CN116219037A - Method for identifying sheep on beach and sheep on non-beach based on genotyping - Google Patents
Method for identifying sheep on beach and sheep on non-beach based on genotyping Download PDFInfo
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Abstract
The invention discloses a method for identifying sheep and non-sheep based on genotyping. The method comprises the following steps: detecting the nucleotide of each SNP locus in a specific SNP locus combination in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected corresponding to the specific SNP locus combination; and then determining whether the sheep to be tested is a Tan sheep according to the following steps: if the genotype of the sheep to be detected corresponding to the specific SNP locus combination accords with the standard genotype of the Tan sheep, the sheep to be detected is or is candidate to be Tan sheep; and if the genotype of the sheep to be detected corresponding to the specific SNP locus combination meets the standard genotype of the non-Tan sheep, the sheep to be detected is or is candidate to be the non-Tan sheep. The invention successfully obtains SNP locus combination for identifying the Tan sheep and the non-Tan sheep, and provides support for the identification, the seed conservation and the genetic breeding of the Tan sheep in the future.
Description
The application is a divisional application with the application number of 201810409670.7 and the application date of 2018, 5 and 2 days, and the invention and creation name of a method for identifying the Tan sheep and the non-Tan sheep based on genotyping
Technical Field
The invention relates to the technical field of biology, in particular to a method for identifying sheep and non-sheep based on genotyping.
Background
The beach sheep is an important animal resource in Ningxia, and the annual output of the beach sheep reaches 600 tens of thousands, and the beach sheep is an important dominant brand in Ningxia because the unique flavor and meat quality of the beach sheep are deeply favored by the market. However, the recently developed filial generation of the sheep and other sheep species is sold as the sheep, which causes great damage to the sheep brands. In order to stabilize the quality of the Tan sheep and the brand image of the heavy-tree Tan sheep, the Tan sheep is manufactured into a national famous brand, and the identification of the Tan sheep and the non-Tan sheep lays a foundation for the seed identification and the genetic breeding of the Tan sheep in the future.
With the recent development of molecular biology, genetic testing techniques have also made a great deal of progress in the identification of biological species. Gene detection technology is widely used for animal and plant species identification by virtue of its ability to identify almost all biological materials. The identification of animal and plant species by gene detection technology is the fastest growing technical means with the highest accuracy in species identification.
Whole genome resequencing is a whole genome-wide sequencing of individuals whose genomic sequences are known, and has become one of the most effective methods in animal and plant breeding studies as sequencing costs decrease and species of known genomic sequences increase. The Sequencom SNP typing technology is carried out by a primer extension reaction principle, combines sensitive PCR amplification and reliable MALDI-TOF mass spectrometry technology to realize genotyping detection, and is particularly suitable for verifying the result found by whole genome research.
Disclosure of Invention
The invention aims to provide a method for identifying sheep and non-sheep based on genotyping.
In a first aspect, the invention claims a method for genotyping-based identification of a sheep to a non-sheep.
The method for identifying the beach sheep and the non-beach sheep based on genotyping provided by the invention can be any one of the following steps:
first, a method for identification based on 11 sets of SNP site combinations. The method specifically comprises the following steps: detecting the nucleotide of each SNP locus in 11 groups of SNP locus combinations in the genome of the sheep to be detected, and determining 11 genotypes of the sheep to be detected corresponding to the 11 groups of SNP locus combinations; and then determining whether the sheep to be tested is a Tan sheep according to the following steps: if the 11 genotypes of the 11 SNP locus combinations correspond to the standard genotypes of the Tan sheep of the corresponding SNP locus combinations, the sheep to be detected is or is candidate to be Tan sheep; and if the 11 genotypes of the 11 SNP locus combinations correspond to the standard genotypes of the non-Tan sheep of the corresponding SNP locus combinations, the sheep to be tested is or is candidate to be the non-Tan sheep.
The 11 sets of SNP sites are combined as follows (a 1) to (a 11); (a1) The non-Tan sheep standard genotypes and Tan sheep standard genotypes corresponding to the 11 SNP locus combinations shown in (a 11) are respectively (b 1) - (b 11) in sequence. Namely, the non-Tan sheep standard genotype and the Tan sheep standard genotype corresponding to the SNP locus combination shown in the (a 1) are (b 1); (a2) The non-Tan sheep standard genotype and Tan sheep standard genotype corresponding to the SNP locus combination are shown as (b 2); … … (a 11) the non-Tan sheep standard genotype and Tan sheep standard genotype corresponding to the SNP locus combination is (b 11).
(a1) Contains 5 SNP loci, namely CM001598.2:69009397, CM001602.2:49927473, CM001603.2:38707088, CM001603.2:46619514 and CM001606.2:34435705;
(a2) Contains 5 SNP loci, namely CM001582.2:86006851, CM001598.2:69009397, CM001600.2:45516740, CM001601.2:47737780 and CM001603.2:46619514;
(a3) Contains 5 SNP loci, namely CM001582.2:86006851, CM001583.2:42344562, CM001584.2:11650210, CM001584.2:12200323 and CM001588.2:9423474;
(a4) Contains 5 SNP loci, namely CM001592.2:57058734, CM001595.2:48968164, CM001598.2:69009397, CM001601.2:47737780 and CM001603.2:38707088;
(a5) Contains 5 SNP loci, namely CM001585.2:30868868, CM001589.2:75561002, CM001590.2:56924859, CM001598.2:69009397 and CM001606.2:34435705;
(a6) Contains 5 SNP loci, namely CM001585.2:30868868, CM001588.2:84941340, CM001598.2:45312379, CM001598.2:69009397 and CM001603.2:46619514;
(a7) Contains 5 SNP loci, namely CM001585.2:30868868, CM001588.2:84941340, CM001594.2:45477903, CM001594.2:80961940 and CM001603.2:38707088;
(a8) Contains 5 SNP loci, namely CM001585.2:30868868, CM001588.2:84941340, CM001593.2:1397868, CM001595.2:14087769 and CM001602.2:49927473;
(a9) Contains 5 SNP loci, namely CM001585.2:30868868, CM001588.2:84941340, CM001592.2:34421625, CM001592.2:53664048 and CM001595.2:48968164;
(a10) Contains 5 SNP loci, namely CM001584.2:79082958, CM001584.2:157466635, CM001601.2:47737780, CM001602.2:49927473 and CM001603.2:38707088;
(a11) Contains 5 SNP loci, namely CM001584.2:79082958, CM001585.2:5250467, CM001588.2:84941340, CM001595.2:43390933 and CM001595.2:48968164;
the physical position of each SNP locus in the 11 sets of SNP locus combinations is determined based on the comparison of the standard sequences of the whole genome of the Tan sheep, and the version number of the standard sequences of the whole genome of the Tan sheep is Ovis. Aries V4.0; a total of 29 SNP sites are specified as follows:
CM001582.2:86006851 is located on chromosome 1 at position 86006851, and its deoxynucleotide is G or T;
CM001583.2:42344562 is located on chromosome 2 at position 42344562, and its deoxynucleotide is T or C;
CM001584.2:11650210 is located on chromosome 3 at 11650210, and its deoxynucleotide is T or C;
CM001584.2:12200323 is located on chromosome 3 at position 12200323, and its deoxynucleotide is C or G;
CM001584.2:79082958 is located on chromosome 3 at position 79082958, and its deoxynucleotide is A or G;
CM001584.2:157466635 is located on chromosome 3 at 157466635, and its deoxynucleotide is C or T;
CM001585.2:5250467 is located on chromosome 4 at position 5250467, and its deoxynucleotide is C or G;
CM001585.2:30868868 is located on chromosome 4 at position 30868868, and its deoxynucleotide is T or C;
CM001588.2:9423474 is located on chromosome 7 at position 9423474, and its deoxynucleotide is A or C;
CM001588.2:84941340 is located on chromosome 7 at position 84941340, and its deoxynucleotide is A or C;
CM001589.2:75561002 is located on chromosome 8 at position 75561002, and its deoxynucleotide is C or G;
CM001590.2:56924859 is located on chromosome 9 at position 56924859, and its deoxynucleotide is A or G;
CM001592.2:34421625 is located on chromosome 11 at position 34421625, and its deoxynucleotide is T or C;
CM001592.2:53664048 is located on chromosome 11 at position 53664048, and its deoxynucleotide is A or G;
CM001592.2:57058734 is located on chromosome 11 at position 57058734, and its deoxynucleotide is C or G;
CM001593.2:1397868 is located on chromosome 12 at position 1397868, and its deoxynucleotide is A or G;
CM001594.2:45477903 is located on chromosome 13 at position 45477903, and its deoxynucleotide is A or G;
CM001594.2:80961940 is located on chromosome 13 at position 80961940, and its deoxynucleotide is A or C;
CM001595.2:14087769 is located on chromosome 14 at position 14087769, and its deoxynucleotide is A or T;
CM001595.2:43390933 is located on chromosome 14 at position 43390933, and its deoxynucleotide is A or G;
CM001595.2:48968164 is located on chromosome 14 at position 48968164, and its deoxynucleotide is T or G;
CM001598.2:45312379 is located on chromosome 17 at position 45312379, and its deoxynucleotide is T or C;
CM001598.2:69009397 is located on chromosome 17 at position 69009397, and its deoxynucleotide is A or C;
CM001600.2:45516740 is located on chromosome 19 at position 45516740, and its deoxynucleotide is A or C;
CM001601.2:47737780 is located on chromosome 20 at position 47737780, and its deoxynucleotide is A or G;
CM001602.2:49927473 is located on chromosome 21 at position 49927473, and its deoxynucleotide is T or C;
CM001603.2:38707088 is located on chromosome 22 at position 38707088, and its deoxynucleotide is T or C;
CM001603.2:46619514 is located on chromosome 22 at position 46619514, and its deoxynucleotide is A or G;
CM001606.2:34435705 is located on chromosome 25 at position 34435705, and its deoxynucleotide is C or G;
Of course, the version number of the whole genome standard sequence of the sheep is not limited to ovis. Aries V4.0, and any kind of the whole genome standard sequence of the sheep is adopted, so long as the SNP locus is the same as the 29 SNP loci described above, the invention falls within the protection scope of the invention.
(b1) The standard genotype of the non-beach sheep 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; the standard genotype of the sheep 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 ggcg; aa_cc_cc ggcc; 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.
(b2) The standard genotype of the non-beach sheep 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; the standard genotype of the sheep 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.
(b3) The standard genotype of the non-beach sheep is any one of the following: TT_TC_TT_CG_AC; TT_TT_GG_AC; TT_TC_TT_GG_CC; tt_cc_ct_gg_cc; tt_tt_ct_gg_cc; TT_TT_GG_CC; tt_cc_tt_gg_cc; TT_TT_CG_CC; tt_tc_ct_gg_cc; TT_TC_TT_CG_CC; TT_TC_TT_GG_AC; GT_TT_TT_GG_CC; the standard genotype of the sheep is any one 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.
(b4) The standard genotype of the non-beach sheep is any one of the following: 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 standard genotype of the sheep is any one of the following: 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_ggcc_ag_tc; cc_gg_ac_ag_tc; cc_tt_cc_ag_tt; cc_gt_cc_ag_cc; cc_ggaa_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.
(b5) The standard genotype of the non-beach sheep 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 standard genotype of the sheep 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; 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.
(b6) The standard genotype of the non-beach sheep 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; the standard genotype of the sheep 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.
(b7) The standard genotype of the non-beach sheep 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; the standard genotype of the sheep 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.
(b8) The standard genotype of the non-beach sheep 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 standard genotype of the sheep 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.
(b9) The standard genotype of the non-beach sheep is any one of the following: 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 standard genotype of the sheep is any one of the following: 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.
(b10) The standard genotype of the non-beach sheep 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 standard genotype of the sheep 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.
(b11) The standard genotype of the non-beach sheep is any one of the following: 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_ggaa_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; the standard genotype of the sheep is any one of the following: 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.
The genotypes shown in (b 1) to (b 11) above are denoted as X 1 X 2 _X 3 X 4 _X 5 X 6 _X 7 X 8 _X 9 X 10 Wherein X is 1 To X 10 All represent A or C or T or G, X 1 X 2 Representing the core at the first SNP site in the SNP site combinationA nucleotide (e.g., AA indicates that the nucleotide AT the SNP site is homozygous for A; e.g., AT indicates that the nucleotide AT the SNP site is heterozygous for A and T), X 3 X 4 Represents the nucleotide at the second SNP site in the SNP site combination … …, X 9 X 10 The nucleotide at the fifth SNP site in the SNP site combination is shown.
Second, a method for identification based on 1 set of SNP site combinations. The method can be specifically any one of the following steps:
(1) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 1) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 1), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 1), the sheep to be detected is or is candidate to be the non-Tan sheep.
(2) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 2) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be tested meets the standard genotype of the Tan sheep in (b 2), the sheep to be tested is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in (b 2), the sheep to be detected is or is candidate to be the non-Tan sheep.
(3) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 3) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be tested meets the standard genotype of the Tan sheep in (b 3), the sheep to be tested is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 3), the sheep to be detected is or is candidate to be the non-Tan sheep.
(4) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 4) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be tested meets the standard genotype of the Tan sheep in (b 4), the sheep to be tested is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 4), the sheep to be detected is or is candidate to be the non-Tan sheep.
(5) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 5) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 5), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 5), the sheep to be detected is or is candidate to be the non-Tan sheep.
(6) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 6) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 6), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 6), the sheep to be detected is or is candidate to be the non-Tan sheep.
(7) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 7) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 7), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 7), the sheep to be detected is or is candidate to be the non-Tan sheep.
(8) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 8) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 8), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 8), the sheep to be detected is or is candidate to be the non-Tan sheep.
(9) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 9) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 9), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 9), the sheep to be detected is or is candidate to be the non-Tan sheep.
(10) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 10) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 10), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be tested accords with the standard genotype of the non-Tan sheep in the step (b 10), the sheep to be tested is or is candidate to be the non-Tan sheep.
(11) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 11) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; determining whether the sheep to be tested is a beach sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 11), the sheep to be detected is or is candidate to be Tan sheep; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the non-Tan sheep in the step (b 11), the sheep to be detected is or is candidate to be the non-Tan sheep.
In a second aspect, the invention claims a method for genotyping-based identification of a beach sheep.
The method for identifying the beach sheep based on genotyping provided by the invention can be any one of the following steps:
first, a method for identification based on 11 sets of SNP site combinations. The method may comprise the steps of: detecting the nucleotide of each SNP locus in 11 groups of SNP locus combinations in the genome of the sheep to be detected, and determining 11 genotypes of the sheep to be detected corresponding to the 11 groups of SNP locus combinations; and if the 11 genotypes of the 11 SNP locus combinations correspond to the standard genotypes of the Tan sheep of the corresponding SNP locus combinations, the sheep to be tested is or is candidate to be Tan sheep.
Wherein the 11 sets of SNP loci are combined as described in (a 1) to (a 11) above; (a1) The standard genotypes of the sheep corresponding to the 11 SNP locus combinations shown in (a 11) are respectively the standard genotypes of the sheep in (b 1) to (b 11) in sequence.
Second, a method for identification based on 1 set of SNP site combinations. The method can be specifically any one of the following steps:
(1) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 1) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 1), the sheep to be detected is or is candidate to be Tan sheep.
(2) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 2) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 2), the sheep to be detected is or is candidate to be Tan sheep.
(3) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 3) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 3), the sheep to be detected is or is candidate to be Tan sheep.
(4) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 4) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 4), the sheep to be detected is or is candidate to be Tan sheep.
(5) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 5) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 5), the sheep to be detected is or is candidate to be Tan sheep.
(6) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 6) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 6), the sheep to be detected is or is candidate to be Tan sheep.
(7) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 7) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 7), the sheep to be detected is or is candidate to be Tan sheep.
(8) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 8) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 8), the sheep to be detected is or is candidate to be Tan sheep.
(9) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 9) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 9), the sheep to be detected is or is candidate to be Tan sheep.
(10) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 10) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 10), the sheep to be detected is or is candidate to be Tan sheep.
(11) The method comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 11) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and (3) if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the step (b 11), the sheep to be detected is or is candidate to be Tan sheep.
In the methods of the first and second aspects, the sheep to be tested may be a sheep or a non-sheep (e.g. mongolian sheep).
In the methods of the first and second aspects, the specific method for detecting a nucleotide at a SNP site in the genome of the sheep to be tested may be whole genome resequencing. Further, the DNA sample for performing the whole genome resequencing method is taken from blood or ear tissue of the sheep to be tested.
In a third aspect, the invention also claims the following applications.
First, the use of a substance for detecting a nucleotide at each SNP site in the 11-set SNP site combination described above in a sheep genome (or a substance for detecting a nucleotide at 29 SNP sites described above in a sheep genome) for identifying a sheep and/or a non-sheep, or for preparing a kit for identifying a sheep and/or a non-sheep.
Second, the use of a substance for detecting a nucleotide at each SNP site of any of the 11 sets of SNP site combinations described above in a sheep genome to identify a sheep and/or a non-sheep, or the use of a kit for preparing a kit for identifying a sheep and/or a non-sheep.
In both applications, the substance may specifically be a PCR primer and/or a sequencing primer designed for the SNP site to be detected.
Further, the primers used for detecting the CM001582.2:86006851 are two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No. 2; primers used for detecting the CM001583.2:42344562 are two single-stranded DNAs shown as SEQ ID No.4 and SEQ ID No. 5; the primers used for detecting the CM001584.2:11650210 are two single-stranded DNA shown as SEQ ID No.7 and SEQ ID No. 8; the primers used for detecting the CM001584.2:12200323 are two single-stranded DNA shown as SEQ ID No.10 and SEQ ID No. 11; the primers used for detecting the CM001584.2:79082958 are two single-stranded DNA shown in SEQ ID No.13 and SEQ ID No. 14; the primers used for detecting the CM001584.2:157466635 are two single-stranded DNA shown as SEQ ID No.16 and SEQ ID No. 17; the primers used for detecting the CM001585.2:5250467 are two single-stranded DNA shown as SEQ ID No.19 and SEQ ID No. 20; the primers used for detecting the CM001585.2:30868868 are two single-stranded DNA shown as SEQ ID No.22 and SEQ ID No. 23; the primers used for detecting the CM001588.2:9423474 are two single-stranded DNA shown as SEQ ID No.25 and SEQ ID No. 26; the primers used for detecting the CM001588.2:84941340 are two single-stranded DNA shown as SEQ ID No.28 and SEQ ID No. 29; the primers used for detecting the CM001589.2:75561002 are two single-stranded DNA shown as SEQ ID No.31 and SEQ ID No. 32; the primers used for detecting the CM001590.2:56924859 are two single-stranded DNA shown as SEQ ID No.34 and SEQ ID No. 35; the primers used for detecting the CM001592.2:34421625 are two single-stranded DNA shown as SEQ ID No.37 and SEQ ID No. 38; the primers used for detecting the CM001592.2:53664048 are two single-stranded DNA shown as SEQ ID No.40 and SEQ ID No. 41; the primers used for detecting the CM001592.2:57058734 are two single-stranded DNA shown as SEQ ID No.43 and SEQ ID No. 44; the primers used for detecting the CM001593.2:1397868 are two single-stranded DNA shown as SEQ ID No.46 and SEQ ID No. 47; the primers used for detecting the CM001594.2:45477903 are two single-stranded DNA shown as SEQ ID No.49 and SEQ ID No. 50; the primers used for detecting the CM001594.2:80961940 are two single-stranded DNA shown as SEQ ID No.52 and SEQ ID No. 53; the primers used for detecting the CM001595.2:14087769 are two single-stranded DNA shown as SEQ ID No.55 and SEQ ID No. 56; the primers used for detecting the CM001595.2:43390933 are two single-stranded DNA shown as SEQ ID No.58 and SEQ ID No. 59; the primers used for detecting the CM001595.2:48968164 are two single-stranded DNA shown as SEQ ID No.61 and SEQ ID No. 62; the primers used for detecting the CM001598.2:45312379 are two single-stranded DNA shown as SEQ ID No.64 and SEQ ID No. 65; the primers used for detecting the CM001598.2:69009397 are two single-stranded DNA shown as SEQ ID No.67 and SEQ ID No. 68; the primers used for detecting the CM001600.2:45516740 are two single-stranded DNA shown as SEQ ID No.70 and SEQ ID No. 71; the primers used for detecting the CM001601.2:47737780 are two single-stranded DNA shown as SEQ ID No.73 and SEQ ID No. 74; the primers used for detecting the CM001602.2:49927473 are two single-stranded DNA shown as SEQ ID No.76 and SEQ ID No. 77; the primers used for detecting the CM001603.2:38707088 are two single-stranded DNA shown as SEQ ID No.79 and SEQ ID No. 80; the primers used for detecting the CM001603.2:46619514 are two single-stranded DNA shown as SEQ ID No.82 and SEQ ID No. 83; the primers used for detecting the CM001606.2:34435705 are two single-stranded DNAs shown as SEQ ID No.85 and SEQ ID No. 86.
Still further, the primers for detecting the CM001582.2:86006851 further comprise a single-stranded DNA shown in SEQ ID No. 3; the primer for detecting the CM001583.2:42344562 also comprises single-stranded DNA shown in SEQ ID No. 6; the primer for detecting the CM001584.2:11650210 also comprises single-stranded DNA shown in SEQ ID No. 9; the primer for detecting the CM001584.2:12200323 also comprises single-stranded DNA shown in SEQ ID No. 12; the primer for detecting the CM001584.2:79082958 also comprises single-stranded DNA shown in SEQ ID No. 15; the primer for detecting the CM001584.2:157466635 also comprises single-stranded DNA shown in SEQ ID No. 18; the primer for detecting the CM001585.2:5250467 also comprises single-stranded DNA shown in SEQ ID No. 21; the primer for detecting the CM001585.2:30868868 also comprises single-stranded DNA shown in SEQ ID No. 24; the primer for detecting the CM001588.2:9423474 also comprises single-stranded DNA shown in SEQ ID No. 27; the primer for detecting the CM001588.2:84941340 also comprises single-stranded DNA shown in SEQ ID No. 30; the primer for detecting the CM001589.2:75561002 also comprises single-stranded DNA shown in SEQ ID No. 33; the primer for detecting the CM001590.2:56924859 also comprises single-stranded DNA shown in SEQ ID No. 36; the primer for detecting the CM001592.2:34421625 also comprises single-stranded DNA shown in SEQ ID No. 39; the primer for detecting the CM001592.2:53664048 also comprises single-stranded DNA shown in SEQ ID No. 42; the primer for detecting the CM001592.2:57058734 also comprises single-stranded DNA shown in SEQ ID No. 45; the primer for detecting the CM001593.2:1397868 also comprises single-stranded DNA shown in SEQ ID No. 48; the primer for detecting the CM001594.2:45477903 also comprises single-stranded DNA shown in SEQ ID No. 51; the primer for detecting the CM001594.2:80961940 also comprises single-stranded DNA shown in SEQ ID No. 54; the primer for detecting the CM001595.2:14087769 also comprises single-stranded DNA shown in SEQ ID No. 57; the primer for detecting the CM001595.2:43390933 also comprises single-stranded DNA shown in SEQ ID No. 60; the primer for detecting the CM001595.2:48968164 also comprises single-stranded DNA shown in SEQ ID No. 63; the primer for detecting the CM001598.2:45312379 also comprises single-stranded DNA shown in SEQ ID No. 66; the primer for detecting the CM001598.2:69009397 also comprises single-stranded DNA shown as SEQ ID No. 69; the primer for detecting the CM001600.2:45516740 also comprises single-stranded DNA shown in SEQ ID No. 72; the primer for detecting the CM001601.2:47737780 also comprises single-stranded DNA shown in SEQ ID No. 75; the primer for detecting the CM001602.2:49927473 also comprises single-stranded DNA shown in SEQ ID No. 78; the primer for detecting the CM001603.2:38707088 also comprises single-stranded DNA shown in SEQ ID No. 81; the primer for detecting the CM001603.2:46619514 also comprises single-stranded DNA shown in SEQ ID No. 84; the primer used for detecting the CM001606.2:34435705 also includes single-stranded DNA shown in SEQ ID No. 87.
Third, the use of polymorphisms in the sheep genome of nucleotides at each SNP site in the 11-set SNP site combination described above (or polymorphisms in the sheep genome of nucleotides at 29 SNP sites described above) for identifying a beach sheep and/or a non-beach sheep.
Fourth, use of a polymorphism of a nucleotide at each SNP site of any of the 11 sets of SNP site combinations described above in sheep genome for identifying a beach sheep and/or a non-beach sheep.
In a fourth aspect, the present invention claims a primer set as shown in (B1) or (B2) below:
(B1) A set of primers for detecting the nucleotide at each SNP site in the 11 sets of SNP site combinations described above in sheep genome;
(B2) A primer set for detecting a nucleotide at each SNP site of any one of the 11 sets of SNP site combinations described above in sheep genome.
Among them, the primers for detecting each SNP site are as described above (see Table 2 for details).
According to the invention, through carrying out whole genome re-sequencing on 50 beach sheep and 50 non-beach sheep, 29 loci are obtained, and then through R programming analysis, 5 typing result genotypes (the identification rate is more than 85%) are obtained. Then, the sequence verification is carried out by 1000 sheep and 1000 sheep which are not sheep, and the results show that the results are consistent with the judgment results. The SNP locus combination for identifying the Tan sheep and the non-Tan sheep is successfully obtained, and the SNP locus combination provides support for developing Tan sheep variety identification chips, and further for the identification, seed conservation and genetic breeding of Tan sheep in the future.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 Whole genome resequencing to obtain 5-typing result genotypes identifying Tan sheep and non-Tan sheep
Sample supply: 50 sheep (from Ningxia salt pool county) and 50 sheep (from Ningxia, inner Mongolia etc.).
The blood or ear tissue of the sheep to be tested is taken as a sample, genomic DNA is extracted, then 29 SNP loci are obtained by adopting a whole genome resequencing method, and then the genotype of the 5 typing result is obtained by R programming analysis (Table 1).
The SNP locus selection method comprises the following steps: all P-value < 8 sites were first marked, and then manually observed for the presence of consecutive chromosomal regions (linkage disequilibrium regions). At least more than 4 consecutive sites are identified as consecutive regions. The site selection principle is as follows: 1) The most prominent of those sites with P values < -8 in the continuous region were selected first. 2) Second select those P values < -8, but not the most prominent ones of the sites of the contiguous region; 3) Those larger contiguous regions were selected, but none of the sites with P values < -8 were the most prominent.
The whole genome resequencing method is specifically as follows: genomic DNA was extracted, randomly disrupted by Covaris, electrophoretically recovered DNA fragments of the desired length (0.2-5 Kb), ligated, cluster preparation (Illumina Hiseq X10) and finally resequenced the inserts by the method of Paired-End (Illumina Hiseq X10).
The physical positions of the 29 SNP loci are determined based on the alignment of the standard sequences of the whole genome of the Tan sheep, and the version number of the standard sequences of the whole genome of the Tan sheep is Ovis. Aries V4.0; the method comprises the following steps:
CM001582.2:86006851 is located on chromosome 1 at position 86006851, and its deoxynucleotide is G or T;
CM001583.2:42344562 is located on chromosome 2 at position 42344562, and its deoxynucleotide is T or C;
CM001584.2:11650210 is located on chromosome 3 at 11650210, and its deoxynucleotide is T or C;
CM001584.2:12200323 is located on chromosome 3 at position 12200323, and its deoxynucleotide is C or G;
CM001584.2:79082958 is located on chromosome 3 at position 79082958, and its deoxynucleotide is A or G;
CM001584.2:157466635 is located on chromosome 3 at 157466635, and its deoxynucleotide is C or T;
CM001585.2:5250467 is located on chromosome 4 at position 5250467, and its deoxynucleotide is C or G;
CM001585.2:30868868 is located on chromosome 4 at position 30868868, and its deoxynucleotide is T or C;
CM001588.2:9423474 is located on chromosome 7 at position 9423474, and its deoxynucleotide is A or C;
CM001588.2:84941340 is located on chromosome 7 at position 84941340, and its deoxynucleotide is A or C;
CM001589.2:75561002 is located on chromosome 8 at position 75561002, and its deoxynucleotide is C or G; CM001590.2:56924859 is located on chromosome 9 at position 56924859, and its deoxynucleotide is A or G; CM001592.2:34421625 is located on chromosome 11 at position 34421625, and its deoxynucleotide is T or C; CM001592.2:53664048 is located on chromosome 11 at position 53664048, and its deoxynucleotide is A or G; CM001592.2:57058734 is located on chromosome 11 at position 57058734, and its deoxynucleotide is C or G; CM001593.2:1397868 is located on chromosome 12 at position 1397868, and its deoxynucleotide is A or G; CM001594.2:45477903 is located on chromosome 13 at position 45477903, and its deoxynucleotide is A or G; CM001594.2:80961940 is located on chromosome 13 at position 80961940, and its deoxynucleotide is A or C; CM001595.2:14087769 is located on chromosome 14 at position 14087769, and its deoxynucleotide is A or T; CM001595.2:43390933 is located on chromosome 14 at position 43390933, and its deoxynucleotide is A or G; CM001595.2:48968164 is located on chromosome 14 at position 48968164, and its deoxynucleotide is T or G; CM001598.2:45312379 is located on chromosome 17 at position 45312379, and its deoxynucleotide is T or C; CM001598.2:69009397 is located on chromosome 17 at position 69009397, and its deoxynucleotide is A or C; CM001600.2:45516740 is located on chromosome 19 at position 45516740, and its deoxynucleotide is A or C; CM001601.2:47737780 is located on chromosome 20 at position 47737780, and its deoxynucleotide is A or G; CM001602.2:49927473 is located on chromosome 21 at position 49927473, and its deoxynucleotide is T or C; CM001603.2:38707088 is located on chromosome 22 at position 38707088, and its deoxynucleotide is T or C; CM001603.2:46619514 is located on chromosome 22 at position 46619514, and its deoxynucleotide is A or G; CM001606.2:34435705 is located on chromosome 25 at position 34435705, and its deoxynucleotide is C or G;
TABLE 1 identification of the genotypes of the parting sites of the Tan sheep and the non-Tan sheep 5
Note that: genotypes of 5 SNP loci in each group are represented as X 1 X 2 _X 3 X 4 _X 5 X 6 _X 7 X 8 _X 9 X 10 Wherein X is 1 To X 10 All represent A or C or T or G, X 1 X 2 Represents the nucleotide AT the first SNP site in the set (e.g., AA represents the homozygous A for the nucleotide AT the SNP site; e.g., AT represents the heterozygous A and T for the nucleotide AT the SNP site), X 3 X 4 Represents the nucleotide at the second SNP site in the set, … …, X 9 X 10 The nucleotide at the fifth SNP site in this set is indicated.
Example 2 verification of the genotyping results genotypes of Tan sheep and non-Tan sheep
Sample for verification: 1000 parts of beach sheep and 1000 parts of non-beach sheep.
Genomic DNA was extracted using sheep blood or ear tissue as a sample, and then subjected to Sequenom validation.
The specific operation steps of the Sequenom verification are as follows:
1. primer design
PCR amplification primers and single base extension primers of SNP loci to be detected are designed by using software of Genotyping Tools and MassARRAY Assay Design of sequencer company, and are synthesized by biological company.
DNA quality inspection
The sample to be tested is quantified by a spectrophotometer, and the agarose gel electrophoresis quality is checked, and the genomic DNA electrophoresis band is usually not smaller than 20kb. The DNA qualified for quality inspection is adjusted to 50 ng/. Mu.l in concentration, transferred to a 96-well plate and stored at-20 ℃ for standby.
3. Primer dilution
(1) Each SNP has three primers (Table 2), and the numbering of these three primers is marked on the tube cap of the primer synthesis tube.
TABLE 2 primer information for each SNP
(2) Forward PCR primer and Reverse PCR primer were centrifuged and air-thrown, then water was added, the water addition amount was correlated to OD value (primer tube and primer design table were labeled), and 36. Mu.l of water was added per 1 OD. Adding water, standing at room temperature for 30min, and shaking and mixing.
(3) The calculation method was calculated by mixing Forward PCR primers and Reverse PCR primers for multiple SNPs in each Well: forward PCR primer and Reverse PCR primer for each SNP were each used in an amount of 2.5. Mu.l added water (. Mu.l) =500-weight.2.5.2 (weight: number of SNPs in well)
PCR amplification
PCR amplification was performed in 384 well plates using multiplex PCR technology, with a total volume of 5. Mu.l per reaction system.
(1) The PCR master mix solution was prepared in a new 2.0ml EP tube, as shown in Table 3.
TABLE 3 PCR Master mix solution
| Volume (mu L) | |
| 10×PCR Buffer | 0.5 |
| MgCl 2 (25mM) | 0.4 |
| dNTP mix(25mM) | 0.1 |
| HotStar Taq(5U/μL) | 0.2 |
| Water and its preparation method | 1.8 |
| PCR primer mix | 1 |
| Total volume of | 4 |
(2) The prepared PCR master mix solution is vibrated and mixed uniformly and then is placed in 8 rows of PCR tubes for standby, an 8-channel sample applicator is used for adjusting the sample application volume to 4 mu L, and the PCR master mix solution is added into each sample application hole of a 384-well plate. The 384 well plate is the PCR reaction plate.
(3) Taking out the prepared DNA sample 96-well plate, adjusting the sample adding volume to 1 mu L by using an 8-channel sample adding device, adding the sample to a corresponding 384PCR reaction plate, attaching a sealing film, shaking and uniformly mixing, and then performing air-shaking.
(4) The PCR conditions were set on a 384 well plate compatible PCR apparatus as shown in Table 4.
TABLE 4PCR reaction conditions
Alkaline phosphatase treatment of PCR products
(1) After the PCR reaction was completed, 384 reaction plates were removed and emptied.
(2) An alkaline phosphatase-treated reaction solution, SAP Mix, was prepared, and is shown in Table 5.
TABLE 5 alkaline phosphatase treatment reaction solution SAP Mix
| SAP Mix | For each reaction (. Mu.L) |
| Water and its preparation method | 1.53 |
| SAP Buffer(10x) | 0.17 |
| SAP enzyme (1.7U/ul) | 0.3 |
| Total volume of | 2 |
(3) And (3) vibrating and uniformly mixing the prepared SAP Mix solution, then placing the mixture in 8 rows of PCR tubes for standby, adjusting the sample adding volume to 2 mu L by using an 8-channel sample adding device, and adding the SAP Mix into a 384-hole PCR reaction plate. Attaching a sealing film, vibrating and uniformly mixing, and then throwing. The total volume of the reaction system was 7. Mu.l (5. Mu.l of PCR product, 2. Mu.l of SAP mix).
(4) The 384 well plates were placed on a 384 well compatible PCR instrument and the PCR reaction conditions were set as shown in table 6.
TABLE 6 PCR reaction conditions
| Temperature (. Degree. C.) | Time (minutes) | Circulation |
| 37 | 40 | 1 |
| 85 | 5 | 1 |
| 4 | ∞ | 1 |
The PCR instrument was started to perform alkaline phosphatase treatment reaction.
6. Single base extension
(1) After the completion of the alkaline phosphatase treatment, the 384-well reaction plate was removed and then was emptied, and a single-base extension reaction was carried out, whereby the total volume of the reaction system was 9. Mu.l.
(2) A single base extension reaction solution, EXTEND Mix, was prepared as shown in Table 7. ( Note that: extend primer Mix and Well number must be correctly corresponding to each other )
TABLE 7 Single base extension reaction solution EXTEND Mix
| EXTEND Mix | For each reaction (. Mu.L) |
| Water | 0.619 |
| Extend primer Mix | 0.94 |
| iPLEX Buffer plus | 0.2 |
| iPLEX terminator | 0.2 |
| iPLEX enzyme | 0.041 |
| Total volume of | 2 |
(3) And (3) vibrating and uniformly mixing the prepared EXTEND Mix solution, then placing the mixed solution in 8 rows of PCR tubes for standby, adjusting the sample adding volume to 2 mu L by using an 8-channel sample adding device, and correspondingly adding the EXTEND Mix into a 384-hole reaction plate. For each reaction well, the single base extension reaction system contained 7. Mu.l of SAP-treated PCR product and 2. Mu.l of EXTEND Mix solution, 9ul overall.
(4) The 384 well plates were placed on a 384 well compatible PCR instrument and PCR reaction conditions were set as shown in table 8.
TABLE 8 PCR reaction conditions
The PCR instrument was started to perform a single base extension reaction.
7. Resin purification
The reaction product was diluted with 16. Mu.l of water, and the diluted product was desalted by using a resin.
8. Chip sample application
And (3) the desalted sample is spotted on a sample target and naturally crystallized.
9. Mass spectrometry detection
And (5) performing mass spectrum detection on the machine, and collecting data.
The results show that the identification rate of the method is more than 85% (which means that 100 sheep to be tested can be identified according to the method, and more than 85 sheep can be identified), and the identification rate is shown in Table 9. The SNP locus combination genotypes shown in Table 1 of the Tan sheep and the non-Tan sheep are within the corresponding genotypes shown in Table 1, and the ratio thereof is shown in Table 10.
TABLE 9 identification rate of the method of the invention
| SNP locus combination | Identification rate of non-Tan sheep | Identification rate of sheep |
| Group 1 | 85％ | 89％ |
| Group 2 | 87％ | 91％ |
| Group 3 | 88％ | 90％ |
| Group 4 | 86％ | 90％ |
| Group 5 | 87％ | 90％ |
| Group 6 | 88％ | 91％ |
| Group 7 | 86％ | 88％ |
| Group 8 | 87％ | 93％ |
| Group 9 | 86％ | 87％ |
| Group 10 | 86％ | 89％ |
| Group 11 | 88％ | 89％ |
Table 10 Sequencom SNP detection results of sheep and non-sheep (proportion of 5 typing site genotype)
Note that: the genotypes of the tables are shown in Table 1.
The results shown in Table 9 demonstrate that the results of the genotyping test of the sheep and the non-sheep shown in Table 1 obtained in example 1 are accurate and reliable in genotype. The invention successfully obtains SNP locus combination for identifying the Tan sheep and the non-Tan sheep, and provides support for the identification, the seed conservation and the genetic breeding of the Tan sheep in the future.
Claims (8)
1. A method for identifying sheep and non-sheep based on genotyping comprises the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the following (a 1) in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; and then determining whether the sheep to be tested is a Tan sheep according to the following steps: if the genotype of the sheep to be detected accords with the standard genotype of the Tan sheep in the following (b 1), the sheep to be detected is or is candidate to be Tan sheep; if the genotype of the sheep to be tested accords with the standard genotype of the non-Tan sheep in the following (b 1), the sheep to be tested is or is candidate to be the non-Tan sheep;
(a1) Contains 5 SNP loci, namely CM001598.2:69009397, CM001602.2:49927473, CM001603.2:38707088, CM001603.2:46619514 and CM001606.2:34435705;
the physical position of each SNP locus is determined based on the comparison of the standard sequence of the whole genome of the Tan sheep, and the version number of the standard sequence of the whole genome of the Tan sheep is Ovis. Aries V4.0;
CM001598.2:69009397 is located on chromosome 17 at position 69009397, and its deoxynucleotide is A or C;
CM001602.2:49927473 is located on chromosome 21 at position 49927473, and its deoxynucleotide is T or C;
CM001603.2:38707088 is located on chromosome 22 at position 38707088, and its deoxynucleotide is T or C;
CM001603.2:46619514 is located on chromosome 22 at position 46619514, and its deoxynucleotide is A or G;
CM001606.2:34435705 is located on chromosome 25 at position 34435705, and its deoxynucleotide is C or G;
(b1) The standard genotype of the non-beach sheep 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; the standard genotype of the sheep 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 ggcg; aa_cc_cc ggcc; 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.
2. A method for identifying a beach sheep based on genotyping, comprising the following steps: detecting the nucleotide at each SNP locus in the SNP locus combination shown in the (a 1) in the claim 1 in the genome of the sheep to be detected, and determining the genotype of the sheep to be detected; if the genotype of the sheep to be tested meets the standard genotype of the Tan sheep in (b 1) in claim 1, the sheep to be tested is or is candidate to be Tan sheep.
3. Use of a substance for detecting a nucleotide at each of the SNP sites in the combination of SNP sites as set forth in claim 1 in sheep genome for identifying a beach sheep and/or a non-beach sheep, or for preparing a kit for identifying a beach sheep and/or a non-beach sheep.
4. A use according to claim 3, characterized in that: the substances are PCR primers and/or sequencing primers designed for SNP sites to be detected.
5. The use according to claim 4, characterized in that: the primers used for detecting the CM001598.2:69009397 are two single-stranded DNA shown as SEQ ID No.67 and SEQ ID No. 68;
the primers used for detecting the CM001602.2:49927473 are two single-stranded DNA shown as SEQ ID No.76 and SEQ ID No. 77;
the primers used for detecting the CM001603.2:38707088 are two single-stranded DNA shown as SEQ ID No.79 and SEQ ID No. 80;
The primers used for detecting the CM001603.2:46619514 are two single-stranded DNA shown as SEQ ID No.82 and SEQ ID No. 83;
the primers used for detecting the CM001606.2:34435705 are two single-stranded DNAs shown as SEQ ID No.85 and SEQ ID No. 86.
6. The use according to claim 5, characterized in that: the primer for detecting the CM001598.2:69009397 also comprises single-stranded DNA shown as SEQ ID No. 69;
the primer for detecting the CM001602.2:49927473 also comprises single-stranded DNA shown in SEQ ID No. 78;
the primer for detecting the CM001603.2:38707088 also comprises single-stranded DNA shown in SEQ ID No. 81;
the primer for detecting the CM001603.2:46619514 also comprises single-stranded DNA shown in SEQ ID No. 84;
the primer used for detecting the CM001606.2:34435705 also includes single-stranded DNA shown in SEQ ID No. 87.
7. A set of primers for detecting a nucleotide at each SNP site in the combination of SNP sites as set forth in claim 1 (a 1) in sheep genome;
the primer for detecting each SNP site is as set forth in claim 5 or 6.
8. Use of a polymorphism of a nucleotide at each SNP site in the combination of SNP sites as set forth in claim 1 in sheep genome for identifying a beach sheep and/or a non-beach sheep.
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