CN108559781B - Method for breeding pigs with high feed utilization efficiency - Google Patents
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Abstract
The invention discloses a method for breeding pigs with high feed utilization efficiency. The method provided by the invention is to detect whether the 200546552 th deoxyribonucleotide on the chromosome 13 of the international pig genome version 11.1 reference sequence of the pig to be detected is A or G, determine whether the genotype of the pig to be detected is AA or GG, and determine that the residual feed intake of the pig with the genotype of GG is lower than that of the pig with the genotype of AA; the GG genotype is a homozygote of G at 200546552 th deoxyribonucleotide on chromosome 13 of the reference sequence of the 11.1 version of the international pig genome; the AA genotype is a homozygote of an 200546552 th deoxyribonucleotide on a chromosome 13 of a reference sequence of the 11.1 version of the international pig genome. The method can reduce breeding cost, effectively improve the utilization efficiency of the pig feed in actual production and improve economic benefit. The method provided by the invention is simple to operate, has high accuracy, can realize automatic detection, and can play a great role in pig breeding.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for breeding pigs with high feed utilization efficiency.
Background
In the pig raising production, the feed input is the largest, which accounts for about 70% of the total breeding cost, and the small improvement of the feed utilization efficiency brings about the great reduction of the breeding cost. Therefore, the feed utilization efficiency is more and more emphasized by people, and is now one of important selection target traits in pig breeding. The feed utilization efficiency evaluation indexes mainly include a feed weight gain ratio, a weight gain feed ratio and a Residual Feed Intake (RFI). The remaining feed intake is the difference between the actual feed intake of the livestock and the predicted feed intake required for maintenance and weight gain, and can reflect the metabolic differences of the animals themselves determined by the genetic background (Foster W, Kilparick D, Heaney I.genetic variation in the efficiency of the efficiency pig. animal science 1983, 37 (3): 387 393.). Therefore, a smaller value of the remaining feed intake represents a higher feed utilization efficiency of the swine. Currently, the remaining feed intake has become the best representative index of feed utilization efficiency.
Estimating the remaining feed intake requires a large amount of average daily feed intake data, which is still expensive and time-consuming, although the updating of electronic feeding equipment and the continuous development of feed intake measuring systems make the collection of feed intake data more convenient. Therefore, it is necessary to identify molecular markers associated with the remaining food consumption.
Disclosure of Invention
An object of the present invention is to provide a method for breeding pigs with high feed utilization efficiency.
The method for breeding the pigs with high feed utilization efficiency detects whether 200546552 th deoxyribonucleotide on chromosome 13 of reference sequence of version 11.1 of international pig genome is A or G, determines the genotype of the pigs to be tested is AA or GG, and the residual feed intake of the pigs with the genotype of GG is obviously lower than that of the pigs with the genotype of AA; the AA genotype is a homozygote of 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the version 11.1 of the international pig genome, which is A; the GG genotype is a homozygote of G at 200546552 th deoxyribonucleotide on chromosome 13 of the international pig genome version 11.1 reference sequence.
The method for determining whether the 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the 11.1 version of the international pig genome of the pig to be detected is A or G can adopt any existing method, such as sequencing analysis, PCR-SSCP, PCR-RFLP method and the like.
The method for determining whether the 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the international pig genome version 11.1 of the pig to be detected is A or G can be specifically sequencing analysis. The sequencing analysis comprises the steps of PCR amplification and sequencing of PCR amplification products; the primer pair used for PCR amplification meets the condition that the product obtained by PCR amplification by taking the genome DNA of the pig as a template contains 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the 11.1 version of the international pig genome.
The primer pair used for PCR amplification can be a primer pair consisting of a single-stranded DNA molecule shown in a sequence 1 of a sequence table and a single-stranded DNA molecule shown in a sequence 2 of the sequence table. When the primer pair is used for PCR amplification, according to the difference of the genome DNA of the pig to be detected, the DNA fragment obtained by amplification is a nucleotide sequence shown in a sequence 3 or a nucleotide sequence shown in a sequence 4.
Another object of the present invention is to provide an agent for raising pigs with high feed efficiency.
The reagent provided by the invention is a substance for detecting whether the 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the 11.1 version of the international pig genome of a pig to be detected is A or G.
In the reagent, the substance is a primer pair consisting of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
The application of the reagent in the auxiliary breeding of the pigs with high feed utilization efficiency is also within the protection range of the invention;
or the application of the reagent in the preparation of the auxiliary breeding pig with high feed utilization efficiency is also within the protection scope of the invention.
The pig to be detected can be a Duroc pig.
A deoxyribonucleotide difference (A/G) exists at 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the international pig genome version 11.1, and the single nucleotide polymorphism is named as g.13.200546552A > G. The residual feed intake of GG homozygous genotype group is about 0.1125 kg/day lower than that of AA homozygous genotype group, so the locus can be used as a molecular breeding marker for the character of the residual feed intake of pigs.
The third purpose of the invention is to provide a method for breeding pigs with high feed utilization efficiency.
The method provided by the invention comprises the steps of selecting pigs with GG genotypes for breeding; the GG genotype is a homozygote of G at 200546552 th deoxyribonucleotide on chromosome 13 of the international pig genome version 11.1 reference sequence.
Experiments prove that the invention uses a gene sequencing method to detect g.13.200546552A > G, can judge the genotype of an individual by sequencing only by carrying out PCR reaction, has very accurate genotype judgment and low detection cost, and has very high breeding practice application value. The method of the invention is used for selecting the residual feed intake traits of the pigs, so that the average residual feed intake of GG genotype pigs is reduced by about 0.1125 kg/day compared with AA genotype pigs, thereby obtaining the pigs with high feed utilization efficiency. The method provided by the invention is used for breeding pigs, and pigs to be selected can be screened early, so that the problem of long time for selecting excellent pigs in actual production is effectively solved, the breeding cost is reduced, and the utilization efficiency of pig feed in actual production is effectively improved. The detection method of the invention has simple operation, low cost and high accuracy, and can realize automatic direct detection. The invention will play a great role in the breeding work of pigs.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The primers in the following examples were synthesized by Shanghai Yingjun Biotechnology Ltd.
Duroc pigs: the pig was purchased from the eagle farming-grazing group shares, ltd.
Example 1 auxiliary identification of pig remaining feed intake traits
Determination of pig g.13.200546552A G polymorphic site
1. PCR amplification
According to the information of the 200546552 th deoxyribonucleotide on the 13 th chromosome of the reference sequence of the 11.1 version of the international pig genome, a pair of primers is designed as follows:
u (upstream primer): 5'-TCCCTTGGCACAACATGGAT-3' (SEQ ID NO: 1 of the sequence Listing);
d (downstream primer): 5'-TTACAAAGAGCAGGTGGAGGC-3' (SEQ ID NO: 2 of the sequence Listing).
Duroc pigs (2 pigs) were selected as experimental material. The genomic DNA of Duroc pigs was used as a template, and PCR amplification was performed using primers U and D.
The amplification system is as follows: 200ng of genome DNA, 5 mul of 10 XPCR amplification buffer, 10mM of dNTPs, 50ng of upstream and downstream primers respectively, 0.75U of Taq DNA polymerase and Mg2+2.5mmol/L from ddH2O made up the reaction to 50. mu.l.
The PCR reaction conditions are as follows: denaturation at 95 deg.C for 5 min; then denaturation at 95 ℃ for 20s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.
The PCR product was detected by agarose gel and stored at-20 ℃. The products amplified by using the genomic DNA of 2 Duroc pigs as templates were named product 1 and product 2, respectively.
2. Clone sequencing and sequence analysis
Respectively recovering and purifying 2 PCR products by using an agarose gel recovery kit (Tiangen Biochemical technology Co., Ltd.), connecting the recovered DNA fragments with a vector pGEM-T (Promega Co.), then transforming the connecting products into escherichia coli DH5 alpha competent cells (Ming Ri Baiao (Beijing) science Co., Ltd.), and screening positive clones according to the carbenicillin resistance marker on the vector to obtain recombinant plasmids containing the recovered fragments. The nucleotide sequences of the T7 and SP6 promoters in the recombinant plasmid vector were determined using the primers (Yinxie fundi (Shanghai) trade Co., Ltd.). The sequencing result shows that: the product 1 is the sequence M1, and the product 2 is the sequence M2; the length of the sequence M1 and the length of the sequence M2 are 817bp, only one difference (A/G) exists between deoxyribonucleotides which are 200546552 th deoxyribonucleotides on the chromosome 13 of the reference sequence of the 11.1 version of the international pig genome, and the single nucleotide polymorphism is named as g.13.200546552A > G. The nucleotide sequence of the sequence M1 is shown in a sequence 3 of the sequence table (the 200546552-position deoxyribonucleotide of the chromosome of the reference sequence 13 of the version 11.1 of the international porcine genome corresponds to A at the 617-position at the 5 'end in the sequence 3), and the nucleotide sequence of the sequence M2 is shown in a sequence 4 of the sequence table (the 200546552-position deoxyribonucleotide of the chromosome of the reference sequence 13 of the version 11.1 of the international porcine genome corresponds to G at the 617-position at the 5' end in the sequence 4). A homozygote genotype in which the 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence No. 11.1 version of the international porcine genome is A is named as AA, a homozygote genotype in which the 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence No. 11.1 version of the international porcine genome is G is named as GG, and a heterozygote genotype thereof is AG.
Second, correlation analysis of 200546552 th deoxyribonucleotide polymorphic site on chromosome 13 of international pig genome version 11.1 reference sequence and residual feed intake of pig
To determine whether the g.13.200546552A > G polymorphic site correlates with the remaining feed intake of pigs, 241 Duroc pigs were used as the experimental material for the following tests: extracting the genome DNA of each pig, carrying out PCR amplification by using the primers U and D, carrying out sequencing analysis on the PCR amplification product of each pig, and determining whether the genotype of each pig is GG, AG or AA. Determining the remaining feed intake of the pig according to the genotype of the pig: the pig residual feed intake of the pig to be detected with the GG genotype is lower than that of the pig to be detected with the AA genotype.
The daily feed intake and daily body weight of 241 Duroc pigs were measured using the American Osthis automatic feeding system, starting at the age of 90 days and ending when the body weight reached 100kg, and the backfat thickness and eye muscle area of the pigs were measured using the Aquila Vet ultrasonic B-ultrasound apparatus at this time. Calculation of Remaining Feed Intake (RFI) in the test reference predecessor methods (Onteru SK, Gorbach DM, Young JM, Garrick DJ, Dekkers JCM, Rothschild MF. wheel genome analysis students of residual feed and related tracks in the pig. PLoS One,2013, 8(6): e61756.), the formula is as follows:
RFI＝ADFI-[1×(OnBW-30)+b2×(OffBW-100)+b3×MetamidBW+b4×ADGA+b5×OffBFA]
in the formula: OnBW means initial measurement of body weight; OffBW means weight measurement; MetamidBW refers to the median metabolic body weight, equal to [ (OnBW + OffBW)/2 ]. times.0.75; ADGA represents the average daily gain correction between 90-180 days; OffBFA refers to the value at the end of the test when the back fat thickness of 10-11 ribs is corrected to 100kg body weight. The correction formula for 100kg backfat thickness and 30-100kg ADGA was derived from the canadian genetic improvement program.
Correlation analysis was performed by least squares for g.13.200546552A > G sites and Remaining Feed Intake (RFI), and least squares analysis was performed using GLM program in SAS9.2 software in combination with the following model to compare the difference in RFI between genotypes. The model used was:
Y＝μ+G+S+B+P+e
wherein Y is a trait measure; μ is the population mean; g is a genotype effect; s is the sex effect; b is measuring batch effect; p is the fetal secondary effect; e is the random error.
The results are shown in Table 1.
TABLE 1 correlation of pig mutation site g.13.200546552A > G genotype with residual feed intake
Note: the same upper label and different letters represent significant differences (P <0.05)
The results are shown in table 1, and show that the residual feed intake of the GG genotype pig is significantly lower than that of the AA genotype pig (P <0.05), and the residual feed intake of the AG genotype pig is not significantly different from that of the GG genotype pig and the AA genotype pig. The invention proves that the method for identifying the residual feed intake of the pig by using the 200546552 th deoxyribonucleotide polymorphism on the chromosome 13 of the international pig genome version 11.1 reference sequence is consistent with the actual measurement result of the residual feed intake of the pig.
Therefore, in actual pig breeding, it is preferable to select pigs of GG genotype for breeding in order to obtain pigs with lower residual feed intake (higher feed utilization efficiency).
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> a method for breeding pigs with high feed utilization efficiency
<160> 4
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 1
tcccttggca caacatggat 20
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 2
ttacaaagag caggtggaggc 21
<210> 3
<211> 817
<212> DNA
<213> pig (Sus scrofa)
<400> 3
ttcatggcag atggtggtca acttgtctgt gggaggaggt gggatcctgc agaggctgag 60
agcagatgct ttgtcttctg tgacttttca cctgtctagg tttttattcc cccagatgag 120
acaatgaatt tccttgaggg ttggaagtat acagtcatac atttcctgta aaatgcacag 180
agaactagag gatcacgcgg gaactctgga acctgagagg acttttggga attaaggatg 240
tagtgtaggc tagaagtgta cttccttcct tggatcttgg tttacttaag cacttagtgt 300
tcttattgtc taccgtatca tagtttaagt gttcagaata tgttaatatg gtatttactg 360
gtcatgagtt aggaagtact tatcttacca aaatggcatt ttaaggacaa gctatcatca 420
tctaacataa tgccaaattg cgatctttcc atttctttgt ttacgacaca ggaaatccgg 480
gatcctctta tgcagtggct ggggaaacac gtggatcctg aaggagttat aacatctagc 540
aagctctccc ttaaattcgt ttcatcctac acatctgagg tggacatcac cccatctgcc 600
atacctgtgg tgactgacac ggcggccttc tcctcagaaa attttaactt tgagatctac 660
cgacagaatt tgcagaccaa gaaacttgga aaaataattc tgtttgccga agtgacatcc 720
accacaatga gtcttctgga cgggtgagtt ctggcccagc aggcttctgt agctgcttgg 780
aggaatccaa gttaggttga ccgttccctt gaaccag 817
<210> 4
<211> 817
<212> DNA
<213> pig (Sus scrofa)
<400> 4
ttcatggcag atggtggtca acttgtctgt gggaggaggt gggatcctgc agaggctgag 60
agcagatgct ttgtcttctg tgacttttca cctgtctagg tttttattcc cccagatgag 120
acaatgaatt tccttgaggg ttggaagtat acagtcatac atttcctgta aaatgcacag 180
agaactagag gatcacgcgg gaactctgga acctgagagg acttttggga attaaggatg 240
tagtgtaggc tagaagtgta cttccttcct tggatcttgg tttacttaag cacttagtgt 300
tcttattgtc taccgtatca tagtttaagt gttcagaata tgttaatatg gtatttactg 360
gtcatgagtt aggaagtact tatcttacca aaatggcatt ttaaggacaa gctatcatca 420
tctaacataa tgccaaattg cgatctttcc atttctttgt ttacgacaca ggaaatccgg 480
gatcctctta tgcagtggct ggggaaacac gtggatcctg aaggagttat aacatctagc 540
aagctctccc ttaaattcgt ttcatcctac acatctgagg tggacatcac cccatctgcc 600
atacctgtgg tggctggcac ggcggccttc tcctcagaaa attttaactt tgagatctac 660
cgacagaatt tgcagaccaa gaaacttgga aaaataattc tgtttgccga agtgacatcc 720
accacaatga gtcttctgga cgggtgagtt ctggcccagc aggcttctgt agctgcttgg 780
aggaatccaa gttaggttga ccgttccctt gaaccag 817
Claims (4)
1. The application of the reagent for detecting whether the 200546552 th deoxyribonucleotide on the chromosome 13 of the international pig genome version 11.1 reference sequence of a pig to be detected is A or G in breeding the pig with high feed utilization efficiency.
2. The use according to claim 1, wherein the detection of whether the 200546552 th deoxyribonucleotide on chromosome 13 of the reference sequence version 11.1 of the international pig genome of the pig to be detected is A or G is a sequencing analysis;
the sequencing analysis comprises two steps of PCR amplification and sequencing of PCR amplification products; the primer pair used for PCR amplification meets the following condition that the product obtained by PCR amplification by taking the genome DNA of the pig to be detected as a template contains 200546552 th deoxyribonucleotide on the chromosome 13 of the reference sequence of the 11.1 version of the international pig genome.
3. Use according to claim 2, characterized in that: the primer pair used for PCR amplification is a primer pair consisting of a single-stranded DNA molecule shown in a sequence 1 in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.
4. A method for breeding pigs with high feed utilization efficiency comprises selecting pigs with GG genotype for breeding; the GG genotype is a homozygote of G at 200546552 th deoxyribonucleotide on chromosome 13 of the international pig genome version 11.1 reference sequence.
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| CN110358840B (en) * | 2019-06-06 | 2023-04-25 | 佛山科学技术学院 | SNP molecular genetic marker of TPP2 gene related to residual feed intake |
| CN110295236B (en) * | 2019-06-06 | 2023-05-30 | 佛山科学技术学院 | SNP molecular genetic marker for pig feed conversion rate |
| CN110218799B (en) * | 2019-06-06 | 2024-03-26 | 佛山科学技术学院 | Molecular genetic marker for pig residual feed intake traits and application thereof |
| CN113584181B (en) * | 2021-07-26 | 2022-04-22 | 温氏食品集团股份有限公司 | SNP molecular marker related to pig residual feed intake and application thereof |
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| CN103937895A (en) * | 2014-04-24 | 2014-07-23 | 中国农业科学院北京畜牧兽医研究所 | Method and special kit for auxiliary detection of pig intramscular fat content |
| CN105219873A (en) * | 2015-11-04 | 2016-01-06 | 中国农业科学院北京畜牧兽医研究所 | A kind of method and primer special identifying pig vertebra data/coherency shape |
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