Molecular markers associated with drip loss in lamb and applications thereof

Abstract

The application 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 a single nucleotide polymorphism of C and T at a position of 42407898 nt of a chromosome numbered 25 of a sheep (Ovis aries) reference genome NC_056078.1. The molecular marker is associated with mutton drip loss, and based on the molecular marker and primers, the mutton drip loss can be detected, and mutton sheep breeding can be carried out.

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 that have a significant impact on the quality, taste, and nutritional value of mutton. Drip loss is an extremely important meat quality trait. It refers to the phenomenon of water seeping out of the meat due to changes in the mutton's own water holding capacity during storage, processing, or transportation. Drip loss is mainly used to measure the water retention performance of mutton. It is an important indicator of mutton quality and economic value. It is usually expressed as a drip loss rate, which is calculated as (weight of meat sample before dripping - weight of meat sample after dripping) / weight of meat sample before dripping × 100%.

[0003] Generally speaking, lamb with a high drip loss rate will have more water seeping out of its surface, making the meat appear moister and possibly lighter in color. In terms of taste, due to this water loss, the meat may become dry and hard, losing its tenderness and juiciness. For example, after cooking, lamb with a high drip loss rate may be tough, while lamb with a low drip loss rate will taste relatively tender. From a nutritional perspective, the loss of a large amount of water means that some of the lamb's water-soluble vitamins, minerals, and other nutrients will also be lost. From an economic perspective, lamb with a high drip loss rate may be sold at a lower price in the market due to its reduced quality. Furthermore, the need to deal with the leaking water during processing increases production costs.

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

[0005] The inventors of this application have discovered a molecular marker associated with drip loss in lamb meat. This molecular marker is a single nucleotide polymorphism (SNP) site with a C / T sequence at position 42407898 on chromosome 25, NC_056078.1, of the sheep (Ovis aries) reference genome. Individuals with the CC genotype at this site have higher drip loss than those with the CC genotype, indicating that the T-type nucleotide at this site may be an allele that helps reduce drip loss in lamb meat.

[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 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 the 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 sequences of the primer pair are shown in 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 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; 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.

[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, the embodiments disclose the application of molecular markers related to mutton drip loss in screening sheep breeds with low drip loss, wherein the molecular markers include a nucleotide sequence formed by a single nucleotide polymorphism of C and T at the 42407898nt position 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 a 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 genomic DNA from double dried mutton blood provided in the example.

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

[0016] Figure 4 The peak graph of the Ovis aries reference genome chromosome 25 number NC_056078.1 position 42407898 nt is T nucleotide for the example. DETAILED DESCRIPTION

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

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

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

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

[0021] The example discloses a primer pair for detecting the molecular marker, and the nucleotide sequences of the primer pair are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0022] The example discloses a method for detecting the molecular marker, and the method includes 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; and based on the sequencing result, judging the polymorphism of the nucleotide site at position 203 in the sequence shown in SEQ ID NO. 1.

[0023] The example discloses a method for breeding a low-drip-loss sheep strain, and the method includes the following steps:

[0024] The embodiment discloses the use of a molecular marker for lamb drip loss in screening lamb strains with low drip loss, wherein the marker comprises 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 lamb (Ovis aries) reference genome.

[0025] The following will illustrate the implementation of the present application with reference to 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 six-month-old male double-bred mutton sheep raised under relevant conditions at the Institute of Animal Husbandry and Veterinary Medicine, Jilin Academy of Agricultural Sciences. Meat quality traits, such as intramuscular fat content, doneness percentage, drip loss, moisture content, pH, and tenderness, were analyzed after slaughter. The fatty acid composition of the longissimus dorsi muscle was also examined.

[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 2.5 cm thick, 70-100 g transverse cut from the longissimus dorsi muscle between the 12th and 13th ribs. Remove the epimysium. After weighing, seal the meat sample in an inflatable plastic bag and suspend it with silk thread in a refrigerator at 4°C. After 48 hours, remove the plastic bag and silk thread, wipe the meat surface with filter paper to remove moisture, and then weigh it again. Drip loss = (weight before suspension - weight after suspension) / weight before suspension × 100%

[0032] 4. Genome extraction and verification

[0033] Before slaughter, blood was collected from the jugular vein of the experimental sheep and stored in a blood collection tube containing anticoagulant. DNA was extracted from 250 mL of blood using a genomic extraction kit. The extracted DNA was subjected to agarose gel electrophoresis to test fragment integrity. The concentration and purity of genomes with good integrity were measured using an ultra-micro spectrophotometer. An OD value of 260 / 280 between 1.8 and 2.1 indicated good purity. DNA with high purity and good integrity was stored in a refrigerator at -20°C for subsequent experiments.

[0034] 5. PCR

[0035] A pool of genomic DNA from 10 randomly selected blood samples from 49 double-dried mutton sheep was amplified by PCR using the primer pair shown in SEQ ID NO. 3 and SEQ ID NO. 4. The 20 μL PCR reaction system contained: 10 μL 2× Taq Master Mix, 0.5 μL DNA of SEQ ID NO. 2, 0.5 μL DNA of SEQ ID NO. 3, 1 μL of pooled DNA, and 8 μL ddH2O. The PCR reaction steps included: Step 1: 94°C denaturation for 5 minutes; Step 2: 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; Step 3: 72°C extension for 8 minutes, and storage of the product at 12°C. The PCR product was analyzed by agarose gel electrophoresis; a single band indicated 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 shows that the blood genomic DNA has a C / T single nucleotide polymorphism at position 42407898nt 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 genotypes of the C / T site at position 42407898 on chromosome 25, NC_056078.1, of the sheep (Ovis aries) reference genome were analyzed. Gene frequency, genotype frequency, heterozygosity, PIC content, and amino acid variation were calculated. Meat quality data from 49 double-dried mutton sheep were compiled and analyzed for association with genotypes. The results are shown in Table 1. Double-dried mutton production data were statistically analyzed using SPSS 17.0 and presented as "mean ± standard deviation." One-way analysis of variance and the LSD method were used for multiple comparisons of different genotypes. P < 0.05 was considered significant.

[0041] As shown in Table 1, the frequencies of individuals with the CC, TT, and CT genotypes were 61.2%, 6.1%, and 32.7%, respectively. This indicates that CT is the dominant genotype, with C being the dominant allele. Synonymous mutations to aspartic acid do not produce a change, indicating moderate polymorphism, consistent with the Hardy-Weingerber theorem.

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

[0043]

[0044]

[0045] 7. Association analysis between mutation sites and meat quality traits

[0046] Table 2 shows the results of an association analysis between the single nucleotide polymorphism (SNP) of C and T at position 42407898 on chromosome 25, NC_056078.1, of the sheep (Ovis aries) reference genome and drip loss. In Table 2, "a," "b," and "ab" are all significant difference markers; identical markers represent no restriction. As shown in Table 2, individuals with the CC genotype had the highest drip loss 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 when no external force is applied but only gravity acts. Drip loss is significantly correlated with pH value, color and marbling score. The lower the drip loss, the better the meat quality. Common parameters for evaluating the quality of lamb after slaughter include meat color, drip loss, marbling, tenderness, juiciness, pH value, fat / lean ratio, etc. Among them, the parameters that consumers are usually most concerned about 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 was the highest, so in the process of breeding meat sheep, avoiding the CC genotype can obtain good drip loss data and thus obtain individuals with high water holding capacity.

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

Claims

1. A method for breeding a low drip loss sheep strain, comprising: Detection of sheep ( Ovis aries ) Refer to the genotype of the 42407898th nucleotide position of chromosome 25, number NC_056078.1 of the reference genome, and select the CT type individuals at the 42407898th nucleotide position as breeding sheep, or detect the individuals whose nucleotide at position 203 in the sequence shown in SEQID NO.1 is T as breeding sheep.

2. Application of a molecular marker for lamb drip loss in screening lamb strains with low drip loss, wherein the molecular marker is a lamb ( Ovis aries ) The nucleotide sequence formed by the single nucleotide polymorphism of C and T at position 42407898 on chromosome 25 of the reference genome, number NC_056078.1, was used to select CT-type individuals at the 42407898th nucleotide site as breeding sheep, or to detect sheep individuals in which the nucleotide at position 203 in the sequence shown in SEQ ID NO.1 was T as breeding sheep.

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