for detecting the CM001592.2:57058734 are two single-stranded DNAs shown in SEQ ID No. 43 and SEQ ID No. 44; the primers for detecting the CM001593.2:1397868 are two single-stranded DNAs shown in SEQ ID No.46 and SEQ ID No. 47; primers for detecting the CM001594.2: 80961940 are the two single-stranded DNAs set forth in SEQ ID No. 52 and SEQ ID No. 53; primers for detecting the CM001595.2: 14087769 are the two single-stranded DNAs set forth in SEQ ID No. 55 and SEQ ID No. 56; primers for detecting the CM001595.2: 43390933 are the two single-stranded DNAs set forth in SEQ ID No. 58 and SEQ ID No. 59; primers for detecting the CM001595.2: 48968164 are the two single-stranded DNAs set forth in SEQ ID No. 61 and SEQ ID No. 62; primers for detecting the CM001598.2: 45312379 are the two single-stranded DNAs set forth in SEQ ID No. 64 and SEQ ID No. 65; primers for detecting the CM001598.2: 69009397 are the two single-stranded DNAs set forth in SEQ ID No. 67 and SEQ ID No. 68; primers for detecting the CM001600.2: 45516740 are the two single-stranded DNAs set forth in SEQ ID No. 70 and SEQ ID No. 71 ; primers for detecting the CM001601.2: 47737780 are the two single-stranded DNAs set forth in SEQ ID No. 73 and SEQ ID No. 74; primers for detecting the CM001602.2: 49927473 are the two single-stranded DNAs set forth in SEQ ID No. 76 and SEQ ID No. 77; primers for detecting the CM001603.2: 38707088 are the two single-stranded DNAs set forth in SEQ ID No. 79 and SEQ ID No. 80; primers for detecting the CM001603.2: 46619514 are the two single-stranded DNAs set forth in SEQ ID No. 82 and SEQ ID No. 83; primers for detecting the CM001606.2: 34435705 are the two single-stranded DNAs set forth in SEQ ID No. 85 and SEQ ID No. 86.

[0100] Further, the primer for detecting the CM001582.2:86006851 further comprises a single-stranded DNA as represented in SEQ ID No. 3; the primer for detecting the CM001583.2:42344562 further comprises a single-stranded DNA as represented in SEQ ID No. 6; the primer for detecting the CM001584.2:11650210 further comprises a single-stranded DNA as represented in SEQ ID No. 9; the primer for detecting the CM001584.2:12200323 further comprises a single-stranded DNA as represented in SEQ ID No. 12; the primer for detecting the CM001584.2:79082958 further comprises a single-stranded DNA as represented in SEQ ID No. 15; the primer for detecting the CM001584.2:157466635 further comprises a single-stranded DNA as represented in SEQ ID No. 18; the primer for detecting the CM001585.2:5250467 further comprises a single-stranded DNA as represented in SEQ ID No. 21; the primer for detecting the CM001585.2:30868868 further comprises a single-stranded DNA as represented in SEQ ID No. 24; the primer for detecting the CM001588.2:9423474 further comprises a single-stranded DNA as represented in SEQ ID No. 27; the primer for detecting the CM001588.2:84941340 further comprises a single-stranded DNA as represented in SEQ ID No. 30; the primer for detecting the CM001589.2:75561002 further comprises a single-stranded DNA as represented in SEQ ID No. 33; the primer for detecting the CM001590.2:56924859 further comprises a single-stranded DNA as represented in SEQ ID No. 36; the primer for detecting the CM001592.2:34421625 further comprises a single-stranded DNA as represented in SEQ ID No. 39; the primer for detecting the CM001592.2:53664048 further comprises a single-stranded DNA as represented in SEQ ID No. 42; the primer for detecting the CM001592.2:57058734 further comprises a single-stranded DNA as represented in SEQ ID No. 45; the primer for detecting the CM001593.2:1397868 further comprises a single-stranded DNA as represented in SEQ ID No. 48; the primer for detecting the CM001594.2:45477903 further comprises a single-stranded DNA as represented in SEQ ID No. 51; the primer for detecting the CM001594.2:80961940 further comprises a single-stranded DNA as represented in SEQ ID No. 54; the primer for detecting the CM001595.2:14087769 further comprises a single-stranded DNA as represented in SEQ ID No.57; the primer for detecting CM001595.2:48968164 further comprises the single-stranded DNA shown as SEQ ID No. 63; the primer for detecting CM001598.2:45312379 further comprises the single-stranded DNA shown as SEQ ID No. 66; the primer for detecting CM001598.2:69009397 further comprises the single-stranded DNA shown as SEQ ID No. 69; the primer for detecting CM001600.2:45516740 further comprises the single-stranded DNA shown as SEQ ID No. 72; the primer for detecting CM001601.2:47737780 further comprises the single-stranded DNA shown as SEQ ID No. 75; the primer for detecting CM001602.2:49927473 further comprises the single-stranded DNA shown as SEQ ID No. 78; the primer for detecting CM001603.2:38707088 further comprises the single-stranded DNA shown as SEQ ID No. 81; the primer for detecting CM001603.2:46619514 further comprises the single-stranded DNA shown as SEQ ID No. 84; the primer for detecting CM001606.2:34435705 further comprises the single-stranded DNA shown as SEQ ID No. 87.

[0101] Thirdly, the polymorphism of the nucleotides at each SNP site in the 11 groups of SNP site combinations described above (or the polymorphism of the nucleotides at each SNP site in the 29 SNP sites described above) in the genome of sheep is used to identify Tan sheep and / or non-Tan sheep.

[0102] Fourthly, the polymorphism of the nucleotides at each SNP site in any one of the 11 groups of SNP site combinations described above is used to identify Tan sheep and / or non-Tan sheep.

[0103] In a fourth aspect, the present application claims the following set of primers shown as (B1) or (B2):

[0104] (B1) a set of primers for detecting the nucleotides at each SNP site in the 11 groups of SNP site combinations described above in the genome of sheep;

[0105] (B2) a set of primers for detecting the nucleotides at each SNP site in any one of the 11 groups of SNP site combinations described above in the genome of sheep.

[0106] Wherein, the primers for detecting each SNP site are as described above (see Table 2 in detail).

[0107] The application obtains 29 sites by whole genome resequencing of 50 Tan sheep and 50 non-Tan sheep, and then obtains 5 typing result genotypes (identification rate is more than 85%) through R programming analysis. Then 1000 Tan sheep and 1000 non-Tan sheep are used for sequenom verification, and the results show that they are consistent with the determination results. The application successfully obtains SNP site combination for identifying Tan sheep and non-Tan sheep, and provides support for developing Tan sheep breed identification chip, and for future identification, conservation and genetic breeding of Tan sheep. DETAILED DESCRIPTION

[0108] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0109] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0110] Example 1, whole genome resequencing obtains 5 typing result genotypes for identifying Tan sheep and non-Tan sheep

[0111] Test sample: 50 Tan sheep (from Yanchi County, Ningxia) and 50 non-Tan sheep (from Ningxia, Inner Mongolia and other places).

[0112] The blood or ear tissue of the test sheep is used as a sample to extract genomic DNA, and then the whole genome resequencing method is used to obtain 29 SNP sites, and then R programming analysis is used to obtain 5 typing result genotypes (Table 1).

[0113] Among them, the SNP site selection method is as follows: first, mark all sites with P value <-8, and then manually observe whether there is a continuous chromosome region (linkage disequilibrium region). At least more than 4 continuous sites are identified as a continuous region. The site selection principle is: 1) first select the most significant sites in the continuous region with P value <-8; 2) secondly, select the most significant sites in the sites with P value <-8 but not in the continuous region; 3) select the most significant sites in the larger continuous region but without P value <-8.

[0114] The method of whole genome resequencing is as follows: genomic DNA is extracted, Covaris is used for random breakage, electrophoresis is used to recover DNA fragments of the required length (0.2-5Kb), adapters are added, cluster preparation (Illumina HiseqX10) is performed, and finally the method of Paired-End (Illumina Hiseq X10) is used to resequence the inserted fragments.

[0115] The physical positions of the 29 SNP sites are determined based on alignment of the whole genome standard sequence of Tan sheep, version number of which is Ovis. Aries V4.0; and the specific information is as follows:

[0116] CM001582.2:86006851 is located at 86006751 on chromosome 1, and its deoxynucleotide is G or T;

[0117] CM001583.2:42344562 is located at 42344462 on chromosome 2, and its deoxynucleotide is T or C;

[0118] CM001584.2:11650210 is located at 11650110 on chromosome 3, and its deoxynucleotide is T or C;

[0119] CM001584.2:12200323 is located at 12200223 on chromosome 3, and its deoxynucleotide is C or G;

[0120] CM001584.2:79082958 is located at 79082958 on chromosome 3, and its deoxynucleotide is A or G;

[0121] CM001584.2:157466635 is located at 157466635 on chromosome 3, and its deoxynucleotide is C or T;

[0122] CM001585.2:5250467 is located at 5250467 on chromosome 4, and its deoxynucleotide is C or G;

[0123] CM001585.2:30868868 is located at 30868868 on chromosome 4, and its deoxynucleotide is T or C;

[0124] CM001588.2:9423474 is located at 9423374 on chromosome 7, and its deoxynucleotide is A or C;

[0125] CM001588.2:84941340 is located at 84941340 on chromosome 7, and its deoxynucleotide is A or C;

[0126] CM001589.2:75561002 is located at 75561002 on chromosome 8, and its deoxynucleotide is C or G;

[0127] CM001590.2:56924859 is located at 56924859 on chromosome 9, and its deoxynucleotide is A or G;

[0128] CM001592.2: 34421625 is located at position 34421625 on chromosome 11, and its deoxynucleotide is T or C;

[0129] CM001592.2: 53664048 is located at position 53664048 on chromosome 11, and its deoxynucleotide is A or G;

[0130] CM001592.2: 57058734 is located at position 57058734 on chromosome 11, and its deoxynucleotide is C or G;

[0131] CM001593.2: 1397868 is located at position 1397868 on chromosome 12, and its deoxynucleotide is A or G;

[0132] CM001594.2: 45477903 is located at position 45477903 on chromosome 13, and its deoxynucleotide is A or G;

[0133] CM001594.2: 80961940 is located at position 80961940 on chromosome 13, and its deoxynucleotide is A or C;

[0134] CM001595.2: 14087769 is located at position 14087769 on chromosome 14, and its deoxynucleotide is A or T;

[0135] CM001595.2: 43390933 is located at position 43390933 on chromosome 14, and its deoxynucleotide is A or G;

[0136] CM001595.2: 48968164 is located at position 48968164 on chromosome 14, and its deoxynucleotide is T or G;

[0137] CM001598.2: 45312379 is located at position 45312379 on chromosome 17, and its deoxynucleotide is T or C;

[0138] CM001598.2: 69009397 is located at position 69009397 on chromosome 17, and its deoxynucleotide is A or C;

[0139] CM001600.2: 45516740 is located at position 45516740 on chromosome 19, and its deoxynucleotide is A or C;

[0140] CM001601.2: 47737780 is located at position 47737780 on chromosome 20, and its deoxynucleotide is A or G;

[0141] CM001602.2:49927473 is located at 49927473 on chromosome 21, and its deoxynucleotide is T or C;

[0142] CM001603.2:38707088 is located at 38707088 on chromosome 22, and its deoxynucleotide is T or C;

[0143] CM001603.2:46619514 is located at 46619514 on chromosome 22, and its deoxynucleotide is A or G;

[0144] CM001606.2:34435705 is located at 34435705 on chromosome 25, and its deoxynucleotide is C or G;

[0145] Table 1, genotype of 5 typing sites of Tan sheep and non-Tan sheep

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] Note: The genotype of each group of 5 SNP sites is represented as X1X2_X3X4_X5X6_X7X8_X9X 10 , wherein X1 to X 10 all represent A or C or T or G, X1X2 represents the nucleotide at the first SNP site in the group (for example, AA represents a homozygote of A at the SNP site; for example, AT represents a heterozygote of A and T at the SNP site), X3X4 represents the nucleotide at the second SNP site in the group, …, X9X 10 represents the nucleotide at the fifth SNP site in the group.

[0152] Example 2, verification of 5 typing results of Tan sheep and non-Tan sheep genotypes

[0153] Verification sample: 1000 Tan sheep and 1000 non-Tan sheep.

[0154] The blood or ear tissue of the verification sheep was used as a sample, genomic DNA was extracted, and then Sequenom verification was performed.

[0155] Specific operation steps of Sequenom verification are as follows:

[0156] 1. Primer design

[0157] PCR amplification primer and single base extension primer of SNP site to be tested were designed using Sequenom Genotyping Tools and MassARRAY Assay Design software, and were synthesized by Biological Company.

[0158] 2. DNA quality inspection

[0159] The sample to be tested was quantified by spectrophotometer, and agarose gel electrophoresis was used for quality inspection. The genomic DNA electrophoresis band was usually not less than 20 kb. The qualified DNA was adjusted to a concentration of 50 ng / μl, transferred to a 96-well plate, and stored at -20°C for standby.

[0160] 3. Primer dilution

[0161] (1) For each SNP, there are three primers (Table 2), and the numbers of the three primers are marked on the tube cap of the primer synthesis tube.

[0162] Table 2, primer information for each SNP

[0163]

[0164]

[0165] (2) The forward PCR primer and the reverse PCR primer were centrifuged and then water was added. The amount of water added was related to the OD value (both the primer tube and the primer design table had marks). For every 1 OD, 36 μl of water was added. After standing at room temperature for 30 min, the mixture was shaken and mixed for standby.

[0166] (3) When mixing the forward PCR primer and the reverse PCR primer of multiple SNPs in each well, the calculation method was as follows: 2.5 μl of the forward PCR primer and the reverse PCR primer of each SNP were taken, and the water amount (μl) = 500-number of multiplicity*2.5*2 (number of multiplicity: number of SNPs in well)

[0167] 4. PCR amplification

[0168] PCR amplification was performed using multiplex PCR technology in a 384-well plate, and the total volume of each reaction system was 5 μl.

[0169] (1) A new 2.0 ml EP tube was prepared to prepare the PCR master mix solution, as shown in Table 3.

[0170] Table 3, PCR master mix solution

[0171] Volume (μL) 10 x PCR Buffer 0.5 MgCl2(25 mM) 0.4 dNTP mix (25 mM) 0.1 HotStar Taq (5 U / μL) 0.2 Water 1.8 PCR primer mix 1 Total volume 4

[0172] (2) The prepared PCR master mix solution was shaken and mixed, and then was placed in 8 continuous PCR tubes for standby. An 8-channel pipette was used to adjust the sample volume to 4 μL, and the PCR master mix was added to each sample well of a 384-well plate. The 384-well plate was the PCR reaction plate.

[0173] (3) The prepared DNA sample 96-well plate was taken out, and an 8-channel pipette was used to adjust the sample volume to 1 μL, which was added to the corresponding 384-PCR reaction plate. A sealing film was attached, and the mixture was shaken and mixed and then was spun down.

[0174] (4) The PCR reaction conditions were set on a PCR instrument compatible with the 384-well plate, as shown in Table 4.

[0175] Table 4, PCR reaction conditions

[0176]

[0177] 5. Alkaline phosphatase treatment of PCR product

[0178] (1) After the PCR reaction was completed, the 384-reaction plate was taken out and was spun down.

[0179] (2) An alkaline phosphatase treatment reaction solution, SAP Mix, was prepared, and the details are shown in Table 5.

[0180] Table 5, Alkaline phosphatase treatment reaction solution SAP Mix

[0181] SAP Mix Per reaction (μL) Water 1.53 SAP Buffer (10x) 0.17 SAP enzyme (1.7 U / ul) 0.3 Total volume 2

[0182] (3) The prepared SAP mix solution was shaken and mixed, and then was placed in 8 continuous PCR tubes for standby. An 8-channel pipette was used to adjust the sample volume to 2 μL, and the SAP Mix was added to the 384-well PCR reaction plate. A sealing film was attached, and the mixture was shaken and mixed and then was spun down. The total volume of the reaction system was 7 μL (of which the PCR product was 5 μL, and the SAP mixture was 2 μL).

[0183] (4) The 384-well plate was placed on a PCR instrument compatible with the 384-well plate, and the PCR reaction conditions were set, as shown in Table 6.

[0184] Table 6, PCR reaction conditions

[0185] Temperature (°C) Time (min) Cycles 37 40 1 85 5 1 4 ∞ 1

[0186] The alkaline phosphatase treatment reaction was started on the PCR instrument.

[0187] 6. Single-base extension

[0188] (1) After the alkaline phosphatase treatment is completed, the 384 reaction plate is taken out and spun down, and a single base extension reaction is performed, with a total reaction volume of 9 μl.

[0189] (2) The single base extension reaction solution, EXTEND Mix, is prepared, as shown in Table 7. (Note: The Extend primer Mix must be correctly matched with the number of wells)

[0190] Table 7, Single base extension reaction solution EXTEND Mix

[0191] EXTEND Mix Per reaction (μL) Water 0.619 Extend primer Mix 0.94 iPLEX Buffer plus 0.2 iPLEX terminator 0.2 iPLEX enzyme 0.041 Total volume 2

[0192] (3) The prepared EXTEND mix solution is shaken and mixed, and then placed in 8 PCR tubes in a row for standby. An 8-channel pipette is used to adjust the sample volume to 2 μL, and the EXTEND Mix is added to the 384-well reaction plate. For each reaction well, the single base extension reaction system contains 7 μl of the SAP-treated PCR product and 2 μl of the EXTEND Mix solution, with a total system of 9 ul.

[0193] (4) The 384-well plate is placed on a PCR instrument compatible with the 384-well plate, and the PCR reaction conditions are set, as shown in Table 8.

[0194] Table 8, PCR reaction conditions

[0195]

[0196] Start the PCR instrument for single base extension reaction.

[0197] 7. Resin purification

[0198] The reaction product is diluted with 16 μl of water, and after dilution, the resin is used for desalting.

[0199] 8. Chip spotting

[0200] The desalted sample is spotted on the sample target, and natural crystallization is performed.

[0201] 9. Mass spectrometry detection

[0202] The instrument is loaded for mass spectrometry detection, and data is collected.

[0203] The results show that the identification rate of this method is more than 85% (meaning: 100 sheep to be tested are identified according to this method, and more than 85 can be identified), as shown in Table 9. The SNP site combination genotypes of Tan sheep and non-Tan sheep shown in Table 1 are within the corresponding genotype range shown in Table 1, and the proportions are shown in Table 10.

[0204] Table 9, Identification rate of the method of the present application

[0205]

[0206]

[0207] Table 10 Sequenom SNP detection results of Tan sheep and non-Tan sheep (proportion of genotypes of 5-locus sites)

[0208]

[0209]

[0210]

[0211] Note: The genotyping method in the table is the same as that in Table 1.

[0212] The results shown in Table 9 show that the genotypes of the 5-locus sites for identifying Tan sheep and non-Tan sheep shown in Table 1 obtained in Example 1 are accurate and reliable. The SNP site combination for identifying Tan sheep and non-Tan sheep is successfully obtained, which provides support for the identification, breeding and genetic breeding of Tan sheep in the future.

Claims

1. A method for identifying Tan sheep and non-Tan sheep based on genotyping, comprising the following steps: detecting the nucleotides at each SNP site in the SNP site combination shown in (a3) ​​below in the genome of the sheep to be tested, and determining the genotype of the sheep to be tested; then determining whether the sheep to be tested is a Tan sheep as follows: if the genotype of the sheep to be tested conforms to the standard genotype of Tan sheep in (b3) below, then the sheep to be tested is or is a candidate for Tan sheep; if the genotype of the sheep to be tested conforms to the standard genotype of non-Tan sheep in (b3) below, then the sheep to be tested is or is a candidate for non-Tan sheep; (a3) Contains 5 SNP sites, namely CM001582.2:86006851, CM001583.2:42344562, CM001584.2:11650210, CM001584.2:12200323 and CM001588.2:9423474; in, The physical location of each SNP locus was determined based on the alignment of the standard sequence of the whole genome of Tan sheep, and the version number of the standard sequence of the whole genome of Tan sheep is Ovis.Aries V4.0; CM001582.2:86006851 is located at position 86006751 on chromosome 1, and its deoxynucleotide is G or T; CM001583.2:42344562 is located at position 42344462 on chromosome 2, and its deoxynucleotide is either T or C; CM001584.2:11650210 is located at position 11650110 on chromosome 3, and its deoxynucleotide is either T or C. CM001584.2:12200323 is located at position 12200223 on chromosome 3, and its deoxynucleotide is C or G; CM001588.2:9423474 is located at position 9423374 on chromosome 7, and its deoxynucleotide is A or C; (b3) The non-standard Tan sheep genotype is any of the following: TT_TC_TT_CG_AC; Tan sheep standard gene TT_TT_TT_GG_AC; The type is any of the following: TT_CC_CT_CG_CC; TT_TC_CC_GG_CC; TT_TT_CT_CC_CC; GT_CC_CT_CG_AC; GG_CC_TT_GG_AC; GT_CC_TT_GG_CC; TT_CC_TT_CG_AC; GG_TT_CC_CC_AA; TT_CC_CT_GG_AC; TT_CC_CC_CG_AA; GT_CC_CT_CG_CC.

2. A method for identifying Tan sheep based on genotyping, comprising the following steps: detecting nucleotides at each SNP site in the SNP site combination shown in claim 1 (a3) ​​in the genome of the sheep to be tested, and determining the genotype of the sheep to be tested; if the genotype of the sheep to be tested conforms to the standard genotype of Tan sheep in claim 1 (b3), then the sheep to be tested is or is a candidate for Tan sheep.

3. The use of a substance for detecting nucleotides at each SNP site in the SNP site combination shown in claim 1 (a3) ​​in the sheep genome for the identification of Tan sheep and / or non-Tan sheep, or for the preparation of a kit for the identification of Tan sheep and / or non-Tan sheep.

4. The application according to claim 3, characterized in that: The substance is a PCR primer and / or sequencing primer designed for the SNP site to be detected.

5. The application according to claim 4, characterized in that: The primers used to detect CM001582.2:86006851 are the two single-stranded DNA molecules shown in SEQ ID No. 1 and SEQ ID No. 2; The primers used to detect CM001583.2:42344562 are the two single-stranded DNA molecules shown in SEQ ID No. 4 and SEQ ID No. 5; The primers used to detect CM001584.2:11650210 are the two single-stranded DNA molecules shown in SEQ ID No. 7 and SEQ ID No. 8; The primers used to detect CM001584.2:12200323 are the two single-stranded DNA molecules shown in SEQ ID No. 10 and SEQ ID No. 11; The primers used to detect CM001588.2:9423474 are the two single-stranded DNA molecules shown in SEQ ID No. 25 and SEQ ID No.

26.

6. The application according to claim 5, characterized in that: The primers used to detect CM001582.2:86006851 further include the single-stranded DNA shown in SEQ ID No. 3; the primers used to detect CM001583.2:42344562 further include the single-stranded DNA shown in SEQ ID No. 6; The primers used to detect CM001584.2:11650210 also include the single-stranded DNA shown in SEQ ID No. 9; The primers used to detect CM001584.2:12200323 also include the single-stranded DNA shown in SEQ ID No. 12; The primers used to detect CM001588.2:9423474 also include the single-stranded DNA shown in SEQ ID No.

27.

7. A set of primers for detecting nucleotides at each SNP site in the SNP site combination shown in claim 1 (a3) ​​in the sheep genome; The primers used to detect each SNP site are as described in claim 5 or 6.

8. The application of nucleotide polymorphisms at each SNP site in the SNP site combination shown in claim 1 (a3) ​​in the sheep genome in the identification of Tan sheep and / or non-Tan sheep.

Images