Application of method for detecting copy number variation of goat LIN28A gene in goat molecular marker-assisted selective breeding
By detecting the copy number variation of the goat LIN28A gene through real-time fluorescence quantitative PCR technology, the problem of slow goat breeding progress in the existing technology was solved, and rapid and low-cost molecular marker-assisted selection of goat growth traits was achieved, significantly improving breeding efficiency.
Patent Information
- Application Number
- CN202510777452.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-09-09
AI Technical Summary
The association between copy number variation of the goat LIN28A gene and growth traits has not been effectively utilized in existing technologies, resulting in slow and inefficient progress in goat molecular breeding.
Real-time fluorescence quantitative PCR technology was used to detect the copy number variation of the goat LIN28A gene. Specific primers were designed to amplify fragments of LIN28A and the internal reference gene MC1R. The -ΔΔCt method was used to determine the copy number variation type. Individuals with excellent growth traits were screened according to the variation type to establish an excellent population.
It has achieved rapid, low-cost, and age- and sex-independent identification of goat LIN28A gene copy number variation types, provided a basis for molecular marker-assisted selection of goat growth traits, and significantly accelerated the goat breeding process.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular genetic breeding, and particularly relates to an application of a method for detecting copy number variation of a goat LIN28A gene in goat molecular marker-assisted selection breeding. Background Art
[0002] Genomic DNA carries all of an organism's genetic information and exhibits extensive variation, ranging from simple single nucleotide variants to complex structural rearrangements. These genetic variations regulate gene expression, resulting in a rich variety of phenotypes in populations. Research has demonstrated that changes in genetic information are the primary source of variation in economically important traits. With the increasing maturity of high-throughput sequencing technology, screening and identifying genes associated with economic traits, as well as discovering and precisely locating sites associated with important phenotypes, have become crucial tasks in advancing molecular breeding.
[0003] Animal molecular breeding combines molecular genetics theory with traditional quantitative genetics theory, utilizing molecular genetic information combined with polygenic information to conduct genetic analysis of desired traits in livestock, enabling rapid and efficient selection and improvement of livestock and poultry breeds. Marker-assisted selection (MAS) has long been a research hotspot in the field of animal molecular breeding. MAS is a modern molecular breeding technique for improving livestock and poultry breeds by identifying molecular markers or functional genes linked to important economic traits and, based on breeding objectives, selecting individuals with genetic markers closely linked to the target traits as breeding stock. This allows for direct selection of target traits at the genotypic level, accelerating the breeding process and improving breeding efficiency.
[0004] Copy number variations (CNVs), also known as copy number polymorphisms (CNPs), are a key component of structural variation (SV) in animal genomes. They have a much higher mutation rate than single-nucleotide polymorphisms (SNPs), involve fewer variant sites, and have longer sequences, making them easier to detect and study. Therefore, CNVs hold great promise in animal genetics and breeding. CNVs are insertions, deletions, and duplications ranging from 1 kilobase (Kb) to several megabases (Mb) in length within the genomic sequence. The integration of overlapping CNVs between individuals in a population is called a CNV region (CNVR). When these CNVs are found in genomic regions encoding protein-coding genes (CNV genes) or miRNAs (CNV-miRNAs), the genetic variation may affect molecular regulatory mechanisms and influence phenotypic diversity and disease susceptibility. Therefore, detecting CNV markers in genes associated with growth traits in goats could help accelerate goat genetic selection.
[0005] The LIN28A gene encodes a LIN-28 family RNA-binding protein, a post-transcriptional regulator of genes involved in embryonic stem cell developmental timing and self-renewal. Recent studies investigating the function and mechanism of action of the LIN28A gene remain preliminary, with no reports linking LIN28A gene copy number variation to goat growth traits. Summary of the Invention
[0006] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for detecting copy number variation of the goat LIN28A gene and its application in goat molecular marker-assisted selection breeding.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] The first aspect of the present invention discloses the application of a method for detecting copy number variation of the goat LIN28A gene in goat molecular marker-assisted selection breeding.
[0009] Preferably, the copy number variation region of the LIN28A gene is located in the goat LIN28A gene candidate region Chr2:9266501-9269500.
[0010] Further preferably, the copy number variation region of the LIN28A gene and a partial fragment of the MC1R gene as an internal reference are amplified by real-time fluorescence quantitative PCR using the goat genomic DNA to be tested as a template, and then the copy number variation type of the goat LIN28A gene is identified based on the quantitative results.
[0011] Further preferably, in the primer pair for amplifying the copy number variation region of the LIN28A gene, the upstream primer F1 sequence is shown as SEQ ID NO.1, and the downstream primer R1 sequence is shown as SEQ ID NO.2; in the primer pair for amplifying the copy number variation region of the MC1R gene, the upstream primer F2 sequence is shown as SEQ ID NO.3, and the downstream primer R2 sequence is shown as SEQ ID NO.4.
[0012] Preferably, the real-time fluorescence quantitative PCR amplification system includes 1 μL 50 ng / μL genomic DNA, 0.5 μL 10 μmol / L upstream primer F1, 0.5 μL 10 μmol / L downstream primer R1, 0.5 μL 10 μmol / L upstream primer F2, 0.5 μL 10 μmol / L downstream primer R2 and 3 μL ddH2O.
[0013] Preferably, the reaction procedure of real-time fluorescence quantitative PCR is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and 39 cycles.
[0014] Preferably, the copy number variation type is based on 2*2 -ΔΔCt The quantitative results were divided into three categories: duplication type, -ΔΔCt>0.5; deletion type, -ΔΔCt<-0.5; normal type, -0.5≤-ΔΔCt≤0.5.
[0015] Preferably, the Taihang black goat population with a normal copy number variation type is superior to the individuals with a repetitive copy number variation type in growth traits; the Guizhou black goat population with a deletion copy number variation type is superior to the individuals with a repetitive copy number variation type or a normal copy number variation type in growth traits.
[0016] Further preferably, the Taihang Black Goat population with normal copy number variation type is superior to individuals with repetitive copy number variation type in terms of growth trait body length.
[0017] Preferably, the Taihang Black Goat population with normal copy number variation type is superior to individuals with repetitive copy number variation type in growth trait height.
[0018] Preferably, the Taihang Black Goat population with normal copy number variation type is superior to individuals with repetitive copy number variation type in the growth trait chest circumference.
[0019] Preferably, the Guizhou black goat population with a deletion-type copy number variation type is superior to individuals with a duplication-type copy number variation type or a normal-type copy number variation type in terms of growth trait weight.
[0020] The second aspect of the present invention discloses a real-time fluorescence quantitative PCR kit for detecting copy number variation of the goat LIN28A gene, the kit comprising a primer pair for amplifying the copy number variation region of the goat LIN28A gene; the copy number variation region of the LIN28A gene is located in the candidate region Chr2:9266501-9269500 of the goat LIN28A gene.
[0021] Preferably, in the primer pair for amplifying the copy number variation region of the LIN28A gene, the sequence of the upstream primer F1 is shown as SEQ ID NO.1, and the sequence of the downstream primer R1 is shown as SEQ ID NO.2.
[0022] Preferably, the kit further comprises a primer pair for amplifying a partial fragment of the cattle MC1R gene as an internal reference.
[0023] Further preferably, in the primer pair for amplifying the copy number variation region of the MC1R gene, the sequence of the upstream primer F2 is shown as SEQ ID NO.3, and the sequence of the downstream primer R2 is shown as SEQ ID NO.4.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The present invention provides a method for detecting goat LIN28A gene copy number variation in goat molecular marker-assisted selection breeding, which associates goat LIN28A gene copy number variation with goat growth traits, and can provide a scientific basis for molecular marker-assisted selection of goat growth traits to a certain extent. Experiments have shown that the Taihang black goat group with normal copy number variation type is superior to individuals with repetitive copy number variation type or deletion type copy number variation type in growth traits (body length, body height, chest circumference); the Guizhou black goat group with deletion type copy number variation type is superior to individuals with repetitive copy number variation type or normal type copy number variation type in growth traits (weight). Compared with high-throughput sequencing methods and gene chip methods, this method is faster, simpler, and has lower cost. It is not limited by individual age and gender, can accurately identify individual copy number variation types, can be used for early goat breeding, and accelerates the goat breeding process.
[0026] Furthermore, using the goat LIN28A gene as the target gene and the MC1R gene as the reference gene, real-time fluorescence quantitative PCR was used to detect the copy number variation of the goat LIN28A gene. The copy number variation type of the individual LIN28A gene can be identified, and the DNA molecular markers (CNV markers) located in the LIN28A gene can be used to assist in detecting individuals with growth trait advantages, quickly establish a dominant population of goat genetic resources, and thus accelerate the breeding process of goats. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a frequency diagram of the copy number distribution of the LIN28A gene of the present invention in five goat breeds. DETAILED DESCRIPTION
[0028] The real-time fluorescence quantitative PCR (qPCR) technology used in this article is a relatively widely used technology among various methods for detecting known CNVs. This method is simple to operate, highly sensitive, and fast. It performs relative quantification of the target gene (with copy number variation) and the internal reference gene (without copy number variation), and then uses -ΔΔCt to determine the copy number variation type of an individual.
[0029] The present invention detects the copy number variation of the LIN28A gene and uses it for goat molecular breeding. The method mainly comprises the following steps: referring to the goat LIN28A gene sequence in the NCBI database, designing primers using the Primer-BLAST website, and testing the primers using ordinary PCR; using real-time fluorescence quantitative PCR technology to detect the copy number variation of a candidate site (Chr2:9266501-9269500) in a population; performing association analysis between the copy number variation type and growth traits, and screening CNV markers related to the growth traits; screening individuals with excellent growth traits according to the copy number variation type, and establishing a goat population for breeding.
[0030] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. Specific examples are described below.
[0031] 1. Goat Sample Collection
[0032] Taihang black goats (TH), Guizhou black goats (GZB), Guizhou white goats (GZW), Huai goats (HA) and Bohuai goats (BH) were used as the test objects. Blood samples were collected and used from 428 goat individuals, and individual growth shape data such as height, body length, weight, chest circumference and tube circumference were recorded for subsequent correlation analysis.
[0033] Table 1. Sample collection information
[0034]
[0035] 2. Extraction of Genomic DNA from Blood Samples
[0036] (1) Thaw the frozen blood sample (mainly blood cells) at room temperature, draw 500 μL of blood into a 1.5 mL centrifuge tube, add an equal volume of phosphate buffered saline (PBS) and mix well, shake gently, centrifuge at 4°C and 12,000 rpm for 5 min, and discard the supernatant; repeat this step until the supernatant is transparent and the precipitate is transparent.
[0037] (2) Add 500 μL of DNA extraction buffer to the centrifuge tube and gently pipette to separate the blood cell pellet from the tube wall. Incubate in a 37°C water bath for 1 hour.
[0038] (3) Add 5 μL of proteinase K (20 mg / mL) and mix well. Digest in a 55°C water bath overnight (about 16 h) until the flocculent precipitate disappears. If it is not clear, add 10 μL of proteinase K and mix well and continue digestion until it is clear. Cool the reaction solution to room temperature.
[0039] (4) Add 500 μL of Tris-saturated phenol and shake gently for 15 minutes to mix thoroughly. Centrifuge at 4°C and 12,000 rpm for 10 minutes. Transfer the upper aqueous phase to another sterile centrifuge tube. Repeat this step once.
[0040] (5) Add 500 μL of chloroform and shake gently for 20 min to mix thoroughly. Centrifuge at 4°C and 12,000 rpm for 15 min. Transfer the upper aqueous phase to another sterilized 1.5 mL centrifuge tube.
[0041] (6) Add 500 μL of a mixture of chloroform and isoamyl alcohol (24:1), mix thoroughly for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, and transfer the supernatant to another 1.5 mL centrifuge tube.
[0042] (7) Add 0.1 volumes of NaAc buffer and 2 volumes of ice-cold ethanol, and rotate the centrifuge tube until a white flocculent precipitate is precipitated.
[0043] (8) Centrifuge at 4°C, 12,000 rpm for 10 min, discard the supernatant, and rinse the DNA pellet twice with 70% ice-cold ethanol.
[0044] (9) Centrifuge at 4°C and 12,000 rpm for 10 min, discard the supernatant, and allow the ethanol to evaporate completely at room temperature.
[0045] (10) The dried DNA was dissolved in 80–100 μL of Tris-EDTA buffer (TE) and stored at 4°C until the DNA was completely dissolved. The quality of the DNA was detected using a UV spectrophotometer and the DNA was stored at −80°C.
[0046] 3. Design of primers specific for target gene and internal reference gene
[0047] Using the goat LIN28A gene (NC_030809.1) published in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as a reference sequence, we searched for the candidate copy number variation region of the LIN28A gene (target gene) identified during resequencing (Chr2:9266501-9269500). Primers (primer pair P1) were designed to amplify a 139-bp sequence near the middle of this region using the Primer-BLAST website. Simultaneously, using the goat MC1R gene sequence (NC_030825.1) published in NCBI as a reference sequence, primers (primer pair P2) were designed using the same method to amplify a 267-bp sequence within the internal reference gene (MC1R gene). Primer pair sequence information is shown in Table 2 (primers synthesized in July 2022).
[0048] Table 2. Primer information for real-time fluorescence quantitative PCR
[0049]
[0050] Note: F1 or F2 is the upstream primer, R1 or R2 is the downstream primer
[0051] 4. Real-time Fluorescence Quantitative PCR
[0052] The qPCR reaction system is shown in Table 3.
[0053] Table 3. qPCR reaction system
[0054]
[0055] The qPCR amplification reaction procedure is as follows:
[0056] (1) Pre-denaturation at 95°C for 10 min, followed by amplification according to (2);
[0057] (2) Denaturation at 95°C for 15 seconds and annealing at 60°C for 1 minute, for a total of 39 cycles.
[0058] 5. Individual CNV type determination
[0059] The cycle threshold (Ct) is used to analyze the experimental results of fluorescence quantitative PCR. The cycle threshold refers to the number of amplification cycles when the fluorescence signal of the amplified product reaches the set threshold during the fluorescence quantitative PCR amplification process. It is used to draw a standard curve to detect the initial copy number of the template in the sample to be tested. The sample 2*2 is calculated based on the qPCR experimental results of the target gene and the internal reference gene. -ΔΔCt :
[0060] ΔΔCt=ΔCt (实验组) -ΔCt (参照组)
[0061] ΔCt (实验组) =Ct (实验组目的基因) -Ct (实验组内参基因) , ΔCt (参照组) =Ct (参照组目的基因) -Ct (参照组内参基因)
[0062] Among them, the experimental group is the individual sample to be tested for copy number variation, and the reference group is the individual sample known to have no copy number variation. The reference group can be selected in the resequencing experiment. Individual goats of various breeds.
[0063] The -ΔΔCt of each individual sample was calculated according to the above formula, and the CNV type of the goat individual was identified according to the following judgment criteria: -ΔΔCt>0.5 was the Duplication type (duplication type; CN≥3); -0.5≤-ΔΔCt≤0.5 was the Normal type (normal type; CN=2); -ΔΔCt<-0.5 was the Deletion type (deletion type; CN≤1).
[0064] The frequency distribution of copy number variation types of LIN28A gene in five goat breeds is as follows Figure 1 As shown, there are no deletion-type individuals in Taihang Black goats, while deletion-type individuals are prevalent in Guizhou Black goats. Guizhou White goats, Huai goats, and Bohuai goats have more duplication-type and deletion-type individuals than normal individuals. Copy number types vary to varying degrees within each breed, likely due to varying genetic conditions. Guizhou White goats and Guizhou Black goats are genetically closely related, but their copy number type distributions differ. This analysis may be due to sample size.
[0065] 6. Association analysis between LIN28A gene CNV sites and growth traits
[0066] Growth traits: height, length, chest circumference and tube circumference.
[0067] CNV types: Deletion, Normal, and Duplication.
[0068] Correlation analysis model: SPSS (23.0) was used for correlation analysis. In data processing, a fixed model was used for analysis based on the different factors affecting growth trait indicators, taking into account environmental effects, age, variety, genetic effects, and their interaction effects. At the same time, simplification was performed based on actual conditions. The final model is as follows:
[0069] Z ijk =μ+M i +N j +CNV k +e ijk
[0070] Among them, Z ijk is the individual phenotypic record; μ is the population mean; M i is (i-th age effect); N j is (the jth variety effect); CNV k is (the kth LIN28A gene copy number variation type effect); e ijk is a random error.
[0071] The results of the association analysis are shown in Tables 4, 5, 6, 7, and 8.
[0072] Table 4. Association analysis between LIN28A gene copy number variation and growth traits in Taihang black goats
[0073]
[0074] Note: The values marked with a, b and * in the same row indicate significant differences (P<0.05); the values marked with A, B and ** in the same row indicate extremely significant differences (P<0.01).
[0075] As can be seen from Table 4, in the Taihang Black Goat population, individuals of the Normal type (CN=2) performed significantly better than other types in terms of body height and chest circumference, and were extremely significantly better than other types in terms of body length.
[0076] Table 5. Association analysis between LIN28A gene copy number variation and growth traits in Guizhou black goats Note: The values marked with a, b and * in the same row indicate significant differences (P<0.05)
[0077] It can be seen from Table 5 that among the Guizhou black goat population, the Deletion (CN≤1) type population is significantly better than other types in terms of body weight.
[0078] Table 6. Association analysis between LIN28A gene copy number variation and growth traits in Guizhou White goats
[0079]
[0080] Table 7. Association analysis between LIN28A gene copy number variation and growth traits in Huai goat
[0081]
[0082] Table 8. Association analysis between LIN28A gene copy number variation and growth traits in Bohuai goats Association analysis results (Tables 4 to 8) indicate that the copy number variation site in the LIN28A gene is significantly associated with important growth traits in goats: weight, height, length, and chest circumference. Therefore, the copy number variation site in the LIN28A gene (Chr2:9266501-9269500) could serve as a candidate molecular genetic marker (CNV) to effectively improve growth traits in goats, thereby accelerating goat breeding.
[0083] The above content is only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.
Claims
1. Application of the method for detecting copy number variation of goat LIN28A gene in goat molecular marker-assisted selection breeding.
2. The use according to claim 1, characterized in that The copy number variation region of the LIN28A gene is located in the goat LIN28A gene candidate region Chr2:9266501-9269500.
3. The use according to claim 2, characterized in that Using the goat genomic DNA as a template, the copy number variation region of the LIN28A gene and a partial fragment of the MC1R gene as an internal reference were amplified by real-time fluorescence quantitative PCR, and the copy number variation type of the goat LIN28A gene was identified based on the quantitative results.
4. The use according to claim 3, characterized in that In the primer pair for amplifying the copy number variation region of the LIN28A gene, the upstream primer F1 sequence is shown as SEQ ID NO.1, and the downstream primer R1 sequence is shown as SEQ ID NO.2; in the primer pair for amplifying the copy number variation region of the MC1R gene, the upstream primer F2 sequence is shown as SEQ ID NO.3, and the downstream primer R2 sequence is shown as SEQ ID NO.
4.
5. The use according to claim 3, characterized in that The reaction procedure of real-time fluorescence quantitative PCR was as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and 39 cycles.
6. The use according to any one of claims 1 to 5, characterized in that: The copy number variation type is based on 2*2 -ΔΔCt The quantitative results were divided into three categories: duplication type, -ΔΔCt>0.5; deletion type, -ΔΔCt<-0.5; normal type, -0.5≤-ΔΔCt≤0.
5.
7. The use according to any one of claims 1 to 5, characterized in that The growth traits of Taihang black goat population with normal copy number variation type were better than those of individuals with duplication copy number variation type; the growth traits of Guizhou black goat population with deletion copy number variation type were better than those of individuals with duplication copy number variation type or normal copy number variation type.
8. A real-time fluorescence quantitative PCR kit for detecting copy number variation of goat LIN28A gene, characterized in that: The kit comprises a primer pair for amplifying a copy number variation region of a goat LIN28A gene; the copy number variation region of the LIN28A gene is located in the goat LIN28A gene candidate region Chr2:9266501-9269500.
9. A real-time fluorescence quantitative PCR kit for detecting goat LIN28A gene copy number variation according to claim 8, characterized in that, In the primer pair for amplifying the copy number variation region of the LIN28A gene, the sequence of the upstream primer F1 is shown as SEQ ID NO.1, and the sequence of the downstream primer R1 is shown as SEQ ID NO.
2.
10. A real-time fluorescence quantitative PCR kit for detecting bovine LIN28A gene copy number variation according to claim 8, characterized in that: The kit also includes a primer pair for amplifying a partial fragment of the cattle MC1R gene as an internal reference.