Molecular marker associated with mutton drip loss and application thereof

Abstract

The invention discloses the technical field of mutton quality, and particularly relates to a molecular marker associated with mutton drip loss and application. The molecular marker comprises a nucleotide sequence formed by single nucleotide polymorphism of C and T at the 42407898 position of a chromosome 25 number NC056078.1 of a sheep (Ovis aries) reference genome, and a nucleotide sequence formed by single nucleotide polymorphism of C and T at the 42407898 position of the chromosome 25 number NC056078.1 of the sheep reference genome. The molecular marker is associated with the mutton drip loss, and can be used for detecting the mutton drip loss and breeding mutton sheep based on the molecular marker and the primer.

Description

Technical Field

[0001] The present application relates to the technical field of mutton drip loss, and in particular to molecular markers associated with mutton drip loss and their applications. Background Art

[0002] The meat quality of mutton is a complex concept, involving multiple characteristics, which have an important impact on the quality, taste and nutritional value of mutton. The drip loss of mutton is an extremely important trait in the meat quality. It refers to the phenomenon that water seeps out of the meat due to changes in the mutton's own water holding capacity during storage, processing or transportation. The drip loss trait is mainly used to measure the water retention performance of mutton. It is an important indicator reflecting the quality and economic value of mutton. It is usually expressed by the drip loss rate, and the calculation method is (weight of meat sample before dripping - weight of meat sample after dripping) / weight of meat sample before dripping × 100%.

[0003] Generally speaking, lamb with high drip loss will have more water seeping out from the surface, making the meat look moister and the color may become lighter. In terms of taste, the meat may become dry and hard and lose its fresh and juicy feeling due to water loss. For example, after cooking, lamb with high drip loss may be relatively dry, while lamb with low drip loss tastes relatively tender. From the perspective of nutritional value, the loss of a large amount of water means that some water-soluble vitamins, minerals and other nutrients in the lamb will also be lost. In terms of economic value, lamb with high drip loss may be sold at a lower price in the market due to its reduced quality, and during the processing, the production cost is increased due to the need to deal with the seeping water.

[0004] Therefore, it is very necessary to breed meat sheep with low drip loss. Summary of the invention

[0005] The inventors of the present application have found a molecular marker associated with the drip loss of mutton through screening. The molecular marker is a single nucleotide polymorphism site with C / T at position 42407898nt of chromosome 25, number NC_056078.1 of the reference genome of sheep (Ovis aries). The drip loss of individuals with CC genotype at this site is higher than that of other individuals, indicating that the T-type nucleotide at this site can serve as an allele that is beneficial to reducing the drip loss of mutton.

[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0007] In a first aspect, the embodiments disclose molecular markers associated with drip loss in mutton, including a nucleotide sequence formed by a single nucleotide polymorphism of C and T at position 42407898 nt of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome.

[0008] In a second aspect, the embodiments disclose a nucleic acid, such as a nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0009] In a third aspect, the embodiment discloses a primer pair for detecting the molecular marker described in the first aspect, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.3 and SEQ ID NO.4.

[0010] In a fourth aspect, an embodiment discloses a method for detecting the molecular marker described in the first aspect, the method comprising the following steps: extracting the genomic DNA of the sheep to be tested; performing PCR amplification on the genomic DNA of the sheep to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; based on the sequencing result, interpreting the polymorphism of the nucleotide site at position 203 in the sequence shown in sheep SEQ ID NO.1.

[0011] In a fifth aspect, an embodiment discloses a method for breeding a low drip loss sheep strain, the method comprising the following steps: detecting the genotype of the 42407898nt nucleotide site of chromosome 25 number NC_056078.1 of the sheep (Ovis aries) reference genome, selecting CT type individuals at the 42407898th nucleotide site as breeding sheep, or detecting individuals whose nucleotide at position 203 in the sequence shown in SEQ ID NO.1 is T as breeding sheep.

[0012] In a sixth aspect, an embodiment discloses the application of a molecular marker related to drip loss of mutton in screening sheep strains with low drip loss, wherein the molecular marker includes a nucleotide sequence formed by a single nucleotide polymorphism of C and T at position 42407898nt of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is an electrophoresis diagram of the PCR products of the mixed sample of sheep blood genomic DNA using primers provided in the example. Lane M is DL2000Maker, and lanes 1, 2, and 3 are all PCR amplification products.

[0014] Figure 2 This is a comparison chart of the sequencing sequences of the PCR products of the mixed sample of double dried mutton sheep blood genomic DNA provided in the example.

[0015] Figure 3 The peak diagram of the sheep (Ovis aries) reference genome chromosome 25, number NC_056078.1, 42407898 nt position, which is a C nucleotide, is provided in the embodiment.

[0016] Figure 4 The peak diagram of the sheep (Ovis aries) reference genome chromosome 25, number NC_056078.1, 42407898 nt position, which is a T nucleotide, is provided in the embodiment. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] The inventors of the present application have found a molecular marker associated with the drip loss of mutton through screening. The molecular marker is a single nucleotide polymorphism site with C / T at position 42407898nt of chromosome 25, number NC_056078.1 of the reference genome of sheep (Ovis aries). The drip loss of individuals with CC genotype at this site is higher than that of other individuals, indicating that the T-type nucleotide at this site can serve as an allele that is beneficial to reducing the drip loss of mutton.

[0019] Based on this, the embodiment discloses a molecular marker associated with drip loss in mutton, including a nucleotide sequence formed by a single nucleotide polymorphism of C and T at position 42407898 nt of chromosome 25 number NC_056078.1 of the sheep (Ovis aries) reference genome.

[0020] The embodiments disclose a nucleic acid, such as the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0021] The embodiment discloses a primer pair for detecting the molecular marker, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.3 and SEQ ID NO.4.

[0022] The embodiment discloses a method for detecting the molecular marker, which comprises the following steps: extracting genomic DNA of the sheep to be tested; performing PCR amplification on the genomic DNA of the sheep to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; and based on the sequencing result, interpreting the polymorphism of the nucleotide site at position 203 in the sequence shown in sheep SEQ ID NO.1.

[0023] The embodiment discloses a method for breeding a low drip loss sheep strain, the method comprising the following steps:

[0024] The embodiment discloses the application of molecular markers related to drip loss of mutton in screening sheep strains with low drip loss, the nucleotide sequence comprising a single nucleotide polymorphism of C and T at position 42407898nt of chromosome 25 numbered NC_056078.1 of the sheep (Ovis aries) reference genome.

[0025] The following will illustrate the implementation of the present application in conjunction with more specific examples, but does not constitute a limitation on the implementation of the present application.

[0026] 1. Experimental Animals

[0027] The experimental animals were 49 6-month-old male double-dry mutton sheep raised by the Institute of Animal Husbandry and Veterinary Medicine of Jilin Academy of Agricultural Sciences, and were raised under relevant conditions. After slaughter, the experimental sheep were analyzed for meat quality traits such as intramuscular fat content, cooked meat rate, drip loss, water content, pH value, tenderness, etc., and the fatty acid composition of the longissimus dorsi muscle was detected.

[0028] 2. Experimental Materials

[0029] The blood genome extraction kit was purchased from Axygen Biotechnology Co., Ltd.; the PCR reaction premix was purchased from Kangwei Century Biotechnology Co., Ltd.; and the Sanger sequencing and primers were provided by Jinweizhi Biotechnology Co., Ltd.

[0030] 3. Determination of drip loss

[0031] After slaughter, take a cross-section of the longissimus dorsi between the 12th and 13th ribs, 2.5 cm thick, weighing about 70-100 g, and remove the epimysium. After weighing, seal the meat sample with an inflatable plastic bag and suspend it with a silk thread in a 4°C refrigerator. After 48 hours, remove the plastic bag and silk thread, wipe the moisture off the meat surface with filter paper, and weigh it again. Drip loss = (weight before hanging - weight after hanging) / weight before hanging × 100%

[0032] 4. Genome extraction and verification

[0033] Blood was collected from the jugular vein of the experimental sheep before slaughter and stored in a blood collection tube containing an anticoagulant. 250 mL of blood was used for DNA extraction according to the genome extraction kit method. The extracted DNA was subjected to agarose gel electrophoresis to detect its fragment integrity. The concentration and purity of the genome with good integrity were measured by an ultra-micro spectrophotometer. The OD value of 260 / 280 was between 1.8 and 2.1, indicating good purity. The DNA with high purity and good integrity was stored in a -20°C refrigerator for the next experiment.

[0034] 5. PCR

[0035] Randomly select 10 blood genomic DNA mixed samples from 49 double dry mutton genomes, and use SEQ ID NO.3 and SEQ ID NO.4 as primer pairs for PCR amplification. The PCR reaction system contains 20 μL: 10 μL 2×Taq Master Mix, 0.5 μL DNA molecules such as SEQ ID NO.2, 0.5 μL DNA molecules such as SEQ ID NO.3, 1 μL mixed pool DNA, and 8 μL ddH2O. The PCR reaction steps include: step 1): 94℃ pre-denaturation for 5min; step 2): 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 35 cycles; step 3) 72℃ extension for 8min, and the product is stored at 12℃. The PCR product is detected by agarose gel electrophoresis, and a single band represents good specificity.

[0036] like Figure 1 As shown, the target fragment was 336 bp (SEQ ID NO: 1), which was consistent with the theoretical size.

[0037] 6. Sanger sequencing

[0038] The PCR amplification products were subjected to Sanger sequencing. Figure 2 , Figure 3 and Figure 3 It is shown that the blood genomic DNA has a C / T single nucleotide polymorphism at the 42407898nt position of chromosome 25 number NC_056078.1 of the sheep (Ovis aries) reference genome, and there are three genotypes of the mutation, namely CC, TT, and CT.

[0039] 7. Statistical analysis of mutation sites

[0040] The genotype of the C / T site at position 42407898nt of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome was used, and the gene frequency, genotype frequency, heterozygosity, PIC content, and amino acid changes were calculated. The meat quality data of 49 double dry meat sheep were sorted out and associated with the genotypes. The results are shown in Table 1. The production data of double dry meat sheep were statistically analyzed by SPSS17.0 and expressed as "mean ± standard deviation". The association analysis of different genotypes with actual production data was performed using one-way analysis of variance and LSD method for multiple comparisons. P<0.05 was considered significant.

[0041] As shown in Table 1, the frequencies of CC, TT, and CT genotype individuals are 61.2%, 6.1%, and 32.7%, respectively. It can be seen that CT is the dominant allele type, and C is the dominant allele. The mutation is a synonymous mutation, aspartic acid, which does not produce changes and is moderately polymorphic, which conforms to the Hardy-Weingerber theorem.

[0042] Table 1 Statistical analysis results of Chr25:42407898 site

[0043]

[0044]

[0045] 7. Analysis of association between mutation sites and meat quality traits

[0046] Table 2 shows the association analysis results of the single nucleotide polymorphisms of C and T at the 42407898 nt position of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome and the drip loss of sheep meat quality. In Table 2, "a", "b" and "ab" are all significant difference markers, and the same marker means no restriction. As shown in Table 2, the drip loss of CC genotype individuals is the highest at 48 and 72 hours.

[0047] Table 2 Association analysis between meat quality traits and genotypes

[0048] Meat quality data\genotype CC CT TT Drip loss 48 hours 2.07±0.13a 1.57±0.10b 1.64±0.54ab Drip loss 72 hours 2.98±0.14a 2.47±0.16b 2.26±0.52ab

[0049] Drip loss refers to the amount of liquid lost by the muscle protein system during measurement without any external force but only under the action of gravity. Drip loss is significantly correlated with pH value, color and marbling score. The lower the drip loss, the better the meat quality. For the evaluation of lamb quality after slaughter, common parameters include meat color, drip loss, marbling, tenderness, juiciness, pH value, fat / lean ratio, etc. Among them, the parameters that consumers usually pay the most attention to are meat color and drip loss. Reducing the drip loss of meat after slaughter can extend the shelf life of meat. The greater the drip loss, the worse the water-binding capacity of the muscle, and the smaller the drip loss, the better the water-binding capacity of the muscle. The measurement results can be evaluated by the comparative ranking method of the same period.

[0050] The present application found that the drip loss of genotype CC is the highest, so in the process of breeding meat sheep, avoiding the CC genotype can obtain good drip loss data, and then obtain individuals with high water holding capacity.

[0051] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Molecular markers associated with drip loss in mutton, including a nucleotide sequence formed by a single nucleotide polymorphism of C and T at position 42407898 of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome.

2. A nucleic acid, such as the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

3. A primer pair for detecting the molecular marker according to claim 1, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. A method for detecting the molecular marker according to claim 1, comprising: Extracting genomic DNA from the sheep to be tested; Performing PCR amplification on the genomic DNA of the sheep to be tested to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the polymorphism of the 42407898nt site of chromosome 25 number NC_056078.1 of the sheep (Ovis aries) reference genome was determined.

5. A method for breeding a low drip loss sheep strain, comprising: The genotype of the 42407898nt nucleotide site of chromosome 25 number NC_056078.1 of the sheep (Ovis aries) reference genome is detected, and the CT type individuals at the 42407898th nucleotide site are selected as breeding sheep, or the individuals whose nucleotide at position 203 in the sequence shown in SEQ ID NO.1 is T are detected as breeding sheep.

6. Application of a molecular marker for drip loss in sheep meat to screening for sheep strains with low drip loss, the molecular marker comprising a nucleotide sequence formed by a single nucleotide polymorphism of C and T at position 42407898 of chromosome 25, number NC_056078.1 of the sheep (Ovis aries) reference genome.

Images