CN106048003A - Method for assisting in identifying sheep growth characters based on rs430810656 SNP locus - Google Patents

Method for assisting in identifying sheep growth characters based on rs430810656 SNP locus Download PDF

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CN106048003A
CN106048003A CN201610374993.8A CN201610374993A CN106048003A CN 106048003 A CN106048003 A CN 106048003A CN 201610374993 A CN201610374993 A CN 201610374993A CN 106048003 A CN106048003 A CN 106048003A
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张莉
马晓萌
杜立新
轩俊丽
魏彩虹
赵福平
朱才业
吴明明
袁泽湖
刘瑞凿
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Institute of Animal Science of CAAS
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Abstract

The invention discloses a method for assisting in identifying sheep growth characters based on an rs430810656 SNP locus. The method comprises the following step of detecting whether the genetype of the rs430810656 SNP locus in genome DNA of a sheep to be detected is AA genetype or GG genetype or GA genetype, wherein the growth character of the sheep with the AA genetype is superior to that of the sheep with the GA genetype, and the growth character of the sheep with the GA genetype is superior to that of the sheep with the GG genetype. The invention further provides a specific primer pair. The specific primer pair is composed of a primer F and a primer R which are as shown in a first sequence of a sequence list and a second sequence of the sequence list respectively. The method can be used for assisting in screening the sheep with good growth characters, has important significance in breeding of the sheep and has important significance in further research of the growth characters of the sheep.

Description

A kind of based on rs430810656 SNP site auxiliary qualification ovine growth character Method
Technical field
The present invention relates to a kind of method identifying ovine growth character based on rs430810656 SNP site auxiliary.
Background technology
SNP (single nucleotide polymorphism single nucleotide polymorphism) refers mainly in genomic level, The polymorphism of the DNA sequence caused by the variation of single core thuja acid, including conversion, transversion, inserts or lacks.Genome divides The SNP that cloth is intensive, because it carries huge hereditary information amount it is considered to be the best genetic marker of application prospect, mankind SNP The detection of labelling has become as the important means of gene diagnosis.The research of the genetic map of SNP marker, has marker assisted selection Huge facilitation, in agricultural animal, the gene of a lot of important characters is the most located.
Sheep is the domestic animal that the world and China have important economical trait, provides substantial amounts of meat, hair, skin etc. for the mankind and produces Product.Utilize traditional breeding method that with raising, the selection-breeding of sheep is achieved significant effect, promote local Sheep Populations Improvement and the formation of various places sheep new varieties, but, use traditional breeding method to the progress of the genetic improvement of sheep very Slowly, the formation of new varieties is accomplished by 10 years even time of decades.Along with Protocols in Molecular Biology appearance with Development, particularly carrying out of human genome research put on new schedule with the research of other species gene groups, have driven sheep The research of genome.Research in terms of the molecular biology of sheep early stage is concentrated mainly on to be labeled as fundamental construction ovine species Genetic linkage maps, the location of the important economical trait based on linkage map, the gene based on candidate gene approach Clone and function research, molecular marker breeding application etc..In recent decades, the achievement in research of ovine genome has Limit, therefore, the research to ovine genome is extremely urgent problem.
Summary of the invention
It is an object of the invention to provide a kind of side identifying ovine growth character based on rs430810656 SNP site auxiliary Method.
What the present invention provided identifies or the method for auxiliary qualification ovine growth character, comprises the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG Genotype or GA genotype;
It is the sheep that body weight is big and/or chest measurement is big if AA genotype or GA genotype, sheep candidate to be measured, if GG genotype, sheep candidate to be measured are the sheep that body weight is little and/or chest measurement is little;When described body weight gross appearance was four monthly age, body weight is big When equal to 28kg and/or six-month-old, body weight is more than or equal to 30kg;When described chest measurement gross appearance was four monthly age, chest measurement is more than or equal to When 78cm and/or six-month-old, chest measurement is more than or equal to 79cm;Body weight that described body weight is little when being presented as four monthly ages is less than 28kg and/or six During the monthly age, body weight is less than 30kg;Described chest measurement is little when being presented as four monthly ages, and chest measurement is less than less than chest measurement when 78cm and/or six-month-old 79cm。
Described sheep concretely 4 monthly ages or the sheep at 6 monthly ages.
Described sheep concretely crow pearl Mu Qin sheep.
The present invention also protects a kind of qualification or the method for auxiliary qualification ovine growth character, comprises the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG Genotype or GA genotype;
It is that height is big and/or Body steep length is big and/or chest breadth is big if AA genotype or GA genotype, sheep candidate to be measured Sheep, be the sheep that height is little and/or Body steep length is little and/or chest breadth is little if GG genotype, sheep candidate to be measured;Described When height gross appearance is six-month-old, height is more than or equal to 60cm;When described Body steep length gross appearance is six-month-old, Body steep length is more than or equal to 64cm;When described chest breadth gross appearance is six-month-old, chest breadth is more than or equal to 15cm;Described height is little to be presented as height during six-month-old Less than 60cm;Described Body steep length is little when being presented as six-month-old Body steep length is less than 64cm;Described chest breadth is little breast when being presented as six-month-old Wide less than 15cm.
The sheep at described sheep concretely 6 monthly ages.
Described sheep concretely crow pearl Mu Qin sheep.
The present invention also protects a kind of qualification or the method for auxiliary qualification ovine growth character, comprises the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG Genotype or GA genotype;
The growth traits of AA genotype sheep is better than GA genotype sheep, and the growth traits of GA genotype sheep is better than GG base Because of type sheep.
Growth traits at least one being presented as in following (a1) to (a5) excellent: (a1) body weight is big;(a2) chest measurement is big;(a3) Height is big;(a4) Body steep length is big;(a5) chest breadth is big;
When growth traits is excellent be presented as that body weight is big time, the body weight of AA genotype sheep be more than GA genotype sheep, GA gene The body weight of type sheep is statistically more than GG genotype sheep.
When growth traits is excellent be presented as that chest measurement is big time, the chest measurement of AA genotype sheep is more than GA genotype sheep, GA base Because the chest measurement of type sheep is statistically more than GG genotype sheep.
When growth traits is excellent be presented as that height is big time, the height of AA genotype sheep be more than GA genotype sheep, GA gene The height of type sheep is more than GG genotype sheep.
When growth traits is excellent be presented as that Body steep length is big time, the Body steep length of AA genotype sheep be more than GA genotype sheep, GA More than GG genotype sheep on the Body steep length of genotype sheep.
When growth traits is excellent be presented as that chest breadth is big time, the chest breadth of AA genotype sheep be more than GA genotype sheep, GA gene The chest breadth of type sheep is more than GG genotype sheep.
Described sheep concretely 4 monthly ages or the sheep at 6 monthly ages.
Described sheep concretely crow pearl Mu Qin sheep.
In method described in any of the above, described " detect in the genomic DNA of sheep to be measured based on rs430810656 SNP The genotype in site " comprise the steps: with the genomic DNA of sheep to be measured as template, use special primer to carrying out PCR expansion Increase, then pcr amplification product is checked order;
Described special primer forms by primers F and primer R;
Described primers F is following (b1) or (b2):
(b1) single strand dna shown in sequence 1 of sequence table;
(b2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had with sequence 1 The DNA molecular of identical function;
Described primer R is following (b3) or (b4):
(b3) single strand dna shown in sequence 2 of sequence table;
(b4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had with sequence 2 The DNA molecular of identical function.
The present invention also protects a species-specific primers pair, is made up of primers F and primer R;
Described primers F is following (b1) or (b2):
(b1) single strand dna shown in sequence 1 of sequence table;
(b2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had with sequence 1 The DNA molecular of identical function;
Described primer R is following (b3) or (b4):
(b3) single strand dna shown in sequence 2 of sequence table;
(b4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had with sequence 2 The DNA molecular of identical function.
The present invention also protects the application of described special primer pair, for as follows (c1) or (c2):
(c1) auxiliary identifies ovine growth character;
(c2) preparation auxiliary identifies the test kit of ovine growth character.
Described growth traits is at least one in following (d1) to (d5): (d1) body weight;(d2) chest measurement;(d3) height; (d4) Body steep length;(d5) chest breadth.Described sheep concretely crow pearl Mu Qin sheep.
The present invention also protects the test kit containing described special primer pair.The purposes of described test kit identifies sheep for auxiliary Growth traits.Described growth traits is at least one in following (d1) to (d5): (d1) body weight;(d2) chest measurement;(d3) height; (d4) Body steep length;(d5) chest breadth.Described sheep concretely crow pearl Mu Qin sheep.
The present invention also protects a kind of DNA containing described rs430810656 SNP site relevant to ovine growth character Fragment, as shown in sequence 3 in sequence table.Described sheep concretely crow pearl Mu Qin sheep.
The present invention also protects method described in any of the above, or, described primer pair, or, described test kit, sheep Application in breeding.The purpose of described breeding is to cultivate the sheep that growth traits is excellent.Growth traits is excellent is presented as following (a1) extremely (a5) at least one in: (a1) body weight is big;(a2) chest measurement is big;(a3) height is big;(a4) Body steep length is big;(a5) chest breadth is big.Institute State sheep concretely crow pearl Mu Qin sheep.
In rs430810656 SNP site concretely ovine genome described in any of the above, the sequence 3 of sequence table is from 5 ' The 25th nucleotide of end.
Present invention finds a rs430810656 SNP site, find rs430810656 SNP site and ovine growth Character has dependency.This SNP as the molecular marker of ovine growth character, can apply the method for the present invention can assist sieve Selecting the sheep that growth traits is excellent, the breeding for sheep is significant, also for the growth traits studying sheep further Significant.
Accompanying drawing explanation
Fig. 1 is that DNA mixes pond sequencing result figure
Fig. 2 is flight mass spectrum analysis result figure
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with three times and repeats experiment, and result is made even Average.
Crow pearl Mu Qin sheep (also known as Ujmuqin sheep): Inner Mongol Dong Wumu oozes sheep seed farm and Zhenglan Banner Yang Chang, implements In example, all growth data of sheep are all from above-mentioned two Yang Chang.
Body steep length: by the air line distance of margin of scapula,anterior to buttocks end.
Circumference of cannon bone: the circumference in left fore pipe portion upper 1/3rd.
Chest depth: by the distance of shoulder blade peak to breastbone bottom surface.
Embodiment 1, SNP site and gene type
Each exon, the control region of sheep TRHDE upstream region of gene 1000bp and sheep for sheep TRHDE gene Some primers pair are designed in the control region of TRHDE downstream of gene 1000bp, carry out each individuality in each Sheep Populations respectively PCR expands and checks order, and excavates SNP site and is associated analyzing for SNP site and various trait.
By above-mentioned lot of experiments, obtain special primer to as follows:
F (sequence 1 of sequence table): 5 '-TCAGTTGTCAGAGCAAAGTGA-3 ';
R (sequence 2 of sequence table): 5 '-TCTCGTCTTGTTTCTCTCCAGT-3 '.
In 343 black pearl Mu Qin sheep test colony, choose wherein 30 individual blood DNA samples, with identical dense The mixed pond DNA of degree composition, uses the primer of F and R composition to carrying out PCR amplification, is checked order by pcr amplification product.To order-checking peak Figure is analyzed, and in conjunction with sheep Meat Traits whole-genome association, finds a phase notable with sheep daily postweaning gain Close SNP site (rs430810656), as shown in Figure 1.
Rs430810656 SNP site is positioned at TRHDE gene exon 14.In crow pearl Mu Qin ovine genome The nucleotide sequence of rs430810656 SNP site periphery as shown in the sequence 3 of sequence table, rs430810656 SNP site position In the 25th, for A or G (samesense mutation).
Flight mass spectrum method is used to detect 343 black pearl Mu Qin sheep bases based on rs430810656 SNP site respectively Because of type typing, result is as in figure 2 it is shown, the idiotype being positioned at a-quadrant is GG, and the idiotype being positioned at B region is GA, position Idiotype in C region is AA.
TRHDE gene is as shown in the sequence 4 of sequence table.
Embodiment 2, association analysis
One, SNP site is analyzed with four monthly age ovine growth trait associations
Experiment material is 277 pearl Mu Qin sheep, raises under the same conditions.
1, the body weight at each individual four monthly ages, height, Body steep length, chest measurement, circumference of cannon bone are recorded respectively.
2, take the blood sample of each individuality respectively and extract genomic DNA, using the primer of F and R composition to carrying out PCR Amplification, then checks order pcr amplification product, obtains each individuality genotype based on rs430810656 SNP site.
3, statistics software growth traits (body weight, height, Body steep length, breast to SNP site with crow pearl Mu Qin sheep is utilized Enclose, circumference of cannon bone) be associated analyze, Tables 1 and 2 is shown in statistical result.
The growth traits of table 14 monthly age crow pearl Mu Qin sheep and the correlation results of SNP site
Note: indicate different lower case person and represent that group difference is notable (P < 0.05);Indicate identical lower case person to represent Group difference is not notable (P > 0.05).
The growth traits statistical result of table 24 monthly age crow pearl Mu Qin sheep
Based on rs430810656 SNP site, in 277 black pearl Mu Qin sheep, 115 is AA genotype, 144 for GA Genotype, 18 be GG genotype.In the sheep of 115 AA genotype, the body weight of 90.98% is both greater than equal to 28kg, averagely Value is 32.33kg;In the sheep of 18 GG genotype, the body weight of 77.78% is both less than 28kg, and meansigma methods is 25.74kg.115 In the sheep of AA genotype, the chest measurement of 80.33% is both greater than equal to 78cm, and meansigma methods is 80.29cm;18 GG genotype In sheep, the chest measurement of 77.78% is both less than 78cm, and meansigma methods is 73.61cm.
Set up following method: detect four monthly age crow pearl Mu Qin sheep genes based on rs430810656 SNP site to be measured Type, four monthly age body weight of AA genotype, chest measurement be all remarkably higher than GA genotype, and four monthly age body weight of GA genotype, chest measurement are the most aobvious Write higher than GG genotype.
Set up following method: detect four monthly age crow pearl Mu Qin sheep genes based on rs430810656 SNP site to be measured Type, if AA genotype, sheep more than or equal to 28kg and/or chest measurement more than or equal to 78cm, if GG genotype, sheep Body weight is less than 78cm less than 28kg and/or chest measurement.
Two, SNP site is analyzed with six-month-old ovine growth trait associations
Experiment material is 221 pearl Mu Qin sheep, raises under the same conditions.
1, each individual body weight of six-month-old, chest measurement, height, Body steep length, circumference of cannon bone, chest breadth, chest depth are recorded respectively.
2, take the blood sample of each individuality respectively and extract genomic DNA, using the primer of F and R composition to carrying out PCR Amplification, then checks order pcr amplification product, obtains each individuality genotype based on rs430810656 SNP site.
3, statistics software growth traits (body weight, chest measurement, body to SNP site with crow pearl Mu Qin sheep six-month-old is utilized Height, Body steep length, circumference of cannon bone, chest breadth, chest depth) it is associated analyzing, statistical result is shown in Table 3 and table 4.
The growth traits of table 36 monthly age crow pearl Mu Qin sheep and the correlation results of SNP site
Note: indicate different lower case person and represent that group difference is notable (P < 0.05);Indicate identical lower case person to represent Group difference is not notable (P > 0.05).
The growth traits statistical result of table 46 monthly age crow pearl Mu Qin sheep
Based on rs430810656 SNP site, in 221 black pearl Mu Qin sheep, 121 is AA genotype, 12 for GG Genotype, 88 be GA genotype.In the sheep of 121 AA genotype, the body weight of 85.95% is both greater than equal to 30kg, averagely Value is 34.56kg;In the sheep of 88 GA genotype, the body weight of 80.68% is both greater than equal to 30kg, and meansigma methods is 34.02kg; In the sheep of 12 GG genotype, the body weight of 75% is both less than 30kg, and meansigma methods is 27.88kg.The sheep of 121 AA genotype In, the height of 76.86% is both greater than equal to 60cm, and meansigma methods is 61.21cm;In the sheep of 88 GA genotype, 76.14% Height is both greater than equal to 60cm, and meansigma methods is 61.15cm;In the sheep of 12 GG genotype, the height of 75% is both less than 60cm, Meansigma methods is 57.92cm.In the sheep of 121 AA genotype, the Body steep length of 78.51% is both greater than equal to 64cm, and meansigma methods is 65.19cm;In the sheep of 88 GA genotype, 72.73% Body steep length both greater than equal to 64cm, meansigma methods is 65.10cm; In the sheep of 12 GG genotype, the Body steep length of 75% is both less than 64cm, and meansigma methods is 60.67cm.The silk floss of 121 AA genotype In sheep, the chest measurement of 89.26% is both greater than equal to 79cm, and meansigma methods is 84.10cm;In the sheep of 88 GA genotype, 84.09% Chest measurement both greater than equal to 79cm, meansigma methods is 83.47cm;In the sheep of 12 GG genotype, the chest measurement of 75% is both less than 79cm, meansigma methods is 75.75cm.In the sheep of 121 AA genotype, the chest breadth of 71.07% is both greater than equal to 15cm, meansigma methods For 15.63cm;In the sheep of 88 GA genotype, 70.45% chest measurement both greater than equal to 15cm, meansigma methods 15.55cm;12 In the sheep of GG genotype, the chest measurement of 83.33% is both less than 15cm, and meansigma methods is 13.17cm.
Set up following method: detect six-month-old crow pearl Mu Qin sheep gene based on rs430810656 SNP site to be measured Type, the sheep six-month-old body weight of AA genotype and GA genotype, height, Body steep length, chest measurement, chest breadth are all remarkably higher than GG gene Type.
Set up following method: detect six-month-old crow pearl Mu Qin sheep gene based on rs430810656 SNP site to be measured Type, if AA genotype or GA genotype, sheep body weight more than or equal to 30kg and/or height more than or equal to 60cm and/or Body steep length more than or equal to 64cm and/or chest measurement more than or equal to 79cm and/or chest breadth more than or equal to 15cm, if GG genotype, The body weight of sheep less than 30kg and/or height less than 60cm and/or Body steep length less than 64cm and/or chest measurement less than 79cm and/or Chest breadth is less than 15cm.

Claims (10)

1. identify or a method for auxiliary qualification ovine growth character, comprise the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG gene Type or GA genotype;
It is the sheep that body weight is big and/or chest measurement is big if AA genotype or GA genotype, sheep candidate to be measured, if GG base Because type, sheep to be measured are the sheep that body weight is little and/or chest measurement is little;When described body weight gross appearance was four monthly age, body weight is more than or equal to When 28kg and/or six-month-old, body weight is more than or equal to 30kg;When described chest measurement gross appearance was four monthly age chest measurement more than or equal to 78cm and/ Or chest measurement is more than or equal to 79cm during six-month-old;Described body weight is little time body weight is less than 28kg and/or six-month-old when being presented as four monthly ages Body weight is less than 30kg;Described chest measurement is little when being presented as four monthly ages chest measurement less than chest measurement when 78cm and/or six-month-old less than 79cm.
2. identify or a method for auxiliary qualification ovine growth character, comprise the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG gene Type or GA genotype;
It is the silk floss that height is big and/or Body steep length is big and/or chest breadth is big if AA genotype or GA genotype, sheep candidate to be measured Sheep, is the sheep that height is little and/or Body steep length is little and/or chest breadth is little if GG genotype, sheep candidate to be measured;Described height When gross appearance is six-month-old, height is more than or equal to 60cm;When described Body steep length gross appearance is six-month-old, Body steep length is more than or equal to 64cm;When described chest breadth gross appearance is six-month-old, chest breadth is more than or equal to 15cm;Height that described height is little when being presented as six-month-old is little In 60cm;Described Body steep length is little when being presented as six-month-old Body steep length is less than 64cm;Chest breadth when being presented as six-month-old that described chest breadth is little Less than 15cm.
3. identify or a method for auxiliary qualification ovine growth character, comprise the steps:
Detecting genotype based on rs430810656 SNP site in the genomic DNA of sheep to be measured is AA genotype, GG gene Type or GA genotype;
The growth traits of AA genotype sheep is better than GA genotype sheep, and the growth traits of GA genotype sheep is better than GG genotype Sheep.
4. method as claimed in claim 3, it is characterised in that: described growth traits is excellent to be presented as in following (a1) to (a5) At least one:
(a1) body weight is big;
(a2) chest measurement is big;
(a3) height is big;
(a4) Body steep length is big;
(a5) circumference of cannon bone is big.
5. the method as described in arbitrary in Claims 1-4, it is characterised in that: in described method, described " detect sheep to be measured Genomic DNA in genotype based on rs430810656 SNP site " comprise the steps: with the genome of sheep to be measured DNA is template, uses special primer to carrying out PCR amplification, is then checked order by pcr amplification product;
Described special primer forms by primers F and primer R;
Described primers F is following (b1) or (b2):
(b1) single strand dna shown in sequence 1 of sequence table;
(b2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had identical with sequence 1 The DNA molecular of function;
Described primer R is following (b3) or (b4):
(b3) single strand dna shown in sequence 2 of sequence table;
(b4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had identical with sequence 2 The DNA molecular of function.
6. the method as described in arbitrary in claim 1 to 5, it is characterised in that: described sheep to be measured is crow pearl Mu Qin sheep.
7. special primer pair, is made up of primers F and primer R;
Described primers F is following (b1) or (b2):
(b1) single strand dna shown in sequence 1 of sequence table;
(b2) sequence 1 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had identical with sequence 1 The DNA molecular of function;
Described primer R is following (b3) or (b4):
(b3) single strand dna shown in sequence 2 of sequence table;
(b4) sequence 2 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and is had identical with sequence 2 The DNA molecular of function.
8. the application of primer pair described in claim 7, for as follows (c1) or (c2):
(c1) auxiliary identifies ovine growth character;
(c2) preparation auxiliary identifies the test kit of ovine growth character.
9. one kind relevant to ovine growth character containing described rs430810656 SNP site arbitrary in claim 1 or 3 DNA fragmentation;As shown in sequence 3 in sequence table.
10. arbitrary described method in claim 1 to 6, or, the special primer pair described in claim 7, in the breeding of sheep Application.
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CN107760793A (en) * 2017-12-08 2018-03-06 锡林郭勒职业学院 Differentiate primer, probe and the method for tin alliance sheep and agriculture district feeding sheep
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CN107760793A (en) * 2017-12-08 2018-03-06 锡林郭勒职业学院 Differentiate primer, probe and the method for tin alliance sheep and agriculture district feeding sheep
CN107760793B (en) * 2017-12-08 2021-02-26 锡林郭勒职业学院 Primer, probe and method for identifying stannum allium sheep and farm-area barn feeding sheep
CN109182543A (en) * 2018-10-18 2019-01-11 北京康普森生物技术有限公司 A kind of identification sheep known for its fine thick wool combines and its applies with 4 kinds of SNP sites of non-sheep known for its fine thick wool
CN109182543B (en) * 2018-10-18 2021-09-17 北京康普森生物技术有限公司 Combination of 4 SNP loci for identifying Tan sheep and non-Tan sheep and application thereof
CN114381531A (en) * 2022-03-11 2022-04-22 浙江省农业科学院 SNPs molecular marker g.43756G & gtA and application thereof in Hu sheep molecular marker-assisted breeding

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