A molecular marker related to sheep body weight traits and application thereof

Abstract

The application provides a molecular marker related to a sheep weight trait and application thereof. MXD3 The application finds that there is an A / G polymorphism site at the 505th position of the sequence shown in SEQ ID NO. 1 of an amplified fragment through PCR amplification and sequence analysis on a sheep MXD3 The gene fragment amplified by the application can be used as a molecular marker related to sheep weight. The molecular marker of the application can be used for selecting AA homozygous sheep into a core group, so as to improve the weight trait of the sheep and help increase economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of sheep molecular marker screening and application technology, specifically relating to a molecular marker related to sheep weight traits and its application. Background Technology

[0002] Marker-assisted selection (MAS) indirectly selects loci controlling a quantitative trait by selecting genetic markers, thereby achieving the purpose of selecting that trait; or it uses genetic markers to predict an individual's genotype or breeding value. Molecular marker technology directly marks genomic DNA, directly reflecting gene inheritance and variation. It can accurately determine whether there is variation in individuals, tissues, organs, and even cells at various growth stages, and has the following advantages: high genetic polymorphism and richness; convenient detection methods, strong operability, and easy automation; many molecular markers exhibit co-dominance; and convenient for intuitive observation and recording. As a useful tool in sheep genetic breeding, molecular marker technology has become a widely used method for breed improvement, providing new ideas and approaches for sheep breeding and genetic improvement.

[0003] MAX dimer protein 3 ( MXD3 It is an atypical member of the MXD family because it has been found to be expressed during the S phase of the cell cycle, while other MXD family members are expressed during cell differentiation (Ngo, Barisone, Lam, & Díaz, 2014). It not only forms a heterodimer with the cofactor MAX (Ayer, Kretzner, & Eisenman, 1993; Ayer, Lawrence, & Eisenman, 1995), but also plays a role in cell proliferation (Barisone et al., 2015). MXD3 Knockdown resulted in a decrease in cell number, indicating that... MXD3 It is essential for the cell cycle process (Barisone et al., 2012; Yun, Rust, Ishimaru, & Díaz, 2007). MXD3 Functionally promotes lipogenesis (Shimada et al., 2014). MXD3 Overexpression of [a specific substance] leads to cell growth and obesity, which in turn exacerbates systemic lipid metabolism dysfunction (Tsai et al., 2021). However, current research on sheep [specific expression of this substance]... MXD3 No causative mutation sites in the gene have been reported.

[0004] The Hu sheep is one of the best-performing sheep breeds, characterized by early sexual maturity, year-round estrus, high milk production, rapid growth, tolerance to roughage, and strong stress resistance (Barisone et al., 2015). Lambs are popular in the Chinese market due to their low fat, low cholesterol, high protein, finer meat texture, and easier digestibility than pork (Zhang et al., 2015). Missense mutations occur in coding regions, leading to translationally defective proteins and are associated with many diseases (Leeet et al., 2008). Synonymous mutations in non-coding regions do not produce altered proteins but rather change DNA and RNA sequences; they are often considered silent mutations and are therefore easily overlooked (Sharma et al.). However, synonymous mutations can alter mRNA stability, splicing regulatory sites, miRNA binding sites, or translation efficiency, resulting in changes in protein levels or protein conformation (Sauna & Kimchi-Sarfaty, 2011). This invention, through... MXD3 The genes were sequenced and analyzed to explore the association between different genotypes and sheep weight, aiming to provide genetic material for improving sheep weight and accelerate the breeding process of a new fast-growing, high-quality meat sheep breed with independent intellectual property rights. Summary of the Invention

[0005] The purpose of this invention is to... MXD3 Gene identification methods were used to locate gene mutation sites and detect gene polymorphisms, resulting in the identification of a molecular marker associated with sheep body weight. This molecular marker was obtained from sheep... MXD3 The gene was amplified, and its specific nucleotide sequence is shown in SEQ ID NO.1. Specifically, it was obtained by amplifying sheep... MXD3 The DNA sequence of the gene is sequenced and used to search for... MXD3 By identifying polymorphic sites in the gene, a method for detecting molecular markers related to sheep body weight can be established, and these molecular markers can be applied to the breeding of new fast-growing, high-quality meat sheep breeds.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In one aspect, the present invention provides a molecular marker associated with sheep weight traits. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1 of the sequence listing. At the 505th base of the nucleotide sequence, there is a SNP mutation site R representing A or G, which leads to A / G polymorphism.

[0007] A second aspect of the present invention provides a PCR primer pair for detecting the above-mentioned molecular marker, the PCR primer pair comprising an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0008] A third aspect of the present invention provides a KASPar primer pair for detecting the above-mentioned molecular marker, wherein the KASPar primer pair comprises: The nucleotide sequence of the forward primer A1 used to detect AlleleA is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer A2 used to detect AlleleG is shown in SEQ ID NO:5, and the nucleotide sequence of the universal reverse primer C is shown in SEQ ID NO:6.

[0009] A fourth aspect of the present invention provides a kit for detecting the above-mentioned molecular markers, the kit comprising PCR primer pairs and KASPar primer pairs.

[0010] The fifth aspect of the present invention provides a method for detecting the above-mentioned molecular markers, characterized by comprising the following steps: (1) PCR amplification of sheep genomic DNA using the PCR primer pair of claim 2 or the KASPar primer pair of claim 3, or the kit of claim 4; (2) The polymorphic sites of the amplification products obtained in step (1) are identified by typing.

[0011] Furthermore, the typing identification method in (2) is self-sequencing method, fluorescent probe method, gene chip method, and high-resolution melting curve method.

[0012] Furthermore, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 59.1℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min.

[0013] The sixth aspect of this invention provides the application of the above-described molecular markers, primer pairs, kits, or detection methods in the detection of sheep weight traits. By utilizing the primer pairs or kits of this invention... MXD3 Genes are amplified and tested to determine the genotype of the sample to be tested, so that fast-growing sheep breeds can be selected from them.

[0014] The seventh aspect of this invention provides the application of the above-described molecular markers, primer pairs, kits, or detection methods in sheep breeding. By utilizing the primer pairs or kits of this invention... MXD3 Genes are amplified and tested to determine the genotype of the sample to be tested, so that fast-growing sheep breeds can be selected from them.

[0015] The beneficial effects of this invention are as follows: The method provided by this invention for detecting molecular markers related to sheep weight traits can be used to screen for fast-growing sheep. When the genotype of the molecular marker is AG or AA, the sheep is a fast-growing breed. When AA type is selected, the breeding process of fast-growing sheep breeds can be greatly accelerated. Attached Figure Description

[0016] Figure 1 Sheep used as molecular markers in this invention MXD3 Gel electrophoresis image of the gene fragment. Lane M: DL 2000 Marker; Lanes 1-10: MXD3 Gene amplification results; Figure 2 The sheep in this invention MXD3 Results of gene mutation sites; Figure 3 For sheep in Embodiment 2 of the present invention MXD3 KASPar SNP genotyping results for the gene g. 3930 A>G mutation site; where the green dot near the left represents the GG genotype, the red dot near the middle represents the AG genotype, and the blue dot near the right represents the AA genotype. Detailed Implementation

[0017] The main growth traits of sheep are quantitative traits, controlled by multiple genes with minor effects. Molecular marker technology can be used to locate and screen loci for specific growth traits, thereby achieving efficient selection of major growth traits and accelerating the breeding of sheep with superior growth traits. Mastranestasisa et al. investigated the association between microsatellite marker polymorphisms and various traits in Lesvos dairy sheep. The results showed that OARJMP29 was related to sheep body weight; OARFCB128 had a significant effect on sheep tail length and width; OARCP38 had a significant effect on sheep body length; and OARJMP29, OARFCB128, and OARCP38 were related to sheep milk production. In this study, the relationship between the MXD3 gene and sheep body weight was examined. At 80 days of age, sheep with the AA genotype had a higher body weight than sheep with other genotypes; at 160 and 180 days of age, sheep with the AA and AG genotypes had a higher body weight than sheep with the GG genotype. In actual production, if sheep with the AG heterozygous genotype are selected for breeding, the genotype of the offspring sheep cannot be stable. Therefore, sheep with the AA homozygous genotype are selected for breeding to stabilize the weight trait and increase economic benefits.

[0018] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0019] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents used in this method are of analytical grade or higher.

[0020] Example 1 MXD3 Gene amplification (1) Primer design sheep MXD3 Using the genetic DNA (GenBank accession number: NC_040256.1) as a template, upstream and downstream primers were designed using Oligo 7.0 software. The primer sequences are as follows. MXD3 : Upstream primer (SEQ ID NO.2) F: 5'-CTTCACGCTGCTCCAGGCATC-3', Downstream primer (SEQ ID NO.3) R: 5'-CGTTTCCCCATCCTCCGAAC-3' (2) MXD3 Gene amplification and sequencing Genomic DNA was extracted from sheep whole blood cells and used as a DNA template for PCR amplification. The total PCR reaction volume was 35 μL, including 1.5 μL DNA template, 17.5 μL 2×PCR Master Mix, 1 μL upstream primer F (10 μmol / L), 1 μL downstream primer R (10 μmol / L), 1 μL DNA template, and 14 μL ddH2O. The PCR amplification program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 59.1℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles, and a final extension at 72℃ for 10 min. The PCR products were detected by 1.5% agarose gel electrophoresis, and the obtained specific amplified fragments were as follows: Figure 1 As shown in the figure. The amplified PCR fragment was sequenced, and the sequencing results showed that the specific nucleotide sequence of the amplified fragment is shown in SEQ ID NO.1. Among them, there is a polymorphic site R at the 505th base of the fragment, which is the amplified... MXD3 The gene fragment exhibits A / G polymorphism at the 717bp site.

[0021] The amplified PCR products were then subjected to Sanger sequencing to detect mutation sites, and the results were analyzed using Chromas to identify the mutation sites. Figure 2 The results are shown in the figure, with the boxed areas indicating mutation sites.

[0022] SEQ ID NO.1:

[0023] DNA sequence homology retrieval and identification: The DNA sequence SEQ ID NO.1 was compared with known physiologically functional genes published in the GenBank database using the BLAST (Basic Local Alignment Search Tool) software on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov) to identify and obtain functional information of the DNA sequence. The search results showed that the sequence was homologous to sheep DNA. MXD3The partial sequence homology of the gene DNA (GenBank accession number: NC_040256.1) reached 99%.

[0024] Example 2 Establishment of genotyping detection methods (1) Primer sequence design KASPar primer pairs were designed for the A / G polymorphic sites of the amplified fragment in Example 1, for the specific detection of these polymorphic sites. The nucleotide sequences of the KASPar primer pairs are as follows: Forward primer A1 (SEQ ID NO.4) used to detect AlleleA: 5′-GAAGGTGACCAAGTTCATGCTCCACACAAGTTACCCAAAATGAGTA- 3′; Forward primer A2 (SEQ ID NO.5) used to detect AlleleG: 5′-GAAGGTCGGAGTCAACGGATTCACACAAGTTACCCAAAATGAGTG- 3′; Universal reverse primer C (SEQ ID NO.6): 5'-ACACCCATTACCCCTCAATTTG-3'.

[0025] The above primers were synthesized by Beijing Sangon Biotech Co., Ltd. Each group of primers in the KASPar primer pair was diluted to 10 μmol / L and mixed in a volume ratio of 12:12:30 (forward primer A1:forward primer A2:universal reverse primer C) to prepare the KASPar primer mixture for later use.

[0026] (2) DNA quality control DNA was extracted from blood using the EasyPure Blood Genomic DNA Kit (TransGen Biotech, Beijing, China) according to the instructions. The concentration and purity of the DNA were tested using Nanodrop ONE (Thermo Fisher Scientific, USA). The qualified DNA was stored at -20°C until use.

[0027] Specifically, the quality of the extracted genomic DNA was assessed using 1% agarose gel electrophoresis and Nanodrop 2100. Acceptable DNA met the following criteria: agarose gel electrophoresis showed a single DNA band, indicating no significant diffusion; Nanodrop 2100 A260 / 280 was between 1.8 and 2.0 (indicating no protein contamination); A260 / 230 was between 1.8 and 2.0 (indicating low salt ion concentration); and there was no significant light absorption at 270 nm (indicating no phenol contamination). Based on the KASP detection technology from LGC (UK) and the conversion of genome size, the required DNA volume was calculated to be 10-20 ng / sample, and the DNA concentration was diluted to 10-20 ng / μL for later use.

[0028] (3) Genotyping First, using a K-pette dispensing workstation, 1.5 µL of diluted DNA template (10-20 ng / μL) and a blank control (using sterile water, denoted as NTC) were added to separate 384-well reaction plates. The plates were then dried at 60°C for 30 min (using a drying oven, LGC) until the DNA became a dry powder. Next, using a Meridian dispensing workstation under the Kraken operating system, 1×Master mix (1536-well microplate, Part No. KBS-1016-011) and KASPar primer mixture were added to each well. Immediately after mixing, the microplates were sealed using a Kube heat sealer and a Fusion laser sealer. High-throughput water bath PCR amplification was then performed using a Hydrocycler. The PCR reaction was carried out in the Hydrocycler high-throughput water bath system, with the following procedure: Pre-denaturation at 94℃ for 15 minutes; 94℃, 20 seconds (denaturation) — 61℃-55℃, 1 minute (annealing & extension), amplify in touch-down order for 10 cycles, decreasing the temperature by 0.6℃ per cycle; 94℃, 20 seconds (denaturation) — 55℃, 60 seconds, continue amplification for 26 cycles.

[0029] After amplification, fluorescence signals were detected and genotyping was performed using a BMG PHERAstar instrument. Specific results are as follows: Figure 3 As shown in the figure, each dot represents a sample to be tested. The green dot near the left indicates that the locus is homozygous genotype "GG"; the blue dot near the right indicates that the locus is homozygous genotype "AA"; the red dot near the middle indicates that the locus is heterozygous genotype "AG" or "GA"; and the black dot represents NTC (not shown in the figure), which is the water control.

[0030] (4) Application of the molecular markers of the present invention in the association analysis of sheep weight trait markers Genomic DNA was extracted from the blood of 1404 sheep in the experimental group. Among them, 1003 individuals had qualified DNA extracted and were successfully genotyped. Their genotypes were determined, and the least squares model described below was established to conduct association analysis between genotype and body weight.

[0031] Y ijk = μ + Genotype i + Batch j + Season k + ε ijk Among them, Y ijlk The observed value is the body weight. μ Genotype is the population mean. i For genotypic effects, Batch j Due to the batch effect, Season k Due to seasonal effects, ε ijk Assuming random error, let ε ijk They are mutually independent and obey N(0, σ). 2 )distributed.

[0032] Genotyping results showed that among 1003 individuals, 421 had the AA genotype, 451 had the AG genotype, and 131 had the GG genotype. The results of the genotype-trait association analysis are shown in Table 1, where BWX represents the sheep's body weight on day X.

[0033] Table 1 Sheep MXD3 Association analysis between gene polymorphism and body weight Note: Different superscript letters between data in the same column indicate significant differences. P <0.05, with the same letter indicating no significant difference ( P >0.05).

[0034] The weight distribution of different genotypes in the sheep population may vary slightly in this experiment. Therefore, this result needs to be considered in conjunction with actual production practices. Although the AG genotype sheep have the highest weight, considering that selecting AG heterozygous sheep as a breeding group in actual production would result in a 1 / 4 probability of GG homozygotes in the offspring, in order to avoid losses caused by the production of GG homozygous sheep in breeding, selecting AA homozygous sheep with higher weight after 160 days as the core breeding group helps to increase economic benefits.

[0035] The results showed that as the measurement period was extended, MXD3The 505 A>G mutation site was significantly associated with sheep body weight. Sheep carrying the AG and AA genotypes had a higher body weight than sheep carrying the GG genotype. P <0.05). This indicates MXD3 g. The 505 A>G mutation site can serve as a potential molecular marker affecting sheep body weight. P <0.05). The molecular markers of this invention can be used to select sheep with the AA homozygous genotype to enter the core group for breeding sheep breeds with excellent weight traits after 160 days, which helps to increase economic benefits.

Claims

1. The application of primer pairs for detecting molecular markers in the preparation of products related to the identification of sheep weight traits, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1 of the sequence listing. There is a SNP mutation site at the 505th base of the nucleotide sequence, where R represents A or G. This mutation leads to A / G polymorphism. Sheep carrying the AG and AA genotypes have a higher body weight than sheep carrying the GG genotype.

2. The application of the primer pair as described in claim 1 in the preparation of products related to the identification of sheep weight traits, characterized in that, The primer pairs include PCR primer pairs and KASPar primer pairs; the PCR primer pairs include an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3; in the KASPar primer pairs: the nucleotide sequence of the forward primer A1 for detecting AlleleA is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer A2 for detecting AlleleG is shown in SEQ ID NO:5, and the nucleotide sequence of the universal reverse primer C is shown in SEQ ID NO:

6.

3. The application of the primer pair as described in claim 1 in the preparation of products related to the identification of sheep weight traits, characterized in that, The product in question is a reagent kit.

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