Haplotype marker for breeding milk fat percentage character of milk production of dairy cow and application of haplotype marker

Abstract

The invention relates to the field of molecular biology, and discloses a haplotype marker for dairy cow milk production butter-fat percentage character breeding and application of the haplotype marker, the haplotype marker is composed of three SNP sites in a strong linkage imbalance state, and the three SNP sites are located in a Block1 haplotype block region of a 10 # chromosome NC037337.1 of a dairy cow ARS-UCD1.2 genome; the three SNP sites are as follows: SNP1: g.59680573 A > C, SNP2: g.59743173 G > C, and SNP3: g.59743414 C > T; the dominant haplotype of the haplotype marker is H2, and the allele combination of the haplotype marker is SNP1-A, SNP2-G and SNP3-C; the homozygous H2H2 of the dominant haplotype H2 is significantly related to the high milk fat percentage character of milk production of dairy cows. The bottleneck of the traditional technology is solved, and the breeding accuracy is remarkably improved; the screening detection technical scheme is stable and reliable and has a wide application range; the milk production quality of groups is improved, and the industrial economic value is enhanced.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a haplotype marker for the selection of milk fat percentage trait in dairy cows and its uses. Background Technology

[0002] Milk is a core livestock product with both nutritional and economic value, occupying a core strategic position in the high-quality development of my country's animal husbandry industry. In recent years, with the upgrading of residents' consumption, the demand for high-end dairy products has continued to rise, and milk quality has become the core focus of industry competition and consumer attention. Milk fat is the core indicator for evaluating milk quality, determining not only the flavor, nutritional value, and processing characteristics of milk, but also serving as a key basis for pricing raw milk purchases. Its content directly determines the core competitiveness of the dairy industry chain. Holstein cows are the most widely raised and highest-producing dairy cattle breed globally, and also the dominant breed in my country's dairy cattle breeding. However, their overall milk fat percentage is relatively low, with an industry average of only about 3.5%. The scarcity of high-fat-percentage breeds has become a core bottleneck restricting the improvement of quality and efficiency in my country's dairy industry and enhancing its international competitiveness. The genetic regulation of milk fat traits and the selection of superior breeds have become research hotspots and key challenges in my country's dairy cattle breeding field.

[0003] Milk production in dairy cows is a complex quantitative trait controlled by multiple genes with minor effects. Genetic factors are the fundamental determinants of core economic traits such as milk fat, and superior heritable traits can be stably passed down through generations. Traditional dairy cow breeding systems, centered on breed registration, DHI (Dairy Hypoglycemic Index) production performance testing, and progeny testing, have played a significant role in improving milk production. However, they suffer from inherent limitations such as long generation intervals, slow genetic progress, and selection accuracy being greatly influenced by the environment, making them unable to meet the breeding needs of my country's dairy industry for rapid improvement of milk fat percentage. Since the 1980s, with the deep integration of molecular genetics and quantitative genetics, molecular breeding technology has developed rapidly, propelling dairy cow breeding from traditional phenotypic selection to precise genotypic selection. Among these, marker-assisted selection (MAS) breeding, with its advantages of directly selecting the genotype of the target trait, significantly shortening the generation interval, and improving selection accuracy, has become one of the core technologies for the genetic improvement of economic traits in dairy cows. The core premise of MAS breeding is to discover and obtain molecular genetic markers that are significantly associated with the target trait, have stable genetic effects, and can explain a high degree of phenotypic variation.

[0004] In recent years, genomic selection (GS) technology has been widely used in dairy cattle breeding. Essentially, it is a high-density genome-wide marker-assisted selection process that enables early and precise selection of young cattle, further improving breeding efficiency. However, existing research shows that the accuracy of GS genomic breeding value estimation is highly dependent on the integration of functional genes and major genetic markers significantly associated with the target trait. Relying solely on randomly distributed genome-wide SNP markers offers limited improvement in the accuracy of selection for low-heritability traits such as milk fat percentage. Single nucleotide polymorphisms (SNPs) are currently the most widely used molecular markers. Haplotype markers composed of multiple tightly linked SNP sites in strong linkage disequilibrium, compared to a single SNP site, more stably reflect the genetic effects of the target gene, have a higher correlation with complex quantitative traits, and can explain a higher proportion of phenotypic variation, making them a more ideal genetic marker for dairy cattle molecular breeding. Currently, there is a severe lack of haplotype markers with significant genetic effects and large-scale validation in large-scale populations for the milk fat percentage trait in Holstein dairy cows in my country, which seriously restricts the efficiency and accuracy of molecular breeding for this trait. Therefore, identifying and validating dominant haplotype markers that are significantly associated with the milk fat percentage trait in Holstein dairy cows, and providing precise and efficient molecular tools for the breeding of high-fat-percentage dairy cow breeds, is a key technical problem that urgently needs to be solved in the field of dairy cow genetic improvement in my country. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a haplotype marker for selecting dairy cows based on milk fat percentage and its applications. This haplotype marker overcomes traditional technical bottlenecks, significantly improving breeding accuracy. The screening and detection technology is stable, reliable, and widely applicable. Its application enhances the quality of milk production in herds and strengthens the economic value of the industry.

[0006] To achieve the above objectives, the present invention provides a haplotype marker for breeding dairy cows' milk fat percentage trait, wherein the haplotype marker consists of three haplotypes in strong linkage disequilibrium (LD, r). 2 The three SNP sites are composed of SNP sites in the state of >0.8, and the three SNP sites are located in the Block 1 haplotype block region of chromosome 10 NC_037337.1 of the bovine ARS-UCD1.2 genome; The three SNP sites are: SNP1: g.59680573 A>C, SNP2: g.59743173 G>C, SNP3: g.59743414 C>T; The dominant haplotype of the haplotype marker is H2, and its allele combination is SNP1-A, SNP2-G, SNP3-C; the homozygous H2H2 of the dominant haplotype H2 is significantly associated with the high milk fat percentage trait in dairy cows.

[0007] Furthermore, the high milk fat percentage trait in dairy cows refers to the milk fat percentage trait in dairy cows during the 305-day lactation period.

[0008] Furthermore, the cow is a Holstein cow.

[0009] A second aspect of the present invention provides a specific primer-probe set for detecting the haplotype marker described in the present invention, comprising upstream primers, downstream primers, and specific probes targeting SNP1, SNP2, and SNP3 sites, as detailed below: (1) For SNP1 site: the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; (2) For SNP2 site: the upstream primer sequence is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6; (3) For SNP3 site: the upstream primer sequence is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9.

[0010] Furthermore, the probe is an SBE probe.

[0011] A third aspect of the present invention provides a kit for detecting haplotype markers according to the present invention, the kit comprising the specific primer and probe set described in the present invention, and optionally comprising PCR reaction buffer, DNA polymerase, dNTP mixture, DNA extraction reagent and genotyping reagent.

[0012] Furthermore, the kit also includes a positive control and a negative control; the positive control is a bovine genomic DNA standard carrying a homozygous haplotype combination of H2H2, and the negative control is nuclease-free ultrapure water.

[0013] The fourth aspect of this invention provides the application of the haplotype markers described in this invention, or the specific primer-probe sets described in this invention, or the kits described in this invention, in marker-assisted selection, genomic selection, or genotypic identification of milk fat percentage trait in dairy cows.

[0014] Furthermore, it includes the following steps: (1) Collect biological samples from the dairy cows to be tested and extract genomic DNA; (2) Genotyping the three SNP sites described in claim 1 in the genomic DNA, constructing haplotypes and determining the haplotype combination of the individual to be tested; (3) Individuals carrying the H2H2 homozygous haplotype combination were selected as breeding individuals for dairy cattle breeding populations to breed dairy cattle breeds with high milk fat content.

[0015] Furthermore, the genotyping method includes any one of Sanger sequencing, time-of-flight mass spectrometry, TaqMan probe method, gene chip method, and high-throughput sequencing.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The genetic effect is significant and stable. The haplotypes formed by three strongly linked SNPs in the Block 1 region of dairy cows were identified, and the H2H2 homozygous type was verified as the dominant genotype. In a large population, its milk fat percentage reached 4.20%, which was significantly better than other genotypes (P<0.05) and more than 20% higher than the industry average, filling the relevant molecular marker gap.

[0017] (2) High selection accuracy. Compared with a single SNP marker, this haplotype captures more complete genetic information and has a higher correlation with milk fat percentage, avoiding the shortcomings of low selection accuracy of single SNP markers and improving the reliability of genotype selection.

[0018] (3) Breeding efficiency is greatly improved. Early selection of calves can be achieved without waiting for lactation phenotyping or progeny testing, shortening the generation interval of breeding from 3-5 years to within 6 months, greatly reducing costs and increasing efficiency, and accelerating the improvement of high milk fat ratio breeds.

[0019] (4) Strong industry adaptability. It can be detected by conventional genotyping technology, and it is easy to develop special reagent kits to adapt to large-scale high-throughput detection. It can also be integrated into the whole genome selection system to help improve the efficiency of the breeding end and enhance the competitiveness of the dairy industry. Attached Figure Description Figure 1 This indicates the results of linkage disequilibrium analysis at gene polymorphism sites; Figure 2 This indicates the results of pooled PCR sequencing of the TRPM7 gene locus 10: g.59680573 A>C in Chinese Holstein cattle; Figure 3 This indicates the TRPM7 gene locus 10: g.59680573 A>C genotyping diagram; Figure 4 This indicates the results of pooled PCR sequencing of the TRPM7 gene locus 10: g.59743173 G>C in Chinese Holstein cattle; Figure 5 This indicates the TRPM7 gene locus 10: g.59743173 G>C genotyping diagram; Figure 6This indicates the results of pooled PCR sequencing of the TRPM7 gene locus 10: g.59743414 C>T in Chinese Holstein cattle; Figure 7 The TRPM7 gene locus 10: g.59743414 C>T genotype is shown. Detailed Implementation

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Phenotypic Data Collection and Bovine Genomic DNA Extraction 1.1 Experimental Materials 1. Experimental Animals: Five hundred healthy lactating Holstein cows from the Pingjibao Dairy Farm No. 2 of Ningxia Helanshan Dairy Company were selected, all under the same feeding conditions (uniform feed formula, work and rest management, and disease prevention standards). The birth years of this experimental group ranged from 2012 to 2021. The production performance records of the experimental cows included individual number, date of birth, farm number, father number, mother number, maternal grandfather number, calving date, parity, 305-day milk yield (MY305), milk fat yield (FY), milk protein yield (PY), fat percentage (FP), and protein percentage (PP). All phenotypic data were standardized (eliminating confounding factors such as parity, calving season, and age). MY305 represents the milk yield trait, while FY, PY, FP, and PP represent four milk component traits, collectively serving as the five milk yield traits in this study.

[0023] 2. Sample Collection: 5 mL of blood was collected from the tail root. DNA was extracted from the bovine blood using a DNA extraction kit. The quality of the extracted DNA was assessed by agarose gel electrophoresis, and the concentration of the extracted DNA was measured using a micro-nucleic acid and protein analyzer. Fifty qualified DNA samples were randomly selected, and 1 μL of each was mixed into a 1.5 mL centrifuge tube to construct a DNA mixing chamber. This chamber was stored at -20°C for subsequent experiments.

[0024] Example 2: Genotyping of target SNP loci Based on previous genome-wide association analysis results, the Block1 genomic region significantly associated with milk fat percentage was identified. Three SNP sites (SNP1, SNP2, and SNP3) meeting the quality control criteria (MAF > 0.05 and HWE P > 1 × 10^-6) were screened, and specific amplification and extension primers were designed using the following methods: 1. Primer design and PCR amplification For the SNP sites screened in Example 1, amplification primers were designed using Primer 5.0 software based on the bovine genome sequence published on NCBI (https: / / www.ncbi.nlm.nih.gov / ) (Table 1), and then sent to Shaanxi Qingke Biotechnology Co., Ltd. for synthesis.

[0025] Table 1. Amplification primer sequence information The gene amplification system consisted of 50 μL: 6 μL cDNA sample, 2 μL each of forward and reverse primers, 25 μL TaqPCRMasterMix, and 15 μL ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, annealing for 40 s, and extension at 72℃ for 1 min, for a total of 35 cycles; 72℃ extension for 5 min, and storage at 4℃. After 1% agarose gel electrophoresis, the products were sent to Shaanxi Qingke Biotechnology Co., Ltd. for sequencing.

[0026] 2. Sequencing results and screening of polymorphic sites The results were compared with the original sequence using SnapGene and compared with the sequencing peak diagram to identify variant sites in the dairy cow population (see...). Figure 2 , Figure 4 , Figure 6 ).

[0027] 3. Genotyping Based on the sequence information of the SNP sites, PCR reactions and single-base expansion primer and probe sets were designed using the software MassARRAY Assay Design SUITE V2.0. The sequences are shown in Table 2 below: Table 2. KASP genotyping primers and probes for mutation sites. The detected SNP loci and complete blood samples from the dairy herd were submitted to Compson for KASP genotyping. Figure 3 , Figure 5 , Figure 7 The primers and probes were combined to prepare a detection kit.

[0028] 4. Experimental Results The association analysis between the genotypes of the aforementioned SNP loci and milk production traits is shown in Table 3 below. Individuals with the AA genotype (SNP1), GG genotype (SNP2), and CC genotype (SNP3) exhibited significantly higher milk fat percentage, milk protein percentage, milk fat content, and milk protein content than individuals with other genotypes, indicating these are dominant genotypes for superior milk quality.

[0029] Table 3 Association analysis between mutation sites and milk production traits Note: P-value indicates the degree of association between each SNP and the milk production trait; different letters indicate the differences between genotypes after multiple comparisons (different lowercase letters indicate significant differences P<0.05, different uppercase letters indicate extremely significant differences P<0.01, and the same letters indicate no significance regardless of case).

[0030] As shown in the table, the TRPM7 gene sites 10:g.59680573 A>C, 10:g.59743173 G>C, and 10:g.59743414 C>T in Chinese Holstein cattle have a significant impact on milk quality (P<0.05), and the dominant genotypes are homozygous AA, homozygous GG, and homozygous CC, respectively.

[0031] Example 3: Linkage disequilibrium analysis, haplotype construction, and trait association analysis Linkage disequilibrium analysis was performed on the three SNP sites using Haploview 4.2 software, such as... Figure 1 As shown, the results indicate that the three sites are located in the Block 1 haplotype block region, and the D' values ​​are all greater than 0.85, indicating a strong linkage disequilibrium state, which meets the requirements for haplotype construction.

[0032] After constructing haplotypes and removing low-frequency haplotypes, three main haplotypes were obtained: H1 (CCT), H2 (AGC), and H3 (AGT). PLINK 1.9 software was used to perform association analysis between haplotype combinations and milk production traits using a general linear model. The results are shown in Table 4 below: Table 4. Results of association analysis between haplotype combinations and milk production traits in Holstein cows (mean ± standard error) Note: P-value indicates the degree of association between each SNP and the milk production trait; different letters indicate the differences between genotypes after multiple comparisons (different lowercase letters indicate significant differences P<0.05, different uppercase letters indicate extremely significant differences P<0.01, and the same letters indicate no significance regardless of case).

[0033] As shown in Table 4, haplotype combinations were significantly associated with milk fat percentage (P<0.05). Comprehensive analysis indicates that the haplotype combination H2H2 is a dominant haplotype combination and can be used for marker-assisted selection in Holstein dairy cows to accelerate the breeding process.

[0034] Example 4: Independent population validation of dominant haplotypes A separate population of 300 Holstein dairy cows from another domestic ranch was selected, with no individuals replicated from the experimental population. Genotyping and haplotype analysis were performed using the methods described above. The results showed that the average milk fat percentage of the H2H2 homozygous individuals (n=42) was 4.17%, significantly higher than that of the H1H2 heterozygous individuals (3.95%) and the H1H1 homozygous individuals (3.94%) (P<0.05), consistent with previous experimental results. This demonstrates that the genetic effect of this dominant haplotype is stable and applicable across different populations.

[0035] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A haplotype marker for selecting milk fat percentage as a trait in dairy cows, characterized in that, The haplotype marker consists of three SNP sites in a strong linkage disequilibrium state, and the three SNP sites are located in the Block 1 haplotype block region of chromosome 10 NC_037337.1 of the bovine ARS-UCD1.2 genome; The three SNP sites are: SNP1: g.59680573 A>C, SNP2: g.59743173 G>C, SNP3: g.59743414 C>T; The dominant haplotype of the haplotype marker is H2, and its allele combination is SNP1-A, SNP2-G, SNP3-C; the homozygous H2H2 of the dominant haplotype H2 is significantly associated with the high milk fat percentage trait in dairy cows.

2. The haplotype marker according to claim 1, characterized in that, The high milk fat percentage trait in dairy cows refers to the milk fat percentage trait produced during the 305-day lactation period.

3. The haplotype marker according to claim 1, characterized in that, The cows in question are Holstein cows.

4. A specific primer-probe set for detecting the haplotype marker of claim 1, characterized in that, This includes upstream primers, downstream primers, and specific probes targeting SNP1, SNP2, and SNP3 sites, as detailed below: (1) For SNP1 site: the upstream primer sequence is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; (2) For SNP2 site: the upstream primer sequence is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6; (3) For SNP3 site: the upstream primer sequence is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.

9.

5. The specific primer-probe set according to claim 4, characterized in that, The probe is an SBE probe.

6. A kit for detecting the haplotype marker of claim 1, characterized in that, The kit comprises the specific primer and probe set as described in claim 4, and optionally includes PCR reaction buffer, DNA polymerase, dNTP mixture, DNA extraction reagent and genotyping reagent.

7. The reagent kit according to claim 6, characterized in that, The kit also includes a positive control and a negative control; the positive control is a bovine genomic DNA standard carrying a homozygous haplotype combination of H2H2, and the negative control is nuclease-free ultrapure water.

8. The application of the haplotype marker according to any one of claims 1-3, or the specific primer and probe set according to any one of claims 4-5, or the kit according to any one of claims 6-7 in molecular marker-assisted selection, genomic selection, or genotypic identification of milk fat percentage trait in dairy cows.

9. The application according to claim 8, characterized in that, Includes the following steps: (1) Collect biological samples from the dairy cows to be tested and extract genomic DNA; (2) Genotyping the three SNP sites described in claim 1 in the genomic DNA, constructing haplotypes and determining the haplotype combination of the individual to be tested; (3) Individuals carrying the H2H2 homozygous haplotype combination were selected as breeding individuals for dairy cattle breeding populations to breed dairy cattle breeds with high milk fat content.

10. The application according to claim 9, characterized in that, The genotyping method includes any one of the following: Sanger sequencing, time-of-flight mass spectrometry, TaqMan probe method, gene chip method, and high-throughput sequencing.

Images