CN110122415B - Breeding method of small pig closed group for high inbred experiment - Google Patents
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Abstract
The invention discloses a breeding method of a small pig closed group for a high inbreeding experiment, which comprises the following steps: A) mating and breeding the male parent and the female parent of the miniature pig in the F0 generation to obtain an F1 generation, and removing bad individuals; B) detecting the OB gene of the F1 generation miniature pig which is not rejected in the step A), eliminating the miniature pig with the BB genotype, and reserving the miniature pig with the AA and AB genotypes for breeding; C) carrying out gene purity detection on the F1 generation reserved in the step B), and reserving the seeds of the breeding boars and the breeding sows according with the inbreeding coefficient range and the genome purity range; D) c), carrying out sibling mating breeding on the reserved F1 generation in the step C) to breed an F2 generation, repeatedly removing bad individuals, and repeating the step B) and the step C); E) and D) repeating the step D) until the number of the closed groups is stable. The invention establishes a breeding method for breeding the small pig closed group for the high inbreeding experiment, and the bred small pig has high inbreeding coefficient and high gene purity.
Description
Technical Field
The invention belongs to the technical field of livestock breeding, and particularly relates to a breeding method of a small pig closed group for a high inbreeding experiment.
Background
In the eighties of the last century, the application of miniature pigs in biomedical research has been tried abroad, the miniature pigs are systematically researched to replace monkeys and dogs to become new experimental small animals, and the comparison system discusses the important position of the miniature pigs as experimental animal models in biomedical research. The miniature pig has greater similarity with human in the aspects of digestive system, skeletal system, skin system and the like, can solve the problem of human organ deficiency, can avoid animal welfare factors, and is a research hotspot for human xenograft development.
In recent years, the biomedical development of China is faster and faster, and the demand of small experimental pigs is gradually increased and urgent. Meanwhile, the application of the experimental miniature pig is more and more extensive, such as human blood type transformation, skin transplantation and the like, and higher requirements are also put forward on the standardization of the experimental miniature pig. Although the breeds of the miniature pigs in China are very rich, most of the breeds are local breeds and are bred naturally for a long time to form local breeds with local characteristics. The pigs of the breeds have the characteristics of the pigs, the hybridization degree is very large, and even the hair color is not uniform. For example, the Hainan Wuzhishan miniature pig has white, black and color hair. The inbred coefficients of the miniature pigs are not high, and the requirements of experimental animals on the inbred coefficients and the high homozygosity of genes are not met, so that the experimental miniature pigs with high inbreeding degree can be bred, and the experimental miniature pigs with high inbred coefficients and high gene homozygosity can be provided for the fields of biomedicine, life science and the like.
Therefore, it is necessary to breed inbred lines and closed groups of the miniature pigs for domestic high-grade inbred experiments, and develop miniature pig resources from multiple aspects such as genetics, microbiology, environment, nutritional standards and the like.
Disclosure of Invention
One of the effective methods for achieving the purpose is to adopt an F0 generation male parent and a female parent to breed an F1 generation, an F1 generation breeds an F2 generation through sib mating, and F2-F15 generations breed groups through sib mating. In each generation, partial bad individuals are eliminated from the traditional breeding perspective, then the breed reserving AA and AB gene type miniature pigs are detected through OB genes, the inbreeding coefficient and the genome purity are calculated, individuals with the inbreeding coefficient range of 0.15-0.32 and the genome purity range of 53% -67% are removed, and the individuals are not included in the range of breeding boars and breeding sows. And repeating breeding and screening until the number of the closed groups is stable.
The invention relates to a breeding method for breeding a small pig closed group for a high inbreeding experiment, which comprises the following steps:
A) mating and breeding the male parent and the female parent of the miniature pig in the F0 generation to obtain an F1 generation, and removing bad individuals;
B) detecting the OB gene of the F1 generation miniature pig which is not rejected in the step A), eliminating the miniature pig with the BB genotype, and reserving the miniature pig with the AA and AB genotypes for breeding;
C) carrying out gene purity detection on the F1 generation reserved in the step B), and reserving the seeds of the breeding boars and the breeding sows according with the inbreeding coefficient range and the genome purity range;
D) c), carrying out sibling mating breeding on the reserved F1 generation in the step C) to breed an F2 generation, repeatedly removing bad individuals, and repeating the step B) and the step C);
E) and D) repeating the step D) until the number of the closed groups is stable.
According to the invention, in the step B), the genotype of the miniature pig is detected by adopting a PCR-RFLP method.
According to the invention, in the step B), a primer pair consisting of the nucleotide sequence of the sequence 1 and the nucleotide sequence of the sequence 2 in the sequence table 1 is utilized for PCR amplification, and a PCR product is detected.
According to the invention, in step B), miniature pigs of the AA genotype are reserved.
According to the invention, the formula for calculating the inbreeding coefficient is as follows:
wherein the content of the first and second substances,
inbred coefficient Froh: the sum of the lengths of long homozygous fragments (ROH) on the genome accounts for the total length of the genome;
ROHk length: sum of length of kth ROH.
According to the invention, the calculation formula of the genome homozygosity is as follows:
wherein the content of the first and second substances,
genome homozygosity Fhom, the proportion of homozygotes in Single Nucleotide Polymorphism (SNP) detected by an individual;
n: the number of detected SNPs;
nhom: number of homozygous SNPs.
Preferably, in the step C), individuals with the inbreeding coefficient ranging from 0.15 to 0.32 and the genome purity ranging from 53% to 67% are reserved as breeding boars and breeding sows.
Preferably, the step C) further comprises a method for judging whether inbreeding exists; the criterion for judging whether inbreeding exists is as follows: the heterozygosity is observed to be less than the desired heterozygosity.
Preferably, the method further comprises the step of recording production performance indexes of each generation, wherein the production performance indexes comprise the number of born pigs, the number of litters, the number of dead pigs, the number of mummy pigs, the number of average born pigs, the survival rate of piglets, the average birth weight and the average 6-month-old weight.
Preferably, in step a), the rejecting of the poor individual refers to: removing weak young, variegated and oversize individuals.
According to the invention, in step E), the number of closed groups is stabilized at about 100.
The invention achieves the positive improvement effects that:
the invention establishes a breeding method for breeding the small pig closed group for the high inbred experiment, can effectively realize the breeding of the small pig inbred line and the closed group for the high inbred experiment, has high inbred coefficient and high gene purity of the bred small pig, and can meet the increasing and urgent requirements of biomedicine on the small pig for the experiment, thereby promoting the development of the biomedicine in China.
Drawings
FIGS. 1, 2 and 3 show the results of detecting RFLP-PCR products of a third generation partial sample by agarose gel electrophoresis, wherein:
FIG. 1 shows the results of digestion of samples 01-10 in the order from right to left;
FIG. 2 shows the results of digestion of samples 11-20 in the order from left to right;
FIG. 3 shows the results of digestion of samples 21-30 in order from right to left;
FIG. 4 is a graph of the inbreeding coefficient and genomic purity of 20 samples from the third generation significant population, with the abscissa representing the sample number, the left ordinate representing the genomic purity Fhom, and the right ordinate representing the inbreeding coefficient Froh.
Detailed Description
Example 1 OB Gene detection method
Detecting the genotypes of multiple sample OB genes of the core group in each generation by the following specific method:
(1) and sampling of a miniature pig sample for each experiment: 0.3mg of porcine ear tissue, which was put into a tissue crusher to be crushed, the crushed tissue sample was put into a 1.5mL EP tube, tissue DNA was extracted using a blood/cell/tissue genomic DNA extraction kit of Tiangen, and the samples were labeled separately.
(2) And designing a primer:
OB-F：5′-GAG CCA ACA TCT CTC TCG CTG AG-3′
OB-R：5′-GAC TCC TGG AAG CTC AGG TTT CTT C-3′
the PCR reaction system was configured according to the parameters of Table 1:
TABLE 1 PCR reaction System
In table 1, 2X Master mix is a commercially available product, purchased from azyme in this example, cat #: and P112.
PCR amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
(3) And preparing a 1.0% agarose gel detection PCR product.
The cleavage reaction system is shown in Table 2.
TABLE 2 digestion reaction System
Agarose gel electrophoresis detection conditions: preparing 3% agarose gel, voltage 120V, time 12 min.
The genotype judgment is carried out according to agarose gel electrophoresis detection bands of the samples. For example, agarose gel electrophoresis is used for detecting the bands, and the sample has three bands which are AB genotypes; one band, AA genotype; two bands, BB genotype.
References to methods for genotyping: Zhi-Hua Jiang, John P.Gibson. genetic polymorphisms in the leptin gene and the upper association with a failure in the four fiber chains [ J ]. Mammalian Genome 10, 191-193 (1999).
Example 2 Gene homology detection method
(1) Calculating the effective population content (Ne) of a certain generation, the polymorphic marker Proportion (PN) of the population, the expected heterozygosity (He) of the population and the observed heterozygosity (Ho). If the observed heterozygosity of the generation is lower than the desired heterozygosity, the population is presumed to have inbred.
The effective population content (Ne), the polymorphic marker ratio (PN) of the population, the expected heterozygosity (He) of the population and the observed heterozygosity (Ho) are conventional techniques in the field, the calculation of the indexes can be realized by sample sequencing and then directly calculated by software, the common software comprises Popgene, GenAlEx and the like, and the calculation is realized by Plunk software.
(2) And in order to further research the inbreeding degree of the population, calculating the inbreeding coefficient of the individual by utilizing the Plink software, and calculating the purity of the genome, wherein the range formula of the inbreeding coefficient is as follows:
wherein the content of the first and second substances,
froh: the sum of the lengths of the ROH fragments accounts for the total length of the genome.
ROHk length: length of kth ROH.
Reference for calculation of inbred coefficients: mcquilan, r.; LEUTENEGGER, a. -l.; abderraran, r.; FRANKLIN, c.s.; PERICIC, M.; BARACLAUC, l.; SMOLEJ-NARANCIC, n.; JANICIJEVIC, b.; POLASEK, o.; TENESA, a.; MACLEOD, a.k.; FARRINGTON, s.m.; RUDAN, p.; HAYWARD, c.; vtart, v.; RUDAN, i.; WILD, s.h.; DUNLOP, m.g.; WRIGHT, a.f.; CAMPBELL, h.; WILSON, J.F. Runs of homozygosis in European publications. American Journal of Human Genetics, v.83, p.359-372,2008.DOI:10.1016/j.ajhg.2008.08.007.
The gene homozygosity range formula is:
wherein the content of the first and second substances,
fhom is the proportion of homozygotes in the SNP detected by the individual.
N: the number of SNPs detected.
Nhom: number of homozygous SNPs.
Reference to calculation of genome homologies: YANG, j.; LEE, s.h.; GODDARD, m.e.; VISCHER, P.M.GCTA: a tool for genome-wide complex track analysis, American Journal of Human Genetics, v.88, p.76-82,2011.DOI:10.1016/j.ajhg.2010.11.011.
Example 3 Breeding method of miniature pig closed group for high inbred experiment
The embodiment specifically provides a breeding method of a small pig closed group for a high inbreeding experiment, which comprises the following steps:
A) f1 generation F0 generation one male two female breeding
The F0 generation of the closed group is originated from one male two female miniature pigs introduced in Guangxi, the hair color is black at two ends, white in the middle, short and small in size, short and short in head, long in the mouth, straight in nose bridge, early in sexual maturity and strong in reproductive capacity. F0 generation is F1 after male and female mating, production performance indexes such as number of born seeds, number of nests, number of dead fetus and number of mummy fetus are recorded, and weak young, mottled and over-large body types are removed.
F0: 1 male and female and 2 male.
B) Performing OB gene detection on the F1 generation miniature pigs, eliminating miniature pigs with BB genotype, and reserving seeds with AA and AB genotypes, wherein the AA genotype is reserved preferentially; 30 sows are kept in the core group, and not less than 6 boars are kept.
C) Detecting the gene purity of the F1 generation miniature pig reserved in the step B), sequencing the whole genome, calculating the purity of the genome, and removing individuals with the inbreeding coefficient range of 0.15-0.32 and the genome purity range of 53-67%, wherein the individuals are not included in the range of the male pig and the female pig;
D) c), carrying out sibling mating breeding on the reserved F1 generation in the step C) to breed an F2 generation, repeatedly removing weak young, variegated and overlarge body types, and repeating the step B) and the step C);
E) and D) repeating the step D) until the number of the closed groups is stabilized to be about 100.
Through the method, the number of the small pig closed groups for the experiment at present is 127, the core group is 35 sows and 6 boars, and the breeding stage of the 16 th generation is carried out. To date, 15 generation population production reproductive performance data is shown in table 3:
TABLE 3, 15 Generation population production reproductive Performance data
As can be seen from Table 3, the survival rate of the enclosed group of miniature pigs is high and all indexes are stable when the pigs are bred to the 15 th generation.
The third generation is taken as an example to concretely describe the application of the gene detection and the gene purity degree detection method in the breeding and screening process of the miniature pig.
(1) OB Gene detection
The gene testing was performed on 30 samples of the third generation core population using the method of example 1. The results of agarose gel electrophoresis are shown in FIGS. 1, 2 and 3.
The columns with many bands at the far right of FIG. 1, far left of FIG. 2, and far right of FIG. 3 are standard protein molecular weights (DNA markers). Placing samples according to a marker in the sample sequence, wherein the marker is on the right, and the sample sequence is from right to left; marker is on the left, sample sequence is from left to right.
The genotype judgment is carried out according to the number of the detection bands of the sample. Three bands, shown as samples 1-3 in FIG. 1, were of the AB genotype; sample 4 in FIG. 1 had a band with the AA genotype; in FIG. 2, there are two bands in samples 11 and 12, which are of the BB genotype. The genotype of the 30 samples from the third generation core population is shown in table 4:
TABLE 4 genotypes of 30 samples of the third Generation core group
In Table 4, miniature pigs of BB genotype were culled, and AA, AB genotypes were reserved for subsequent detection of gene-purity.
(2) Gene homozygosity detection
Calculating the effective population content (Ne) of the AA and AB genotype miniature pigs reserved in the step (1), the polymorphism marker ratio (PN) of the population, the expected heterozygosity (He) of the population and the observed heterozygosity (Ho), and if the observed heterozygosity is 0.373 and is lower than the expected heterozygosity by 0.491, the effective population of the third generation is presumed to have inbreeding.
The inbreeding coefficients of the individuals were calculated using Plink software and the inbreeding coefficients and genomic homologies were determined for 20 samples in the third generation significant population as shown in table 5:
TABLE 5 inbreeding coefficient
The inbreeding coefficient and genomic homozygosity for 20 samples in the third generation significant population are shown in figure 4. From the results of Table 5 and FIG. 4, it can be seen that the inbreeding coefficient of the third generation ranged from 0.15 to 0.32, and the genomic purity ranged from 53% to 67%. Individuals outside this range were excluded and not included in the boar and sow range.
Sequence listing
<110> Shanghai city academy of agricultural sciences
Breeding method of small pig closed group for <120> high inbred experiment
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> miniature pig (Sus scrofa)
<400> 1
gagccaacat ctctctcgct gag 23
<210> 2
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<212> DNA
<213> miniature pig (Sus scrofa)
<400> 2
gactcctgga agctcaggtt tcttc 25
Claims (9)
1. The breeding method of the small pig closed group for the high inbreeding experiment comprises the following steps:
A) mating and breeding the male parent and the female parent of the miniature pig in the F0 generation to obtain an F1 generation, and removing bad individuals;
B) detecting the OB gene of the F1 generation miniature pig which is not rejected in the step A), eliminating the miniature pig with the BB genotype, and reserving the miniature pig with the AA and AB genotypes for breeding;
C) carrying out gene purity detection on the F1 generation reserved in the step B), and reserving the seeds of the breeding boars and the breeding sows according with the inbreeding coefficient range and the genome purity range; specifically, individuals with an inbred coefficient range of 0.15-0.32 and a genome purity range of 53% -67% are reserved as breeding boars and breeding sows;
D) c), carrying out sibling mating breeding on the reserved F1 generation in the step C) to breed an F2 generation, repeatedly removing bad individuals, and repeating the step B) and the step C);
E) and D) repeating the step D) until the number of the closed groups is stable.
2. The breeding method as claimed in claim 1, characterized in that: and in the step B), detecting the genotype of the miniature pig by adopting a PCR-RFLP method.
3. The breeding method as claimed in claim 2, characterized in that: in the step B), a primer pair consisting of the nucleotide sequence of the sequence 1 and the nucleotide sequence of the sequence 2 in the sequence table 1 is utilized for PCR amplification, and a PCR enzyme digestion product is detected.
4. The breeding method as claimed in claim 1, characterized in that: and B), reserving the AA genotype miniature pig.
5. The breeding method as claimed in claim 1, characterized in that: the calculation formula of the inbreeding coefficient is as follows:
wherein the content of the first and second substances,
froh: the sum of the lengths of the ROH fragments accounts for the total length of the genome;
ROHk length: length of kth ROH.
6. The breeding method as claimed in claim 1, characterized in that: the calculation formula of the genome purity is as follows:
wherein the content of the first and second substances,
fhom is the proportion of homozygotes in the SNP detected by an individual;
n: the number of detected SNPs;
nhom: number of homozygous SNPs.
7. The breeding method as claimed in claim 1, characterized in that: the step C) also comprises a method for judging whether inbreeding exists; the criterion for judging whether inbreeding exists is as follows: the heterozygosity is observed to be less than the desired heterozygosity.
8. The breeding method as claimed in claim 1, characterized in that: the method also comprises the step of recording the production performance indexes of each generation, wherein the production performance indexes comprise the number of born piglets, the number of litters, the number of dead births, the number of mummy births, the number of average born piglets, the survival rate of piglets, the average birth weight and the average 6-month-old weight.
9. The breeding method as claimed in any one of claims 1 to 8, wherein: in the step A), the rejecting of the poor individuals refers to: removing weak young, variegated and oversize individuals.
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