A molecular marker related to broiler carcass traits and meat quality traits, an amplification primer and application thereof

Abstract

This invention discloses a molecular marker, amplification primer, and their applications related to broiler carcass and meat quality traits, belonging to the field of molecular biology. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.3; SNP1, with an A / C mutation, exists at base 334; SNP2, with an A / G mutation, exists at base 362; SNP3, with a C / T mutation, exists at base 518; SNP4, with a C / A mutation, exists at base 652; SNP5, with an A / G mutation, exists at base 840; and SNP6, with a C / T mutation, exists at base 863. This invention, through analysis of the LCA5L gene, discovered multiple SNP sites significantly associated with chicken carcass and meat quality traits, providing new SNP molecular markers for broiler carcass and meat quality traits, and simultaneously providing a scientific basis for chicken breeding.

Description

Molecular markers, amplification primers and their applications related to broiler carcass and meat quality traits Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a molecular marker, amplification primer, and their applications related to broiler carcass traits and meat quality traits. Background Technology

[0002] In the modern poultry industry, chickens, as one of the most widely farmed poultry species globally, occupy an extremely important position in the human diet. With the continuous improvement of living standards, consumer demand for chicken has shifted from simply satisfying quantity to demanding stringent quality requirements. This is reflected not only in the pursuit of tender, juicy, and flavorful chicken, but also in a high level of attention to its nutritional value and safety and health attributes. Against this backdrop, in-depth research into the carcass and meat characteristics of chickens is of great significance for the high-quality development of the poultry farming industry.

[0003] Single nucleotide polymorphisms (SNPs) are variations in DNA sequences within a genome caused by the insertion, deletion, inversion, and transformation of a single nucleotide. As a common type of heritable variation in animals, SNPs are widely distributed throughout the animal genome, possess stable genetic characteristics, and are easily detected. In animal production, numerous studies have shown that SNPs in gene structure have a significant impact on actual animal production performance and can serve as an important tool for molecular marker-assisted selection (MAS) to improve the efficiency and effectiveness of breeding and selection.

[0004] In other organisms, the LCA5L gene is predicted to be involved in ciliary transport and function within the axonal filament. Many tissues and organs in birds contain ciliated cells; for example, the epithelial cells of the respiratory tract, reproductive tract, and oviduct all have cilia on their surface. Therefore, the LCA5L gene may be expressed in these ciliated tissues to maintain normal ciliary function. However, the association between the avian LCA5L gene and carcass traits and meat quality traits in chickens has not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide molecular markers, amplification primers, and their applications related to broiler carcass and meat quality traits, thereby addressing the problems existing in the prior art. This invention, through analysis of the LCA5L gene, discovered multiple SNP sites significantly associated with chicken carcass and meat quality traits, providing novel SNP molecular markers for MAS (Magnetic Analytic Hierarchy Process). The molecular markers provided by this invention can accurately identify chicken carcass and meat quality traits, thus providing a scientific basis for chicken breeding.

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

[0007] This invention provides a molecular marker related to broiler carcass traits and meat quality traits, the nucleotide sequence of which is shown in SEQ ID NO.3; SNP1 has an A / C mutation at base 334; SNP2 has an A / G mutation at base 362; SNP3 has a C / T mutation at base 518; SNP4 has a C / A mutation at base 652; SNP5 has an A / G mutation at base 840; and SNP6 has a C / T mutation at base 863.

[0008] The genotypes of SNP1 include AA, AC, and CC; the genotypes of SNP2 include AA, AG, and GG; the genotypes of SNP3 include CC, CT, and TT; the genotypes of SNP4 include CC, CA, and AA; the genotypes of SNP5 include AA, AG, and GG; and the genotypes of SNP6 include CC, TT, and CT.

[0009] The present invention also provides a primer pair for amplifying the aforementioned molecular marker, comprising an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a kit for identifying chicken carcass traits and meat traits, comprising the primer pair described above.

[0011] The present invention also provides the application of the primer pair or the kit described herein in identifying chicken carcass traits and meat quality traits, wherein the chicken carcass traits and meat quality traits include intramuscular fat width, leg muscle L value, leg muscle pH value, breast muscle L value, breast muscle pH value, and subcutaneous fat thickness.

[0012] The present invention also provides a method for identifying chicken carcass traits and meat quality traits, comprising the following steps:

[0013] Take the genomic DNA of the broiler chicken to be tested, and perform PCR amplification using the primer pair or the kit described above to obtain the amplification product;

[0014] The amplified products were sequenced to detect the genotype of the corresponding SNP loci;

[0015] At the SNP1 locus, individuals with the AC genotype had a greater intramuscular fat width than those with the CC genotype.

[0016] At the SNP2 locus, individuals with the AG genotype had a greater intermuscular fat width than those with the GG genotype; individuals with the AG genotype had a greater leg muscle L value than those with the AA genotype.

[0017] At the SNP3 locus, individuals with the CT genotype had a higher L value in their pectoral muscles than those with the CC genotype.

[0018] At the SNP4 locus, individuals with the CC genotype had greater subcutaneous fat thickness than those with the CA and AA genotypes; individuals with the CC genotype had a higher leg muscle pH than those with the CA genotype.

[0019] At the SNP5 locus, the leg muscle L value of individuals with the GG and AG genotypes was higher than that of individuals with the AA genotype.

[0020] At the SNP6 locus, individuals with the TT genotype had a higher pectoral muscle pH than those with the CT genotype.

[0021] Optionally, the PCR amplification reaction system consists of 2 μL template DNA, 15 μL 2x Rapid Taq Master Mix, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0022] Optionally, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; 72℃ final extension for 5 min; storage at 4℃.

[0023] The present invention also provides the application of the primer pair or the kit described herein in marker-assisted breeding of chickens.

[0024] The present invention discloses the following technical effects:

[0025] This invention, through analysis of the LCA5L gene, discovered multiple SNP loci significantly associated with chicken carcass and meat quality traits, providing novel SNP molecular markers for MAS (meat quality assay). The molecular markers provided by this invention can accurately identify chicken carcass and meat quality traits, thus providing a scientific basis for chicken breeding. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 shows a schematic diagram of primer pairing positions and product length for LCA5L;

[0028] Figure 2 shows the genotyping diagram of the SNP sites in the LCA5L gene sequence. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] This invention selected 318 small white-feathered broilers, slow-growing yellow-feathered broilers, and fast-growing white-feathered broilers aged 49 days. 2 mL of subcutaneous venous blood was collected from each broiler and stored at -80℃ for DNA extraction. The following traits of the selected population were recorded for subsequent analysis: breast angle, breast depth, breast width, live weight, shank length, shank circumference, body length, keel length, comb height, carcass weight, subcutaneous fat thickness, intramuscular fat width, semi-eviscerated weight, fully eviscerated weight, abdominal fat weight, wing weight, breast muscle weight, leg muscle weight, foot weight, breast muscle shear force, leg muscle shear force, drip loss rate, cooking loss rate, breast muscle pH value, leg muscle pH value, breast muscle L value, breast muscle a value, breast muscle b value, leg muscle L value, leg muscle a value, leg muscle b value, dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, abdominal fat percentage, breast muscle percentage, and leg muscle percentage.

[0035] Example

[0036] I. Experimental Methods

[0037] 1. Primer design

[0038] Based on the published sequence of the Gallus (Gallus) LCA5L gene (NC_052532.1), primers were designed using Primer Premier5, with primer synthesis services provided by Guangzhou Qingke Biotechnology Co., Ltd. Primer sequence information is shown in Table 1, and the primer pairing positions on the LCA5L gene are shown in Figure 1.

[0039] Table 1. Primer sequences for PCR amplification

[0040]

[0041] 2. Blood sample DNA extraction

[0042] Blood DNA was extracted using a blood sample DNA extraction kit (brand: OMEGA; catalog number: D3392; Guangzhou Feiyang Biotechnology Co., Ltd.), following the kit's instruction manual.

[0043] 3. PCR amplification of partial LCA5L gene sequence

[0044] Using the genomic DNA from the blood samples of the above 318 chickens as templates, the reaction was carried out according to the following system: 2 μL template DNA, 15 μL 2xRapidTaq Master Mix, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

[0045] Reaction procedure: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; final extension at 72℃ for 5 min; storage at 4℃. PCR products were sent to Guangzhou Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0046] 4. SNP identification and genotyping

[0047] The Sanger sequencing results of PCR products were analyzed using SnapGene software to identify potential SNP sites. The sequencing data of each sample were then compared with the SnapGene software to perform genotyping.

[0048] 5. Association analysis between genotype and carcass traits

[0049] The phenotypic data of SNP loci and corresponding individuals were analyzed using SPSS 27.0.

[0050] II. Experimental Results

[0051] 1. LCA5L gene sequence PCR amplification and SNP screening

[0052] The above 318 chicken individuals were selected, and PCR amplification was performed using blood sample DNA from each individual as a template. The obtained PCR products (nucleotide sequences shown in SEQ ID NO.3) were subjected to Sanger sequencing. The peak diagrams after sequencing were compared and analyzed, and a total of 6 SNP sites were detected, namely: NC_052532.1: g108106369A>C, NC_052532.1: g.108106397A>G, NC_052532.1: g.108106553C>T, NC_052532.1: g.108106687C>A, NC_052532.1: g.108106875A>G, NC_052532.1: g.108106898C>T, as shown in Figure 2.

[0053] SEQ ID NO.3:

[0054] 。

[0055] The corresponding positions and polymorphism information of the above 6 SNP sites in SEQ ID NO.3 are as follows: there is an A / C mutation at the 334th base, an A / G mutation at the 362nd base, a C / T mutation at the 518th base, a C / A mutation at the 652nd base, an A / G mutation at the 840th base, and a C / T mutation at the 863rd base.

[0056] 2. Association analysis of LCA5L gene sequence SNP sites with carnivorous traits

[0057] Association analysis was performed on the above six SNP loci and traits (breast angle, breast depth, breast width, live weight, tibia length, tibia circumference, body oblique length, keel length, comb height, carcass weight, subcutaneous fat thickness, intramuscular fat width, semi-eviscerated weight, fully eviscerated weight, abdominal fat weight, wing weight, breast muscle weight, leg muscle weight, chicken foot weight, breast muscle shear force, leg muscle shear force, drip loss rate, cooking loss rate, breast muscle pH value, leg muscle pH value, breast muscle L value, breast muscle a value, breast muscle b value, leg muscle L value, leg muscle a value, leg muscle b value, dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, abdominal fat percentage, breast muscle percentage, and leg muscle percentage). According to the slaughtering process, meat quality traits were measured only after the carcass trait assessment was completed. This resulted in measurements for some samples exceeding the one-hour post-slaughter timeframe, therefore the sample size for some meat quality traits was less than 318.

[0058] As shown in Table 2, the NC_052532.1:g108106369A>C site was significantly correlated with the width of intramuscular fat (p<0.05). Among them, the intramuscular fat width of individuals with the AC heterozygous genotype was significantly higher than that of individuals with the CC mutation homozygous genotype (p<0.05).

[0059] As shown in Table 3, the NC_052532.1:g.108106397A>G site was significantly correlated with intramuscular fat width and leg muscle L value (p<0.05). Among them, the intramuscular fat width of AG heterozygous individuals was significantly higher than that of GG mutant homozygous individuals (p<0.05); the leg muscle L value of AG heterozygous individuals was significantly higher than that of AA wild-type homozygous individuals (p<0.05).

[0060] As shown in Table 4, the NC_052532.1:g.108106553C>T site was significantly correlated with the L value of the pectoral muscle (p<0.05). Among them, the L value of the pectoral muscle of individuals with the CT heterozygous genotype was significantly higher than that of individuals with the CC wild-type homozygous genotype (p<0.05).

[0061] As shown in Table 5, the NC_052532.1:g.108106687C>A site was significantly correlated with subcutaneous fat thickness and leg muscle pH (p<0.05). Subcutaneous fat thickness was significantly higher in individuals with the CC wild-type homozygous genotype than in those with the CA heterozygous genotype and the AA wild-type homozygous genotype, while there was no significant difference between CA and AA (p>0.05). Leg muscle pH was significantly higher in individuals with the CC wild-type homozygous genotype than in those with the CA heterozygous genotype (p<0.05).

[0062] As shown in Table 6, the molecular marker NC_052532.1:g.108106397A>G site was significantly correlated with leg muscle L value (p<0.05). Among them, the leg muscle L value of individuals with homozygous GG mutant genotype and heterozygous AG genotype was significantly higher than that of individuals with homozygous AA wild-type genotype, while there was no significant difference between AG and GG (p>0.05).

[0063] As shown in Table 7, the NC_052532.1:g.108106898C>T site was significantly correlated with pectoral muscle pH (p<0.05). Among them, the pectoral muscle pH of individuals with homozygous TT mutation was significantly higher than that of individuals with heterozygous CT mutation (p<0.05).

[0064] Table 2 Association between NC_052532.1:g108106369A>C site and carnivorous traits

[0065]

[0066] Table 3. Associations between the NC_052532.1:g.108106397A>G site and carcass and meat quality traits.

[0067]

[0068] Table 4. Association between NC_052532.1:g.108106553C>T site and meat quality traits

[0069]

[0070] Table 5. Associations between the NC_052532.1:g.108106687C>A site and carcass and meat quality traits.

[0071]

[0072] Table 6. Association between NC_052532.1:g.108106875A>G site and meat quality traits.

[0073]

[0074] Table 7. Association between NC_052532.1:g.108106898C>T site and meat quality traits.

[0075]

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a primer pair or a kit containing the primer pair in identifying carcass traits and meat traits in chickens, characterized in that, The carcass traits and meat quality traits of the chicken are: wide intramuscular fat, leg muscle L value, leg muscle pH value, breast muscle L value, breast muscle pH value, and thick subcutaneous fat; the primer pair includes an upstream primer with the sequence shown in SEQ ID NO. 1 and a downstream primer with the sequence shown in SEQ ID NO. 2; the primer pair is used to amplify molecular markers related to the carcass traits and meat quality traits of broilers; the nucleotide sequences of the molecular markers are shown in SEQ ID NO. 3; in SEQ ID NO... The sequence shown in .3 contains SNP1 with an A / C mutation at base 334; SNP2 with an A / G mutation at base 362; SNP3 with a C / T mutation at base 518; SNP4 with a C / A mutation at base 652; SNP5 with an A / G mutation at base 840; and SNP6 with a C / T mutation at base 863. The genotypes at the sites of SNP1 include AA, AC, and CC; SNP2 includes AA, AG, and GG; SNP3 includes CC, CT, and TT; SNP4 includes CC, CA, and AA; SNP5 includes AA, AG, and GG; and SNP6 includes CC, TT, and CT.

2. A method for identifying the carcass characteristics and meat quality characteristics of chickens, characterized in that, Includes the following steps: Genomic DNA of the broiler to be tested was collected and amplified by PCR using the primer pair or the kit described in claim 1 to obtain molecular marker amplification products; the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 3; SNP1 with an A / C mutation exists at base 334 of the sequence shown in SEQ ID NO. 3; SNP2 with an A / G mutation exists at base 362; SNP3 with a C / T mutation exists at base 518; SNP4 with a C / A mutation exists at base 652; SNP5 with an A / G mutation exists at base 840; and SNP6 with a C / T mutation exists at base 863; the amplification products were sequenced to detect the genotype of the corresponding SNP sites; the carcass traits and meat quality traits of the chicken are: wide intramuscular fat, leg muscle L value, leg muscle pH value, breast muscle L value, breast muscle pH value, and thick subcutaneous fat. At SNP1, individuals with the AC genotype had a wider intermuscular fat layer than those with the CC genotype; at SNP2, individuals with the AG genotype had a wider intermuscular fat layer than those with the GG genotype; individuals with the AG genotype had a higher leg muscle L value than those with the AA genotype; at SNP3, individuals with the CT genotype had a higher pectoral muscle L value than those with the CC genotype; at SNP4, individuals with the CC genotype had a thicker subcutaneous fat layer than those with the CA and AA genotypes; individuals with the CC genotype had a higher leg muscle pH value than those with the CA genotype; at SNP5, individuals with the GG and AG genotypes had a higher leg muscle L value than those with the AA genotype; at SNP6, individuals with the TT genotype had a higher pectoral muscle pH value than those with the CT genotype.

3. The method according to claim 2, characterized in that, The PCR amplification reaction system consisted of 2 μL template DNA, 15 μL 2xRapid Taq Master Mix, 1.2 μL upstream primer, 1.2 μL downstream primer, and 10.6 μL ddH2O.

4. The method according to claim 2, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, 34 cycles; 72℃ final extension for 5 min; and storage at 4℃.

5. The application of a primer pair or a kit containing the primer pair in marker-assisted breeding of chickens, characterized in that, The primer pair or the kit is used to screen for chicken carcass traits and meat quality traits, which are: wide intramuscular fat, leg muscle L value, leg muscle pH value, breast muscle L value, breast muscle pH value, and thick subcutaneous fat; the primer pair includes an upstream primer with the sequence shown in SEQ ID NO. 1 and a downstream primer with the sequence shown in SEQ ID NO. 2; the primer pair is used to amplify molecular markers related to broiler carcass traits and meat quality traits; the nucleotide sequences of the molecular markers are shown in SEQ ID NO. 3; in SEQ ID NO... The sequence shown in .3 contains SNP1 with an A / C mutation at base 334; SNP2 with an A / G mutation at base 362; SNP3 with a C / T mutation at base 518; SNP4 with a C / A mutation at base 652; SNP5 with an A / G mutation at base 840; and SNP6 with a C / T mutation at base 863. The genotypes at the sites of SNP1 include AA, AC, and CC; SNP2 includes AA, AG, and GG; SNP3 includes CC, CT, and TT; SNP4 includes CC, CA, and AA; SNP5 includes AA, AG, and GG; and SNP6 includes CC, TT, and CT.

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