KASP molecular marker related to sheep milk fat percentage character, detection primer group and application of KASP molecular marker

Abstract

The invention relates to the technical field of molecular detection, and aims to provide a method for detecting sheep Chr15: g.12674721 site Cgt; the primer group is composed of the following three primers: a site C allele typing primer FAM, the nucleotide sequence of which is shown as SEQ ID NO: 1, a site C allele typing primer FAM, a site C allele typing primer FAM, a site C allele typing primer FAM, a site C allele typing primer FAM and a site C allele typing primer FAM, the nucleotide sequence of the locus T allele typing primer VIC is as shown in SEQ ID NO: 2; and the nucleotide sequence of the universal downstream primer is as shown in SEQ ID NO: 3. Wherein the Chr15: g.12674721 locus is based on a sheep reference genome version AR-UIRamv3.0, and the recombinant plasmid can be used for preparing a recombinant plasmid. The invention discovers and discloses a Cgt of a Chr15: g.12674721 site in a sheep genome for the first time; the T single nucleotide polymorphism (SNP) is obviously associated with the milk fat percentage character. Association analysis of 140 Hu sheep proves that the cream-fat percentage of a CC genotype individual is obviously higher than that of a CT type individual and a TT type individual. The discovery provides a direct and reliable molecular target for genetic improvement of the fat percentage of sheep.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a KASP molecular marker, detection primer set, and application related to sheep milk fat percentage trait. Background Technology

[0002] Approximately 50% of the fatty acids in goat milk are synthesized from acetic acid produced by fiber fermentation in the rumen of the ewe. A stable or high milk fat percentage usually indicates sufficient available fiber in the diet, suggesting that acetic acid fermentation is dominant in the rumen and that subacute rumen acidosis has not occurred. A sudden or significant drop in milk fat percentage is often an early warning sign of rumen dysfunction and an unbalanced diet (such as excessive concentrate and insufficient fiber). Therefore, maintaining a high milk fat percentage is, to some extent, equivalent to maintaining the health of the ewe. Thus, milk fat percentage is an effective indicator of rumen health.

[0003] For breeding work, milk fat percentage is an important selective trait. Numerous genes regulate milk fat percentage, often working synergistically through complex signaling pathway networks. Previous studies have shown that the mTOR, PI3K-Akt, MAPK, AMPK, and PPAR signaling pathways are involved in milk fat synthesis and metabolism. In dairy cows, several candidate regulatory genes for milk fat percentage have been identified, including the discovery of novel genes such as EFNA1 and HSTN.

[0004] Like other economic traits, sheep milk fat percentage is regulated by multiple minor genes, making it difficult to determine the major genes. However, by identifying molecular markers closely linked to the milk fat percentage trait, marker-assisted selection can be achieved, improving breeding efficiency. This patent discloses a rapid KASP detection method for the C>T variant at the Chr15:g.12674721 locus in sheep and its application in marker-assisted selection breeding for the sheep milk fat percentage trait. Summary of the Invention

[0005] To address the technical problems mentioned in the background section, this invention provides a KASP molecular marker, detection primer set, and its application related to sheep milk fat percentage.

[0006] This invention is achieved using the following technical solution: One of the objectives of this invention is to propose a specific primer set for detecting the C>T variant at the Chr15:g.12674721 locus in sheep, wherein the primer set consists of the following three primers:

[0007] The nucleotide sequence of the C allele typing primer FAM is shown in SEQ ID NO:1;

[0008] The nucleotide sequence of the site T allele typing primer VIC is shown in SEQ ID NO:2;

[0009] The universal downstream primer has the nucleotide sequence shown in SEQ ID NO:3.

[0010] The Chr15:g.12674721 locus is based on sheep reference genome version ARS-UI_Ramb_v3.0.

[0011] The second objective of this invention is to propose the application of the aforementioned specific primer set in the preparation of reagents or kits for identifying or assisting in the identification of the fat content of sheep milk.

[0012] The third objective of this invention is to propose the application of the specific primer set in molecular marker-assisted selection breeding of sheep.

[0013] Furthermore, the molecular marker-assisted selection breeding involves selecting sheep with the genotype CC at the Chr15:g.12674721 locus and / or culling sheep with the genotype TT at the Chr15:g.12674721 locus to obtain or breed a sheep population with increased milk fat percentage.

[0014] The fourth objective of this invention is to provide a method for detecting the genotype at the Chr15:g.12674721 locus in sheep, comprising the following steps:

[0015] (1) Using the genomic DNA of the sheep to be tested as a template, KASP genotyping PCR amplification was performed using the specific primer set described in claim 1;

[0016] (2) Detect the fluorescence signal of the PCR amplification product in step (1);

[0017] (3) Based on the type and intensity of the fluorescence signal, determine whether the genotype of the sheep to be tested at the Chr15:g.12674721 site is CC, CT or TT.

[0018] Furthermore, the procedure for KASP genotyping PCR amplification in step (1) is as follows: pre-denaturation at 94℃ for 15 min; followed by 10 cycles of landing PCR: denaturation at 94℃ for 20 s, annealing at 61℃ for 1 min, with the annealing temperature decreasing by 0.6℃ in each cycle; and finally 26 cycles: denaturation at 94℃ for 20 s, annealing at 55℃ for 1 min.

[0019] The fifth objective of this invention is to propose a molecular marker for identifying or assisting in the identification of high or low milk fat percentage in sheep. The molecular marker is a single nucleotide polymorphism at the Chr15:g.12674721 site in the sheep genome, where the nucleotide is C or T. Sheep with the genotype CC have a higher milk fat percentage than sheep with the genotypes CT or TT.

[0020] The sixth objective of this invention is to provide a kit for detecting the genotype of sheep Chr15:g.12674721 locus, comprising the aforementioned specific primer set.

[0021] The seventh objective of this invention is to propose a method for screening sheep with high milk fat percentage, comprising the following steps: detecting the genotype of the candidate sheep based on the Chr15:g.12674721 locus, and selecting sheep with the genotype CC.

[0022] The eighth objective of this invention is to propose a sheep breeding method, which includes using the aforementioned molecular markers, or employing methods such as detecting the genotype of the sheep Chr15:g.12674721 locus or screening sheep with high milk fat percentage to select sheep populations in order to improve the milk fat percentage trait of the population.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] First, it provides a novel, scientifically validated molecular marker. This invention is the first to discover and disclose a significant association between the C>T single nucleotide polymorphism (SNP) at the Chr15:g.12674721 locus in the sheep genome and the milk fat percentage trait. Association analysis of 140 Hu sheep confirmed that individuals with the CC genotype had a significantly higher milk fat percentage than those with the CT and TT genotypes. This discovery provides a direct and reliable molecular target for the genetic improvement of sheep milk fat percentage.

[0025] Secondly, an efficient, accurate, and economical early detection technology system has been established. Based on this marker, this invention designed a specific KASP primer set and an optimized detection method, achieving a genotyping accuracy rate exceeding 99.8%. This technical solution has significant advantages: it only requires the synthesis of ordinary primers, resulting in low cost; it requires a small amount of DNA sample and is suitable for detection needs of varying throughputs; more importantly, it enables genotyping and trait prediction in early sheep life stages (such as the lambing stage), significantly advancing the selection process and overcoming the traditional breeding bottleneck that relies on phenotypic determination.

[0026] Third, it creates clear breeding application value and socio-economic benefits. Based on this molecular marker, a precision breeding strategy of "preferring CC phenotypes and eliminating TT phenotypes" can be formulated to achieve efficient and targeted improvement of the milk fat percentage trait. This not only helps to improve the nutritional value and commercial quality of goat milk products, but also indirectly promotes rumen health in sheep flocks by selecting individuals with high milk fat percentages. This invention provides the sheep breeding industry with a complete set of technical support, from core markers and detection tools to complete solutions, and powerfully promotes the transformation and upgrading of traditional breeding to modern molecular design breeding. Attached Figure Description

[0027] Figure 1This is the KASP genotyping map of the sheep Chr15: g.12674721 locus proposed in this invention;

[0028] Figure 2 This is a bar chart showing the correlation between the sheep milk fat percentage trait and the target site proposed in this invention. Detailed Implementation

[0029] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0031] Example 1:

[0032] The design and synthesis of the KASP primer set for detecting the sheep Chr15:g.12674721 locus are as follows:

[0033] Based on the sheep reference genome version ARS-UI_Ramb_v3.0, competitive allele-specific PCR (KASP) genotyping primers were designed targeting the C>T single nucleotide polymorphism (SNP) site at physical location g.12674721 on chromosome 15. The genomic region sequence context information for this site was obtained from public databases.

[0034] The designed specific KASP primer set contains three primers:

[0035] 1. C-allele-specific forward primer (FAM marker): This primer has a 3' end that perfectly matches the C allele, and its 5' end is attached with a universal adapter sequence corresponding to the FAM fluorescent reporter gene. Its complete sequence is:

[0036] 5'-gaaggtgaccaagttcatgctAACCTGTATTTCTTACCTTCTCTTTC-3' (SEQ ID NO: 1).

[0037] The lowercase letters represent the common adapter sequence (FAM), while the uppercase letters represent the site-specific sequence.

[0038] 2. Allele T-specific forward primer (VIC marker): This primer has a 3' end base that perfectly matches the T allele, and its 5' end is attached with a universal adapter sequence corresponding to the VIC fluorescent reporter gene. Its complete sequence is:

[0039] 5'-gaaggtcggagtcaacggattAACCTGTATTTCTTACCTTTCTCTTTT-3' (SEQ ID NO: 2).

[0040] The lowercase letters represent universal adapter sequences (VIC), while the uppercase letters represent site-specific sequences.

[0041] 3. Universal reverse primer: Its sequence is as follows:

[0042] 5'-GGATAGAATCCTATGGGTCTTCCCA-3' (SEQ ID NO: 3).

[0043] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and purified by PAGE to ensure quality.

[0044] Example 2: Extraction of sheep genomic DNA, the specific implementation of which is as follows:

[0045] The experiment used 140 healthy primiparous Hu sheep replacement ewes (all from Yongfeng Sheep Farm in Jinghai District, Tianjin) as experimental subjects. All experimental sheep were bred under the same conditions and fed and managed after giving birth.

[0046] Two mL of anticoagulated blood was collected from the jugular vein of each sheep. Genomic DNA was extracted using the phenol-chloroform method, with the following simplified steps: Blood samples were mixed with cell lysis buffer, centrifuged to remove the supernatant; protein K and buffer GB were added, and the mixture was digested in a 55°C water bath until the solution became clear; buffer BD was added to precipitate proteins; the supernatant was transferred to an adsorption column, washed twice with buffer GD and wash buffer PW, and then eluted with elution buffer TB to obtain high-purity genomic DNA. The DNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer, and all sample DNA concentrations were uniformly diluted to 20-50 ng / μL and stored at -20°C for later use.

[0047] Example 3:

[0048] The specific implementation of sheep Chr15:g.12674721 locus genotyping based on KASP primers is as follows:

[0049] 1. Preparation of the KASP reaction system:

[0050] The reaction system consisted of approximately 20-50 ng of sheep genomic DNA template, 0.14 μL of KASP primer mixture (with allele C and T specific primers each at a final concentration of 12 μM and universal reverse primer at a final concentration of 30 μM), 5 μL of 2×KASPMasterMix (purchased from LG C Biosearch Technologies), and the remaining volume was made up with sterile deionized water.

[0051] 2. PCR amplification procedure:

[0052] The reaction was performed on a real-time quantitative PCR instrument (model: QuantStudio5). The reaction procedure is as follows:

[0053] Pre-denaturation at 94℃ for 15 minutes;

[0054] Then, 10 cycles of "touchdown PCR" were performed: each cycle included denaturation at 94°C for 20 seconds, annealing and extension at 61°C for 1 minute, with the annealing temperature decreasing by 0.6°C per cycle.

[0055] Finally, 26 cycles of amplification were performed: each cycle consisted of denaturation at 94°C for 20 seconds and annealing at 55°C for 1 minute.

[0056] Fluorescence signals from the FAM and VIC channels were collected throughout the reaction.

[0057] 3. Genotype determination:

[0058] After the reaction, the endpoint fluorescence signal was analyzed using the instrument's accompanying genotyping analysis software (such as QuantStudioDesign&AnalysisSoftware). Figure 1 As shown, the data points in the scatter plot clearly cluster into three clusters:

[0059] Clusters showing strong FAM fluorescence (strong X-axis, weak Y-axis) correspond to CC homozygous genotypes;

[0060] Clusters showing strong VIC fluorescence (strong Y-axis, weak X-axis) correspond to TT homozygous genotypes;

[0061] Clusters displaying both FAM and VIC fluorescence (located in the middle) correspond to CT heterozygous genotypes.

[0062] Verification showed that using the primer set and method of this embodiment, the genotyping accuracy of this site was greater than 99.8%.

[0063] Example 4:

[0064] The association analysis between different genotypes at the Chr15:g.12674721 locus and sheep milk fat percentage was conducted as follows:

[0065] Milk fat percentage was measured and recorded in all 140 experimental sheep. Milk fat percentage data are expressed as mean ± standard error. One-way ANOVA was performed using SPSS 22.0 software to compare differences in milk fat percentage among different genotypes, and Duncan's method was used for multiple comparisons. The significance level was set at P < 0.05.

[0066] Association analysis results as follows Figure 2 And as shown in the table below:

[0067] Chr15:g.12674721 genotype Number of individuals (n) Milk fat percentage (%) (mean ± standard error) CC 35 5.08±0.61a CT 68 3.74±0.21b TT 37 3.20±0.17b

[0068] Note: Different letters in the same column indicate significant differences (P<0.05).

[0069] The results showed that sheep carrying the CC genotype had a significantly higher milk fat percentage (5.08%) than sheep carrying the CT genotype (3.74%) and TT genotype (3.20%) (P<0.05). This conclusively demonstrates that the C allele at the Chr15:g.12674721 locus in the sheep genome is the dominant allele for increasing milk fat percentage, and the CC genotype can be used as a molecular marker for screening sheep with high milk fat percentage.

[0070] Example 5:

[0071] Based on the conclusions of Example 4, the present invention can be practically applied in sheep (especially Hu sheep) breeding to achieve early and accurate selection. The specific implementation is as follows:

[0072] 1. Screening for individuals with high milk fat percentage:

[0073] For any candidate sheep (such as newborn lambs), only a small blood or tissue sample needs to be collected to extract genomic DNA. Using the specific KASP primer set provided in Example 1 and the detection method described in Example 3, the genotype at the Chr15:g.12674721 locus can be determined within hours. Individuals with the CC genotype are directly selected as core breeding sheep for breeding high-fat-percentage lines.

[0074] 2. Guiding population selection and variety improvement:

[0075] In the existing sheep flock, this technology will be used to conduct genotyping screening of all members. The following breeding strategy will be developed:

[0076] Priority selection: Ewes and rams with the CC genotype will be selected as the core breeding group.

[0077] Gradual elimination: systematically eliminating individuals with the TT genotype to rapidly increase the frequency of the dominant C allele in the population.

[0078] Monitoring and mating: For CT heterozygous individuals, mating with CC individuals can fix the C allele in offspring.

[0079] The aforementioned marker-assisted selection (MAS) can overcome the limitations of traditional phenotypic determination, enabling precise selection early in an individual's life, significantly shortening the generation interval in breeding, increasing selection intensity and genetic progress, and thus efficiently breeding new sheep populations with improved milk fat percentage traits.

[0080] Example 6: This example proposes a detection kit containing the aforementioned KASP primer set; its components include:

[0081] Component A: Specific KASP primer dry powder or premix as shown in SEQ ID NO: 1, 2 and 3.

[0082] Component B: 2×KASPMasterMix (containing DNA polymerase, dNTPs, Mg²⁺ and optimized buffer).

[0083] Component C: Positive control DNA samples (one sample each of sheep genomic DNA known to be CC, CT and TT genotypes).

[0084] Component D: Sterile deionized water.

[0085] Optional components: Genomic DNA extraction reagent, instruction manual. The instruction manual details the reaction system and procedure as described in Example 3, and the result interpretation criteria as described in Example 4.

[0086] This kit integrates the core components required for detection. Users only need to add the DNA template to be tested to conduct experiments. It is easy to operate, produces stable results, and is suitable for large-scale sample screening in laboratories and breeding farms.

[0087] In summary, this invention discloses for the first time the significant association between the C>T variant at the Chr15:g.12674721 locus in sheep and the milk fat percentage trait, and provides a specific, cost-effective KASP primer set and a mature and reliable genotyping method for detecting this locus. Using this invention, early, rapid, and accurate molecular identification of the sheep milk fat percentage trait can be achieved, providing a powerful tool for sheep genetic improvement and efficient breeding, and has significant industrial application value.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A specific primer set for detecting the C>T variant at the Chr15:g.12674721 locus in sheep, characterized in that, The primer set consists of the following three primers: The nucleotide sequence of the C allele typing primer FAM is shown in SEQ ID NO:1; The nucleotide sequence of the site T allele typing primer VIC is shown in SEQ ID NO:2; A universal downstream primer, the nucleotide sequence of which is shown in SEQ ID NO:3; The Chr15:g.12674721 locus is based on sheep reference genome version ARS-UI_Ramb_v3.

0.

2. The use of the specific primer set of claim 1 in the preparation of reagents or kits for identifying or assisting in the identification of the fat content of sheep milk.

3. The application of the specific primer set described in claim 1 in molecular marker-assisted selection breeding of sheep.

4. The application according to claim 3, characterized in that, The marker-assisted selection breeding involves selecting sheep with the genotype CC at the Chr15:g.12674721 locus and / or culling sheep with the genotype TT at the Chr15:g.12674721 locus to obtain or breed a sheep population with increased milk fat percentage.

5. A method for detecting the genotype at the Chr15:g.12674721 locus in sheep, characterized in that, Includes the following steps: (1) Using the genomic DNA of the sheep to be tested as a template, KASP genotyping PCR amplification was performed using the specific primer set described in claim 1; (2) Detect the fluorescence signal of the PCR amplification product in step (1); (3) Based on the type and intensity of the fluorescence signal, determine whether the genotype of the sheep to be tested at the Chr15:g.12674721 site is CC, CT or TT.

6. The method according to claim 5, characterized in that, The procedure for KASP genotyping PCR amplification in step (1) is as follows: pre-denaturation at 94℃ for 15 min; followed by 10 cycles of landing PCR: denaturation at 94℃ for 20 s, annealing at 61℃ for 1 min, with the annealing temperature decreasing by 0.6℃ in each cycle; and finally 26 cycles: denaturation at 94℃ for 20 s, annealing at 55℃ for 1 min.

7. A molecular marker for identifying or assisting in the identification of sheep milk fat content, characterized in that, The molecular marker is a single nucleotide polymorphism at the Chr15:g.12674721 site in the sheep genome, where the nucleotide is C or T; sheep with the genotype CC have a higher milk fat percentage than sheep with the genotypes CT or TT.

8. A kit for detecting the genotype at the Chr15:g.12674721 locus in sheep, characterized in that, It includes the specific primer set as described in claim 1.

9. A method for screening sheep with high milk fat content, characterized in that, Includes the following steps: The genotype of the candidate sheep was determined based on the Chr15:g.12674721 locus, and sheep with the genotype CC were selected.

10. A sheep breeding method, characterized in that, This includes selecting sheep populations using the molecular markers described in claim 7, or the methods described in claim 5 or 6, or the methods described in claim 9, to improve the milk fat percentage trait of the population.

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