CN111172241A - Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof - Google Patents

Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof Download PDF

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CN111172241A
CN111172241A CN201911255618.1A CN201911255618A CN111172241A CN 111172241 A CN111172241 A CN 111172241A CN 201911255618 A CN201911255618 A CN 201911255618A CN 111172241 A CN111172241 A CN 111172241A
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nucleotide sequence
primer
sequence
microsatellite
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CN111172241B (en
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马惠海
闫守庆
胡明月
赵中利
曹阳
刘正喜
鲍志鸿
巩俊明
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Jilin Academy of Agricultural Sciences
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Jilin Academy of Agricultural Sciences
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The invention provides a sheep four-base repeated microsatellite marker, a screening method, a primer set and application thereof, and relates to the technical field of molecular biology DNA marker technology and genetics application. The screening method comprises the steps of searching microsatellite markers with four-base repetition on all chromosomes in a sheep whole genome, and then designing primers for amplifying the microsatellite markers; and (3) reserving a single and clear microsatellite marker of an amplification band after amplification, then amplifying the microsatellite locus of the sheep sample by using a forward primer with a 5 'end fused with a forward universal primer, a forward universal primer with a 5' end labeled with a fluorescent marker and a reverse primer, and carrying out polymorphism identification on the microsatellite marker to obtain the sheep four-base repeated microsatellite marker. The screening method can obtain the sheep four-base microsatellite markers with good polymorphism.

Description

Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof
Technical Field
The invention relates to the technical field of molecular biology DNA marking technology and genetics application, in particular to a sheep four-base repeated microsatellite marker and a screening method, a primer group and application thereof.
Background
Microsatellite markers (Microsatellite markers) are known for length polymorphism, have the advantages of good stability, rich polymorphic information content, co-dominant inheritance, good repeatability, easy realization of analysis automation and the like, and cannot be replaced by other genetic markers, so that the Microsatellite markers are widely applied to the aspects of genetic map construction, genetic diversity evaluation, quantitative character positioning, paternity test, individual identification and the like of animals and plants.
Regarding the microsatellite molecular genetic marker technology, the most important thing is how to obtain microsatellite loci which have a large number of markers, high genetic polymorphism and can be stably amplified, and the application of the microsatellite loci in population genetics research is extremely important. At present, the sheep have more microsatellite loci, but basically all the microsatellite loci are two-base repeat units, and the sheep four-base repeat microsatellite markers are not reported at present. Compared with a two-base or three-base repeated microsatellite locus, the four-base repeated microsatellite locus has the greatest advantage that shadow bands generated by slippage of a PCR amplification chain are not easy to generate in the PCR amplification process, so that the four-base repeated microsatellite locus is easier to read alleles from an electrophoresis peak image for statistical analysis. The sheep microsatellite marker plays an important role in the aspects of sheep genetic diversity analysis, genetic relationship identification, individual identification, paternity identification, marker-assisted selection and the like, so that the development of the four-base repetitive microsatellite marker is necessary.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a method for screening a four-base repeat microsatellite marker in sheep, which enables screening of a four-base repeat microsatellite marker having a good polymorphism.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides a method for screening a sheep four-base repeat microsatellite marker, which comprises the following steps:
(a) performing BLAST comparison in a sheep whole genome, and searching for microsatellite markers with four base repeats on all chromosomes;
(b) designing primers for amplifying the microsatellite markers screened in step (a), wherein the primers comprise a forward primer and a reverse primer;
(c) performing PCR amplification on the microsatellite marker obtained by screening in the step (a), and screening out the microsatellite marker with single and clear PCR amplification product band;
(d) selecting at least 20 sheep samples, and performing three-primer PCR amplification on the microsatellite marker obtained by screening in the step (c), wherein the three-primer PCR amplification comprises the following steps of amplifying the microsatellite marker by using the following primers: the 5 'end of the forward primer is fused with a forward universal primer, and the 5' end of the forward universal primer and the reverse primer are marked with fluorescent labels;
(e) performing polymorphism identification on the microsatellite marker by analyzing the amplification product of the step (d) to obtain the sheep four-base repetitive microsatellite marker.
Compared with the prior art, the invention has the following beneficial effects:
the screening method of the sheep four-base repeated microsatellite marker provided by the invention starts from the existing sheep whole genome sequence, screens a plurality of candidate microsatellite markers, and then screens the four-base repeated microsatellite markers by analyzing the specificity of the bands of the amplification products of the candidate sites and the polymorphism of the candidate sites. The microsatellite markers obtained by screening by the method have good polymorphism, and lay a foundation for the researches such as paternity test, population genetic diversity, sex identification and the like of sheep. And by using the three-primer PCR, the condition that each forward primer is marked with a fluorescent label is avoided, the analysis cost is reduced, and the analysis flux is improved. Compared with the sheep four-base repeated microsatellite marker provided by the invention, the four-base repeated microsatellite marker PCR amplification is not easy to generate shadow bands, and the data is convenient and accurate to read.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an agarose gel electrophoresis of a portion of a sheep DNA sample extracted according to an embodiment of the present invention;
FIG. 2 is a partial agarose gel electrophoresis of the microsatellite marker PCR amplification of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, the invention provides a method for screening a sheep four-base repeat microsatellite marker, which comprises the following steps:
(a) performing BLAST alignment in a sheep whole genome, searching potential four-base repeat microsatellite markers on all chromosomes, preferably microsatellite markers with the number of the repetitive units of the core sequence being more than or equal to 7, and counting the distribution condition of the chromosomes. In order to ensure the application of the screened microsatellite markers in the aspects of genetic diversity and paternity test, the problems that the microsatellite markers on a single chromosome are too many, the distance between two microsatellite markers on the chromosome is too close and the like are avoided, and the number of the microsatellite markers on the same chromosome is preferably less than or equal to 3; the distance between any two microsatellite markers on the same chromosome is preferably at least 200 kb.
(b) Designing primers for amplifying the microsatellite markers screened in step (a), wherein the primers comprise a forward primer and a reverse primer. The tools used to design primers are not limiting in the present invention, and can be implemented using software design tools that are conventional in the art. When designing the primer, the following aspects need to be noted: (1) conservative analysis is carried out on two side sequences of a four-base repeated sequence of a microsatellite locus, aiming at each region containing four-base repetition, the two side sequences are required to be compared to determine that the region where a primer is located is a single sequence in the whole genome, namely a highly repeated sequence is required to be eliminated, and the design of the primer by taking the highly repeated sequence as a target sequence can cause a large amount of PCR non-specific amplification. (2) The parameters of the primers are preferably as follows: the length of the primer is preferably 18-30 bp; the GC content of the primer is preferably 35-60%; the annealing temperature of the primer is preferably 50-65 ℃; the length of the amplified fragment of the primer is preferably 100-400 bp. In addition, the internal structure of the primer, such as hairpin structure, primer dimer and the like, is considered.
(c) And (b) carrying out PCR amplification on the microsatellite marker obtained by screening in the step (a), carrying out electrophoresis amplification on the amplified product, imaging in a gel imaging system, removing sites without amplified bands, unclear amplified bands and non-specific amplified bands, and reserving the microsatellite marker with a single and clear amplified band for next analysis. Preferably, the amplification product of the microsatellite locus with a single and clear PCR amplification band is recovered, the amplification product is sequenced after recovery to obtain the sequence information of the locus, and the sequence information is compared with the sheep genome to determine that the sequence of the screened locus is consistent with the sequence of the sheep genome.
(d) Selecting at least 20 sheep samples, and performing three-primer PCR amplification on the microsatellite marker obtained by screening in the step (c) in each sheep sample, wherein three primers used in the three-primer PCR amplification are as follows: the forward primer designed in the step (b), and a forward universal primer sequence is fused at the 5' end of the forward primer; a single forward universal primer, and the 5' end of the forward universal primer is marked by a fluorescent label; and the reverse primer designed in the step (b). It should be noted that, the forward primer used in the amplification in step (c) may not be fused with the forward universal primer sequence at the 5' end, but a forward primer fused with the forward universal primer sequence at the 5' end may be directly synthesized in the screening process, and then a forward primer fused with the forward universal primer at the 5' end is also used in the amplification in step (b), so as to reduce the number of primers to be synthesized in the screening process. The principle of three-primer PCR amplification is as follows: in the PCR process, a forward primer with a 5 'end fused with a forward universal primer is used for fusing a forward universal primer sequence into a target DNA fragment, so that the 5' end of an amplification product is fused with a forward universal primer sequence; then, the forward universal primer marked with fluorescence at the 5 'end takes a DNA fragment fused with the forward universal primer sequence at the 5' end as a template, so that the amplified product is marked with fluorescence.
The forward universal primer may be selected from universal primers conventional in the art, and the present invention is not limited thereto, and some examples of the forward universal primer include M13 universal primer, and the M13 universal primer may be, for example, but not limited to, M13(-47), M13(-40), or M13 (-21). The forward universal primer used in some embodiments includes M13(-21), and the sequence of M13(-21) is shown in SEQ ID NO.97 (5'-TGTAAAACGACGGCCAGT-3').
When the forward universal primers used to amplify all microsatellite markers are labeled with only one color of fluorescent label, only one site can be analyzed per channel at a subsequent analysis step. Therefore, in some preferred embodiments, in order to increase the throughput in the subsequent analysis, different fluorescent labels are labeled at the 5' end of the forward universal primer, i.e., a single forward universal primer is labeled with only one color fluorescent label, but the fluorescent labels of the forward universal primers for amplifying different sites are different. Preferably, 2-5 fluorescent markers with different colors are used, and in the embodiment, 2-5 sites can be analyzed simultaneously in each channel, so that the screening efficiency is improved. Preferably, 3 fluorescent labels of different colors are used, such as FAM, Tamra or Hex labeling the forward universal primer, respectively, to both improve the throughput of the assay and ensure the accuracy of the assay.
In order to analyze the polymorphism of each site subsequently, a plurality of sites of the sheep sample need to be amplified in the step, the sheep sample size is preferably 25-35, the screening workload is increased when the sample size is too large, and the accuracy of the subsequent polymorphism analysis is reduced when the sample size is too small.
During three-primer PCR amplification, the molar ratio of the forward primer with the forward universal primer fused to the 5 'end, the forward universal primer with the 5' end labeled with the fluorescent label and the reverse primer in a three-primer PCR amplification reaction system is preferably (0.8-1.2): (1.2-2): 2. examples of preferred reaction systems are as follows: 2 XPCR mix 7. mu.L, forward primer 0.2. mu.L (12.5. mu.M) with forward universal primer fused to the 5' end, fluorescence-labeled universal primer 0.2. mu.L (25. mu.M) and reverse primer 0.2. mu.L (25. mu.M), made up to a total volume of 15. mu.L with double distilled water.
The amplification procedure of the three-primer PCR amplification comprises the steps of firstly amplifying for 25-35 cycles at the annealing temperature of the forward primer, and then amplifying for 5-15 cycles at the annealing temperature of the forward universal primer. Wherein the annealing temperature of the forward primer is preferably 57-59 ℃; the annealing temperature of the forward universal primer is preferably 52-54 ℃. Examples of preferred reaction procedures are as follows: firstly, pre-denaturing the template at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 45s, and circulating for 30 times; then continuing to denature at 94 ℃ for 30s, renaturate at 53 ℃ for 45s, extend at 72 ℃ for 45s, and circulating for 8 times; finally, extension at 72 ℃ is maintained for 10min, and the amplification product is stored at 4 ℃.
(e) Analyzing the amplification product in the step (d), and carrying out polymorphism identification on the microsatellite marker to obtain the sheep four-base repeated microsatellite marker. Preferably, the amplified product in the step (d) is subjected to capillary electrophoresis and data is collected, then the collected data is subjected to fluorescence color separation, the size of an allele is calculated by using a typing standard substance to obtain the genotyping data of each genome, and then polymorphism identification is carried out on the sheep four-base repeat microsatellite. Wherein capillary electrophoresis is preferably performed and data is collected using an ABI3730 DNA sequencer; fluorescence color separation of the collected data is preferably performed using GeneMaker software; the typing standard is preferably a GeneScan 600 molecular internal standard. Polymorphism identification of sheep four-base repeat microsatellites involves analysis of one or more of allele factors, polymorphic information content, expected heterozygosity, observed heterozygosity, and Hardy-Weinberg equilibrium at the microsatellite locus. Examples of polymorphism identification optionally include analysis of the allelic factors, polymorphic information content, and expected heterozygosity at satellite sites; optionally including analyzing the polymorphic information content of the satellite sites, the desired heterozygosity, the observed heterozygosity, and the Hardy-Winberg equilibrium; optionally including allelic factors, polymorphic information content, expected heterozygosity, observed heterozygosity, and Hardy-Winberg equilibrium testing of the microsatellite loci to ensure that the screened loci have sufficient polymorphism. The number of alleles is preferably at least 5; the polymorphic information content is preferably at least 0.5; preferably, the observed heterozygosity is at least 0.5; the desired heterozygosity is preferably at least 0.5; the P value of the Hardy-Weinberg equilibrium is preferably at least 0.05.
According to another aspect of the present invention, the present invention further provides a set of sheep four-base repeat microsatellite markers, wherein the sheep four-base repeat microsatellite markers provided by the present invention mainly comprise microsatellite markers selected by the screening method of the sheep four-base repeat microsatellite markers, and comprise one or more of the microsatellite markers selected from the group consisting of Ovr1402, Ovr1602, Ovr2001, Ovr0501, Ovr2101, Ovr0201, Ovr0702, Ovr0503, Ovr1002, Ovr2204, Ovr0502, Ovr1401, Ovr2304, Ovr1901, Ovr2604, Ovr2601, Ovr1101, Ovr1003, Ovr1601, Ovr2401, Ovr2202, Ovr0602, Ovr2103, Ovr1702, Ovr1501, Ovr2606, Ovr1001, Ovr1801, Ovr0801, Ovr0802 and 0902:
the microsatellite repeat motif of Ovr1402 is (ATAG)9The nucleotide sequence of Ovr1402 is the sequence shown in SEQ ID NO.1, or the sequence with homology of more than 90% with SEQ ID NO. 1; the microsatellite repeat motif of Ovr1602 is (CTAT)7The nucleotide sequence of Ovr1602 is shown as SEQ ID NO.2, or is a sequence with homology of more than 90% with SEQ ID NO. 2; ovr2001 has a microsatellite repeat motif of (ATAG)6The nucleotide sequence of Ovr2001 is shown as SEQ ID NO.3, or is a sequence with homology of more than 90% with SEQ ID NO. 3; the microsatellite repeat motif of Ovr0501 is(ATAG)12The nucleotide sequence of Ovr0501 is shown as SEQ ID NO.4, or is a sequence with homology of more than 90% with SEQ ID NO. 4; the microsatellite repeat motif of Ovr2101 is (CTAT)5The nucleotide sequence of Ovr2101 is the sequence shown in SEQ ID NO.5, or the sequence with homology of more than 90% with SEQ ID NO. 5; the microsatellite repeat motif of Ovr0201 is (CTAT)8The nucleotide sequence of Ovr0201 is a sequence shown in SEQ ID NO.6, or a sequence with homology of more than 90% with SEQ ID NO. 6; the microsatellite repeat motif of Ovr0702 is (ATAG)15The nucleotide sequence of Ovr0702 is a sequence shown in SEQ ID NO.7, or a sequence with homology of more than 90% with SEQ ID NO. 7; the microsatellite repeat motif of Ovr0503 is (CTAT)7The nucleotide sequence of Ovr0503 is a sequence shown as SEQ ID NO.8, or a sequence with homology of more than 90% with SEQ ID NO. 8; the microsatellite repeat motif of Ovr1002 is (ATAG)13The nucleotide sequence of Ovr1002 is a sequence shown as SEQ ID NO.9, or a sequence with homology of more than 90% with SEQ ID NO. 9; the microsatellite repeat motif of Ovr2204 is (CTAT)9The nucleotide sequence of Ovr2204 is a sequence shown in SEQ ID NO.10, or a sequence which has more than 90 percent of homology with SEQ ID NO. 10; the microsatellite repeat motif of Ovr0502 is (ATAG)7The nucleotide sequence of Ovr0502 is a sequence shown by SEQ ID NO.11, or a sequence with homology of more than 90% with SEQ ID NO. 11; the microsatellite repeat motif of Ovr1401 is (CTAT)7The nucleotide sequence of Ovr1401 is a sequence shown in SEQ ID NO.12, or a sequence with homology of more than 90% with SEQ ID NO. 12; the microsatellite repeat motif of Ovr2304 is (ATAG)11The nucleotide sequence of Ovr2304 is a sequence shown in SEQ ID NO.13, or a sequence with homology of more than 90% with SEQ ID NO. 13; the microsatellite repeat motif of Ovr1901 is (CTAT)7The nucleotide sequence of Ovr1901 is a sequence shown in SEQ ID NO.14, or a sequence with homology of more than 90% with SEQ ID NO. 14; the microsatellite repeat motif of Ovr2604 is (CTAT)10The nucleotide sequence of Ovr2604 is shown as SEQ ID NO.15, or is a sequence with homology of more than 90% with SEQ ID NO. 15; the microsatellite repeat motif of Ovr2601 is (CTAT)10The nucleotide sequence of Ovr2601 is shown as SEQ ID NO.16, or is the same as SEQ ID NO.16Sequences with a source of more than 90%; the microsatellite repeat motif of Ovr1101 is (CTAT)15The nucleotide sequence of Ovr1101 is shown as SEQ ID NO.17, or is a sequence with homology of more than 90% with SEQ ID NO. 17; the microsatellite repeat motif of Ovr1003 is (ATAG)10The nucleotide sequence of Ovr1003 is a sequence shown in SEQ ID NO.18, or a sequence with homology of more than 90% with SEQ ID NO. 18; the microsatellite repeat motif of Ovr1601 is (ATAG)10The nucleotide sequence of Ovr1601 is shown as SEQ ID NO.19, or is a sequence with homology of more than 90% with SEQ ID NO. 19; the microsatellite repeat motif of Ovr2401 is (ATAG)11The nucleotide sequence of Ovr2401 is a sequence shown in SEQ ID NO.20, or a sequence with homology of more than 90% with SEQ ID NO. 20; the microsatellite repeat motif of Ovr2202 is (ATAG)12The nucleotide sequence of Ovr2202 is shown in SEQ ID NO.21, or is a sequence with homology of more than 90% with SEQ ID NO. 21; the microsatellite repeat motif of Ovr0602 is (ATAG)14The nucleotide sequence of Ovr0602 is a sequence shown in SEQ ID NO.22, or a sequence which has more than 90 percent of homology with SEQ ID NO. 22; the microsatellite repeat motif of Ovr2103 is (CTAT)6The nucleotide sequence of Ovr2103 is a sequence shown in SEQ ID NO.23, or a sequence which has more than 90 percent of homology with SEQ ID NO. 23; the microsatellite repeat motif of Ovr1702 is (CTAT)12The nucleotide sequence of Ovr1702 is shown as a sequence shown in SEQ ID NO.24, or a sequence which has more than 90 percent of homology with SEQ ID NO. 24; the microsatellite repeat motif of Ovr1501 is (CTAT)13The nucleotide sequence of Ovr1501 is shown as SEQ ID NO.25, or is a sequence with homology of more than 90% with SEQ ID NO. 25; the microsatellite repeat motif of Ovr2606 is (ATAG)6The nucleotide sequence of Ovr2606 is shown as SEQ ID NO.26, or is a sequence with homology of more than 90% with SEQ ID NO. 26; the microsatellite repeat motif of Ovr1001 is (ATAG)11The nucleotide sequence of Ovr1001 is a sequence shown as SEQ ID NO.27, or a sequence with homology of more than 90% with SEQ ID NO. 27; the microsatellite repeat motif of Ovr1801 is (CTAT)13The nucleotide sequence of Ovr1801 is a sequence shown in SEQ ID NO.28, or a sequence with homology of more than 90% with SEQ ID NO. 28; the microsatellite repeat motif of Ovr2102 is (ATAG)10The nucleotide sequence of Ovr2102 is a sequence shown in SEQ ID NO.29, or a sequence which has more than 90 percent of homology with SEQ ID NO. 29; ovr0801 microsatellite repeat motif (ATAG)15The nucleotide sequence of Ovr0801 is a sequence shown in SEQ ID NO.30 or a sequence with homology of more than 90% with SEQ ID NO. 30; ovr0802 microsatellite repeat motif (ATAG)13The nucleotide sequence of Ovr0802 is a sequence shown in SEQ ID NO.31, or a sequence with homology of more than 90% with SEQ ID NO. 31; the microsatellite repeat motif of Ovr0902 is (ATAG)8The nucleotide sequence of Ovr0902 is shown as SEQ ID NO.32, or is a sequence which has more than 90 percent of homology with SEQ ID NO. 32.
Wherein "homology" refers to the similarity of a nucleotide sequence to a target sequence. Differences between a sequence having more than 90% homology to the target sequence and the target sequence include, but are not limited to, deletions, mutations, deletions or additions of one or more nucleotides, such as additions or deletions of one or more nucleotides to the flanking sequence of the microsatellite locus, which changes result in a nucleotide sequence which differs from the nucleotide sequence of the target sequence by more than 90%, for example, but not limited to, 90%, 92%, 95%, 98% or 99% similarity, with the same repeat motif. The homology between the nucleotide sequence and the target sequence can be achieved by software routine in the art, and the present invention is not limited thereto.
The group of sheep four-base repetitive microsatellite markers provided by the invention at least comprises one of Ovr1402, Ovr1602, Ovr2001, Ovr0501, Ovr2101, Ovr0201, Ovr0702, Ovr0503, Ovr1002, Ovr2204, Ovr0502, Ovr1401, Ovr2304, Ovr1901, Ovr2604, Ovr2601, Ovr1101, Ovr1003, Ovr1601, Ovr2401, Ovr2202, Ovr0602, Ovr2103, Ovr1702, Ovr1501, Ovr2606, Ovr1001, Ovr1801, Ovr2102, Ovr0801, Ovr0802 and Ovr 0902; multiple microsatellite markers can also be used in combination to improve the detection accuracy, such as 20 microsatellite loci Ovr1402, Ovr1602, Ovr0501, Ovr0201, Ovr0702, Ovr1002, Ovr2204, Ovr0502, Ovr2304, Ovr1901, Ovr2601, Ovr1101, Ovr1003, Ovr2401, Ovr1702, Ovr1501, Ovr1801, Ovr2102, Ovr0801 and Ovr0802 are used in combination, and the like. Preferably, the kit comprises at least thirteen sheep four-base repetitive microsatellite markers Ovr1402, Ovr1602, Ovr0501, Ovr0201, Ovr0702, Ovr1002, Ovr2204, Ovr0502, Ovr2401, Ovr1702, Ovr1501, Ovr1801 and Ovr2102, and has better paternity test effect.
According to another aspect of the invention, the invention also provides a primer group for amplifying the sheep four-base repeat microsatellite marker, wherein one or more of the following primer pairs can be selected according to the specific microsatellite marker to be amplified.
Primer pair for amplifying Ovr 1402: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 33; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 34; primer pair for amplifying Ovr 1602: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 35; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 36; primer pair for amplification of Ovr 2001: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 37; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 38; primer pair for amplification of Ovr 0501: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 39; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 40; primer pair for amplifying Ovr 2101: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 41; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 42; primer pair for amplifying Ovr 0201: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 43; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 44; primer pair for amplifying Ovr 0702: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 45; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 46; primer pair for amplification of Ovr 0503: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 47; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 48; primer pair for amplifying Ovr 1002: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 49; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 50; primer pair for amplifying Ovr 2204: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 51; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 52; primer pair for amplification of Ovr 0502: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 53; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 54; primer pair for amplification of Ovr 1401: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 55; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 56; primer pair for amplifying Ovr 2304: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 57; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 58; primer pair for amplifying Ovr 1901: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 59; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 60; primer pair for amplification of Ovr 2604: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 61; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 62; primer pair for amplifying Ovr 2601: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 63; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 64; primer pair for amplification of Ovr 1101: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 65; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 66; primer pair for amplifying Ovr 1003: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 67; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 68; primer pair for amplifying Ovr 1601: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 69; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 70; primer pair for amplifying Ovr 2401: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 71; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 72; primer pair for amplification of Ovr 2202: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 73; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 74; primer pair for amplification of Ovr 0602: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 75; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 76; primer pair for amplifying Ovr 2103: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 77; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 78; primer pair for amplification of Ovr 1702: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 79; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 80; primer pair for amplifying Ovr 1501: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 81; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 82; primer pair for amplification of Ovr 2606: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 83; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 84; primer pair for amplifying Ovr 1001: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 85; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 86; primer pair for amplifying Ovr 1801: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 87; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 88; primer pair for amplifying Ovr 2102: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 89; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 90; primer pair for amplifying Ovr 0801: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 91; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 92; primer pair for amplifying Ovr 0802: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 93; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 94; primer pair for amplification of Ovr 0902: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 95; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 96.
In some preferred embodiments, the primer pairs in the primer set are labeled with a label. The primer with the label means that at least one of the forward primer and the reverse primer in the primer pair has the label, or the forward primer and the reverse primer can have the label at the same time. The purpose of labeling the primer pair with a label is to label the amplification product for convenient subsequent detection, and the specific label may be selected from labels conventional in the art, which is not limited by the present invention. It is understood that when a plurality of primer pairs are included in a primer set, the labels labeled by the different primer pairs may be the same or different. Preferably, a pair of fluorescently labeled primers is used for convenient detection.
According to another aspect of the present invention, the present invention also provides a kit comprising the above primer set. The kit can be used for amplifying the sheep four-base repeat microsatellite markers. The kit can also comprise buffer solution for PCR reaction, including but not limited to PCR buffer solution, dNTP, salt, DNA polymerase and other reaction reagents, and can also contain conventional experiment consumables and the like.
According to another aspect of the invention, the invention also provides a screening method of the sheep four-base repeated microsatellite marker, the primer group or the kit in paternity test of sheep. The screening method and the four-base repetitive microsatellite obtained by screening have good polymorphism, the amplified band is not easy to generate shadow band, the data is convenient and accurate to read, and the method is suitable for being used as a genetic molecular marker to be applied to paternity test of sheep.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
Examples
1 materials and methods
1.1 Experimental materials: the DNA sample in the invention is from QINGLIN sheep farming and animal husbandry Limited liability company, and 40 small tailed Han sheep blood samples are collected.
1.2 Main reagents and instruments for the experiment
1.2.1 Main test reagents: DNA extraction kit (beijing tiangen biotechnology); 50 XTAE buffer solution preparation: weigh Tris 242g, Na2EDTA·2H2Adding 800mL of deionized water into a 1L beaker by using 37.2g of O, fully stirring uniformly, adding 57.1mL of glacial acetic acid to fully dissolve, adding deionized water to a constant volume of 1L, and storing at room temperature (when agarose gel electrophoresis is needed, diluting a small amount to 1 XTAE for later use).
1.2.2 Main laboratory instruments: a pipettor, a table type high-speed centrifuge, a PCR amplification instrument, a gel imaging system, an electrophoresis instrument, a 4 ℃ refrigerator, an autoclave, an electric heating blowing drying box, an electronic balance, a microwave oven, an agarose horizontal electrophoresis tank and a vortex oscillator.
1.3 Experimental methods
1.3.1 extraction of blood DNA: the DNA of the small-tailed Han sheep blood is extracted by adopting a blood DNA extraction kit of Beijing Tiangen Biotech company and is preserved at the temperature of-20 ℃.
1.3.2 screening of four base microsatellite sequences: performing BLAST comparison in a sheep whole genome, searching a four-base repetitive sequence chromosome region existing in the sheep genome, obtaining a site with the number of core sequence repetitive units more than or equal to 7 and two end sequences being non-specific repetitive sequences, and counting the distribution condition of the chromosome.
1.3.3 microsatellite primer design: software Primer 5.0 was used to design primers flanking the microsatellite four base repeat sequence, taking care to design the primers: conservative analysis of sequences on two sides of a four-base repetitive sequence of a microsatellite locus requires that for each region containing four-base repeats, the sequences on two sides are compared to determine that the region where a primer is located is a single sequence in the whole genome, namely a highly repetitive sequence needs to be eliminated, because designing the primer by using the highly repetitive sequence as a target sequence can cause a large amount of PCR (polymerase chain reaction) non-specific amplification.
The length of the designed primer is 18-30 bp, the GC content is 35-60%, the annealing temperature is 50-65 ℃, and the length of the amplified fragment is 100-400 bp. Wherein the 5' end of the forward primer at each site is added with M13(-21) universal primer sequence. Further, M13(-21) universal primers were synthesized individually, each of which had a fluorescent label added to the 5' end, and the fluorescent labels were 3 types, such as FAM, Tamra, and Hex.
In order to ensure the application of the microsatellite marker developed in the embodiment in the aspects of genetic diversity and paternity test, the problems that the microsatellite loci on a single chromosome are too many, the distance between two microsatellite loci on the chromosome is too short and the like need to be avoided. In addition, the internal structure of the primer, such as hairpin structure, primer dimer and the like, is considered.
1.3.4 PCR amplification of fragments of interest: randomly selecting 2 samples for PCR amplification, wherein the reaction system is as follows: 2 XPCRmix 10. mu.L, forward and reverse primers 0.2. mu.L each (25. mu.M), made up to 20. mu.L with double distilled water. The amplification procedure was: first, pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s and annealing at 58 ℃ for 30 s; stretching at 72 ℃ for 30s, and circulating for 30 times; final extension at 72 deg.c for 5min and storing PCR amplified product at 4 deg.c.
1.3.5 identification of PCR amplification specificity of microsatellite marker primers, namely separating and analyzing a PCR product by 2.5 percent agarose gel electrophoresis, imaging in a gel imaging system, removing sites without an amplification band, unclear amplification band and non-specific amplification band, recovering an amplification product of a microsatellite site with a single and clear PCR amplification band, connecting the recovered product with a pMD18-T plasmid, transforming DH5 α escherichia coli, identifying the size of an insert fragment by PCR amplification, sequencing positive clones, and obtaining 32 microsatellite sites with amplification specificity.
1.3.6 amplification and detection of fluorescence-labeled microsatellite primers: and carrying out PCR amplification on 30 sheep samples by using the selected microsatellite loci with PCR amplification specificity. The reaction system is as follows: 2 XPCR mix 7. mu.L, forward primer 0.2. mu.L (12.5. mu.M), M13(-21) fluorescent-labeled universal primer 0.2. mu.L (25. mu.M), reverse primer 0.2. mu.L (25. mu.M), made up to a total volume of 15. mu.L with double distilled water. The amplification procedure was: firstly, pre-denaturing the template at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 45s, and circulating for 30 times; then continuing to denature at 94 ℃ for 30s, renaturate at 53 ℃ for 45s, extend at 72 ℃ for 45s, and circulating for 8 times; finally, extension at 72 ℃ is maintained for 10min, and the amplification product is stored at 4 ℃. And (3) performing ABI3730 DNA sequencer capillary electrophoresis on the PCR product, and automatically detecting the separated fluorescence labeling PCR amplified DNA fragment by equipment and storing data.
1.3.7 statistical analysis of data: fluorescence color separation is carried out on the collected data by using GeneMaker software, and the size of the allele is calculated with a molecular internal standard of a typing standard GeneScan 600, so that the genotyping data of each locus are determined. Allele counts (n), Polymorphic Information Content (PIC), expected heterozygosity (He) and observed heterozygosity (Ho) at each site were analyzed using the CERVUS3.0 software, among others. The Hardy-Weinberg equilibrium (HWE) test was performed for each microsatellite locus using Genpop 3.4 software.
1.3.8 use of microsatellite markers for paternity testing: the microsatellite markers are used for genotyping 2 offspring (S1 and S2), 2 female parents (M1 and M2) and 6 ram (P1 and P2) individuals in 2 families, the relativity identification is carried out through Cervus3.0 software, and the paternity identification result is compared with a breeding farm pedigree to verify the accuracy of the paternity.
2 results
2.1 sheep genome DNA extraction: after DNA is extracted from the collected sheep blood, the result is observed by a gel imaging system by using an agarose gel electrophoresis method. In this embodiment, the quality and integrity of DNA of 40 sheep samples are detected, as shown in FIG. 1, lane M in FIG. 1 is DL15000 DNA Marker, and lanes 1-24 are sheep individual numbers. The result shows that the DNA quality of the sheep sample is better, and the next experiment can be carried out.
2.2 primer design and amplification identification: through four-base microsatellite marker whole genome screening, primer design, PCR amplification and cloning, and sequence analysis, 32 pairs of microsatellite marker primers capable of specific amplification are screened out in the embodiment, as shown in Table 1. The agarose gel electrophoresis image of the partial microsatellite marker PCR amplification is shown in FIG. 2, wherein a lane M is DL15000 DNAmarker, and microsatellite markers in lanes 1-16 are Ovr1402, Ovr1602, Ovr2001, Ovr0501, Ovr2101, Ovr0201, Ovr0702, Ovr0503, Ovr1002, Ovr2204, Ovr0502, Ovr1401, Ovr2304, Ovr1901, Ovr2604 and Ovr2601 in sequence.
TABLE 132 characterisation of sheep four base repeat microsatellite marker primers
2.3 genetic diversity analysis of microsatellite markers
The analysis result of the screened four-base repeated microsatellite marker genetic information parameters of the sheep is shown in Table 2, the allele factors of 32 microsatellite markers are all larger than 5, the allele factors are between 5 and 15, 272 alleles are co-amplified by 32 markers, and the average allele factor is 8.500; the content of Polymorphic Information (PIC) is between 0.566 and 0.898, the observed heterozygosity (Ho) ranges from 0.548 to 0.903, the expected heterozygosity (He) ranges from 0.631 to 0.921, and the average expected heterozygosity is 0.7799; the Hardy-Winberg equilibrium test result shows that 32 sites are in a genetic equilibrium state. In conclusion, the 32 microsatellite markers are highly polymorphic and can be applied to identification analysis and genetic diversity analysis of sheep genetic relationship.
Polymorphic information parameter analysis of the 232 microsatellite markers in Table
Note: k is the allelic factor, N is the number of genotyping individuals, Ho is the observed heterozygosity, He is the expected heterozygosity, and PIC is the polymorphic information content.
2.4 paternity test results
The CERVUS software identification result shows that the offspring S1 is in maternal-child relationship with M2, the offspring S2 is in maternal-child relationship with M1, the offspring S1 and S2 is in paternal-child relationship with ram P1, and the offspring S1 and S2 are not in genetic relationship with ram P2 and are matched with the paternity female parent and the male parent in the pedigree.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Jilin province academy of agricultural sciences
<120> sheep four-base repeated microsatellite marker and screening method, primer set and application thereof
<160>97
<170>PatentIn version 3.5
<210>1
<211>139
<212>DNA
<213> sheep (Ovis aries)
<400>1
tgtaaaacga cggccagttt cggtatggta tgcagtaggt atctcctctt atagacagat 60
agatagatag atagatagat agatagatag atagacagag tcataaaatg acgaaaggct 120
aagagggtcc caagtttgt 139
<210>2
<211>145
<212>DNA
<213> sheep (Ovis aries)
<400>2
tgtaaaacga cggccagtat tccccatctc tatctagcag gctagctagt tagctattgt 60
ctatcatcta ttctctctcc atcatctatc tatctatcta tctatctatc tatcacttca 120
tggtaaacag atgtaggaaa cagtg 145
<210>3
<211>130
<212>DNA
<213> sheep (Ovis aries)
<400>3
gacctgcata catctctgtc tgcaaatata gatagataga tagatagata gagcaacagt 60
tttgggaaaa gtaaaccatg gcatctactg tgtgacatag tggccatgtc atagacagca 120
ggagggagat 130
<210>4
<211>160
<212>DNA
<213> sheep (Ovis aries)
<400>4
ttcctagtca gagatgtcac aaaacatcct ttaaatgtga agattcatta aaaatacaga 60
tgaaaagatg aatggataga tagatagata gatagataga tagatagata gatagataga 120
tagagatgta attgtagcgt tttcaaatga ataatgtcca 160
<210>5
<211>149
<212>DNA
<213> sheep (Ovis aries)
<400>5
aatgcctcta cctggagtga tgctgggagg aggcaaccct ggtaatggtt tcatatattt 60
tcctgacttc tgagatatta ttcaagtcat ttctaattga aatatctatc tatctatcta 120
tctatattcc ttagcttccc caatggctc 149
<210>6
<211>160
<212>DNA
<213> sheep (Ovis aries)
<400>6
gcagcctaag cgtgaatgtg aaagatgttt gaagaggcac tgtttgttaa tacctgaaac 60
tgaatcacac atctctgaat ccttagttgt tttatatcta tctatctatc tatctatcta 120
tctatctatc tgtagtttga ccagattgat ttcttgccaa 160
<210>7
<211>202
<212>DNA
<213> sheep (Ovis aries)
<400>7
caatactttg tttttgaggc tggcatgacc cgatagatag atagatagat agatagatag 60
atagatagat agatagatag ataggtagat agctggctgg ctgctggtgc aaacaggaca 120
taaatgtgag gaaatttacc ttaaagacca ctaacttttg gaggggaaga cgtttatata 180
ccaagggata cctgttttca ac 202
<210>8
<211>196
<212>DNA
<213> sheep (Ovis aries)
<400>8
tggttgaaca gcctgtggat ttgaagaaat gagcacaaat cacatttttc tgtctatgta 60
tgtatgtatg tatctatcta tctatctatc tatctatcta tcttctatct ttttatggta 120
gctgcagtct ggttgccagc tgggacaagt gggtagatta atatttgagt ctcagtaaca 180
ttgccaagga ctgggt 196
<210>9
<211>179
<212>DNA
<213> sheep (Ovis aries)
<400>9
aatcaggtgt tgttgtttca ggtgtatagt gtaataattt gatactcact tagatagata 60
gatagataga tagatagata gatagataga tagatagata gatagatata gtacaaaatg 120
atcaacaata aatcattatc atcatacata aacaattatc agcttagatt gatcatagc 179
<210>10
<211>163
<212>DNA
<213> sheep (Ovis aries)
<400>10
aatcaccctt ctcacattca ccctctccca attagaaaaa tatgtaatca tatatctatc 60
tatctatcta tctatctatc tatctatcta tctcctggga tttggcatct gcttcataat 120
tatttaggaa catgattcag cagtttggaa ggaggttgga gga 163
<210>11
<211>228
<212>DNA
<213> sheep (Ovis aries)
<400>11
ttctgttaca tgctagaggc catgtatctc tatgtttata ggtaggtagg taggtaggta 60
ggtaggtagg tagataggta ggtaggtagg taggtaggta ggtagataga tagatagata 120
gatagataga tagatatgta tagacaactt ctggcagggt actcaagaaa ctaggaaagt 180
tagtagtcaa tggatagtgt tactgcaggg acagggagca aaatgtaa 228
<210>12
<211>221
<212>DNA
<213> sheep (Ovis aries)
<400>12
tcaatcaaag tctgtaatgg aatcactaca gcaactgtcc accatttact ttttctctaa 60
cagttaatat atctatctat ctatctatct atctatctat gtatgtatag agagacgatc 120
caattattct actacatttc caattctata tttcaaaccc aaattgccag aaaattctac 180
ctaagtttta tatttcctct gatttggcaa ggacagtctt t 221
<210>13
<211>237
<212>DNA
<213> sheep (Ovis aries)
<400>13
cataactgtt ttactacctg gaccactgtg agcatgtcca accctcagcg accacatgga 60
ctgcagccta ccaggctcct ccgtccatgg gattttccag gcaagagtac tggagtgggg 120
tgccattgcc ttctccgaga tatatatata tagatagata gatagataga tagatagata 180
gatagataga tagatataga tagatataaa agactcattg tgctgtatac ttgaaac 237
<210>14
<211>224
<212>DNA
<213> sheep (Ovis aries)
<400>14
tgccagttcc tcagacacca tcgaagcgga agtggagtgg ctgcggctag cttgatattt 60
ctcatgtgat aattgttctg gccacttgaa gtcagaacct tttctagaac tctcagtatg 120
ggttgtcttt atctatcatt tatctatcta tctatctatc tatctatcta tctgtgactc 180
tagtccaggc caggctgatc aggacaatag tgggcaggat tcta 224
<210>15
<211>255
<212>DNA
<213> sheep (Ovis aries)
<400>15
gccagagaga cctattggga gctattaaat tagcccagga ttgtaataac aaacagatga 60
tataaatggg tggcatgcca acatcagaaa ccctggaaaa agaaaagtta ggttgatgtg 120
aaaagtaccc cattgtagct agctgtatta tatctatcta tctatctatc tatctatcta 180
tctatctatc tatctcttta tttataaatc tgattctgag acatgagatc tgagctgggt 240
attttggctt agtca 255
<210>16
<211>248
<212>DNA
<213> sheep (Ovis aries)
<400>16
tattcctgaa taaactcatc tttactgaag aaatatttgg cagtctattt gtttcaggtc 60
aaatatatct ctatctatct atctatctat ctatctatct atctatctat catctctcta 120
gctctaggag aatcaactaa gacaatcagc aacaaaacta cagatggaga aagttaggtt 180
tattgaccca aagcaacaag agaaagcaca cctaaaagcc acccagggac atttcactca 240
aaacggaa 248
<210>17
<211>271
<212>DNA
<213> sheep (Ovis aries)
<400>17
ctctacaaaa tgttccgcca agttggtgca tcattgtcaa gaagcatact atttccatta 60
agaccttcca ttcattagaa agtggctttt cctcctgtca aacttttaca tagatgaata 120
tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc tatctatcta 180
tatctatata tatatctcca aaagggacag tgttgagagt aaaagaacat actaatcata 240
attataccaa gacacctggg actttcctgg a 271
<210>18
<211>271
<212>DNA
<213> sheep (Ovis aries)
<400>18
ccccaggaag agagggtatc acatatgtga aatattgctg agagatcaag gatcatagca 60
taaactacag actttttgtt tggaaacaga tagactagat agatagatag atagatagat 120
agatagatag atagatagat agatatagag agacagtatg gttacaatgc acaagtttgt 180
ttagttggaa tgttggactt taaataacat tactatggat gctatcaaca acatacaaaa 240
caaaatggca ttcaaaagca agtgtattag g 271
<210>19
<211>272
<212>DNA
<213> sheep (Ovis aries)
<400>19
ccagctgagc taccagtata taaaactccc atatagatgg atagatagat agatagatag 60
atagatagat agataaatag atagatagat atagacagtt tcagtggttt taaacaggat 120
ttggtgtaga attggtgaca atttttagaa aagtacaaaa tattctcaat tgactttggt 180
tattctacat ttttaaattt agattattga aaagtaaaag aaaattactg aagaagcaaa 240
tttccataat ggtttacaat gagacttgaa ag 272
<210>20
<211>314
<212>DNA
<213> sheep (Ovis aries)
<400>20
cacagtcaga gctgggagtt agagctataa tacgtgaatt aggggtgcaa ttcagtccat 60
aaaataattg ttctcaagga caaactgcat ataaagatag atagatagat agatagatag 120
atagatagat agatagatag ataaacacac attgagaata cagtatgtct caggacagag 180
gacaggtcca gaaactaata aaaagaacca aatggaaatt ctagaaaaac ataatttaac 240
taaaaattaa tttgatggac ttgagagcag atgaaacact gtagaagaaa agatgagtga 300
accagaagac aggg 314
<210>21
<211>122
<212>DNA
<213> sheep (Ovis aries)
<400>21
ggacacaact gagtgactaa gcacacacgc acagacacac agacacacat agatagatag 60
atagatagat agatagatag atagatagat agatagatgg atggatttcc ttgctggcac 120
ca 122
<210>22
<211>148
<212>DNA
<213> sheep (Ovis aries)
<400>22
ccaggggagg cattttcaac ttacacacaa tagatagata gatagataga tagatagata 60
gatagataga tagatagata gataggtgga tggatcgatc ccagtaactc acaggtctag 120
ctcatgttac ttgccttgga gtgagttt 148
<210>23
<211>279
<212>DNA
<213> sheep (Ovis aries)
<400>23
gtttctgtca agtgtgtgat gctgatgtat ggattcagga cagtttgtac caggagagat 60
aagtatctga ttatgagaca tgggatatga gattataaga acatggccat agtcgtttaa 120
cctttaagaa cagagtatgg aaaactagta gctctgaatc tgagacctta ttctatctat 180
ctatctatct atctatcatc atcatctatc aaacacacac atgcacacac agccatagtt 240
agggcccaat caggattgac atccgcaaaa ggaacataa 279
<210>24
<211>316
<212>DNA
<213> sheep (Ovis aries)
<400>24
actttttgat ttgcccaggt tcaggaaata tttctgttat ttgtcattgt gccattaaaa 60
ttatcacaga ttttcctaac aggtctgtct atctatctat ctatctatct atctatctat 120
ctatctatct atctatctaa tcacagtttt agaatcattt cctcttttcc ctcttgcaat 180
ctttgatcta tatgtttcag gtatgtattt ggacataaaa tagtgatcat accagcaagg 240
aaatttataa gactggatcc ttcagagctc aagagtgaga agcagcaaga gtagtcagtt 300
ggacacaggg aaagca 316
<210>25
<211>296
<212>DNA
<213> sheep (Ovis aries)
<400>25
tgatttagaa aaagtggttg gagtccatct atctatctat ctatctatct atctatctat 60
ctatctatct atctatctat ctaattggtt cttgtcctca ataatttgtt caggggaagc 120
catataatat ttatgtaagg aaattgtgct aaagattcca tgttatgcagtgctattaaa 180
tttctttaaa cactatacca gagacagtct tgctccctgt atggatgtgt agcttagaat 240
tgctgtagcc actttgaaaa cctgtggaaa tttgtgtgtg gaggaagtct aaaacc 296
<210>26
<211>298
<212>DNA
<213> sheep (Ovis aries)
<400>26
agacccagtg tatgtcagcc ttctctgaga acaactactt gtcacaacag ccttaaaaat 60
acaaaggcaa tagaagttca caccagaaat agagaaggca ttcccaggcc tcttatcagg 120
atgactcact ttcaagaaag atgactaaaa atcaagaaag tggaattaca gatacttcca 180
gcatcatgcc atgtgttctc atcctttgaa gaagaaagta ggagaggtct gtagaaggat 240
ggatggatag atagatagat agatagatag atacagagga aagaagacag ggatgacg 298
<210>27
<211>309
<212>DNA
<213> sheep (Ovis aries)
<400>27
ctccccaaaa tcaagagggt tagttttatg ctgagagaat gaatagatag atagatagat 60
agatagatag atagatagat agatagatat taatatagca aaccaatcct gaaagttgga 120
aggtgaagaa cgctttaaaa tctaaagaac atttattttc caatataata gttattagcc 180
ttcccatatt atctgatact gaccagagtg cttggccttc tccagttaat aggaatgaat 240
aaatgccaga caagaaattc aggcaaggct ttattggggc ccttgctgaa gcacagggaa 300
atgataaca 309
<210>28
<211>330
<212>DNA
<213> sheep (Ovis aries)
<400>28
ctggagttat tatgtgactg tgtggtgcta agtcacttca atcagtcttt ttgcaatact 60
atggactgta acacgccagc cttgtctgtc catggggttc tccaggcaag aatactggag 120
tggttgccat tcccttcttc agggaatcct cctgacccat tgatcaaacc caaccctctt 180
acctctcttg cattggcagg cgagttcttt actgctagcg ccacctggga agagtcacta 240
tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc taaatacaag 300
gaccacagtt cgtgtaattg gctctaagga 330
<210>29
<211>409
<212>DNA
<213> sheep (Ovis aries)
<400>29
ccctggcacc caatctaaag tagcccctca ggcactctcc ctcccttctc ccacaagata 60
tatagataga tagatagata gatagataga tagatataga tatagatata gatagataga 120
tagatagaca gagatataga tagatagata gatagataga tagatagata gataggctga 180
gcatttatct ctaccagata ttacatgctt atctgttcat caccgccact ggagtttaaa 240
ctcactgagg gtaggaactt tttctatctt gtgcccagtt atgtctcgag cacgtaaata 300
atacttatct catagtgtgt gttcagtaaa cacctgctag agggagagag aaagggaggt 360
aaaaatatgt tggatgggag gtcatttaac cctccaaaat gaagaaggc 409
<210>30
<211>382
<212>DNA
<213> sheep (Ovis aries)
<400>30
ctctttttga gacaagccac cttcaaaatt gtgctaatct tcactttgtt acttcactta 60
tcagaaacat ttccacgtct aattcaaact gtattctata atttcttcaa catacaagaa 120
agactcactt tgatttatgt tgatccatga ccacctctcc ttgctttcag ttccttcaac 180
atttgtaaat aaagttgctt aactttatac atcttcagca tatagctttg gagttaaact 240
tcaaatgttc cttgtgccat aaaggactta aatatataga tagatagata gatagataga 300
tagatagata gatagataga tagatagata gatagattct agttttcttt aggacaataa 360
ctgttctttc tgtgtcttgg ga 382
<210>31
<211>468
<212>DNA
<213> sheep (Ovis aries)
<400>31
gcttctgtca gtagtggcat cttacaaagt acactatcca aaccaggata taggtattgg 60
caaatgagag ttaactagac ccagtcatca aatactgcca gtcaccaaaa ctcaagatga 120
aatagataat ctgaattctt tggtaaatat tagaaaaatc aatattttat ttgaaagctt 180
ctacaaaaca cactccccag tggtttctct ggagaattcc accaagtatt taaagaataa 240
ttaagatcag ttttacataa tttcttccag agaacaaaag agaaggaaac acttaccaat 300
actttgtttt tgaggctggc atgacccgat agatagatag atagatagat agatagatag 360
atagatagat agatagatag gtagatagct ggctggctgt tggtgcaaac aggacataaa 420
tgtgaggaaa tttaccttaa agaccactaa cttttggagg ggaagacg 468
<210>32
<211>448
<212>DNA
<213> sheep (Ovis aries)
<400>32
ccagtaaaat ccaggacatt ctcatggctt ctataagagt tatactcaga tactcacctt 60
ctgtgatcac cagcaccaga gctaccaaga ccatgataaa aaggagaaaa gcagttttct 120
caaaagactc cagatactgt gattaaaatt cagtgcagaa aagctaaaca atcacaactc 180
ttctattgat gaccatctta aactatctct tttctattag acagactgac agattgaata 240
gggatcagat agactgctag gccctaccgc ttatcaactc cttcctgaca gagggcacat 300
cactcaacat ttctcagcct caattcctcc tttgcaaata gggatgaccg ttttatatag 360
atagatagat agatagatag atagatagat atgcacctgc tgtgaggaaa ataacacatt 420
aaacgtaaag cacttggtag tcaacatt 448
<210>33
<211>25
<212>DNA
<213> Artificial sequence
<400>33
ttcggtatgg tatgcagtag gtatc 25
<210>34
<211>22
<212>DNA
<213> Artificial sequence
<400>34
acaaacttgg gaccctctta gc 22
<210>35
<211>23
<212>DNA
<213> Artificial sequence
<400>35
attccccatc tctatctagc agg 23
<210>36
<211>26
<212>DNA
<213> Artificial sequence
<400>36
cactgtttcc tacatctgtt taccat 26
<210>37
<211>23
<212>DNA
<213> Artificial sequence
<400>37
gacctgcata catctctgtc tgc 23
<210>38
<211>22
<212>DNA
<213> Artificial sequence
<400>38
atctccctcc tgctgtctat ga 22
<210>39
<211>25
<212>DNA
<213> Artificial sequence
<400>39
ttcctagtca gagatgtcac aaaac 25
<210>40
<211>25
<212>DNA
<213> Artificial sequence
<400>40
tggacattat tcatttgaaa acgct 25
<210>41
<211>23
<212>DNA
<213> Artificial sequence
<400>41
aatgcctcta cctggagtga tgc 23
<210>42
<211>21
<212>DNA
<213> Artificial sequence
<400>42
gagccattgg ggaagctaag g 21
<210>43
<211>22
<212>DNA
<213> Artificial sequence
<400>43
gcagcctaag cgtgaatgtg aa 22
<210>44
<211>22
<212>DNA
<213> Artificial sequence
<400>44
ttggcaagaa atcaatctgg tc 22
<210>45
<211>23
<212>DNA
<213> Artificial sequence
<400>45
caatactttg tttttgaggc tgg 23
<210>46
<211>25
<212>DNA
<213> Artificial sequence
<400>46
gttgaaaaca ggtatccctt ggtat 25
<210>47
<211>21
<212>DNA
<213> Artificial sequence
<400>47
tggttgaaca gcctgtggat t 21
<210>48
<211>22
<212>DNA
<213> Artificial sequence
<400>48
acccagtcct tggcaatgtt ac 22
<210>49
<211>22
<212>DNA
<213> Artificial sequence
<400>49
aatcaggtgt tgttgtttca gg 22
<210>50
<211>27
<212>DNA
<213> Artificial sequence
<400>50
gctatgatca atctaagctg ataattg 27
<210>51
<211>22
<212>DNA
<213> Artificial sequence
<400>51
aatcaccctt ctcacattca cc 22
<210>52
<211>22
<212>DNA
<213> Artificial sequence
<400>52
tcctccaacc tccttccaaa ct 22
<210>53
<211>22
<212>DNA
<213> Artificial sequence
<400>53
ttctgttaca tgctagaggc ca 22
<210>54
<211>22
<212>DNA
<213> Artificial sequence
<400>54
ttacattttg ctccctgtcc ct 22
<210>55
<211>26
<212>DNA
<213> Artificial sequence
<400>55
tcaatcaaag tctgtaatgg aatcac 26
<210>56
<211>22
<212>DNA
<213> Artificial sequence
<400>56
aaagactgtc cttgccaaat ca 22
<210>57
<211>27
<212>DNA
<213> Artificial sequence
<400>57
cataactgtt ttactacctg gaccact 27
<210>58
<211>27
<212>DNA
<213> Artificial sequence
<400>58
gtttcaagta tacagcacaa tgagtct 27
<210>59
<211>22
<212>DNA
<213> Artificial sequence
<400>59
tgccagttcc tcagacacca tc 22
<210>60
<211>24
<212>DNA
<213> Artificial sequence
<400>60
tagaatcctg cccactattg tcct 24
<210>61
<211>22
<212>DNA
<213> Artificial sequence
<400>61
gccagagaga cctattggga gc 22
<210>62
<211>22
<212>DNA
<213> Artificial sequence
<400>62
tgactaagcc aaaataccca gc 22
<210>63
<211>26
<212>DNA
<213> Artificial sequence
<400>63
tattcctgaa taaactcatc tttact 26
<210>64
<211>22
<212>DNA
<213> Artificial sequence
<400>64
ttccgttttg agtgaaatgt cc 22
<210>65
<211>23
<212>DNA
<213> Artificial sequence
<400>65
ctctacaaaa tgttccgcca agt 23
<210>66
<211>21
<212>DNA
<213> Artificial sequence
<400>66
tccaggaaag tcccaggtgt c 21
<210>67
<211>22
<212>DNA
<213> Artificial sequence
<400>67
ccccaggaag agagggtatc ac 22
<210>68
<211>24
<212>DNA
<213> Artificial sequence
<400>68
cctaatacac ttgcttttga atgc 24
<210>69
<211>26
<212>DNA
<213> Artificial sequence
<400>69
ccagctgagc taccagtata taaaac 26
<210>70
<211>25
<212>DNA
<213> Artificial sequence
<400>70
ctttcaagtc tcattgtaaa ccatt 25
<210>71
<211>24
<212>DNA
<213> Artificial sequence
<400>71
cacagtcaga gctgggagtt agag 24
<210>72
<211>23
<212>DNA
<213> Artificial sequence
<400>72
ccctgtcttc tggttcactc atc 23
<210>73
<211>22
<212>DNA
<213> Artificial sequence
<400>73
ggacacaact gagtgactaa gc 22
<210>74
<211>20
<212>DNA
<213> Artificial sequence
<400>74
tggtgccagc aaggaaatcc 20
<210>75
<211>20
<212>DNA
<213> Artificial sequence
<400>75
ccaggggagg cattttcaac 20
<210>76
<211>24
<212>DNA
<213> Artificial sequence
<400>76
aaactcactc caaggcaagt aaca 24
<210>77
<211>24
<212>DNA
<213> Artificial sequence
<400>77
gtttctgtca agtgtgtgat gctg 24
<210>78
<211>23
<212>DNA
<213> Artificial sequence
<400>78
ttatgttcct tttgcggatg tca 23
<210>79
<211>22
<212>DNA
<213> Artificial sequence
<400>79
actttttgat ttgcccaggt tc 22
<210>80
<211>22
<212>DNA
<213> Artificial sequence
<400>80
tgctttccct gtgtccaact ga 22
<210>81
<211>25
<212>DNA
<213> Artificial sequence
<400>81
tgatttagaa aaagtggttg gagtc 25
<210>82
<211>25
<212>DNA
<213> Artificial sequence
<400>82
ggttttagac ttcctccaca cacaa 25
<210>83
<211>23
<212>DNA
<213> Artificial sequence
<400>83
agacccagtg tatgtcagcc ttc 23
<210>84
<211>24
<212>DNA
<213> Artificial sequence
<400>84
cgtcatccct gtcttctttc ctct 24
<210>85
<211>23
<212>DNA
<213> Artificial sequence
<400>85
ctccccaaaa tcaagagggt tag 23
<210>86
<211>23
<212>DNA
<213> Artificial sequence
<400>86
tgttatcatt tccctgtgct tca 23
<210>87
<211>25
<212>DNA
<213> Artificial sequence
<400>87
ctggagttat tatgtgactg tgtgg 25
<210>88
<211>24
<212>DNA
<213> Artificial sequence
<400>88
tccttagagc caattacacg aact 24
<210>89
<211>22
<212>DNA
<213> Artificial sequence
<400>89
ccctggcacc caatctaaag ta 22
<210>90
<211>23
<212>DNA
<213> Artificial sequence
<400>90
gccttcttca ttttggaggg tta 23
<210>91
<211>22
<212>DNA
<213> Artificial sequence
<400>91
ctctttttga gacaagccac ct 22
<210>92
<211>22
<212>DNA
<213> Artificial sequence
<400>92
tcccaagaca cagaaagaac ag 22
<210>93
<211>23
<212>DNA
<213> Artificial sequence
<400>93
gcttctgtca gtagtggcat ctt 23
<210>94
<211>23
<212>DNA
<213> Artificial sequence
<400>94
cgtcttcccc tccaaaagtt agt 23
<210>95
<211>25
<212>DNA
<213> Artificial sequence
<400>95
ccagtaaaat ccaggacatt ctcat 25
<210>96
<211>25
<212>DNA
<213> Artificial sequence
<400>96
aatgttgact accaagtgct ttacg 25
<210>97
<211>18
<212>DNA
<213> Artificial sequence
<400>97
tgtaaaacga cggccagt 18

Claims (10)

1. A screening method of sheep four-base repeated microsatellite markers is characterized by comprising the following steps:
(a) performing BLAST comparison in a sheep whole genome, and searching for microsatellite markers with four base repeats on all chromosomes;
(b) designing primers for amplifying the microsatellite markers screened in step (a), wherein the primers comprise a forward primer and a reverse primer;
(c) performing PCR amplification on the microsatellite marker obtained by screening in the step (a), and screening out the microsatellite marker with single and clear PCR amplification product band;
(d) selecting at least 20 sheep samples, and performing three-primer PCR amplification on the microsatellite marker obtained by screening in the step (c), wherein the three-primer PCR amplification comprises the following steps of amplifying the microsatellite marker by using the following primers: the 5 'end of the forward primer is fused with a forward universal primer, and the 5' end of the forward universal primer and the reverse primer are marked with fluorescent labels;
(e) performing polymorphism identification on the microsatellite marker by analyzing the amplification product of the step (d) to obtain the sheep four-base repetitive microsatellite marker.
2. The screening method according to claim 1, wherein in step (a), the number of repeat units in the core sequence of the microsatellite marker is not less than 7;
preferably, the distance between any two microsatellite markers on the same chromosome is more than or equal to 200 kb;
preferably, the number of microsatellite markers on the same chromosome is ≦ 3.
3. The screening method according to claim 1, wherein in the step (b), the region where the primer is located has no homologous sequence in the whole genome;
preferably, the length of the primer is 18-30 bp;
preferably, the GC content of the primer is 35-60%;
preferably, the annealing temperature of the primer is 50-65 ℃;
preferably, the length of the amplified fragment is 100-400 bp.
4. The screening method according to claim 1, wherein at least two sheep samples are subjected to PCR amplification in step (c);
preferably, step (c) further comprises sequencing the microsatellite markers that are unique and distinct from the PCR amplification product band.
5. The screening method of claim 1, wherein the forward universal primer comprises a M13 forward universal primer;
preferably, the M13 forward universal primer comprises M13(-47), M13(-40) or M13 (-21);
preferably, the M13 forward universal primer comprises M13(-21), and the sequence of the M13(-21) is shown in SEQ ID NO. 97;
preferably, the fluorescent markers comprise 2-5 fluorescent markers with different colors, the 5' end of each forward universal primer is marked with a fluorescent marker with one color, and each site is amplified by using the universal primer marked with the fluorescent marker with the same color;
preferably, the fluorescent markers comprise 3 different colored fluorescent markers;
preferably, fluorescent labels include FAM, Tamra, and Hex;
preferably, 25-35 sheep samples are selected, and the microsatellite marker obtained by screening in the step (c) is subjected to three-primer PCR amplification;
preferably, in the reaction system for three-primer PCR amplification, the molar ratio of the forward primer fused with the forward universal primer at the 5 'end, the forward universal primer labeled with the fluorescent label at the 5' end and the reverse primer is (0.8-1.2): (1.2-2): 2;
preferably, the amplification procedure of the three-primer PCR amplification comprises annealing and amplifying at 57-59 ℃ for 25-35 cycles, and then annealing and amplifying at 52-54 ℃ for 5-15 cycles;
preferably, annealing and amplifying at 58 ℃ for 25-35 cycles;
preferably, annealing and amplifying at 53 ℃ for 5-15 cycles.
6. The screening method of claim 1, wherein the identifying of polymorphisms in the microsatellite marker comprises analyzing one or more of the allele count, polymorphic information content, desired heterozygosity, observed heterozygosity, and Hardy-Weinberg equilibrium of the microsatellite marker;
preferably, the allelic factors are at least 5:
preferably, the polymorphic information content is at least 0.5;
preferably, the observed heterozygosity is at least 0.5;
preferably, it is desirable for the heterozygosity to be at least 0.5;
preferably, the P value of the Hardy-Weinberg equilibrium is at least 0.05;
preferably, the amplified product of step (d) is subjected to capillary electrophoresis, then the collected data is subjected to fluorescence color separation, the size of the allele is calculated by using a typing standard, the genotyping data of each genome is obtained, and then polymorphism identification is carried out on the sheep four-base repeat microsatellite.
7. A set of ovine four base repeat microsatellite markers comprising one or more microsatellite markers selected from the group consisting of Ovr1402, Ovr1602, Ovr2001, Ovr0501, Ovr2101, Ovr0201, Ovr0702, Ovr0503, Ovr1002, Ovr2204, Ovr0502, Ovr1401, Ovr2304, Ovr1901, Ovr2604, Ovr2601, Ovr1101, Ovr1003, Ovr1601, Ovr2401, Ovr2202, Ovr0602, Ovr2103, Ovr1702, Ovr1501, Ovr2606, Ovr1001, Ovr1801, Ovr2102, Ovr0801, Ovr0802 and Ovr 0902;
the microsatellite repeat motif of Ovr1402 is (ATAG)9The nucleotide sequence of Ovr1402 is a sequence shown in SEQ ID NO.1, or a sequence with homology of more than 90% with SEQ ID NO. 1;
the microsatellite repeat motif of Ovr1602 is (CTAT)7The nucleotide sequence of Ovr1602 is a sequence shown in SEQ ID NO.2, or a sequence with homology of more than 90% with SEQ ID NO. 2;
the microsatellite repeat motif of Ovr2001 is (ATAG)6The nucleotide sequence of Ovr2001 is a sequence shown in SEQ ID NO.3, or a sequence with homology of more than 90% with SEQ ID NO. 3;
the microsatellite repeat motif of the Ovr0501 is (ATAG)12The nucleotide sequence of the Ovr0501 is a sequence shown by SEQ ID NO.4, or a sequence with homology of more than 90% with the SEQ ID NO. 4;
the microsatellite repeat motif of the Ovr2101 is (CTAT)5The nucleotide sequence of the Ovr2101 is a sequence shown by SEQ ID NO.5, or a sequence with homology of more than 90% with SEQ ID NO. 5;
the microsatellite repeat motif of Ovr0201 is (CTAT)8The nucleotide sequence of Ovr0201 is a sequence shown in SEQ ID NO.6, or a sequence with homology of more than 90% with SEQ ID NO. 6;
the microsatellite repetitive motif of Ovr0702 is (ATAG)15The nucleotide sequence of Ovr0702 is a sequence shown in SEQ ID NO.7, or a sequence with homology of more than 90% with SEQ ID NO. 7;
the microsatellite repetitive motif of the Ovr0503 is (CTAT)7The nucleotide sequence of the Ovr0503 is a sequence shown by SEQ ID NO.8, or a sequence with homology of more than 90% with SEQ ID NO. 8;
the microsatellite repeat motif of Ovr1002 is (ATAG)13The nucleotide sequence of Ovr1002 is a sequence shown in SEQ ID NO.9, or a sequence with homology of more than 90% with SEQ ID NO. 9;
the microsatellite repeat motif of Ovr2204 is (CTAT)9The nucleotide sequence of Ovr2204 is a sequence shown in SEQ ID NO.10,or a sequence with more than 90 percent of homology with SEQ ID NO. 10;
the microsatellite repeat motif of the Ovr0502 is (ATAG)7The nucleotide sequence of the Ovr0502 is a sequence shown by SEQ ID NO.11, or a sequence with homology of more than 90 percent with the SEQ ID NO. 11;
the microsatellite repeat motif of Ovr1401 is (CTAT)7The nucleotide sequence of Ovr1401 is a sequence shown in SEQ ID NO.12, or a sequence with homology of more than 90% with SEQ ID NO. 12;
the microsatellite repeat motif of Ovr2304 is (ATAG)11The nucleotide sequence of Ovr2304 is a sequence shown in SEQ ID NO.13, or a sequence with homology of more than 90% with SEQ ID NO. 13;
the microsatellite repeat motif of Ovr1901 is (CTAT)7The nucleotide sequence of Ovr1901 is a sequence shown in SEQ ID NO.14, or a sequence with homology of more than 90% with SEQ ID NO. 14;
the microsatellite repeat motif of Ovr2604 is (CTAT)10The nucleotide sequence of Ovr2604 is a sequence shown in SEQ ID NO.15, or a sequence with homology of more than 90% with SEQ ID NO. 15;
the microsatellite repetitive motif of Ovr2601 is (CTAT)10The nucleotide sequence of Ovr2601 is a sequence shown in SEQ ID NO.16, or a sequence with homology of more than 90% with SEQ ID NO. 16;
the microsatellite repeat motif of Ovr1101 is (CTAT)15The nucleotide sequence of Ovr1101 is shown as SEQ ID NO.17, or is a sequence with homology of more than 90% with SEQ ID NO. 17;
the microsatellite repeat motif of Ovr1003 is (ATAG)10The nucleotide sequence of Ovr1003 is a sequence shown in SEQ ID NO.18, or a sequence with homology of more than 90% with SEQ ID NO. 18;
the microsatellite repeat motif of Ovr1601 is (ATAG)10The nucleotide sequence of Ovr1601 is shown in SEQ ID NO.19, or is a sequence with homology of more than 90% with SEQ ID NO. 19;
the microsatellite repeat motif of Ovr2401 is (ATAG)11Core of said Ovr2401The nucleotide sequence is shown in SEQ ID NO.20, or is a sequence with homology of more than 90% with SEQ ID NO. 20;
the microsatellite repeat motif of the Ovr2202 is (ATAG)12The nucleotide sequence of the Ovr2202 is a sequence shown in SEQ ID NO.21, or a sequence which has more than 90 percent of homology with SEQ ID NO. 21;
the microsatellite repetitive motif of Ovr0602 is (ATAG)14The nucleotide sequence of Ovr0602 is a sequence shown in SEQ ID NO.22, or a sequence which has more than 90 percent of homology with SEQ ID NO. 22;
the microsatellite repeat motif of Ovr2103 is (CTAT)6The nucleotide sequence of Ovr2103 is a sequence shown by SEQ ID NO.23, or a sequence with homology of more than 90% with SEQ ID NO. 23;
the microsatellite repeat motif of Ovr1702 is (CTAT)12The nucleotide sequence of the Ovr1702 is a sequence shown in SEQ ID NO.24, or a sequence which has more than 90 percent of homology with SEQ ID NO. 24;
the microsatellite repetitive motif of Ovr1501 is (CTAT)13The nucleotide sequence of Ovr1501 is a sequence shown in SEQ ID NO.25, or a sequence with homology of more than 90% with SEQ ID NO. 25;
the microsatellite repeat motif of Ovr2606 is (ATAG)6The nucleotide sequence of Ovr2606 is a sequence shown in SEQ ID NO.26, or a sequence with homology of more than 90% with SEQ ID NO. 26;
the microsatellite repeat motif of Ovr1001 is (ATAG)11The nucleotide sequence of Ovr1001 is a sequence shown in SEQ ID NO.27, or a sequence with homology of more than 90% with SEQ ID NO. 27;
the microsatellite repetitive motif of Ovr1801 is (CTAT)13The nucleotide sequence of Ovr1801 is a sequence shown in SEQ ID NO.28, or a sequence with homology of more than 90% with SEQ ID NO. 28;
the microsatellite repeat motif of Ovr2102 is (ATAG)10The nucleotide sequence of the Ovr2102 is a sequence shown in SEQ ID NO.29, or a sequence which has more than 90 percent of homology with SEQ ID NO. 29;
the microsatellite repetitive motif of Ovr0801 is(ATAG)15The nucleotide sequence of Ovr0801 is a sequence shown in SEQ ID NO.30 or a sequence with homology of more than 90% with SEQ ID NO. 30;
the microsatellite repetitive motif of Ovr0802 is (ATAG)13The nucleotide sequence of Ovr0802 is a sequence shown in SEQ ID NO.31, or a sequence with homology of more than 90% with SEQ ID NO. 31;
the microsatellite repeat motif of Ovr0902 is (ATAG)8The nucleotide sequence of Ovr0902 is a sequence shown by SEQ ID NO.32, or a sequence which has more than 90 percent of homology with SEQ ID NO. 32;
preferably, the ovine four-base repeat microsatellite markers comprise at least Ovr1402, Ovr1602, Ovr0501, Ovr0201, Ovr0702, Ovr1002, Ovr2204, Ovr0502, Ovr2401, Ovr1702, Ovr1501, Ovr1801 and Ovr 2102;
preferably, the ovine four-base repeat microsatellite markers include Ovr1402, Ovr1602, Ovr0501, Ovr0201, Ovr0702, Ovr1002, Ovr2204, Ovr0502, Ovr2304, Ovr1901, Ovr2601, Ovr1101, Ovr1003, Ovr2401, Ovr1702, Ovr1501, Ovr1801, Ovr2102, Ovr0801 and Ovr 0802.
8. A primer set for amplifying the sheep four base repeat microsatellite marker of claim 7 comprising at least one of the following primer pairs:
primer pair for amplifying Ovr 1402: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 33; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 34;
primer pair for amplifying Ovr 1602: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 35; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 36;
primer pair for amplification of Ovr 2001: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 37; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 38;
primer pair for amplification of Ovr 0501: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 39; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 40;
primer pair for amplifying Ovr 2101: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 41; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 42;
primer pair for amplifying Ovr 0201: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 43; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 44;
primer pair for amplifying Ovr 0702: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 45; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 46;
primer pair for amplification of Ovr 0503: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 47; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 48;
primer pair for amplifying Ovr 1002: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 49; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 50;
primer pair for amplifying Ovr 2204: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 51; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 52;
primer pair for amplification of Ovr 0502: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 53; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 54;
primer pair for amplification of Ovr 1401: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 55; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 56;
primer pair for amplifying Ovr 2304: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 57; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 58;
primer pair for amplifying Ovr 1901: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 59; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 60;
primer pair for amplification of Ovr 2604: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 61; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 62;
primer pair for amplifying Ovr 2601: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 63; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 64;
primer pair for amplification of Ovr 1101: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 65; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 66;
primer pair for amplifying Ovr 1003: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 67; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 68;
primer pair for amplifying Ovr 1601: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 69; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 70;
primer pair for amplifying Ovr 2401: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 71; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 72;
primer pair for amplification of Ovr 2202: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 73; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 74;
primer pair for amplification of Ovr 0602: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 75; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 76;
primer pair for amplifying Ovr 2103: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 77; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 78;
primer pair for amplification of Ovr 1702: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 79; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 80;
primer pair for amplifying Ovr 1501: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 81; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 82;
primer pair for amplification of Ovr 2606: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 83; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 84;
primer pair for amplifying Ovr 1001: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 85; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 86;
primer pair for amplifying Ovr 1801: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 87; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 88;
primer pair for amplifying Ovr 2102: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 89; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 90;
primer pair for amplifying Ovr 0801: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 91; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 92;
primer pair for amplifying Ovr 0802: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 93; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 94;
primer pair for amplification of Ovr 0902: the nucleotide sequence of the forward primer is shown as SEQ ID NO. 95; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 96;
preferably, the primer pairs in the primer set are labeled with a label;
preferably, the label comprises a fluorescent label.
9. A kit comprising the primer set according to claim 8.
10. Use of the method for screening a four-base repeat in sheep microsatellite marker according to any one of claims 1 to 6, the four-base repeat in sheep microsatellite marker according to claim 7, the primer set according to claim 8 or the kit according to claim 9 for paternity testing in sheep.
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