A molecular marker associated with sheep growth traits and application thereof

Abstract

The application provides a molecular marker related to sheep growth traits and application thereof, the molecular marker is designed by primers according to the sequence of the LGALS3 gene, DNA is extracted from sheep blood, and through PCR amplification, DNA sequencing and sequence analysis, it is found that a T / C polymorphism site exists at the 298th position of the amplified fragment, further, the polymorphism site of 849 Hu sheep is detected by using AQP primers, a least square model is established, genotype and growth traits are associated, finally, it is determined that Y at the 298th bp of the amplified LGALS3 gene fragment shown in SEQ ID NO. 1 is T or C, the mutation leads to T / C polymorphism of the molecular marker, and the molecular marker can be used as a molecular marker related to sheep growth traits. The molecular marker of the application can be used for screening a new variety of fast-growing high-quality mutton sheep, and can be used for screening assisted breeding, cultivating fast-growing sheep, and helping to increase economic benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening and application of molecular markers, and in particular relates to a molecular marker related to sheep growth traits and an application thereof. Background Art

[0002] With China's rapid economic development and adjustments in the national diet, mutton, thanks to its low cholesterol, lean meat, high nutritional value, and tender texture, has become increasingly popular and important in the market. Hu sheep, with their rapid growth, high meat quality, and strong adaptability, hold a significant position in the mutton market. Furthermore, with the advancement of modern breeding technology, traditional breeding methods are no longer able to meet the demand for efficient and precise Hu sheep breeding. Against this backdrop, the application of molecular marker technology has provided new opportunities for Hu sheep breeding. Molecular markers allow for in-depth analysis of the genetic structure of Hu sheep, identifying genes associated with economic traits and enabling precision breeding.

[0003] LGALS3 (Galectin 3) is a gene encoding a member of the galectin family of carbohydrate-binding proteins. The protein encoded by this gene functions extracellularly to regulate glycoprotein secretion and turnover and is involved in protein chaperones and mRNA splicing intracellularly. The encoded protein has an N-terminal proline-rich tandem repeat domain and a C-terminal carbohydrate recognition domain. The N-terminal domain self-associates with this domain, enabling it to bind multivalent sugar ligands. This protein is localized to the extracellular matrix, cytoplasm, and nucleus. It also plays a role in numerous cellular functions, including apoptosis, innate immunity, cell adhesion, and T cell regulation, and exhibits antimicrobial activity against bacteria and fungi. LGALS3 is expressed in multiple tissues, particularly the immune system, muscle, and adipose tissue, demonstrating its multifunctionality in diverse physiological and pathological conditions. Furthermore, the LGALS3 protein has significant effects on fat metabolism and muscle development, potentially promoting growth by regulating related signaling pathways. However, the relationship of the LGALS3 gene to traits in sheep remains unclear.

[0004] The present invention sequences and analyzes the LGALS3 gene to explore the correlation between its different genotypes and sheep growth-related traits, aiming to provide a reference basis for the breeding of sheep and a genetic engineering method for cultivating excellent sheep breeds. Summary of the Invention

[0005] The present invention aims to provide a molecular marker associated with sheep growth traits and its application. The molecular marker of the present invention is amplified from the sheep LGALS3 gene, and its specific nucleotide sequence is shown in SEQ ID NO. 1. By amplifying and sequencing the DNA sequence of the sheep LGALS3 gene and screening for polymorphic sites in the LGALS3 gene, a method for detecting molecular markers associated with sheep growth traits can be established. This molecular marker can then be applied to the breeding of fast-growing, high-quality meat sheep.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A molecular marker related to sheep growth traits, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein Y at the 298th bp is T or C, and the mutation leads to T / C polymorphism of the molecular marker.

[0008] The molecular markers described above are used in screening fast-growing sheep and breeding. When the genotype of the polymorphic site is CC, the body weight of the sheep is significantly higher than that of the sheep with TT genotype.

[0009] A primer pair for detecting the above-mentioned molecular markers related to sheep growth traits is used in screening fast-growing sheep. Preferably, the sequences of the primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0010] An application of AQP primers for detecting the above-mentioned molecular markers related to sheep growth traits in screening fast-growing sheep. Preferably, the sequences of the AQP primers are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0011] A kit for detecting the above-mentioned molecular markers related to sheep growth traits is used in screening fast-growing sheep. Preferably, the kit comprises a common PCR primer pair or an AQP sequence pair, the nucleotide sequence of the common PCR primer pair is shown as SEQ ID NO.2 and SEQ ID NO.3; the nucleotide sequence of the AQP primer is shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0012] A method for detecting the above-mentioned molecular markers related to sheep growth traits comprises the following steps:

[0013] 1) amplifying sheep blood genomic DNA using the above-mentioned common PCR primer pair, AQP primer pair, or a kit containing the above-mentioned primer pair;

[0014] 2) Typing and identifying the polymorphic sites of the amplified product obtained in step 1).

[0015] Wherein, in step 2), the above-mentioned typing and identification methods include but are not limited to direct sequencing, fluorescent probe method, gene chip method, and high-resolution melting curve method.

[0016] Furthermore, preferably, when amplification is performed using a common PCR primer pair, the polymorphic sites of the amplified products are identified by direct sequencing.

[0017] Furthermore, preferably, when the AQP primer pair is used for amplification, the typing result is checked by detecting the fluorescence signal.

[0018] The above method is used in screening fast-growing sheep. By analyzing the type of polymorphic sites, the growth weight of the sheep can be determined, and sheep with faster weight gain and higher weight can be screened out. The genotype of the amplified product at the 298th bp position shown in SEQ IN NO. 1 is identified as T or C. The weaning weight and body weight of sheep carrying the CC genotype and the CT genotype are significantly higher than those of sheep carrying the TT genotype. The daily weight gain of sheep carrying the CC genotype at 160 days of age and 180 days of age is significantly higher than that of sheep carrying the TT genotype and the TC genotype.

[0019] The molecular markers, PCR primer pairs for detecting molecular markers, AQP primers or detection methods of the kit described above are used in sheep breeding. By using the primer pairs or kits described above to amplify and detect the genomic DNA of sheep, the genotype of the molecular markers of the sheep to be tested is determined, so that fast-growing, high-weight sheep breeds can be bred therefrom. When the weaning weight and body weight of sheep carrying the CC genotype and the CT genotype are significantly higher than those of sheep carrying the TT genotype, sheep with the CC genotype are screened for breeding.

[0020] The beneficial effects of the present invention are:

[0021] The present invention performs PCR amplification and sequencing on the sheep LGALS3 gene, discovers a T / C polymorphic site at position 298 of the sequence shown in the amplified fragment SEQ ID NO.1, and determines a molecular marker associated with sheep growth traits by detecting the polymorphism of 849 Hu sheep and establishing a least squares model. The molecular marker can determine the genotype of the polymorphic site of the sheep to be tested, and is used to select sheep with homozygous CC genes as breeding sheep for breeding, so as to cultivate fast-growing high-quality meat sheep. The invention provides genetic material for the genetic improvement of sheep growth traits, and has significant practical application value.

[0022] The molecular markers related to sheep growth traits and their T / C polymorphic sites provided by the present invention can effectively identify whether the sheep are fast-growing and heavy-weight sheep by detecting the genotype of the polymorphism, providing an effective detection method for the selection and breeding of fast-growing and fast-weight sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a gel electrophoresis diagram of the sheep LGALS3 gene fragment used as a molecular marker in the present invention; wherein, lane M: DL 3000Plus Marker, lanes 1-12: LGALS3 gene amplification results.

[0024] Figure 2 This is the sequencing result of the sheep LGALS3 gene mutation site in the present invention.

[0025] Figure 3 This is the AQPTM typing result of the g.65688711C>T mutation site of the sheep LGALS3 gene in the present invention; among them, the blue point near the left represents the TT genotype, the green point near the middle represents the TC genotype, and the orange point near the right represents the CC genotype. DETAILED DESCRIPTION

[0026] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all fall within the scope of the present invention.

[0027] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are of analytical grade or above.

[0028] Example 1 Amplification of the LGALS3 gene

[0029] 1) Primer design

[0030] Using sheep LGALS3 gene DNA (GenBank accession number: NC_056060.1) as a template, a pair of primers MF and MR were designed using Oligo7.0 software. The primer sequences are as follows:

[0031] LGALS3:

[0032] MF (SEQ ID NO.2): 5'-CTATGGCATCCCTTCTGGAC-3',

[0033] MR(SEQ ID NO.3):5'-ATAAGCAAGCACTCAGCTGGTA-3'

[0034] 2) Amplification and sequencing of the LGALS3 gene

[0035] The PCR reaction system used a total volume of 35.14 μL, including: 17.5 μL of 2× PCR Master Mix, 1.12 μL of upstream primer MF at a concentration of 10 μmol / L, 1.12 μL of downstream primer M-R at a concentration of 10 μmol / L, 1.4 μL of DNA template, and 14 μL of ddH₂O. The DNA template was genomic DNA extracted from sheep blood, and the samples were blood samples from 10 sheep.

[0036] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56.7°C for 30 s, extension at 72°C for 60 s, 35 cycles, and a final extension at 72°C for 10 min.

[0037] The PCR reaction products were detected by 1.5% agarose gel electrophoresis, and the results showed that a specific amplified fragment was obtained, such as Figure 1 The amplified PCR fragment was sequenced, and the sequencing results showed that the specific nucleotide sequence of the amplified fragment was as shown in SEQ ID NO.1, wherein there was a polymorphic site Y in the 298 bp fragment of the sequence, which was represented by T or C, that is, the amplified LGALS3 gene fragment had a T / C polymorphism at the 298 bp site ( Figure 2 ).

[0038] SEQ ID NO. 1: CTATGGCATCCCTTCTGGACCCCTGGTAAGATGGA.

[0039] (3) DNA sequence homology search and identification:

[0040] The DNA sequence obtained after sequencing was compared with the known physiological function genes published in the GenBank database using BLAST (Basic Local Alignment Search Tool) software on the website of the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov) to identify and obtain functional information of the DNA sequence. The search results showed that the sequence was 99% identical to the partial sequence of the sheep LGALS3 gene DNA (GenBank accession number: NC_056060.1). Figure 2 shown.

[0041] Example 2 Establishment of Genotyping Detection Method

[0042] (1) Primer sequence design

[0043] An AQP™ primer pair was designed for the T / C polymorphic site of the amplified fragment in Example 1, thereby being used for specific detection of the polymorphic site. The nucleotide sequence of the AQP™ primer pair is:

[0044] Forward primer A1 for detecting AlleleC (SEQ ID NO.4):

[0045] 5′-GAAGGTGACCAAGTTCATGCTTGCTCTGACTGGAAGCTGGC-3′;

[0046] Forward primer A2 (SEQ ID NO.5) for detecting AlleleT:

[0047] 5′-GAAGGTCGGAGTCAACGGATTTAGTGCTCTGACTGGAAGCTG GT-3′;

[0048] Universal reverse primer C (SEQ ID NO. 6): 5'-GCCCTCTGCAGGTCACCAG T-3'.

[0049] The above primers were synthesized by Beijing Sangon Biotechnology Co., Ltd. Each primer in the AQPTM primer pair was diluted to 100 μmol / L and mixed in a volume ratio of 12:12:30:46 (primer A1:primer A2:primer C:sterile water) for later use.

[0050] (2) Extracted genomic DNA and performed quality control

[0051] Genomic DNA can be extracted from sheep blood using a DNA extraction kit. The quality of the extracted genomic DNA is tested using 1% agarose gel electrophoresis and Nanodrop 2100. Qualified DNA requires: (1) agarose gel electrophoresis shows a single DNA band with no obvious diffusion. (2) Nanodrop 2100 detection shows A260 / 280 between 1.8-2.0; A260 / 230 between 1.8-2.0; and no obvious light absorption at 270nm. If it does not meet the requirements, the genomic DNA should be re-extracted. Based on the AQPTM detection technology of Beijing Jiacheng Biotechnology Co., Ltd. and the genome size, the DNA dosage is calculated to be 2-50 ng / sample. The extracted genomic DNA is diluted to a concentration of 2-50 ng / μL and used as a DNA template.

[0052] (3) Genotyping

[0053] First, each primer (100 μmol / L) in the above-mentioned AQPTM primer pair was mixed with sterile water in a volume ratio of 12:12:30:46 (primer A1: primer A2: primer C: sterile water) to prepare a primer mixture for use.

[0054] Then, use a pipette to add 0.07 μL of primer mix, 0.5 μL of sterile water, 2.5 μL of HiGeno 2xProbe Mix, and 2 μL of diluted DNA template (2-50 ng / μL) to each well of a 384-well plate. After addition, seal the plate, centrifuge with vortexing, and perform PCR amplification on a C1000Touch™ Thermal Cycler.

[0055] The specific procedures are:

[0056] Pre-denaturation at 95°C for 10 minutes;

[0057] 95°C, 20 seconds (denaturation) - 61°C - 55°C, 40 seconds (annealing & extension), 10 cycles, decreasing 0.6°C per cycle;

[0058] Amplification was continued for 34 cycles from 95°C, 20 seconds (denaturation) to 55°C, 40 seconds.

[0059] After amplification, the fluorescence signal was detected and the typing was checked using a C1000 TouchTM Thermal Cycler instrument at 37°C. Some of the test results are as follows: Figure 3 As shown. HEX is the horizontal axis and FAM is the vertical axis. Each figure in the figure represents a test material. The blue square near the left side indicates that the site is the homozygous genotype "CC"; the green triangle near the middle indicates that the site is the heterozygous genotype "CT"; the orange dot near the right side indicates that the site is the homozygous genotype "TT".

[0060] (4) Application of the molecular markers of the present invention in sheep growth trait marker-trait association analysis

[0061] A total of 849 Hu sheep were tested for polymorphism, their genotypes were determined, and a least squares model was established as described below to conduct association analysis between genotypes and growth traits.

[0062] Y ijkl =μ+Genotype i +P j +F k +M l +ε ijkl

[0063] Among them, Y ijkl is the observed value of the trait, μ is the overall mean, and Genotypei is the genotype effect, P j is the batch effect, F k is the paternal effect, M l is the maternal effect, ε ijkl is a random error, assuming that ε ijlmk Independent of each other, obey N(0, σ 2 )distributed.

[0064] Genotyping results showed that among the 849 individuals, 79 had the TT genotype, 408 had the TC genotype, and 362 had the CC genotype. The results of the genotype-trait association analysis are shown in Table 1, where BW80 represents the weight of sheep at 80 days of age, BW100 represents the weight of sheep at 100 days of age, and so on. ADG80-100 represents the average daily weight gain of sheep from 80 to 100 days of age, and ADG80-120 represents the average daily weight gain of sheep from 80 to 120 days of age. The calculation formula is ADG80-100 = (BW100 - BW80) / (100 - 80), ADG80-120 = (BW120 - BW80) / (120 - 80), and so on. That is, ADG initial age - final age = (BW final age - BW initial age) / (final age - initial age).

[0065] Table 1 Association analysis between sheep LGALS3 gene polymorphism and growth traits

[0066]

[0067] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05), while data with the same lowercase letters indicate no significant differences (P>0.05).

[0068] The results showed that the LGALS3 g.65688711C>T mutation site, also known as the mutation site at position 298 shown in SEQ ID NO.1, was significantly correlated with sheep growth traits (birth weight, weaning weight, body weight, and daily weight gain) (P<0.05). Sheep carrying the CC genotype and the CT genotype had significantly higher weaning weight and body weight than those carrying the TT genotype. The daily weight gain of sheep carrying the CC genotype at 80-100 days, 80-120 days, 80-160 days, and 80-180 days was significantly higher than that of sheep carrying the TT genotype. This shows that the growth performance of sheep carrying the CC genotype is better than that of sheep carrying the TT genotype (P<0.05). This shows that the C allele is the dominant allele. During breeding, sheep with the CC genotype can be used as breeding sheep and crossed with other sheep to obtain a dominant flock with faster growth rate and higher body weight.

Claims

1. Application of a primer pair for detecting molecular markers related to Hu sheep growth traits in Hu sheep breeding, characterized in that: The purpose of breeding is to screen fast-growing Hu sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein Y at the 298th bp is T or C, and the mutation causes the T / C polymorphism of the molecular marker; the weaning weight and weight at 80-180 days of age of Hu sheep carrying the CC genotype and the CT genotype are significantly higher than those of Hu sheep carrying the TT genotype; the average daily weight gain of Hu sheep carrying the CC genotype at 80-100 days of age, 80-120 days of age, 80-160 days of age, and 80-180 days of age are significantly higher than those of Hu sheep carrying the TT genotype.

2. The use according to claim 1, characterized in that The sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.

3.

3. The use according to claim 1, characterized in that The sequences of the primer pairs are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

4. Application of a kit for detecting molecular markers related to Hu sheep growth traits in Hu sheep breeding, characterized in that: The purpose of breeding is to screen fast-growing Hu sheep. The kit includes a common PCR primer pair or an AQP primer pair, the sequence of the common PCR primer pair is shown in SEQ ID NO.2 and SEQ ID NO.3; the sequence of the AQP primer pair is shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein Y at the 298th bp is T or C, and the mutation causes the T / C polymorphism of the molecular marker; the weaning weight and the weight at 80-180 days of age of Hu sheep carrying the CC genotype and the CT genotype are significantly higher than those of Hu sheep carrying the TT genotype; the average daily weight gain of Hu sheep carrying the CC genotype at 80-100 days of age, 80-120 days of age, 80-160 days of age, and 80-180 days of age are significantly higher than those of Hu sheep carrying the TT genotype.

5. Application of a method for detecting molecular markers related to Hu sheep growth traits in Hu sheep breeding, characterized in that: The purpose of breeding is to select fast-growing Hu sheep, and the method comprises the following steps: 1) Amplify Hu sheep blood genomic DNA using the common PCR primer pairs shown in SEQ ID NO. 2 and SEQ ID NO. 3, or the AQP primer pairs shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, or a kit containing any of the above primer pairs; 2) Typing and identifying the polymorphic sites of the amplified product obtained in step 1); when the nucleotide sequence of the amplified product is shown in SEQ ID NO.1, wherein Y at the 298th bp is T or C, the mutation causes the T / C polymorphism of the molecular marker; the weaning weight and the weight at 80-180 days of age of the Hu sheep carrying the CC genotype and the CT genotype are significantly higher than those of the Hu sheep carrying the TT genotype; the average daily weight gain of the Hu sheep carrying the CC genotype at 80-100 days of age, 80-120 days of age, 80-160 days of age, and 80-180 days of age are significantly higher than those of the Hu sheep carrying the TT genotype.

6. The use according to claim 5, characterized in that When common PCR primer pairs are used for amplification, the polymorphic sites of the amplified products are identified by direct sequencing.

7. The use according to claim 5, characterized in that When using the AQP primer pair for amplification, the typing results are checked by detecting the fluorescence signal.

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