CN110317880B - Molecular marker related to pig feed conversion rate, identification and application thereof - Google Patents
Molecular marker related to pig feed conversion rate, identification and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP marker related to the pig feed conversion rate, wherein the SNP marker is positioned in an intron region of a DHRS4 gene, in particular to a 7524571 base of a No.7 chromosome of a pig genome version Ensembl Scrofa11.1, and the SNP marker has A/T polymorphism; the three genotypes corresponding to the SNP marker are TT, TA and AA respectively, and compared with the pigs with the genotypes of TA and TT, the pigs with the genotypes of AA have lower feed conversion rate. The determination of the DHRS4 gene and the SNP marker related to the pig feed conversion rate can be used for identifying or assisting in identifying pig varieties with low feed conversion rate, shortening the breeding period and improving the breeding efficiency.
Description
Technical Field
The invention relates to the technical field of pig breeding, in particular to molecular markers related to pig feed conversion rate, identification and application thereof.
Background
The animal husbandry plays a very important role in the development of modern agriculture, and the ratio of the animal husbandry in agricultural production is an important index for measuring the development degree of national and regional laws. Pork is the most important meat source in China, so that the live pig breeding industry occupies a significant position in the livestock industry in China. In the production of the pig industry, the economic traits of the pigs refer to traits influencing the productivity of the pigs, the economic production total value of the pig industry is mainly influenced, and the feed cost accounts for 65-75% of the total cost of the pig industry, so that the reduction of the feed consumption and the feed cost in the live pig breeding is of great importance. The feed conversion rate as one of the important indexes of the economic traits of the pork pigs becomes an important direction of research at present.
The feed conversion rate refers to the amount of feed consumed per 1KG of weight of product added during the performance measurement period, and the lower the feed conversion rate, which means that the less the amount of feed consumed per unit weight of product produced, the production cost can be remarkably reduced, and the production efficiency of the farm can be increased. The Feed Conversion Rate (FCR) is influenced by different factors such as the ingestion process and activity of the pork pigs, the digestion and absorption capacity, the environmental temperature regulation, the animal metabolism, the body composition and the like, is related to the growth performance and meat quality characteristics such as Average Daily Gain (ADG), backfat thickness (BF), residual Feed Intake (RFI), system water power, carcass lean meat percentage and the like, and greatly influences the production efficiency of the pork pigs. The gene related to the feed conversion rate participates in a plurality of metabolic activities of an animal body, such as synthesis of triglyceride, potassium ion metabolism, adjustment of distribution conditions of fat cells, metabolic activity of the liver, development of animal digestive tracts and the like, so that the identification of the gene related to the feed conversion rate of the pork pigs can provide an important theoretical basis for genetic improvement of growth traits of the pork pigs.
The gene marker assisted selective breeding is a modern breeding method for carrying out genotype selection on target traits by using DNA molecular markers closely linked with the genes of the target traits, has the advantages of strong operation stability, difficult interference by environmental factors and the like, and can greatly improve the breeding efficiency. The method is a key link for establishing an auxiliary selective breeding technology by screening molecular marker loci obviously associated with target traits. Among the common molecular marker technologies, single Nucleotide Polymorphism (SNP) is most widely used. SNP is a third-generation genetic marker, mainly refers to DNA sequence polymorphism caused by variation of single nucleotide on the genome level, has the advantages of large quantity, wide distribution, high frequency, low mutation rate and the like, is suitable for rapid and large-scale screening, and is applied to aspects such as genome analysis, biological information automatic detection, genetic research of simple and complex diseases, livestock breeding genetic marker research and the like.
Therefore, the discovery of a gene related to the pig feed conversion rate, the screening of a molecular marker related to the pig feed conversion rate, the identification of the molecular marker and the application thereof are problems to be solved at present.
Disclosure of Invention
In order to overcome the problems, the inventor of the present invention has conducted intensive research, and first detected the expression of the DHRS4 gene in skeletal muscle of pigs at different growth stages, then detected the polymorphism of the DHRS4 gene and genotypic test on the polymorphic site, and then performed association analysis on the polymorphic site and important growth traits of pigs, and first obtained the SNP molecular marker related to feed conversion rate in the pig DHRS4 gene for identifying or assisting in identifying pig breeds with low feed conversion rate, and improving breeding efficiency, thereby completing the present invention.
Specifically, the present invention aims to provide the following:
in a first aspect, a SNP marker associated with pig feed conversion ratio is provided, wherein the SNP marker is located in an intron region of the DHRS4 gene, in particular at base 7524571 of chromosome 7 of the pig genomic version Ensembl Sscrofa11.1, and the SNP marker has an a/T polymorphism.
In a second aspect, a method for obtaining the SNP marker related to pig feed conversion ratio in the first aspect is provided, wherein the method includes the following steps:
step 1, obtaining pig genome DNA;
step 2, detecting to obtain SNP loci;
step 3, carrying out genotyping on the SNP locus;
and 4, carrying out association analysis on the SNP locus and the pig growth traits.
In a third aspect, there is provided a method of identifying or aiding in the identification of feed conversion ratio in pigs, wherein the method comprises the step of detecting the base type at position 7524571 of chromosome 7 of the porcine genomic version Ensembl Scrofa11.1 to determine whether the genotype at that locus is TT, TA or AA.
In a fourth aspect, there is provided a use of the SNP marker of the first aspect or the SNP marker obtained by the method of the second aspect in identification or in assistance in identification of feed conversion rate of pigs.
In a fifth aspect, there is provided a use of the SNP marker of the first aspect or the SNP marker obtained by the method of the second aspect in molecular marker assisted breeding of swine.
The invention has the advantages that:
(1) The SNP marker related to the pig feed conversion rate can be used for early breeding of pigs, reduces the breeding cost, shortens the middle breeding period and can remarkably promote the breeding process of the pigs;
(2) The SNP marker related to the pig feed conversion rate provided by the invention has high identification accuracy, can realize automatic detection and improve economic benefits;
(3) The SNP marker related to the pig feed conversion rate provides scientific basis for the molecular marker-assisted selection of pig growth traits.
Drawings
FIG. 1 shows the fluorescence quantitative primer-specific assay results obtained in a preferred embodiment of the present invention, wherein A is the dissolution curve of the GAPDH reference gene and B is the dissolution curve of the DHRS4 gene;
FIG. 2 shows the expression of DHRS4 gene in skeletal muscle of a growing white pig at different growth stages according to a preferred embodiment of the present invention, wherein A shows the expression of DHRS4 gene in breast muscle of a growing white pig, B shows the expression of DHRS4 gene in leg muscle of a growing white pig, and C shows the expression of DHRS4 gene in back muscle of a growing white pig;
FIG. 3 shows a positional diagram of SNP sites obtained by screening according to a preferred embodiment of the present invention, wherein A shows the Chr7: 7524571 SNP site (NCBI reference number rs 334250151) of the porcine genome version Ensembl Sdecrofa 11.1, B shows the Chr7: 7524589 SNP site (NCBI reference number rs 342446613) of the porcine genome version Ensembl Sdecrofa 11.1, and C shows the Chr7:75253401SNP site (NCBI reference number rs 326982309) of the porcine genome version Ensembl Sdecrofa 11.1.
Detailed Description
The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.
The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
The inventor researches and discovers that DHRS4 (dehydrogenases/reductases 4, dehydrogenase/Reductase 4) is a dehydroreductase widely existing in various mammals and catalyzing interconversion between retinol and retinal, and also has various splicing subtypes, so that different functional localization and expression are realized. DHRS4 plays an important role in retinoic acid synthesis, steroid metabolism and benzyl metabolism, and can regulate and control proliferation and differentiation, cell signal conduction and tumorigenesis of cells by influencing retinoic acid synthesis. In addition, the enzyme coded by the DHRS4 gene and the derivative thereof are closely related to the reproductive performance of animals, and the all-trans retinoic acid is closely related to the proliferation and differentiation of germ cells in testis and ovary of animals.
In terms of growth performance, retinoic acid is involved in Myogenic differentiation of skeletal muscle cells, induces further differentiation of embryoid bodies into skeletal muscle cells, and increases expression activities of MyoG (Myogenin), myf5 (Myogenic factor5, myogenic determinant), myHC (Myogenin heavy chain), and other myogenesis-associated transcription factors. From the above, the DHRS4 gene is closely related to the growth and development of muscle, but its molecular signaling pathway and mechanism in muscle development still need to be further studied. Moreover, DHRS4 gene studies have focused mainly on mouse and human tumor cells, with less research in porcine muscle development.
Therefore, in the present invention, it is preferable to select the DHRS4 gene as a target gene and further screen the SNP site related to the feed conversion rate on the gene.
In a first aspect of the invention, a SNP marker related to the pig feed conversion rate is provided, and the SNP marker is located in an intron region of a DHRS4 gene, in particular the Chr7: 7524571 (the base of chromosome 7524571 of 7 number chromosome 7524571) of a pig genome version Ensembl Scrofa 11.1.
The NCBI of the SNP marker is in a reference number of rs334250151, has A/T polymorphism, and has three genotypes which correspond to the SNP site, namely TT, TA and AA, wherein the TT genotype is a homozygote of T basic groups of the SNP site of the pig, the TA genotype is a heterozygote of T basic groups and A basic groups of the SNP site of the pig, and the AA genotype is a homozygote of A basic groups of the SNP site of the pig.
In the present invention, the swine with the SNP marker genotype AA have a lower feed conversion rate than the swine with the genotypes TA and TT.
According to a preferred embodiment of the present invention, the SNP marker sites are genotyped using the Sequenom MassARRAY technique.
Wherein the Sequenom MassARRAY technology is a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technology, and comprises the following steps: firstly, PCR amplifies the target sequence, then adds SNP sequence specific extension primer, and extends 1 base on the SNP site. The prepared sample analytes were co-crystallized with the chip matrix and then passed through the vacuum tube of the mass spectrometer for transient nanoseconds (10) -9 s) strong laser excitation, nucleic acid molecule desorption and conversion into metastable state ion, ion flight time in electric field is inversely proportional to ion mass, and the flight time of nucleic acid molecule in vacuum tube is detected by flight time detector to obtain accurate molecular weight of sample analyte, thereby detecting SNP site information.
As can be seen from the above, the process of genotyping the SNP site includes the steps of extraction of porcine genomic DNA, PCR amplification reaction, and single base extension reaction.
In a further preferred embodiment, the nucleotide sequences of the amplification primers P1 and P2 in the PCR amplification reaction are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively;
the nucleotide sequence of the extension primer P3 in the single base extension reaction is shown as SEQ ID NO. 3.
The invention also provides a method for genotyping the SNP locus related to the pig feed conversion rate, which comprises the following steps:
(1) Extracting the genomic DNA of the pig;
(2) Sequenom MassARRAY detection is carried out by taking the genome DNA of the pig extracted in the step (1) as a template and utilizing PCR amplification primers P1 and P2 and an extension primer P3 of single-base extension reaction, and the genotype of the genome version Ensembl Sscrifa 11.1 of the pig at 7524571 bases of a chromosome 7 is determined.
Wherein, the genome DNA of the pig is extracted by a method commonly used in the prior art, and the nucleotide sequences of the primers P1, P2 and P3 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
According to another preferred embodiment of the present invention, the genotyping of the SNP sites associated with pig feed conversion ratio is determined using direct sequencing or by means of a genotyping kit.
In a further preferred embodiment, the genotyping kit comprises PCR amplification primers P1, P2 and extension primer P3 for a single base extension reaction.
In a still further preferred embodiment, the genotyping kit further comprises PCR amplification buffer, dNTP, pfuDNA polymerase, SNaPshot polymerization solution, and PCR product purification reagents,
wherein the PCR product purification reagent comprises exonuclease, shrimp-alkaline phosphatase and a buffer solution for purification.
According to a preferred embodiment of the present invention, the genotyping kit is used in a method comprising: extracting a genomic DNA sample from pig ears or other tissues; performing PCR amplification and purifying PCR products; carrying out single base extension reaction; performing capillary electrophoresis analysis on the extension product; and analyzing SNP loci to obtain genotyping information.
The SNP locus analysis can adopt, but is not limited to, an ABI3730XL automatic sequencer.
In a second aspect of the present invention, there is provided a method for obtaining the SNP marker related to pig feed conversion rate, the method comprising the following steps:
step 1, obtaining pig genome DNA;
step 2, detecting to obtain SNP loci;
step 3, carrying out genotyping on the SNP locus;
and 4, carrying out association analysis on the SNP locus and the growth traits of the pig.
Optionally, prior to step 1, the expression of the DHRS4 gene in skeletal muscle of pigs at different growth stages is examined.
The method of obtaining the SNP sites is further described below:
step 1, obtaining pig genome DNA.
The research of the inventor finds that the research of the DHRS4 gene is mainly focused on mouse and human tumor cells, and the function of the DHRS4 gene in the growth and development of pig skeletal muscle is rarely researched. Thus, optionally, prior to step 1, DHRS4 gene expression in skeletal muscle of pigs at different growth stages is tested.
According to a preferred embodiment of the present invention, the expression of DHRS4 gene in skeletal muscle of pig at different growth stages is detected by fluorescent quantitative PCR, which is performed according to the following steps:
(1) Extracting RNA of different skeletal muscle (leg muscle, dorsal muscle and pectoralis muscle) tissues of the pig, and performing reverse transcription to obtain cDNA;
(2) Designing a primer for RT-qPCR, selecting a proper reference gene, and carrying out RT-qPCR reaction;
(3) And (4) counting the amplification results, and analyzing the expression condition of the DHRS4 gene in skeletal muscles of pigs at different growth stages.
In the invention, detection and analysis show that the DHRS4 gene is expressed in different development stages of the breast muscle, the leg muscle and the dorsal muscle of the pig, which indicates that the DHRS4 gene participates in the growth and development of the postnatal skeletal muscle of the pig, lays a foundation for the subsequent detection of SNP sites related to the growth traits of the pig in the gene, and provides data support.
Wherein, the pig genome DNA is extracted by a method or a kit commonly used in the prior art, and preferably, the pig genome DNA of a certain population is extracted.
In the present invention, it is preferable to perform quality inspection on the extracted porcine genomic DNA.
And 2, detecting to obtain the SNP locus.
Wherein, step 2 comprises the following substeps:
and 2-1, designing a primer, and amplifying by taking the obtained pig genome DNA as a template to obtain a pig DHRS4 gene sequence.
Among them, primers were designed using Primer permier 5.0 software in consideration of the principles of Primer design by referring to the pig DHRS4 genome sequence (NM-214019.2) published in GeneBank and based on the reported SNP site information.
In the present invention, it is preferable to design 6 pairs of primers, which are denoted as P A ～P F Wherein P is A The forward and reverse primer sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, P B The forward and reverse primer sequences are respectively shown as SEQ ID NO.6 and SEQ ID NO.7, P C The forward and reverse primer sequences are respectively shown as SEQ ID NO.8 and SEQ ID NO.9, P D The forward and reverse primer sequences are respectively shown as SEQ ID NO.10 and SEQ ID NO.11, P E The forward and reverse primer sequences are respectively shown as SEQ ID NO.12 and SEQ ID NO.13, P F The forward and reverse primer sequences are respectively shown in SEQ ID NO.14 and SEQ ID NO. 15.
The method comprises the steps of taking pig DNA as a template, carrying out PCR amplification by adopting the primers, wherein 100 equal sample DNAs are randomly selected and mixed together to construct a mixed pool, carrying out segmented cloning on DHRS4 gene by using 6 segments of primers in the mixed pool, preparing 1% agarose gel electrophoresis to detect the fragment size of a PCR product and the specificity of the primers, collecting the PCR product (5 mu L) and the primers (1 mu L) of a target band for bidirectional sequencing, directly sequencing a peak diagram, wherein the background of the peak diagram is clean and easy to read, the peak diagram contains a nested peak, and the SNP with the heterozygosity of more than or equal to 0.10 is obtained.
In the present invention, sanger sequencing analysis is preferably performed on the PCR amplification products.
And 2-2, carrying out sequence comparison to obtain SNP loci.
In the present invention, it is preferable to obtain SNP sites by comparing the sequencing results with the porcine DHRS4 gene sequences published in GeneBank using the Chromas Pro software.
And 3, carrying out genotyping on the SNP locus.
Among them, it is preferable to use the Sequenom MassARRAY technique to genotype the SNP site.
And 4, carrying out association analysis on the genotyping data of the SNP locus and the growth traits of the pig.
Wherein, step 4 comprises the following substeps:
and 4-1, acquiring the phenotype data of the growth traits of the pigs.
In the present invention, the growth traits of the swine include average daily gain, feed conversion ratio and back fat thickness, wherein,
calculating the average daily gain and the feed conversion rate by measuring the initial age of the day, the final age of the day, the initial weight (g), the final weight (g) and the final consumption (g), wherein the formula is as follows:
average daily gain (g) = [ knot weight (g) -initial weight (g) ]/[ knot age-initial age ];
feed conversion (%) = knot loss (g)/[ knot body weight (g) -initial body weight (g) ];
measurement of backfat thickness: and (3) measuring the backfat thickness of the individual pigs in the test swinery by adopting a high ultrasonic backfat determinator. When the measurement is started, the pig is naturally stood still and kept still, hair is sheared at a position 5cm above the back center line at the last rib position of the pig, a couplant is coated, a backfat instrument probe is placed at the position, and the measurement is recorded after the reading of the instrument is stable; the measurements were taken 3 times in succession, averaged, and the data recorded.
And 4-2, carrying out correlation analysis on the genotyping data of the SNP locus and the growth traits of the pig to obtain the SNP marker related to the feed conversion rate of the pig.
In the invention, popGene3.2 is preferably adopted to calculate the genotype frequency and the allele frequency of the SNP locus, and then GLM in SAS9.2 software is adopted to analyze the relevance of the SNP locus and the pig growth traits so as to obtain the SNP marker which is obviously related to the pig feed conversion rate. Meanwhile, the genotype of the pig feed with low conversion rate can be obtained.
In a third aspect of the invention, there is provided a method of identifying or aiding in the identification of feed conversion efficiency in pigs, the method comprising the step of detecting the base type at Chr7: 7524571 of the porcine genomic version Ensembl sscrofa11.1 to determine whether the genotype at that locus is TT, TA or AA.
Wherein, the feed conversion rate of the pigs is determined according to the genotype detected as the gene locus, and the pigs with the genotype of AA have lower feed conversion rate compared with the pigs with the genotypes of TA and TT.
In a fourth aspect of the invention, the invention provides a use of the SNP marker of the first aspect or the SNP marker obtained by the method of the second aspect in identification or auxiliary identification of the feed conversion rate of pigs.
In a fifth aspect of the present invention, there is provided an application of the SNP sites of the first aspect in pig molecular marker-assisted breeding, the application including the following steps:
step I, extracting the genome DNA of a pig to be detected;
step II, detecting the genotype of the pig to be detected by a Sequenom MassARRAY technology;
and III, breeding the dominant variety of the pig with low feed conversion rate according to the genotype.
In the step II, amplification primer pairs adopted in the detection process of the Sequenom MassARRAY technology are P1 and P2, an extension primer adopted is P3, and the primer sequences are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
Examples
The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.
Test animals used in the examples:
in the experiment, tissue samples of dorsal muscles, pectorals and leg muscles of long white pigs of 30 days old (30 d), 180 days old (180 d) and 300 days old (300 d) are collected, 3 samples are collected in each period as biological repetition, the samples are placed into a freezing storage tube, liquid nitrogen is rapidly added, the samples are transported to a laboratory and placed into a refrigerator at the temperature of-80 ℃ for storage and standby application.
The pig ear samples of 235 healthy adult blue Si white pigs were collected and placed in 75% alcohol and stored at-20 ℃. Meanwhile, production files are arranged, and the production files comprise performance indexes such as initial measurement of day age (day), final measurement of day age (day), initial measurement of body weight (g), final measurement of material consumption (g), backfat thickness (mm) and the like.
The test samples are all collected from Shandong blue Si species industry Co., ltd, and the pig raising process and the experiment operation method in the research all follow the raising and operation requirements of the experimental animals. The Lansi white pig is a special new population which is directionally bred and has the advantages of high growth speed, strong adaptability, stable reproductive performance and meat quality through 6 centuries and 17 years of subculture breeding, and contains 12.5% of kindred black pigs, wherein Yimeng black pigs are parents of Yimeng white pigs and Yimeng black pigs of domestic excellent local variety, which are researched by Beijing animal husbandry veterinary science of Shandong Lansi province GmbH and Chinese agricultural science school.
The reagent sources and instrumentation used in the examples:
reagent: (1) DNA extraction kit (animal tissue DNA extraction): TIANGEN, tiangen Biochemical technology (Beijing) Ltd; (2) Chloroform (Beijing Chemicals), absolute ethanol (Beijing Chemicals), isopropanol (Beijing Chemicals); (3) RNAioso Plus: taKaRa, japan; (4) SYBR Primer script RT-PCR Kit: taKaRa, japan; (5) SYBR Premix EX Taq: tli RNaseH Plus, taKaRa, japan.
An instrument device: (1) a full-automatic gel imaging system: bio-rad; (2) electrophoresis tank: bio-rad; (3) nucleic acid protein concentration determinator (Nano-100): hangzhou Osheng company; (4) PCR apparatus (C1000 Touch TM): bio-Rad company; (5) Fluorescent quantitative PCR instruments (Applied Biosystems 7500, ABI 7500); (6) Centrifuge (Centrifuge 5810R) Eppendorf Co.
Example 1 acquisition of SNP marker
1. The expression of the DHRS4 gene in skeletal muscle of pigs at different growth stages is detected.
1.1 extraction and reverse transcription of RNA
Collecting leg muscle, dorsal muscle and pectoral muscle tissues (3 biological replicates in each group, total 27 samples) of 30d, 180d and 300d of long white pig, extracting total RNA according to the instruction of animal tissue total RNA extraction Kit, measuring RNA concentration and OD (optical Density) value with nucleic acid protein concentration meter, and making qualified RNA sample refer to RevertAid First Strand cDNA Synthesis KitRecording kit instruction to synthesize first strand cDNA, taking 2000ng RNA solution, adding random primer Oligo (dT) 1 μ L according to determined total RNA concentration, supplementing volume to 12 μ L with double distilled water, shaking, mixing, heating at 70 deg.C for 5min, taking out ice, standing for 3-5 min, centrifuging briefly, adding 5 × RT Buffer 4 μ L, dNTP 2 μ L, riboLock TM 1μL、RevertAid TM M-MLV 1 muL, 20 muL of the total system, mixing uniformly and putting into a PCR instrument, setting the program to heat at 42 ℃ for 1 hour, 70 ℃ and 10min; at 4 ℃ and an infinite value. After the reaction is finished, the mixture is put into a refrigerator at the temperature of 20 ℃ below zero for storage and standby.
1.2 real-time fluorescent quantitative PCR (RT-qPCR)
Using GAPDH as an internal reference gene, RT-qPCR primers P4 and P5 with nucleotide sequences shown in SEQ ID No.16 and SEQ ID No.17, respectively, and internal reference primers P6 and P7 with nucleotide sequences shown in SEQ ID No.18 and SEQ ID No.19, respectively, were designed using Permier primer5.0 software, and the RT-qPCR reaction was performed according to the instructions of the SYBR Premix Ex TaqTM kit (TaKaRa ra) in ABI prism 7500PCR system with an overall reaction of 15 μ L:2 × SYBR Premix EX Taq 7.2 μ L, ROXReference Dye II 0.3 μ L, primer sense 0.3 μ L, cDNA template 0.8 μ L, ddH2O 6.1 μ L, reaction conditions of 95 deg.C 5min,95 deg.C 5s,60 deg.C 1min, cycle 40 times and then dissolution curve analysis, DHRS4 gene and GAPDH reference gene in the same reaction conditions, each sample using 3 technical repeat, finally taking the average value.
The primer specificity detection result of the fluorescence quantification is shown in figure 1, and the expression condition of the DHRS4 gene in skeletal muscle of the growing white pig at different growth stages is shown in figure 2.
A in FIG. 1 shows the lysis curve of the GAPDH reference gene, and B in FIG. 1 shows the lysis curve of the DHRS4 gene, wherein 27 muscle tissue samples of a long white pig were specifically amplified using the primers, resulting in 27 corresponding amplification curves.
As can be seen from fig. 1, the dissolution curves of GAPDH reference gene (27 curves of 27 samples) are all single peak type, and no other peaks; the solubility curves of the DHRS4 gene (27 curves from 27 samples) were also all single peak type, with no other peaks. The results show that the amplification products of the two pairs of primers are single, are specific products, and have no non-specific amplification such as primer dimer, and the like, and the RT-qPCR primers adopted in the embodiment have high specificity and can be used for subsequent expression detection of target gene DHRS4 genes.
A, B and C in FIG. 2 show the expression of DHRS4 gene in the breast, leg and back muscles of the long white pig, respectively, and it can be seen from FIG. 2 that DHRS4 gene is expressed in the breast, leg and back muscles of the long white pig at different development stages, and the expression level is the highest at 30 d. The expression patterns of DHRS4 genes in the chest muscle and the leg muscle are similar, and the expression is obviously reduced at 180d and 300 d; the expression level of DHRS4 gene in dorsal muscle did not change significantly with age.
The meat yield and meat quality of pigs are closely related to the process of skeletal muscle development, the skeletal muscle development comprises the steps of formation of embryonic period muscle fibers, development of postnatal muscle fibers, regeneration of adult period skeletal muscles and the like, the quantity of the postnatal muscle fibers is not changed, and part of slow muscle fibers are gradually converted into fast muscle fibers along with the time, the conversion sequence of the slow muscle fibers is slow muscle, fast muscle, intermediate fast muscle and fast white muscle, and the form of the slow muscle fibers shows the change of hypertrophy of the muscle fibers. FIG. 2 shows that the DHRS4 gene has high gene expression in the muscle tissue of the long and white pig 30d, and the expression level is sharply reduced along with the increase of the muscle, so that the DHRS4 gene is presumed to be involved in the development process of the skeletal muscle of the pig, and the expression level of the DHRS4 gene is in a descending trend in the process of muscle fiber type transformation, namely the process of slow muscle to fast muscle transformation.
Therefore, the DHRS4 gene is considered to be involved in the growth and development of the postnatal skeletal muscle of the pig.
2. Extraction and quality detection of pig genome DNA
(1) Shearing 30 mg-sized pig ear tissues, and placing the cut pig ear tissues into a 1.5ml centrifuge tube;
(2) Taking a 50ml centrifuge tube, uniformly mixing proteinase K with the final concentration of 0.4mg/ml and lysis buffer solution, and adding 0.5ml of lysis solution into a 1.5ml centrifuge tube filled with pig ear tissues for lysis;
(3) Placing the centrifuge tube on a constant temperature shaking bed (sealing the tube cover tightly to prevent liquid from leaking outwards), placing the centrifuge tube at 55 ℃ overnight, and digesting until no tissue sample can be observed by naked eyes;
(4) Taking out the centrifuge tube after the sample is fully cracked, adding 0.3ml of saturated sodium chloride solution into each tube, reversing and fully mixing for a plurality of times, then placing on ice, and carrying out ice bath for 15min;
(5) After ice-cooling, centrifuge at 12000rpm for 15min at room temperature, carefully and slowly transfer the supernatant to a new 1.5ml centrifuge tube, carefully avoid pouring out the precipitate together;
(6) Adding isopropanol with the same volume as the supernatant, and reversing until flocculent precipitate appears in the solution (if no flocculent precipitate exists, the solution can be placed in a refrigerator at-20 ℃ for 2h or a refrigerator at 4 ℃ for overnight);
(7) Centrifuging at 12000rpm for 15min at room temperature, and removing supernatant (during this process, observing the white DNA precipitate at the bottom of the centrifuge tube, and pouring off the precipitate together with the supernatant);
(8) 0.5ml of 70% ethanol was added to each centrifuge tube, and the mixture was inverted gently to wash the precipitated DNA sufficiently;
(9) 10000rmp centrifugation for 30s, using a pipette to suck off ethanol in the centrifuge tube, and keeping the precipitated DNA in the tube (when the DNA precipitate in the tube is not sucked away, the pipette tip used in the step can be operated between centrifuge tubes without replacement);
(10) Naturally drying the extracted DNA for 10min;
(11) Taking 0.1ml of TE buffer solution by a pipette gun, dissolving the DNA precipitate again, placing the DNA precipitate at 55 ℃ for 2 hours, and shaking for several times to fully dissolve the DNA;
(12) After the DNA is fully dissolved, sucking 1 mu LDNA, detecting the concentration of the DNA and the OD260/OD280 value by using an ultramicro spectrophotometer, and if the OD260/OD280 value is more than 1.9, indicating that RNA pollution exists; if the OD260/OD280 value is less than 1.6, it indicates that phenol or protein contamination may be present; the OD260/OD280 value is approximately equal to 1.8, and the DNA is pure DNA;
meanwhile, agarose gel electrophoresis is used for detecting the quality of the DNA, and a single band indicates that the quality of the DNA meets the requirement.
3. Detection of SNP sites
Referring to the pig DHRS4 genome sequence (NM _ 214019.2) published in GeneBank, the principles of Primer design are comprehensively considered, and 6 pairs of primers are designed by using Primer permier 5.0 software according to the reported SNP site information and are marked as P A ～P F Wherein, P A The forward and reverse primer sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, P B The forward and reverse primer sequences are respectively shown as SEQ ID NO.6 and SEQ ID NO.7, P C The forward and reverse primer sequences are respectively shown as SEQ ID NO.8 and SEQ ID NO.9, P D The forward and reverse primer sequences are respectively shown as SEQ ID NO.10 and SEQ ID NO.11, P E The forward and reverse primer sequences are respectively shown as SEQ ID NO.12 and SEQ ID NO.13, P F The forward and reverse primer sequences of (1) are respectively shown in SEQ ID NO.14 and SEQ ID NO. 15.
The method comprises the steps of taking pig DNA as a template, carrying out PCR amplification by adopting the primers, wherein 100 equal sample DNAs are randomly selected and mixed together to construct a mixed pool, carrying out segmented cloning on DHRS4 gene by using 6 segments of primers in the mixed pool, preparing 1% agarose gel electrophoresis to detect the fragment size of a PCR product and the specificity of the primers, collecting the PCR product (5 mu L) and the primers (1 mu L) of a target band for bidirectional sequencing, directly sequencing a peak diagram, wherein the background of the peak diagram is clean and easy to read, the peak diagram contains a nested peak, and the SNP with the heterozygosity of more than or equal to 0.10 is obtained.
The PCR product is subjected to Sanger sequencing, the obtained sequencing result is compared with the pig DHRS4 gene sequence published in GeneBank through DNAMAN and Chromas Pro software, 3 SNPs sites are screened out altogether, and the result is shown in figure 3.
A in FIG. 3 shows the Chr7: 7524571 SNP site (NCBI reference number rs 334250151) of the porcine genomic version Ensembl Scrofa 11.1;
b in FIG. 3 shows the Chr7: 7524589 SNP site (NCBI reference number rs 342446613) of the porcine genomic version Ensembl Scrofa 11.1;
c in FIG. 3 shows the Chr7:75253401SNP site (NCBI reference number rs 326982309) of the porcine genomic version Ensembl Scrofa 11.1.
As can be seen from FIG. 3, the 3 selected SNP sites are respectively located at the base positions 7524571, 7524589 and 75253401 of the pig genome version Ensembl Sscofa 11.1, and the annotation of gene elements shows that the 3 SNP sites are all located in the intron region of the DHRS4 gene.
4. SNP locus genotyping and quality control
Designing a PCR amplification primer and a base extension primer according to the detected sequence information before and after the SNP locus, wherein nucleotide sequences P1 and P2 of the PCR amplification primer of rs334250151 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the base extension primer P3 is shown as SEQ ID NO. 3; the nucleotide sequences P8 and P9 of the PCR amplification primer of rs342446613 are shown as SEQ ID NO.20 and SEQ ID NO.21, and the base extension primer P10 is shown as SEQ ID NO. 22; the nucleotide sequences P11 and P12 of the PCR amplification primer of rs326982309 are shown as SEQ ID NO.23 and SEQ ID NO.24, and the base extension primer P13 is shown as SEQ ID NO. 25.
And (2) carrying out PCR amplification reaction by taking a DNA sample of the 235-head blues white pig as a template, purifying a PCR product to ensure the accuracy of base extension, then co-crystallizing the PCR product with a high-sensitivity mass spectrum chip, and carrying out genotyping on the SNP locus on the DHRS4 gene in a population by utilizing a Sequenom MassARRAY technology.
The typing results are shown in table 1, wherein the genotype frequency and the allele frequency of the SNP sites are calculated by using popgene 3.2:
TABLE 1
As can be seen from table 1, three genotypes were detected at locus rs 334250151: TT, TA and AA, and TT genotype is the dominant genotype with genotype frequency (0.668) higher than that of TA (0.302) and AA genotype (0.030); three genotypes were detected at the rs342446613 site: TT, TC and CC, wherein the frequency of the TC genotype (0.494) is higher than the frequency of the TT genotype (0.327) and the CC genotype (0.179); three genotypes were detected at rs326982309 site: AA. TA and TT, AA genotype frequency (0.698) was higher than the frequencies of TA genotype (0.285) and TT genotype (0.017).
5. Analyzing the correlation between the 3 SNP loci and the growth traits of the pigs
5.1 obtaining growth trait phenotype data of pigs
Calculating the average daily gain and the feed conversion rate by measuring the initial age of the day, the final age of the day, the initial weight (g), the final weight (g) and the final consumption (g), wherein the formula is as follows:
average daily gain (g) = [ knot weight (g) -initial weight (g) ]/[ knot age-initial age ]
Feed conversion ratio (%) = knot-measuring consumed material (g)/[ knot-measuring body weight (g)/[ initial body weight (g) ]
Measurement of backfat thickness: and (3) measuring the backfat thickness of the individual pigs in the test swinery by adopting a high ultrasonic backfat determinator. When the measurement is started, the pig is naturally stood still and kept still, hair is sheared at the position 5cm above the back center line at the last rib position of the pig, a couplant is coated, a backfat instrument probe is placed at the position, and the record is carried out after the reading of the instrument is stable. Measurements were taken 3 times in succession, averaged, and the data recorded. The above phenotypic data were then statistically analyzed using Excel (Microsoft Office 2017).
5.2 Association analysis
The GLM process in SAS9.2 software is used for analyzing the correlation between the genotype of the SNP locus and the growth traits of the pig, and the adopted fixed effect model is as follows:
y＝μ+gi+mk+e；
wherein y is a phenotypic record of growth traits; μ is the overall average of the trait; gi is the genotype effect; mk is the production month effect; e is the random error.
Data are expressed as probability values and mean ± standard deviation, with P values <0.05 indicating significant differences.
The correlation analysis results are shown in table 2:
TABLE 2
Where data are expressed as mean ± standard deviation, and the difference in the lower case letters marked on the shoulder indicates significant difference (p < 0.05).
As can be seen from table 2, the rs334250151 site was significantly related to feed conversion rate (P < 0.05), and the feed conversion rate (2.24) of AA genotype individuals was significantly lower than TA type (2.40) and TT type (2.45), and was not significantly related to average daily gain and backfat thickness. And the sites rs342446613 and rs326982309 have no obvious relationship with average daily gain, feed conversion rate, average back fat thickness and the like.
In conclusion, the polymorphic site rs334250151 on the DHRS4 gene is expected to be used as a candidate genetic marker site related to the feed conversion rate traits of the Lansius white pigs and used for guiding the breeding work of the meat production traits of the Lansius white pigs. The genotype of a pig individual can be determined by determining the base type of the rs334250151 site of the pig DHRS4 gene, so that the feed conversion rate of the pig can be identified in an auxiliary manner, and the AA genotype individual has lower feed conversion rate.
The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the invention. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.
SEQUENCE LISTING
<110> Tianjinnuo cereal-based bioinformatics science and technology Co., ltd
<120> molecular marker related to pig feed conversion rate, identification and application thereof
<130> 2019
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> amplification primer P1 (Artificial sequence)
<400> 1
acgttggatg gtgaaagtag cagagaaggg 30
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<212> DNA
<213> amplification primer P2 (Artificial sequence)
<400> 2
acgttggatg tgtacttttt ccagaggcgg 30
<210> 3
<211> 24
<212> DNA
<213> extension primer P3 (Artificial sequence)
<400> 3
ccttttcctc cgttggagcc tacc 24
<210> 4
<211> 20
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<213> Forward primer (Artificial sequence) of amplification primer PA
<400> 4
tccctccttg gctatctgct 20
<210> 5
<211> 20
<212> DNA
<213> reverse primer (Artificial sequence) of amplification primer PA
<400> 5
agccaggact agtctccctg 20
<210> 6
<211> 20
<212> DNA
<213> Forward primer (Artificial sequence) of amplification primer PB
<400> 6
gagtgtgtgc tgtcttcgga 20
<210> 7
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<213> reverse primer (Artificial sequence) of amplification primer PB
<400> 7
gccttgaatc ttccctgcct 20
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<213> Forward primer (Artificial sequence) of amplification primer PC
<400> 8
agagagtcag ggagccagag 20
<210> 9
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<212> DNA
<213> reverse primer (Artificial sequence) of amplification primer PC
<400> 9
gaccctaagg ggcatttgct 20
<210> 10
<211> 20
<212> DNA
<213> Forward primer (Artificial sequence) for amplification primer PD
<400> 10
ggtcaagagt tgctggctct 20
<210> 11
<211> 20
<212> DNA
<213> reverse primer (Artificial sequence) for amplification primer PD
<400> 11
agcagatagc caaggaggga 20
<210> 12
<211> 20
<212> DNA
<213> Forward primer (Artificial sequence) of amplification primer PE
<400> 12
ctgatgggga aatgcctggt 20
<210> 13
<211> 20
<212> DNA
<213> reverse primer (Artificial sequence) of amplification primer PE
<400> 13
cctgtgacca tgggaacctc 20
<210> 14
<211> 20
<212> DNA
<213> Forward primer (Artificial sequence) of amplification primer PF
<400> 14
gccaggcagt tcaccctaat 20
<210> 15
<211> 20
<212> DNA
<213> reverse primer (Artificial sequence) of amplification primer PF
<400> 15
aagtgctgga acaaccccaa 20
<210> 16
<211> 20
<212> DNA
<213> RT-qPCR amplification primer P4 (Artificial sequence)
<400> 16
gccgtcaacc cattctttgg 20
<210> 17
<211> 20
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<213> RT-qPCR amplification primer P5 (Artificial sequence)
<400> 17
gcaccactgc ctttgtcatc 20
<210> 18
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<213> internal reference Gene amplification primer P6 (Artificial sequence)
<400> 18
agggcatcct gggctacact 20
<210> 19
<211> 20
<212> DNA
<213> internal reference Gene amplification primer P7 (Artificial sequence)
<400> 19
tccaccaccc tgttgctgta 20
<210> 20
<211> 30
<212> DNA
<213> amplification primer P8 (Artificial sequence)
<400> 20
acgttggatg gtgaaagtag cagagaaggg 30
<210> 21
<211> 30
<212> DNA
<213> amplification primer P9 (Artificial sequence)
<400> 21
acgttggatg tgtacttttt ccagaggcgg 30
<210> 22
<211> 16
<212> DNA
<213> extension primer P10 (Artificial sequence)
<400> 22
gctcagtgtt gatcgt 16
<210> 23
<211> 30
<212> DNA
<213> amplification primer P11 (Artificial sequence)
<400> 23
acgttggatg aggtggttga aacatggtgg 30
<210> 24
<211> 30
<212> DNA
<213> amplification primer P12 (Artificial sequence)
<400> 24
acgttggatg tgggtattcc cactatgagc 30
<210> 25
<211> 24
<212> DNA
<213> extension primer P13 (Artificial sequence)
<400> 25
aactgtttat tcatctttta aagt 24
Claims (3)
1. A method for identifying or assisting in identifying feed conversion efficiency in pigs, the method comprising the step of detecting the base type at position 7524571 of chromosome 7 of the porcine genomic version Ensembl Sscrofa11.1 to determine that the genotype at the locus is TT, TA or AA.
2. The method of claim 1, wherein the swine with genotype AA have a lower feed conversion ratio than the swine with genotypes TA and TT.
3. The method of claim 1, wherein the method of genotyping the site at position 7524571 of chromosome 7 of the porcine genomic version Ensembl Sscofa 11.1 comprises the steps of a PCR amplification reaction and a single base extension reaction,
the nucleotide sequences of amplification primers P1 and P2 in the PCR amplification reaction are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the nucleotide sequence of the extension primer P3 in the single base extension reaction is shown as SEQ ID NO. 3.
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