Copy number variation markers related to growth traits of nanyang cattle, primer combination, kit and application

Abstract

The application discloses a copy number variation marker related to growth traits of Nanyang cattle, a primer combination, a kit and application. MAX The copy number variation marker is located at 77227001 bp-77230500 bp of the 10th autosome of a bovine ARS-UCD1.2 reference genome, is a copy number variation marker located on the upstream of a gene which is discovered and verified for the first time and is significantly related to growth traits. A primer combination and a kit developed based on the copy number variation marker can rapidly and accurately determine the type of the copy number variation marker of the Nanyang cattle, the detection is based on genomic DNA, can be carried out on Nanyang cattle individuals of different ages and genders, is not dependent on adult phenotype determination, is suitable for early screening, can be used as a molecular marker for growth trait improvement of the Nanyang cattle, provides a clear detection site and application standard for molecular marker assisted selection of the Nanyang cattle, and is helpful to improve seed selection efficiency and accelerate construction of an excellent Nanyang cattle population.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding, and in particular relates to a copy number variation marker, primer combination, kit and application related to the growth traits of Nanyang cattle. Background Technology

[0002] Copy number variation (CNV) is an important component of genomic structural variation, typically referring to copy number differences resulting from deletions, duplications, or complex rearrangements of DNA fragments ranging from approximately 50 bp to several Mb in length. Compared to single nucleotide polymorphisms (SNPs), CNVs cover a wider range of the genome and have stronger genetic effects. They can regulate gene expression by altering gene dosage, affecting cis-regulatory elements such as promoters or enhancers, disrupting coding sequences, or changing chromatin spatial conformation. This, in turn, affects important economic traits such as animal growth and development, reproductive performance, disease resistance, environmental adaptability, and breed formation, making them crucial genetic markers for current functional genomics and molecular breeding research. In recent years, numerous studies have shown that CNVs are widely present in the bovine genome and are closely related to various traits, demonstrating significant breeding application value.

[0003] Molecular marker-assisted selection (MAS) is a modern breeding technique that utilizes DNA molecular markers closely linked to or directly related to target traits to screen for superior genotypes early in an individual's life. Compared to traditional phenotypic selection, MAS offers advantages such as being unaffected by environmental factors, accurate selection, shorter breeding cycles, and improved genetic progress. With the continuous discovery of CNVs (Central Vibration Variables) related to important economic traits in cattle, CNVs have become a promising new type of molecular marker after SNPs, providing new technological avenues for the genetic improvement and precision breeding of local cattle breeds.

[0004] MAX Gene( MYC-associated factor X It encodes a basic helical-cyclic-helical leucine zipper (bHLH-LZ) transcription factor, which is a core component of the MYC-MAX-MXD transcriptional regulatory network. MAX It can form heterodimers with proteins such as MYC and MXD, and bind to E-box sequences to regulate the transcription of downstream genes, playing a crucial role in cell proliferation, differentiation, metabolism, apoptosis, and development. However... MAX The potential impact of gene expression or genetic variation on economic traits in cattle, and the relevant CNV sites, are currently unclear. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a copy number variation marker related to the growth traits of Nanyang cattle; the second purpose is to provide primer combinations and kits for detecting the copy number variation marker; the third purpose is to provide the application of the above products in the screening of growth traits and molecular marker-assisted selection of Nanyang cattle.

[0006] Technical solution: The copy number variation marker related to growth traits in Nanyang cattle described in this invention is located at 77227001 bp to 77230500 bp on chromosome 10 of the bovine ARS-UCD1.2 reference genome, with a length of 3500 bp. MAX Upstream regulatory regions of genes.

[0007] The primer combination described in this invention is used to detect the aforementioned copy number variation markers associated with the growth traits of Nanyang cattle.

[0008] Preferably, the primer combination consists of an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO: 2.

[0009] The kit described in this invention contains the aforementioned primer combination.

[0010] Preferably, the kit further contains primer pairs for detecting a bovine internal reference gene; more preferably, the bovine internal reference gene is... BTF3 The primer pair for detecting the gene consists of an upstream primer with a sequence as shown in SEQ ID NO: 3 and a downstream primer with a sequence as shown in SEQ ID NO: 4.

[0011] The application of the copy number variation marker, primer combination, or kit described in this invention in the screening of growth traits in Nanyang cattle.

[0012] Preferably, the application steps include: (1) Extract genomic DNA from the Nanyang cattle to be tested; (2) Using the genomic DNA obtained in step 1 as a template, perform real-time quantitative PCR detection using the aforementioned primer combination or the aforementioned kit; (3) Determine the growth traits of the Nanyang cattle to be tested based on the absolute copy number or the relative copy number.

[0013] More preferably, in step 3, the determination is made based on the relative copy number; wherein, when 2 −ΔCt When the ratio is 4, the Nanyang cattle being tested are individuals with excellent growth traits; when 2 −ΔCt <4 or 2 −ΔCt When the value is >4, the Nanyang cattle to be tested are individuals with non-ideal growth traits.

[0014] The application of the copy number variation markers, primer combinations, or kits described in this invention in molecular marker-assisted selection of Nanyang cattle.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to discover and verify the presence of chromosome 10 in Nanyang cattle. MAX 1. A copy number variation marker upstream of the gene that is significantly associated with growth traits; 2. Primer combinations and kits developed based on this copy number variation marker can quickly and accurately determine the type of this copy number variation marker in Nanyang cattle. This detection is based on genomic DNA and can be performed on Nanyang cattle individuals of different ages and sexes. It does not depend on phenotypic determination after adulthood, is suitable for early screening, and can be used as a molecular marker for improving the growth traits of Nanyang cattle. It provides a clear detection site and application standard for molecular marker-assisted selection of Nanyang cattle, which helps to improve breeding efficiency and accelerate the construction of a superior Nanyang cattle population. Attached Figure Description

[0016] Figure 1 For real-time quantitative PCR detection MAX Upstream copy number variation markers (blue) and BTF3 Amplification curve of the gene (red); Figure 2 For real-time quantitative PCR detection MAX upstream copy number variation markers and BTF3 Melting curve of a gene; Figure 3 For MAX Template-free control amplification curves of upstream copy number variation markers of genes; Figure 4 for MAX Representative amplification curves of different copy number types of upstream copy number variation markers. Detailed Implementation

[0017] The technical solution of the present invention will be further described below.

[0018] Example 1: Development and validation of copy number variation (CNV) markers related to growth traits in Nanyang cattle 1. Sample collection from Nanyang cattle This invention focuses on Nanyang cattle as the key testing object. The Nanyang cattle used in the test came from Henan Province, with clear individual numbers, traceable genotyping data and corresponding growth trait records, and no kinship for at least three generations. The test cattle were weaned at 6 months of age and raised under relatively consistent feeding and management conditions. Growth trait records were collected from birth to 3 years of age. At 2 years of age, 90 blood samples were collected and treated with 2% heparin anticoagulation.

[0019] 2. Isolation, extraction, and purification of genomic DNA For the aforementioned blood samples from Nanyang cattle, whole blood genomic DNA was extracted using the phenol-chloroform method. Specifically, 1 mL of whole blood from Nanyang cattle was taken, leukocytes were separated, and proteinase K was added for lysis. The samples were then treated with Tris-saturated phenol and chloroform to remove proteins. DNA was subsequently precipitated with anhydrous ethanol, collected by centrifugation, and washed with 70% ethanol to obtain whole blood genomic DNA. After the DNA quality and concentration were determined using a spectrophotometer, the whole blood genomic DNA was diluted to 50 ng / μL and stored at -80℃ for later use.

[0020] 3. Amplification of the target sequence and internal reference sequence Using the bovine ARS-UCD1.2 reference genome (i.e., the bosTau9 version in the UCSC database) as a reference, the detection methods shown in Table 1 were designed and synthesized. MAX (MYC-associated factor X) gene upstream CNV markers and BTF3 Primers for real-time quantitative PCR of the (BasicTranscription Factor 3) gene.

[0021] Table 1 Primer sequences for real-time quantitative PCR

[0022] Using the obtained whole blood genomic DNA as a template, real-time quantitative PCR was performed using the aforementioned primers and Novizan ChamQ Blue Universal SYBR qPCR Master Mix reagent (catalog number Q312). The 10 μL reaction mixture consisted of: 1 μL whole blood genomic DNA, 0.5 μL each of the forward and reverse primers corresponding to primer pair P1 or primer pair P2 (10 pmol / L), 5 μL 2×ChamQ Blue Universal SYBR qPCR Master Mix, and 3 μL ddH2O. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. After the reaction, melting curve analysis was performed, and a blank control without template was included to confirm the primer amplification specificity.

[0023] By amplification curve ( Figure 1 ), melting curve ( Figure 2 ) and template-free control amplification curves ( Figure 3 The primers were verified to be suitable for qPCR analysis. The melting curves of each sample showed a single peak shape and a smooth trend, with no obvious primer dimers or non-specific amplification peaks.

[0024] 4. Inference of copy number variation Each of the aforementioned whole blood genomic DNA samples was subjected to real-time quantitative PCR detection using primer pair P1 or primer pair P2, with two technical replicates for each primer pair. Sample numbers were retained from the original individual numbers recorded in previous sampling and growth trait records; no new consecutive numbering was performed.

[0025] According to 2 -ΔCt The method performs copy number analysis, where ΔCt = Ct CNV标记 - Ct BTF3 .

[0026] Table 2. 90 samples of Nanyang cattle MAX 2 upstream CNV markers of the gene -ΔCt value

[0027] Based on the results in Table 2, when 2 -ΔCt = 4 is considered intermediate; when 2 -ΔCt When <4, it is determined to be a decreasing type; when 2 -ΔCt If the value is greater than 4, it is classified as an increasing type.

[0028] To visually demonstrate the amplification of samples with different copy number types, a graph is drawn. MAX Representative amplification curves for the upstream copy number variation markers of the gene, including the increased, intermediate, and decreased types, are shown below. Figure 4 As shown, all three types of samples exhibited typical S-shaped amplification curves with stable baselines and good amplification trends.

[0029] 5. Association analysis between CNV markers and growth traits Production data: Collect and record birth weight, as well as weight, height, body length, chest circumference, and ischial tuberosity width at 6 months, 12 months, 18 months, 2 years, and 3 years of age.

[0030] Association analysis model: First, descriptive analysis was performed on the data to check for outliers, and then least squares analysis was used to correct the data. Based on the data characteristics, SPSS 27.0 software was used to analyze the differences in growth traits among different copy number variation types. A fixed model was used when analyzing genotype effects.

[0031] in, Y ijk These are observed trait values. μ The population mean A i Age effect, S j For gender effect, CNV k For the fixed effect of the k-th type of copy number variation, eijk The error is random. Differences between groups were analyzed using the LSD multiple comparison test, and the results are expressed as Mean ± SE.

[0032] In this embodiment, a total of 90 Nanyang cattle were tested. Each cattle had corresponding growth trait observation records at different age stages, theoretically resulting in 540 observation records. After removing records that failed to be detected, had missing data, could not be matched with individual numbers, or did not meet the requirements for statistical analysis, 203 valid observation records were obtained. MAX Association analysis between upstream CNVs and growth traits.

[0033] The contribution of genotype to phenotypic variation was determined using partial correlation analysis in SPSS 27.0 software.

[0034] Table 3 MAX Association analysis between upstream CNV genes and growth traits of Nanyang cattle

[0035] In the table, 'n' represents the number of valid observations included in the association analysis, with 115 being of the decreasing type, 31 of the intermediate type, and 57 of the increasing type. Different capital letters in the same row indicate significant differences. P <0.01, different lowercase letters in the same row indicate significant differences. P <0.05.

[0036] Table 4 MAX Phenotypic Explanation Rate of Upstream CNVs

[0037] The results of the association analysis are shown in Table 3. (Nanyang cattle) MAX The upstream CNV locus of the gene significantly affects an individual's chest circumference and ischial end width. P <0.05), the phenotypic values ​​of chest circumference and ischial end width in intermediate individuals were higher than those in reduced and increased individuals, indicating that this locus can serve as a molecular genetic marker to improve growth traits in Nanyang cattle. Further analysis, as shown in Table 4, shows that this CNV can explain 13.6% of the phenotypic variation in chest circumference and 12.2% in ischial end width, and can be used for marker-assisted selection in Nanyang cattle.

Claims

1. A copy number variation marker associated with growth traits of Nanyang cattle, characterized in that, The copy number variation marker is located at 77227001 bp to 77230500 bp on chromosome 10 of the bovine ARS-UCD1.2 reference genome.

2. A primer combination, characterized in that, The primer combination detects the copy number variation markers associated with the growth traits of Nanyang cattle as described in claim 1.

3. The primer combination according to claim 2, characterized in that, The primer combination consists of an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO:

2.

4. A reagent kit, characterized in that, The kit contains the primer combination as described in claim 2 or 3.

5. The reagent kit according to claim 4, characterized in that, The kit also contains primer pairs for detecting bovine internal reference genes.

6. The reagent kit according to claim 5, characterized in that, The primer pair for detecting the bovine internal reference gene consists of an upstream primer with a sequence as shown in SEQ ID NO: 3 and a downstream primer with a sequence as shown in SEQ ID NO:

4.

7. The application of the copy number variation marker of claim 1, or the primer combination of claim 2 or 3, or the kit of any one of claims 4 to 6 in the screening of growth traits in Nanyang cattle.

8. The application according to claim 7, characterized in that, The steps of the application include: (1) Extract genomic DNA from the Nanyang cattle to be tested; (2) Using the genomic DNA obtained in step 1 as a template, perform real-time quantitative PCR detection using the primer combination described in claim 3 or the kit described in any one of claims 4 to 6; (3) Determine the growth traits of the Nanyang cattle to be tested based on the absolute copy number or the relative copy number.

9. The application according to claim 8, characterized in that, In step 3, the determination is made based on the relative copy number; wherein, when 2 −ΔCt When the ratio is 4, the Nanyang cattle being tested are individuals with excellent growth traits; when 2 −ΔCt < 4 or 2 −ΔCt At 4 o'clock, the Nanyang cattle to be tested were individuals with non-ideal growth traits.

10. The application of the copy number variation marker of claim 1, or the primer combination of claim 2 or 3, or the kit of any one of claims 4 to 6 in molecular marker-assisted selection of Nanyang cattle.

Images