A molecular marker related to feed conversion rate of sheep, detection method and application thereof

Abstract

The application provides a molecular marker related to feed conversion rate of sheep, a detection method and application thereof. Through PCR amplification and sequence analysis of a sheep GFRA2 gene, it is found that there is an A / G polymorphic site at the 196th site of the amplified fragment, further detection of the polymorphic site of 1004 Hu sheep is carried out by using KASPar primers, a least square model is established, genotype and feed conversion rate are associated, and finally it is determined that the amplified GFRA2 gene fragment can be used as a molecular marker related to feed conversion rate of sheep. The molecular marker can be used for selecting and reserving GG homozygous sheep into a core group, so as to improve the feed conversion rate of sheep and help to increase economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a molecular marker related to sheep feed conversion rate, its detection method, and its application. Background Technology

[0002] Glial cell line-derived neurotrophic factor (GDNF) and neuron (NTN) are two structurally related and potent neurotrophic factors that play a crucial role in controlling neuronal survival and differentiation. GFRA2, encoded by this gene, is a member of the GDNF receptor family. It is a cell surface receptor for the glycosylphosphatidylinositol (GPI) linker of GDNF and NTN, mediating the activation of RET tyrosine kinase receptors. Compared to other family members, such as GDNF family receptor α1, this encoded protein preferentially acts as a receptor for NTN (RefSeq, Sep 2009). Mice with GFRA2 knockout exhibit impaired digestion, salivation, and intestinal motility, grow slower than wild-type mice, and have an increased basal metabolic rate (Rossi J, 2003).

[0003] According to data from the National Bureau of Statistics, in 2017, my country's sheep and goat population reached 302 million, with mutton production reaching 4.7107 million tons, making it the world's largest producer and consumer of mutton (Zhang Xiaoxue. Study on the production performance, rumen microbiota, and liver transcriptome of lambs with different residual feed intake [D]. Lanzhou University, 2019). Hu sheep are mainly distributed along the Yangtze River in China. Missense mutations occur in coding regions, leading to translationally defective proteins and are associated with many diseases (Lee et al., 2008). Synonymous mutations in non-coding regions do not produce altered proteins but rather change DNA and RNA sequences; they are 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, leading to changes in protein levels or protein conformation (Sauna & Kimchi-Sarfaty, 2011). Residual feed intake (RFI) is an indicator of feed efficiency, while feed conversion ratio (FCR) measures the relationship between feed intake and weight gain in sheep farming, serving as a crucial indicator for evaluating feed efficiency. Genome-wide analysis identified alleles negatively correlated with RFI as being associated with increased GFRA2 expression in the liver. GFRA2 influences basal metabolic rate, suggesting that genetic variations may contribute to the mechanism of RFI (Higgins MG, 2018). Phenotypic correlation analysis among feed efficiency-related traits showed a highly significant positive correlation between residual feed intake and both feed conversion ratio and feed intake (P < 0.01) (Zhang Deyin, 2020). However, the nature and nature of the relationship between the GFRA2 gene and feed efficiency remain unclear.

[0004] This invention explores the association between different genotypes of the GFRA2 gene and sheep feed conversion ratio by sequencing and analyzing the gene, aiming to provide genetic material for improving the genetic conversion ratio of sheep and accelerate the breeding process of new high-quality meat sheep breeds with independent intellectual property rights. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a molecular marker related to sheep feed conversion ratio and its application. The molecular marker of this invention is amplified from the sheep GFRA2 gene, and its specific nucleotide sequence is shown in SEQ ID NO.1. By amplifying and sequencing the DNA sequence of the sheep GFRA2 gene, polymorphic sites in the GFRA2 gene can be identified, thereby establishing a method for detecting molecular markers related to sheep feed conversion ratio. This molecular marker can then be applied to the breeding of new high-quality meat sheep breeds with high feed conversion ratios.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A molecular marker associated with sheep feed conversion ratio, the nucleotide sequence of which is shown in SEQ ID NO.1, wherein the R at position 196 represents A or G. Due to a G / A mutation at position 196 of the above sequence, the sheep GFRA2 gene has a G / A polymorphism at this site.

[0008] The method for detecting molecular markers related to sheep feed conversion ratio as described above includes detection of the sheep GFRA2 gene, and the specific detection method includes the following steps:

[0009] S1. Extract sheep genomic DNA and amplify the GFRA2 gene containing the sequence shown in SEQ ID NO.1;

[0010] S2. Identify the polymorphic sites in the amplification products obtained in step S1.

[0011] Furthermore, sheep genomic DNA is DNA extracted from sheep blood, and identification methods include direct sequencing, probe method, gene chip method, and high-resolution melting curve method.

[0012] As described above, in step S1, when the primer pair used for amplification is shown in SEQ ID NO.2 and SEQ ID NO.3, PCR amplification is performed, and the obtained amplification product is directly sequenced to obtain the genotype of the molecular marker.

[0013] As described above, in step S1, when the primer pair used for amplification is the KASPar primer pair, specifically the nucleotide sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, high-throughput water bath PCR amplification is performed. After amplification, the fluorescence signal is detected and the genotyping results are viewed using a BMG PHERAstar instrument.

[0014] Primer pairs for detecting molecular markers associated with sheep feed conversion ratio as described above, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0015] More preferably, the KASPar primer pair for detecting molecular markers related to sheep feed conversion ratio as described above includes a forward primer A1 for detecting AlleleA, a forward primer A2 for detecting AlleleG, and a universal reverse primer C, wherein the nucleotide sequence of the forward primer A1 is shown in SEQ ID NO.4, the nucleotide sequence of the forward primer A2 is shown in SEQ ID NO.5, and the nucleotide sequence of the universal reverse primer C is shown in SEQ ID NO.6.

[0016] A kit for detecting molecular markers associated with sheep feed conversion ratio as described above, comprising primer pairs or KASPar primer pairs as described above.

[0017] The molecular markers or methods for detecting feed conversion ratio-related molecular markers in sheep, as described above, or the primer pairs or kits described above, can be used in breeding to screen for high-quality, feed-saving sheep with low feed conversion ratios. By amplifying and detecting the GFRA2 gene using the primer pairs or kits of this invention, the genotype of the sample can be determined, thereby allowing the selection of sheep breeds with low feed conversion ratios. When the genotype is GG, it has a low feed conversion ratio; therefore, selecting GG homozygous sheep for the core flock can improve the feed conversion ratio and contribute to increased economic benefits.

[0018] The beneficial effects of this invention are as follows:

[0019] The molecular marker provided by this invention is related to the feed conversion rate of sheep. The molecular marker is the 196th base of the fragment shown in SEQ ID NO.1 in the sheep GFRA2 gene, which has A / G polymorphism. When the genotype of this site is GG, it is used for breeding and has a lower feed conversion rate. That is, efficient feed utilization can save feed, thereby reducing the production cost of farms and improving the economic benefits of sheep farming. Attached Figure Description

[0020] Figure 1 This is a gel electrophoresis image from Example 1 of the present invention, where lane M represents the DL 5000 Marker, and lanes 1-10 represent the GFRA2 gene amplification results.

[0021] Figure 2 The sequencing results of the sheep GFRA2 gene mutation site in this invention.

[0022] Figure 3 The results of KASPar SNP typing for the g.87957A>G mutation site in the sheep GFRA2 gene. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] Example 1

[0026] (1) Primer design

[0027] Using sheep GFRA2 gene DNA (GenBank accession number: NC_040253) as a template, a pair of primers, MF and MR, were designed using Oligo 7.0 software. The primer sequences are as follows:

[0028] MF (SEQ ID NO.2): 5′-AACCTGCTTCTCCATGGTC-3′,

[0029] MR(SEQ ID NO.3): 5′-CACTCAGTTTCCCTAAGCCTCA-3′

[0030] (2) Amplification and sequencing of the GFRA2 gene

[0031] Genes were extracted from sheep blood and used as DNA templates. The total volume of the PCR reaction was 35 μL, including 1.5 μL of DNA template, 17.5 μL of 2×PCR Master Mix, 1 μL of upstream primer MF (10 μmol / L), 1 μL of downstream primer MR (10 μmol / L), and 14 μL of ddH2O.

[0032] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0033] The PCR products were detected by 1.5% agarose gel electrophoresis, and the results showed a 462bp specific amplified fragment (see...). Figure 1 The amplified PCR fragment was sequenced, and the sequencing results showed that the nucleotide sequence of the amplified fragment is as shown in SEQ ID NO.1. A polymorphic site exists in this 462bp fragment, specifically an A / G polymorphism at position 196 of the amplified GFRA2 gene fragment (see [link to PCR]). Figure 2 In SEQ ID NO.1, the R at position 196 represents A or G.

[0034] SEQ ID NO.1:AACCTGCTTCTCCATGGTCTTTCTCCTGACCCCAGG TCCCGGCTGGCGGACTTCCATGCCAACTGCCGTGCCTCCTACCAGACGCTCACCAGCTGCCCCACCGACAATTACCAGGCGTGTCTGGGCTCCTACGCTGGCATGATCGGTAAGCCACCCCAGCCTGGGATCTCAGTGGCTCCCGGCTGCCCTGGCCCRGTTAGGGACTGAAGTCCAGTTCCTCCTCTGCCTGGTTTCAGATAAGAGCTGTG ATGGGAGCTGGGGTTTTATCACAGGGGAGGGAGCCCCAAGAATCTTAGGAAGCTTCCAAAATAAGAAGGGTGATGTTCATTGTGTGACATTCTAGGTTCCCCACTGAGCAGTTTACGTGGACTGTCTTATGTAAATCTCAAAACAGAGCTTTCTGAGTGGGGGTTAGGGCTCCACTTTACAGATGGGAAAACTGAGGCTTAGGGAAACTGAGTG.

[0035] DNA sequence homology retrieval and identification:

[0036] The DNA sequence obtained after sequencing 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 (NCBI, http: / / www.ncbi.nlm.nih.gov) to identify and obtain functional information of the DNA sequence. The search results showed that the sequence obtained had 99% homology with a partial sequence of the sheep GFRA2 gene DNA (GenBank accession number: NC_040253).

[0037] Example 2: Establishment of a Genotyping Detection Method

[0038] (1) Primer sequence design

[0039] KASPar primer pairs were designed for the C / T polymorphism sites of the amplified fragment in Example 1, for the specific detection of these polymorphism sites. The nucleotide sequences of the KASPar primer pairs are as follows:

[0040] Forward primer A1 (SEQ ID NO.4) used to detect AlleleA:

[0041] GAAGGTGACCAAGTTCATGCTGGAACTGGACTTCAGTCCCTAACT;

[0042] Forward primer A2 (SEQ ID NO.5) used to detect AlleleG:

[0043] GAAGGTCGGAGTCAACGGATTGAACTGGACTTCAGTCCCTAACC;

[0044] Universal reverse primer C (SEQ ID NO.6): GCTGGCATGATCGGTAAGC.

[0045] The above primers were synthesized by Beijing Sangon Biotech Co., Ltd. Each primer in the KASPar primer pair was diluted to 10 μmol / L and mixed in a volume ratio of 12:12:30 for forward primer A1:forward primer A2:reverse primer C.

[0046] (2) DNA quality control

[0047] The quality of the extracted genomic DNA was assessed using 1% agarose gel electrophoresis and Nanodrop 2100, respectively. Acceptable DNA met the following criteria: agarose gel electrophoresis showed a single DNA band without 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.

[0048] (3) Genotyping

[0049] First, using a K-pette dispensing workstation, 1.5 μL of diluted DNA template (10–20 ng / μL) and a blank control (No template control, NTC) were added to separate 384-well reaction plates. The plates were then dried at 60°C for 30 min (using an LGC drying oven) until the DNA powder was ready for use. Next, using a Meridian dispensing workstation under the Kraken operating system, 1×Master mix (part no. KBS-1016-011 for 1536-well microplates) and 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 conducted in the Hydrocycler high-throughput water bath system, and the specific procedure was as follows:

[0050] Pre-denaturation at 94℃ for 15 minutes;

[0051] 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;

[0052] 94℃, 20 seconds (denaturation) — 55℃, 60 seconds, continue amplification for 26 cycles.

[0053] 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, which is the water control.

[0054] (4) Application of the molecular markers of the present invention in marker-associated analysis of sheep feed conversion rate

[0055] The experiment examined the polymorphism of 1004 Hu sheep, determined their genotypes, and established the least squares model as described below to conduct a correlation analysis between genotype and feed conversion ratio.

[0056] Y ijk =μ+Genotype i +P j +S k +ε ijk

[0057] Where Yijk is the observed value of feed conversion ratio, μ is the population mean, and Genotype is the genetic standard.i For genotype effect, P j Due to the batch effect, S k Due to seasonal effects, ε ijk Assuming random error, let ε ijk They are mutually independent and follow N(0, σ). 2 )distributed.

[0058] Genotyping results showed that among 1004 individuals, 184 had the AA genotype, 492 had the AG genotype, and 328 had the GG genotype. The results of the genotype-trait association analysis are shown in Table 1. Using 80 days of age as the starting age, sheep body weight and feed intake at 100, 120, 140, 160, and 180 days of age were measured. The predicted feed intake in this experiment was calculated using a multiple regression model of actual daily feed intake (AFI) with average daily gain (ADG) and average mid-term metabolic body weight (MBW). ADG and MBW were used as fixed effects, and the calculation method followed the regression model constructed by Mo Futao.

[0059] ADG = (BW 180 -BW 80 ) / N

[0060] MBW = [1 / 2 × (BW)] 180 +BW 80 )] 0.75

[0061] Y i =β0+β1(ADG i )+β2(MBW i )+e i

[0062] FCR = FI / (BW) 180 -BW 80 )

[0063] Where ADG is the average daily weight gain, and MBW is the average mid-metabolous body weight (W). 0.75 ), BW 80 BW was the initial body weight for the experiment. 180 FI is the body weight at the end of the experiment, N is the number of feeding days, and Y is the weight at the end of the experiment. i β0 represents the actual dry matter intake of animal i, β1 is a fixed value representing the influence of ADG on feed intake, and β2 represents the influence of MBW on feed intake, which is a fixed value. iLet RFI represent the random error of the i-th animal, i.e., RFI = actual feed intake - predicted feed intake. RFI80-120 represents the remaining feed intake from 80 to 120 days. FCR80-100, FCR80-120, FCR80-140, FCR80-160, FCR80-180, FCR100-160, FCR100-180, and FCR120-180 represent the feed conversion efficiency from 80 to 100 days, 80 to 120 days, 80 to 140 days, 80 to 160 days, 80 to 180 days, 100 to 160 days, 100 to 180 days, and 120 to 180 days, respectively.

[0064] Table 1. Association analysis between GFRA2 gene polymorphism and feed conversion ratio in sheep.

[0065]

[0066] Note: Different superscript letters in the same row indicate significant differences (P<0.05), while the same letter or no letter indicates no significant differences (P>0.05).

[0067] The results showed that with the extension of the measurement period, the A>G mutation site at position 196 of the GFRA2 gene (as shown in SEQ ID NO.1) was significantly associated with sheep feed conversion ratio (FCR). Sheep carrying the GG genotype had a higher FCR than sheep carrying the AA genotype (P<0.05). This indicates that the G allele is the dominant allele. This suggests that the A>G mutation site at position 196 of the GFRA2 gene (as shown in SEQ ID NO.1) can serve as a potential molecular marker affecting sheep FCR (P<0.05). FCR is the ratio of feed consumption to weight gain. A lower FCR means less feed consumption for the same weight gain. Selecting individuals carrying the GG genotype helps reduce breeding costs, improve economic efficiency, and also has a positive impact on environmental protection and sustainable development.

[0068] In large-scale flocks, phenotypic selection requires manual measurement of body weight and feed intake, which is time-consuming, labor-intensive, costly, and time-consuming. However, the technology of this invention can quickly screen large-scale flocks at the molecular level using efficient and economical methods, effectively screening high-quality sheep with low feed conversion rates, greatly saving time and improving economic benefits.

Claims

1. The application of a method for detecting molecular markers related to feed conversion ratio in Hu sheep breeding, characterized in that, The specific testing method includes the following steps: S1. Extract sheep genomic DNA and amplify the GFRA2 gene containing the sequence shown in SEQ ID NO.1; S2. Identify the polymorphic site at the 196th base of the amplification product obtained in step S1 as shown in SEQ ID NO.1; the feed conversion efficiency of sheep carrying the GG genotype was significantly lower than that of sheep carrying the AA genotype on days 80-120, 80-140, 80-160, and 100-160. The purpose of the breeding is to screen for feed-saving sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where R at position 196 represents A or G. Because there is a G / A mutation at position 196 of the above sequence, the G / A polymorphism of the GFRA2 gene of Hu sheep at this site is caused.

2. The application according to claim 1, characterized in that, In step S1, the nucleotide sequences of the primer pairs used for amplification are shown in SEQ ID NO.2 and SEQ ID NO.

3. PCR amplification is performed, and the amplification products are directly sequenced to obtain the genotype of the molecular marker.

3. The application according to claim 1, characterized in that, In step S1, the primer pair used for amplification is the KASPar primer pair, whose nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6. High-throughput water bath PCR amplification is performed. After amplification, the fluorescence signal is detected and the genotyping results are viewed.

4. The application of primer pairs for detecting molecular markers related to feed conversion ratio in Hu sheep breeding, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where R at position 196 represents A or G. Due to a G / A mutation at position 196 of the above sequence, the GFRA2 gene of the Hu sheep exhibits G / A polymorphism at this site. Sheep carrying the GG genotype showed significantly lower feed conversion efficiency than sheep carrying the AA genotype on days 80-120, 80-140, 80-160, and 100-160. The purpose of this breeding is to screen for feed-saving sheep.

5. A KASPar primer pair for detecting molecular markers related to feed conversion ratio in Hu sheep, characterized in that, The nucleotide sequences of the KASPar primer pairs are shown in SEQ ID NO.4-6; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where R at position 196 represents A or G. Due to a G / A mutation at position 196 of the above sequence, the GFRA2 gene of Hu sheep exhibits G / A polymorphism at this site. Sheep carrying the GG genotype showed significantly lower feed conversion efficiency than sheep carrying the AA genotype on days 80-120, 80-140, 80-160, and 100-160. The purpose of this breeding is to screen for feed-saving sheep.

6. The application of a kit for detecting molecular markers related to feed conversion ratio in Hu sheep breeding, characterized in that, The kit includes primer pairs with sequences as shown in SEQ ID NO.2 and SEQ ID NO.3 or KASPar primer pairs with sequences as shown in SEQ ID NO.4-6; the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where R at position 196 represents A or G. Due to a G / A mutation at position 196 of the above sequence, the GFRA2 gene of Hu sheep exhibits G / A polymorphism at this site; sheep carrying the GG genotype have significantly lower feed conversion efficiency than sheep carrying the AA genotype on days 80-120, 80-140, 80-160, and 100-160. The purpose of this breeding is to screen for feed-saving sheep.

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