InDel marker of pig growth rate related gene RPS27L and application thereof

Abstract

The application discloses application of an InDel molecular marker or a substance for detecting the InDel molecular marker in identification or auxiliary identification of pig growth rate, and the InDel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1. The application has beneficial effects, including: (1) the application finds that an insertion / deletion site in a pig RPS27L gene promoter region can be used as a genetic selection marker for pig molecular breeding, and is beneficial to screening of pig breeds with excellent growth traits and acceleration of speed of selection and breeding of fine breeds; (2) the application uses a primer pair P1 designed according to a whole genome of a reference pig as a primer, uses pig genomic DNA as a template, and detects a genotype of an insertion / deletion polymorphism site (Chr1:108,880,114-108,880,126bp) in a pig RPS27L gene promoter region in a pig population through sequence amplification, electrophoretic identification and Sanger sequencing, and the detection is efficient, accurate and low in cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pig breeding, and particularly relates to an InDel marker of a pig growth rate related gene RPS27L and application thereof. BACKGROUND

[0002] China is the largest pig breeding and pork consumption country in the world, and the pork production and consumption account for more than 50% of the world. In addition, China has rich pig breed resources, accounting for 1 / 3 of the world, but more than 90% of the commercial pigs bred in China are western lean-type breeds. Therefore, excavating and utilizing high-quality characteristic gene resources of local pig breeds, cultivating excellent pig breeds with independent intellectual property rights in China, and realizing "localization of good pig breeds" are not only the needs of healthy and sustainable development of China's pig industry, but also the fundamental guarantee of pork supply safety. Carrying out breeding must rely on a solid theoretical basis, therefore, studying the genetic basis of important economic traits such as pig growth rate is the need of China's pig industry development, and has important economic value and strategic significance.

[0003] The key factor affecting the phenotypic characteristics of animals at the genome level is the variation of genome sequence. At present, common genome variations include insertion / deletion (InDels), single nucleotide polymorphisms (SNPs) and structural variations (SVs) and the like. InDel refers to a certain number of nucleotide insertions or deletions in the genome. With the in-depth research of comparative genomics, InDel provides a large amount of biological information for genetic breeding application, and as a new generation of genetic identification marker, it has the advantages of SNP. And compared with other variations, InDel is easier to be directly identified by PCR amplification and agarose gel electrophoresis technology.

[0004] In summary, it is of great significance to excavate molecular markers related to pig growth rate for the improvement of pig growth traits and the cultivation of excellent pig breeds. SUMMARY

[0005] The application provides an application of an InDel molecular marker or a substance for detecting the InDel molecular marker in identifying or assisting in identifying pig growth rate, and the InDel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1.

[0006] The molecular marker can be a 13-bp insertion and / or deletion polymorphism site located at 108,880,114-108,880,126 bp of a pig reference genome Sscrofa11.1 version sequence Chr1.

[0007] The insertion and / or deletion sequence at 108,880,114-108,880,126 bp of Chr1 is SEQ ID NO. 1, which is located in the promoter region of the RPS27L gene.

[0008] Further, in the above application, the substance can contain a PCR primer for amplifying a pig genomic DNA fragment containing the InDel molecular marker.

[0009] Further, in the above application, the PCR primer can consist of a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID No. 2, and the sequence of the reverse primer is shown in SEQ ID No. 3.

[0010] The present application provides a PCR primer for identifying or assisting in identifying the growth rate of pigs, which can be used to amplify a pig genomic DNA fragment containing the above-mentioned InDel molecular marker.

[0011] Further, the PCR primer can consist of a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID No. 2, and the sequence of the reverse primer is shown in SEQ ID No. 3.

[0012] The present application also provides a kit for identifying or assisting in identifying the growth rate of pigs, which contains the above-mentioned PCR primer.

[0013] The present application provides a pig breeding method, which can include breeding pigs using pigs with a homozygous genotype of RPS27L as parents, and the pigs with a homozygous genotype of RPS27L are pigs with a homozygous genotype of the InDel molecular marker of the nucleotide sequence shown in SEQ ID No. 1.

[0014] Further, in the method, the purpose of the pig breeding can include breeding pigs with high growth rate.

[0015] The pig can be specifically the offspring of a cross between a Large White and a Minzhu, such as an F2 generation.

[0016] Further, the present application also provides the above-mentioned PCR primer and / or the above-mentioned kit for identifying or assisting in identifying the growth rate of pigs and / or for pig breeding.

[0017] The above-mentioned growth rate of pigs can be manifested as the body weight at 240 days of age.

[0018] The above-mentioned InDel molecular marker is also within the protection scope of the present application.

[0019] In order to improve the breeding efficiency, the present application uses modern molecular biology and bioinformatics technology to deeply study the pig genetic information, and obtains a molecular marker related to the growth rate of pigs, which is a 13-bp insertion / deletion polymorphism site existing at the sequence Chr1:108,880,114-108,880,126bp of the pig genome Sscrofa11.1 version. The pigs with genotype of deletion type at the site have higher growth rate than the pigs with genotype of insertion, and the growth traits of pigs can be judged and improved by identifying different genotypes of the molecular marker, so as to shorten the breeding cycle, accelerate the improvement process, and improve the economic benefit and social value of pig breeding.

[0020] The beneficial effects of the present application include: (1) the present application finds that the insertion / deletion site in the promoter region of the pig RPS27L gene can be used as a genetic selection marker for pig molecular breeding, which is beneficial to screen pigs with excellent growth traits and accelerate the speed of breeding superior varieties; (2) the present application uses the primer P1 designed according to the whole genome of the reference pig as the primer, uses pig genomic DNA as the template, and through sequence amplification, electrophoresis identification and Sanger sequencing, the genotype of the insertion / deletion polymorphism site (Chr1:108,880,114-108,880,126bp) in the promoter region of the pig RPS27L gene in the pig population is detected, identified, and determined efficiently, accurately and at low cost. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The InDel allele frequency of the insertion / deletion site (InDel) in the promoter region of the pig RPS27L gene in different pig breeds in Europe and Asia in the present application.

[0022] Figure 2 The sequencing map of the upstream and downstream PCR amplification products of the insertion / deletion site (InDel) in the promoter region of the RPS27L gene of Tongcheng pigs and Landrace pigs; wherein the part marked by the black square box represents a 13-bp insertion sequence.

[0023] Figure 3 The genotype of the insertion / deletion site (InDel) in the promoter region of the RPS27L gene and the correlation analysis of the body weight at 240 days in the F2 generation population of 557 hybrid pigs of Large White pigs and Min pigs. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in combination with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0025] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0026] Example 1: Development of an InDel molecular marker related to pig growth rate

[0027] The present application provides a molecular marker related to pig growth rate and applications thereof.

[0028] The molecular marker is a 13-bp insertion / deletion polymorphism site at the sequence of Chr1: 108,880,114-108,880,126 bp in the Sscrofa11.1 version of pig genome.

[0029] The insertion / deletion sequence at Chr1: 108,880,114-108,880,126 bp is SEQ ID NO. 1, which is located in the promoter region of the RPS27L gene.

[0030] According to a preferred embodiment of the present application, the molecular marker is characterized in that the qq, i.e. deletion / deletion genotype of the 13-bp insertion / deletion polymorphism site can be used as a DNA molecular marker for improving pig growth rate.

[0031] The verification method for the application of the molecular marker related to pig growth rate comprises the following steps:

[0032] The method uses a primer pair designed according to the whole genome of a reference pig as primers, and uses pig genomic DNA as a template to detect the genotype of the insertion / deletion polymorphism site in the promoter region of the pig RPS27L gene in a pig population through sequence amplification, electrophoresis identification, and Sanger sequencing, wherein the sequence amplification primer pair is P1.

[0033] The primer pair P1 comprises the following upstream primer and downstream primer:

[0034] The upstream primer P1-F is 5'-AGATTTGACAGAGCTGGTTGGT-3' (SEQ ID NO. 2);

[0035] The downstream primer P1-R is 5'-CCCATTGTGGCTCAGTGATTA-3' (SEQ ID NO. 3).

[0036] The primer pair P1 is used for priming, the pig genomic DNA is used as a template, sequence amplification, electrophoresis identification, Sanger sequencing are carried out, and the genotype of the insertion / deletion polymorphism site (Chr1:108,880,114-108,880,126 bp) in the promoter region of the pig RPS27L gene in a pig population is detected in a high-efficiency, accurate and low-cost manner.

[0037] The genotype and gene frequency of the insertion / deletion polymorphism site in the promoter of the pig RPS27L gene are analyzed, and the gene frequency of the insertion / deletion polymorphism site in the slow growth fat type and the fast growth lean type pig population is analyzed, and the results show that the 13-bp insertion / deletion polymorphism site detected in the application can be used as a molecular marker of pig growth rate, which is beneficial to accelerate the breeding speed of good breed pigs.

[0038] In Example 2, the association analysis of the InDel molecular marker related to the growth rate of pigs and the phenotype of the natural population is carried out by using the PCR method to detect the insertion / deletion polymorphism generated by the variation of the promoter region (Chr1:108,880,114-108,880,126 bp) of the pig RPS27L gene in the pig population, and the genotype distribution of the insertion / deletion polymorphism in different pig breeds is analyzed to verify whether the insertion / deletion polymorphism can be used as a molecular marker for assisted selection in molecular breeding.

[0039] 2.1, Experimental reagents and reagents

[0040] Taq DNA polymerase (purchased from Takara Company); protease K (purchased from Huawme Biological Engineering Company); Marker I (purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.); agarose (purchased from Aikewei Biological); TAE buffer (purchased from Beijing Ruibo Xingke Biological Technology Co., Ltd.); animal genomic DNA extraction kit (purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.).

[0041] The primer pair P1 is:

[0042] The upstream primer P1-F is 5'-AGATTTGACAGAGCTGGTTGGT-3' (SEQ ID NO. 2);

[0043] The downstream primer P1-R is 5'-CCCATTGTGGCTCAGTGATTA-3' (SEQ ID NO. 3);

[0044] The PCR amplification is carried out on the target fragment in the promoter region of the pig RPS27L gene.

[0045] Collection of pig ear samples and collection of breed information:

[0046] A total of 903 animal samples were used in the experiment. Individual ear tissue samples were collected, preserved in 70% ethanol, brought back to the laboratory in ice boxes, and then frozen at -80°C. At the same time, the breed information of the pigs was recorded.

[0047] Extraction and isolation of genomic DNA from tissue samples:

[0048] Ear tissue DNA was extracted according to the instructions of the Tiangen Tissue Genomic DNA Extraction Kit.

[0049] 2.2 Screening of InDel sites

[0050] We selected lean-type European pig breeds such as Berkshire, European wild boar, Yucatan, Duroc, Large White, Landrace, and Pietrain, as well as fatty-type Asian pig breeds such as Debao, Meishan, Huai, Baoshan, Tibetan, Minbei Spotted Pig, Wuzhishan, Huanjiang Xiang, Tunchang, Erhualian, Tongcheng, Diannan Small-eared Pig, Luchuan, Wannan, Bama, Guizhou, Gaoligong Mountain, Jinhua, Large White, Dongshan, Guanzhuang Spotted Pig, Guangdong Small-eared Pig, Lantang, Laiwu, and Mingguang Small-eared Pig, and integrated our own sequencing data. The obtained whole-genome resequencing data and the whole-genome resequencing data published in public databases totaled 903 individuals. The sample number for each breed is shown in Table 1. The above resequencing data were compared with the pig reference genome (Sscrofa11.1) to extract InDel site information. A 13bp insertion / deletion (InDel) site was found at Chr1:108,880,114-108,880,126bp. This InDel is located in the promoter region of the RPS27L gene.

[0051] 2.3 Allele frequency calculation:

[0052] P Q =(2N) QQ +N Qq ) / 2N

[0053] In the formula, P Q N represents the frequency of allele Q. QQ N represents the number of individuals in the population with the QQ genotype. Qq This represents the number of individuals with the Qq genotype in the population, and N represents the total number of individuals.

[0054] Q represents the insertion allele (13-bp insertion), and the PCR product is shown in SEQ ID No. 4; q represents the deletion allele (13-bp deletion), and the PCR product is shown in SEQ ID No. 5.

[0055] The allele frequencies are shown in Table 1:

[0056] Table 1. Polymorphism parameters of the InDel site in the promoter region of the porcine RPS27L gene

[0057]

[0058]

[0059] As shown in Table 1, the insertional allele Q is mainly distributed in Asian fat-type pigs, while the deletional allele q is mainly distributed in European lean-type pigs.

[0060] 2.4 Verification of InDel sequence information

[0061] 2.4.1 PCR amplification:

[0062] Fatty Tongcheng pigs and lean Landrace pigs were selected as representatives, and InDel and its flanking sequences were amplified using PCR technology.

[0063] The PCR reaction system consisted of 10 μL of 2×Taq PCR SuperMix (containing Taq DNA polymerase, dNTPs, and optimized reaction buffer at a concentration of 2×); 1 μL of the upstream primer P1-F from Example 1; 1 μL of the downstream primer P1-R from Example 1 (with upstream and downstream primer concentrations of 10 pmol / μL); 1 μL of genomic DNA (concentration of 30 ng / μL porcine genomic DNA); and 7 μL of deionized water, for a total PCR amplification volume of 20 μL.

[0064] PCR reaction procedure:

[0065] 1) Pre-denaturate at 95.0℃ for 5 minutes, then proceed to step 2);

[0066] 2) Denaturation at 95.0℃ for 30 seconds,

[0067] 3) Refold at 60.0℃ for 30 seconds.

[0068] 4) Extend at 72.0℃ for 30 seconds, then proceed to step 2), for a total of 34 cycles;

[0069] 5) Extend at 72.0℃ for 10 minutes

[0070] 2.4.2 Agarose gel electrophoresis detection

[0071] The steps are as follows:

[0072] 1) Prepare a 2.0% agarose gel, stain with nucleic acid dye, spot 4.5 μL of the sample, and electrophoresis at 120V for 40-50 min after spotting;

[0073] 2) Once DNA fragments of different molecular weights are clearly separated, image them using the BIO-RAD Gel Doc 2000 gel imaging system;

[0074] 3) Preliminary analysis of InDel polymorphism based on agarose gel electrophoresis results.

[0075] 2.4.3 Sanger sequencing

[0076] Sanger sequencing was performed using PCR amplification primer pair P1 as sequencing primers. The sequence information obtained is as follows: Figure 2 As shown, the sequencing genotype of Tongcheng pig is Q, and the sequencing genotype of Landrace pig is q, which is consistent with the results of whole-genome resequencing.

[0077] Example 3: Correlation Analysis between InDel Molecular Markers and Growth Rate

[0078] The association analysis of genotype and growth rate (body weight at 240 days of age) at the InDel site of the RPS27L gene promoter region in a F2 population of 557 pigs obtained by crossing Large White (European) pigs as the sire and Min pigs (Asian) pigs as the dam is shown in Table 2. Figure 3 .

[0079] Q represents the insertion allele (13-bp insertion), and the PCR product is shown in SEQ ID No. 4; q represents the deletion allele (13-bp deletion), and the PCR product is shown in SEQ ID No. 5.

[0080] Table 2: Association analysis between genotype of the InDel site in the promoter region of the RPS27L gene and body weight at 240 days of age in a F2 generation population of 557 crossbred Large White pigs and Min pigs.

[0081] Genotype Number of samples Weight at 240 days of age (kg) qq 411 109.63±15.78 Qq 110 108.48±16.11 QQ 36 103.69±16.15

[0082] Table 2 and Figure 3 The results showed that the growth rate of the pigs to be identified with the qq homozygous genotype was significantly higher than that of the pigs to be identified with the QQ homozygous genotype.

[0083] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences <120> InDel Marker of Porcine Growth Rate-Related Gene RPS27L and Its Application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 13 <212> DNA <213> Pig (Sus scrofa) <400> 1 gccaccggcc tac 13 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 agatttgaca gagctggttg gt 22 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 cccattgtgg ctcagtgatt a 21 <210> 4 <211> 640 <212> DNA <213> Pig (Sus scrofa) <400> 4 cccaaagggg ggtactttca ttattgtatt cgttttctat tttcactata acaaattgcc 60 ataaagttag tggctttttt attttattgt ctttttgcca tttcttgggc cgctcccacg 120 gcacatggag gttcccaggt taggggtcta atcggagctt tagccaccgg cctacgccac 180 agcaaggagg gatcagagcc gcatctgcaa cctacactgc agctcaccac aacgtcagat 240 ccttaaccca ctgagcaagg cgagggatca aacctgcaac ctcatggttc ctagtcagat 300 tcgttaatca ctgagccaca atgggctcga gtctagagtc gacctgcagg catgcaagct 360 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 420 taactagcat aaccccttgg ggcggccgct tcgagcagac atgataagat acattgatga 480 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 540 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 600 cattcatttt atgtttcagg ttcagggggg agatgtggga 640 <210> 5 <211> 627 <212> DNA <213> Sus scrofa <400> 5 ggaaggggg atctttcatt attgtattcg tttctattt tcactataac aaattgccat 60 aaagttagtg gcttttttat tttattgtct ttttgccatt tcttgggccg ctcccacggc 120 acatggaggt tcccaggtta ggagtctaat cggagcttta gccacagcaa ggagggatca 180 gagccgcatc tgcaacctac actgcagctc accacaacgc cagatcctta acccactgag 240 caaggccagg gatcaaacct gcaacctcat ggttcctagt cagattcgtt aatcactgag 300 ccacaatggg ctcgagtcta gagtcgacct gcaggcatgc aagctgatcc ggctgctaac 360 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 420 cttggggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 480 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 540 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 600 tcaggttcag ggggagatgt gggaggt 627

Claims

1. The application of substances that detect InDel molecular markers in the identification or auxiliary identification of pig growth rate, characterized in that, The InDel molecular marker is a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1; the InDel molecular marker is a 13bp insertion and / or deletion polymorphism site located at the sequence Chr1:108,880,114-108,880,126bp in the porcine reference genome Sscrofa11.1 version.

2. The application as described in claim 1, characterized in that, The substance contains PCR primers for amplifying porcine genomic DNA fragments including the InDel molecular marker.

3. The application as described in claim 2, characterized in that, The PCR primers consist of a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID No. 2, and the sequence of the reverse primer is shown in SEQ ID No.

3.

4. A pig breeding method, characterized in that, The method includes breeding pigs with the RPS27L homozygous genotype as parents, wherein the RPS27L homozygous pigs are homozygous pigs lacking the InDel molecular marker described in claim 1; the purpose of the pig breeding is to cultivate pigs with high growth rates.

5. Application of PCR primers or kits containing said PCR primers in pig breeding; The PCR primers are used to amplify porcine genomic DNA fragments containing the InDel molecular marker as described in claim 1. The purpose of the pig breeding program is to develop pigs with high growth rates.

6. The application according to claim 5, characterized in that, The PCR primers consist of a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID No. 2, and the sequence of the reverse primer is shown in SEQ ID No. 3.

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