CN101343663B - Molecular marker clone correlated with pig growth rate and application thereof - Google Patents

Abstract

The invention belongs to the technical field of livestock genetic engineering, and in particular relates to a molecular marker related to growth rate traits used as pig marker-assisted selection and its application. The molecular marker is obtained by cloning the COPB1 gene, and its DNA sequence is as described in SEQ ID NO: 1 in the sequence table. There is a C744-T744 base mutation at the 744th bp of SEQ ID NO: 1 in the sequence table, resulting in RsaI-RFLP polymorphism. The invention also discloses the primer used for amplifying the partial DNA sequence of the COPB1 gene and the polymorphism detection method, which provides a new molecular marker for marker-assisted selection of pigs.

Description

Cloning and Application of Molecular Markers Related to Pig Growth Velocity

technical field

The invention belongs to the technical field of pig molecular marker-assisted selection, and specifically relates to the cloning and application of a molecular marker related to growth rate as a pig marker-assisted selection. The molecular marker is related to the COPB1 gene. Association analysis was carried out for traits related to pig growth rate. the

Background technique

Since the 1980s, the research on molecular markers has developed rapidly, and molecular marker-assisted selection and introgression molecular breeding techniques have been formed. These techniques, combined with conventional breeding methods, have greatly improved the breeding efficiency of pigs. The genes or markers that can be applied to molecular marker-assisted selection must have a large genetic contribution to the target traits, that is, the main effect genes or markers, so finding these main effect genes and molecular markers linked to them has become the premise and basis of molecular marker-assisted selection. It is also the research focus and urgent problem to be solved in the field of porcine molecular biology at present and in the future. the

At present, a number of functional gene molecular markers closely linked to growth traits and carcass traits have been discovered and applied for patents: 1) Lin et al. detected that polymorphisms in the 5' flanking region of heat stress protein 70.2 (hsp70.2) can improve the performance of pigs. The production performance of this gene has been applied for a patent (patent number is US, US2003104392-A1); 2) Rothschild et al. found the role of CAST gene and applied marker-assisted selection in pig growth and meat quality improvement (patent number US, WO2003060151 -A2); 3) Rothschild et al. detected the variation of CKM, SCN4 alpha, and LDH alpha genes related to muscle growth and meat quality traits, and provided molecular markers for breeding (patent number US, WO2004081194-A2); 4) Rothschild found that the pig melanocortin-4 receptor (MC4R) gene is related to growth rate, fat content and feed consumption (patent number US, WO200175161-A2); , meat quality and genetic markers of feed consumption ratio (Patent No. CN, 03806119.8); 6) Greger et al. found that the CYP11a1 gene was associated with growth and reproductive traits (Patent No. US, WO200069882); 7) Hiendleder et al. established the relationship between growth, reproduction and carcass The detection method of trait-related INHA and INHBA gene polymorphisms (Patent No. DE, DE10121225-A1); 8) Gerbens et al. found that the polymorphism of the H-FABP gene was related to production performance (body weight, intramuscular fat) (Patent No. EP, WO9735878-A); 9) Kojima et al. found that the polymorphism of the pLEPR gene can improve the genetic traits of pigs and the yield of meat (patent number US, WO2005017204-A2); 10) Li Kui et al. disclosed a pig production traits-related protein and its Coding gene CA3 and its application (Patent No. CN, 200610066856.4); 11) Li Kui et al. found the correlation between the variation of PSMB8 gene and growth rate and immune traits (Patent No. CN, 200410096857.4). the

The genetic basis of pig economic traits is very complex. Although scientists and researchers in the field of animal genetics and breeding have carried out a lot of research work, the identification of functional genes and molecular markers that can be used in breeding practice are still very limited; The competition for gene property rights is ongoing in the world, and some important genes have been patented by foreign counterparts. In order to protect my country's precious genetic resources, it is necessary to accelerate the research speed of functional gene identification and molecular markers. For the purpose of the above research, our laboratory carries out the isolation, mapping, SNP screening and detection of candidate genes for pig economic traits, and the association analysis with traits. the

Research progress of the target gene in this patent COPB1 (coatomer protein complex, subunit beta 1) gene encodes the subunit of the coat protein complex. At present, there have been reports on the structure, expression and function of the COPB1 gene in mice and rats. The human COPB1 gene is located at 11p15, encoding 953 amino acids. The mouse COPB1 gene is located at 53.3cM of chromosome 7, and the full-length cDNA is 3173bp, encoding 953 amino acids. Human, mouse, and rat COPB1 genes all encode 953 amino acids, and the sequences are highly conserved. The COPB1 gene is expressed in the heart, liver, spleen, lung, and kidney of mice. the

The current research on this gene is mainly manifested in: in 1994, it was found that the COPB1 gene is necessary to maintain the viability of yeast cells, and its mutation can lead to defects in its ability to transport certain proteins to the endoplasmic reticulum (Letourneur et al., Coatomeris essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 1994, 79: 1199-1207). In 2000, it was revealed that the COPB1 gene may be a candidate gene for ruby-eye-2 (ru2), and ru2 is one of the human Hermansky-Pudlak Syndrome (HPS) mouse models (Wei et al., cDNA sequence and mapping of the mouse copb gene encoding the beta subunit of the COPI coatomercomplex. Somat Cell Mcl Genet, 1999, 25: 177-183). The transport of Opioid receptor (KOR) mRNA needs the participation of COPB1 gene, and COPB1 gene promotes cell body and axon to translate kor mRNA (Jing etc., Copb1-facilitated axonal transport and translation of kappa opioid-receptor mRNA.Proc Natl Acad Sci USA, 2007, 104:13810-13815). the

From the above data, we can conclude that the COPB1 gene plays an important role in protein transport. But its function research is not very thorough, and the research on porcine COPB1 gene is still blank at home and abroad. Finding the variation sites in genes and discovering the relationship between genes and traits through association analysis with traits is an important means of studying gene functions and the basis for marker-assisted selection. To this end, the cloning, mapping, SNP detection and association analysis of pig COPB1 gene were carried out. the

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, to clone a molecular marker related to the growth rate as the marker-assisted selection of pigs, and to use the molecular marker as the marker-assisted selection of pigs. the

The present invention is realized through the following technical solutions:

The applicant cloned a growth rate-related molecular marker as a marker-assisted selection for pigs from the pig COPB1 gene fragment, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table. There is a C744-T744 base mutation at the 744th base of the sequence shown in SEQ ID NO: 1, resulting in Rsa I-RFLP polymorphism. the

The DNA sequence of the primer pair for detecting the base mutation of C744-T744 at the 744th base of the sequence shown in SEQ ID NO: 1 is as follows:

Forward: 5'-GGGCTTACTGGACTCCAACAT-3',

Reverse: 5'-TGGTCTTGATACATGTGTGAAACA-3'. the

The method for preparing the above-mentioned molecular markers, according to the following steps:

DNA was extracted from the pig blood genome, and human COPB1 gene cDNA was used as an information probe for homologous sequence screening to obtain an expressed sequence tag (EST) with a homology of more than 80%; then the porcine EST-contig was spliced and primers were designed for amplification Middle unknown fragment, PCR product purification, cloning, sequencing, obtained the DNA sequence as described in the sequence table SEQ ID NO: 1. 

The applicant successfully applied the molecular markers of the above clones to the association analysis of marker-assisted selection of pig growth speed-related traits, thus completing the present invention. the

Description of drawings

Sequence listing SEQ ID NO: 1 is the DNA fragment of the porcine COPB1 gene cloned by the present invention;

Fig. 1: is the flowchart of COPB1 gene preparation of the present invention;

Fig. 2: is the DNA fragment used for cloning of the porcine COPB1 gene in the present invention. The primer sequences used are underlined;

Figure 3: is the DNA fragment used for positioning of the porcine COPB1 gene in the present invention. The primer sequences used are underlined;

Fig. 4: is the DNA fragment used for positioning of the porcine COPB1 gene in the present invention. The primer sequences used are underlined;

Figure 5: is the DNA fragment of the pig COPB1 gene used in PCR-RFLP detection in the present invention. The primer sequences used are underlined;

Fig. 6: is the electrophoresis result of three genotypes (CC CT TT) of porcine COPB1 gene RsaI-RFLP in the present invention. M in the figure: DNA molecular weight standard (100bp ladder). the

Detailed ways

Embodiment 1, the cloning of COPB1 gene

1. Primer design

Using human COPB1 gene cDNA (GenBank accession number: NM_016451) as an information probe, using the BLAST tool in NCBI to screen homologous sequences in the GenBank pig EST database, a series of ESTs with a homology of more than 80% (fragment length greater than 100bp), use ENTREZ (http://www.ncbi.nlm.nih.gov/Web/Search/index.html) to query the corresponding sequence in NCBI with the accession numbers of these ESTs, and then use the ASSEMBLY program in GeneTool to construct Pig EST-contigs. Design a pair of primers P1-CF and P1-CR according to the EST splicing sequence, the sequence is as follows

COPB1: P-CF 5′-GCACTTTCTGGTTATTGTGGCT-3′

P-CR 5′-TGGTCTTGATACATGTGTGAAACA-3′

2. Purification, cloning and sequencing of PCR products

Purification of PCR products: Cut the gel containing the target fragment from the low-melting point agarose gel under ultraviolet light, put it into a 1.5ml Ependorff tube, incubate at 70°C until the gel is completely melted, and then purify it with the PCR product The kit (purchased from Promega) was used to purify the PCR product, and the operation was performed according to the kit instructions. The specific steps were to add 1ml Resin to every 300μl of melted gel, mix for 20s, put the Resin/DNA mixture into the syringe, and let the slurry pass through the Minicolumn extrude. Then add 2ml of 80% isopropanol to the syringe, gently push the plunger to squeeze out the isopropanol through the Minicolumn, remove the Minicolumn and put it into a 1.5ml Ependorff tube, centrifuge at 10,000g for 2min to dry the Resin, put the Minicolumn into another Add 30-50μl sterilized water to a clean 1.5ml Ependorff tube, let it stand for 1min, and centrifuge at 10,000g for 20s to elute the DNA and store it in the Ependorff tube. the

Ligation reaction: Ligate the purified PCR product with the pGEM-T vector, the total volume of the ligation reaction is 5 μl, including 2.5 μl 2×buffer, 0.5 μl of T vector (purchased from Promega), 0.5 μl of purified PCR product, 0.5 μl of T4 ligase, and finally add 1 μl of sterilized water and place in a water bath at 4°C overnight. the

Preparation of competent cells: Pick a single colony of DH5α from a fresh plate cultured at 37°C for 16-20 hours and inoculate it in 2ml LB, culture it with shaking at 37°C for 3 hours, transfer 1ml of the bacterial solution to a saline bottle containing 30ml LB , and continue shaking culture at 37°C for about 4 hours. When the OD600 reaches 0.3-0.4, take the saline bottle out of the shaker and place it in an ice bath to cool for 10-15 minutes. For cells, turn the centrifuge tube upside down to discard the culture medium, resuspend the pellet with 10ml of ice-cold 0.1mol/L CaCl ₂ , ice-bath for 30min, repeat centrifugation at 4,000g at 4°C for 10min once, and use 4ml of ice-cold 0.1mol/L CaCl 2 /L of CaCl ₂ to resuspend the pellet and store at 4°C for later use.

Transformation: Take 100-120 μl of competent cells in a sterile 1.5ml Ependorff tube, add 5 μl of the ligation product and mix well, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, do not shake the Ependorff tube during the process, take it out and ice After bathing for 3 to 4 minutes, add 400 μl LB liquid medium without antibiotics, and culture with shaking at 37°C for 45 minutes. Take 100 μl and spread it on the agar plate that has been coated with IPTG (Isopropylthio-β-D-galactoside, Chinese name is isopropylthio-β-D-galactoside) and X-gal 4 hours in advance. After 1 h, culture upside down. the

Small amount of plasmid preparation: pick a single colony on the plate, inoculate in 2-3ml LB, and culture overnight at 37°C and 300r/min. Use a 1.5ml EP tube to centrifuge at 12000r/min for a few seconds to collect the bacteria. Add 100 μl of ice-precooled solution I [50 mM glucose, 25 mM Tris.Cl (pH 8.0), 10 mM EDTA (pH 8.0)] to each tube, and vortex until the cells are fully suspended. Add 200 μl of newly prepared solution II [0.2M NaOH, 1% SDS], quickly invert and mix, and ice-bath for 5 minutes, then add 150 μl of pre-cooled solution III [5M potassium acetate, glacial acetic acid 11.5ml, H2O 28.5ml], mix After uniformity, ice bath for 5 minutes, centrifuge at 12000r/min for 5 minutes, transfer the supernatant to another EP tube, add phenol: chloroform: isoamyl alcohol 500μl, vortex, carefully absorb the upper aqueous phase after centrifugation, add 2 times the volume of Absolute ethanol, precipitate at -20°C for 30 min, centrifuge at 12,000 r/min for 5 min, wash the precipitate twice with 70% ethanol, drain, and add 20 μl of TE containing RNase. the

Enzyme digestion identification of recombinant plasmids: Mix 3 μl of plasmid DNA with appropriate amount of double distilled water to make the total volume 15 μl, add 2-3U restriction endonuclease EcoR I and 2 μl corresponding 10X restriction endonuclease reaction buffer , lightly flick the tube wall to mix and centrifuge, place in a 37°C water bath for 1-2 hours, take 2-3 μl of the reaction solution for agarose gel electrophoresis detection, and if the digestion result is exactly the same as expected, it is the target recombinant plasmid. The recombinant plasmid was sequenced on a DNA automatic sequencer by the dideoxy terminal termination method, and the sequence determination was completed by Beijing Aoke Biotechnology Co., Ltd. Splicing was carried out with the ASSEMBLY program in the GeneTool1.0 software to obtain a DNA sequence (seeing SEQ ID NO: 1) with a length of 919bp. the

DNA sequence homology search and identification:

Through the BLAST (Basic Local Alignment Search Tool) software of the National Center for Biotechnology Information (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) website, the DNA sequences obtained after sequencing The known physiological function genes published in the GenBank database were compared for sequence homology to identify and obtain the functional information of the DNA sequence. The search results showed that the sequence homology between the measured sequence and human COPB1 gene DNA (GenBank accession number: NC 000011) was 77%. the

Embodiment 2, the localization of COPB1 gene

1. Primer sequences for gene mapping

COPB1: P-MF 5′-GTGTTTTGTGCATTGGAAGCA-3′

P-MR 5′-ACCACATCAACCATGAAACCA-3′

The length of the fragment amplified by the primers is 285bp. the

2. Experimental materials for gene mapping

Chromosome precise mapping was performed using the INRA-Minnesota porcine radiation hybrid panel (IMpRH) constructed by the University of Minnesota, France, which was donated by Dr. Martin Yerle (Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, France). the

The radiation dose used by IMpRH is 7,000-rad. IMpRH includes 118 pig × hamster radiation hybrid cell lines, as well as positive controls of hamster and pig genomic DNA. The identification results of 757 markers show that the average marker retention rate in IMpRH is 29.3%. It contains 128 linkage groups, covering 18 For autosomes and X chromosomes, the kb/cR ratio used to estimate the distance between markers is ~70kb/cR (1Ray=100cR), and the theoretical resolution is 145kb. Information on porcine chromosomes and chromosome fragments contained in each cell line can be obtained from WWW (http://www.toulouse.inra.fr/lgc/lgc.html/). the

3. PCR typing conditions

The total volume of the PCR reaction for amplification is 10 μl, in which the template DNA is 20ng, containing 1×buffer (Promega), 1.5mmol/L MgCl ₂ , the final concentration of dNTP is 150μmol/L, and the final concentration of primers is 0.4μmol/L, 2U Taq DNA polymerase (Promega). The PCR amplification program was: 94°C for 3min, 35 cycles of 94°C for 30s, 62°C for 30s, then 72°C for 20s, and finally 72°C for 5min. PCR reaction products were detected by 2% agarose gel electrophoresis. The IMPRH typing result is 0000010000 01000010001001101001000000000 10010010001010001010010010010010011 0000010001000101011001100 1101110 (of which 0 and 1 expresses the expansion results are negative and positive). Submit the above test results to the website ( http://www.toulouse.inra.fr/lgc/lgc.html/ ), statistical analysis results show that the COPB1 gene is closely linked to the gene PTH3 on pig chromosome 2, and the LOD value is 17.51 , the RH image distance is 0.2Ray.

Embodiment 3, establishment of PCR-RFLP diagnostic method

1. Primer sequence

COPB1P-SF 5′-GGGCTTACTGGACTCCAACAT-3′

P-SR 5′-TGGTCTTGATACATGTGTGAAACA-3′

2. PCR amplification conditions

The total volume of the PCR reaction is 20μl, including about 100ng of porcine genomic DNA, containing 1×buffer (Promega), 1.5mmol/L MgCl ₂ , the final concentration of dNTP is 150μmol/L, the final concentration of primers is 0.4μmol/L, and 2U Taq DNA polymerase (Promega). The PCR amplification program was: 94°C for 3min, 35 cycles of 94°C for 30s, 59°C for 30s, then 72°C for 20s, and finally 72°C for 5min. PCR reaction products were detected by 2% agarose gel electrophoresis. A 205bp specific amplified fragment was obtained, which was located in exon 22 (as shown in Figure 2). As a result of sequencing, it was found that there was a RsaI restriction site (GT↓AC) in the 205bp fragment, and the 28th bp was a polymorphic cutting site, which was located in exon 22.

3. PCR-RFLP detection conditions

The volume of PCR product digestion reaction is 10μl, including 1×buffer 1μl, PCR product 3-5μl, restriction endonuclease RsaI 0.3μl (10U), make up 10μl with H ₂ O, mix the sample and centrifuge at 37℃ Bath in water for 4 hours, use 2% agarose gel electrophoresis to detect the digestion result, record the genotype, and take pictures under ultraviolet light. The sequencing results of the two homozygotes at this site show that when the 28th bp position is T28, the RsaI restriction site does not exist, and the detection result after RsaI digestion is only 1 fragment with a length of 205 bp (determined as equal However, when there is a substitution of C28→T28, the result leads to the generation of a Rsal restriction site at the 28th bp, and two fragments are obtained, the lengths of which are 177bp and 28bp respectively (determined as allele G). The genotypes CC, CT, and TT are described in Figure 5.

4. Distribution of PCR-RFLP-RsaI polymorphism in various pig breeds

Six different Chinese and foreign pig breeds were detected by PCR-RFLP-RsaI: the pig breeds with foreign pig blood were Large White pig and Duroc pig, and the pig breeds with Chinese local pig blood were Tongcheng pig, Qingping pig, Dahua pig, etc. White pigs and small Meishan pigs. The genotype and gene frequency of the mutation site in different pig breeds are shown in Table 1. The results show that each genotype of the COPB1 gene is distributed in Tongcheng pigs, large white pigs, Xiaomeishan pigs, Large White pigs and Duroc pigs , no TT genotype was detected in Qingping pigs. The difference of genotype frequency among 6 different Chinese and foreign pig breeds by × ² test is shown in Table 1. The results show that there are certain differences in the distribution of gene frequency among the 6 different breeds.

Table 1: Genotype and gene frequency of COPB1 gene RsaI polymorphism in several pig breeds

Table 2: Difference test results of the distribution of pig COPB1 gene RsaI-RFLP polymorphism sites in different breeds

Note: ^** means P<0.01, ^* means P<0.05.

5. The application test of the molecular marker of the present invention in the correlation analysis of pig growth rate marker traits detected a total of 169 pig individuals: 27 purebred pigs from China, 25 purebred large white pigs, and 26 purebred Landrace pigs , the polymorphism of 48 Dachangtong (Dabai×(Changbai×Tongcheng)) and 43 Dachangtong (Landrace×(Largebai×Tongcheng)) hybrid pigs of Chinese local pigs and exotic pigs, and their genotypes were determined. And the genotype and production traits (average daily gain from birth to market, carcass straight length, carcass oblique length, leg-hip ratio, leg-hip bone ratio, suet percentage, sebum weight, water loss rate, intramuscular fat content, flesh color , eye muscle area, hip fat thickness) correlation analysis. Build a least squares model as described below:

y _ijk ＝μ+GENOTYPE _i +SEX _j +COMBINATION _k +ε _ijk , where y _ijk is the observed value of the trait, μ is the overall mean, GENOTYPE _i is the genotype effect, SEX _j is the gender effect, and COMBINATION _k is the combined Effect, ε _ijk is random error, assumed to obey N(0, σ ² ) distribution.

The results of genotype detection showed that among 169 individuals, there were 60 individuals with CC genotype, 72 individuals with CT genotype, and 37 individuals with TT genotype. The results of the association analysis between genotypes and traits are shown in Table 3. the

Table 3 Association analysis between different genotypes of COPB1 gene RsaI-RFLP polymorphism and some production traits

Note: ^** means P<0.01, ^* means P<0.05.

It can be seen from Table 3 that the SNP site of COPB1 gene has a significant effect on weight gain and carcass length from birth to market date (P<0.05), but this site has no significant effect on other production traits. the

Table 4 shows the least squares mean of the SNP site genotypes of the COPB1 gene that have a significant effect (P<0.05) on the weight gain from birth to the date of market and carcass length. the

Table 4: Least squares mean of average daily gain and carcass length of SNP loci (COPB1) genotypes from birth to market

Note: ^** means P<0.01, ^* means P<0.05.

Table 4 shows that the daily weight gain and carcass length of CT genotype individuals are significantly higher than those of CC genotype individuals, and the birth-to-market weight gain of TT genotype individuals is in the middle. Therefore, it can be seen that the growth rate of CT genotype individuals is faster. the

sequence listing

<110> Huazhong Agricultural University

<120> Cloning and application of a molecular marker related to growth rate as a marker-assisted selection for pigs

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gcactttctg gttattgtgg ctttatggca gccaacctct atcgaaaagc caattcagca 60

gggaccagag gcccctgtta ctggccacat aagaattcgt gcaaagagtc aggtaacact 120

tctttatttg agatgctctc ttaactagat aaaaagaaaa agaattttg tctctcttat 180

ttctgtatgt ggtatcgtac tagtgttttg tgcattggaa gcatatatat ttataataat 240

aatatccttg aatagaagta taattttcct tcattaaaat ggcccacttt gagggccttt 300

tgttctgatt tacttgctta aaatcagatt aattcctgtg gggtgtttgt gaaatggatt 360

cttcagagat tttcaaaatc caagctgcaa atctagtgac tttgtaaata tagcactaaa 420

agtacttaat acagtaatgt tatggtgagt ctattttgta tatccttggt ttcatggttg 480

atgtggtgtc attgaatggg ggcttggtta aaatagaata acctaccggt ataaatataa 540

acatatata aatatattat tatgtaataa taattataaa tatcttcatt ctttcttaca 600

gggaatggcc ttaagtcttg gagataaaat caacttgtct cagaagaaaa ctagtatata 660

agaataaaca agttcttgca gctttacagt taagatttag gtatgggtag gtatgggctt 720

actggactcc aacatctttt gtaytctttc atgcttta

tg ttatatagaa tctgagttca 780

tgctgaatgc attccagcca ataatttata gcctttccct taaatcaaga ttgattttaa 840

aattatagtt gtcttttgtc ttaacagttc tgaatgctgt cctcaaagta tataatgttt 900

cacacatgta tcaagacca 919

Claims (4)

1. molecule marker relevant as the pig marker assisted selection with the speed of growth; Its nucleotide sequence is shown in sequence table SEQ ID NO:1; The base mutation of a C744-T744 is arranged at the 744th bit base place of sequence shown in the sequence table SEQ ID NO:1, cause Rsa I-RFLP polymorphum.

2. it is right that test right requires the primer of 1 said base mutation, and its dna sequence dna is as follows:

Forward: 5 '-GGGCTTACTGGACTCCAACAT-3 ',

Oppositely: 5 '-TGGTCTTGATACATGTGTGAAACA-3 '.

3. the preparation method of a growth speed of pigs related molecular marker, according to following steps:

From the pig blood genome, extract DNA, personnel selection COPB1 gene cDNA is the information probe, does the homologous sequence screening, obtains the EST (EST) of homology more than 80%; Splice pig EST-contig then, unknown fragment in the middle of the design primer amplification, purifying, the clone, order-checking obtains like the described dna sequence dna of sequence table SEQ ID NO:1.

4. the application of the described molecule marker of claim 1 in the growth speed of pigs assisted Selection.

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