AU2020104202A4 - Application of a molecular marker assisted selection in number of piglets born alive - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention provides an application of a molecular marker assisted
selection in number of piglets born alive, the molecular marker is located
in the partial coding sequence of porcine DKK2 gene, and the nucleotide
sequence of the molecular marker is shown in SEQ ID NO.1. The
sequence length of the molecular marker is 631 bp, and there is a
mutation of A>G at 261 bp of the SEQ ID NO. 1. The invention also
provides a detection kit for identifying the SNP associated with number
of piglets born alive, which comprise the pair of primers shown in SEQ
ID NO.2- 3. The invention firstly discovered the molecular markers of
porcine DKK2 gene associated with number of piglets born alive, which
provide a new molecular breeding marker for marker assisted selection
breeding of piglets born alive trait.
Description
The invention relates to the field of the screening of molecular
markers, and in particular to the application of a molecular marker assisted
selection in number of piglets born alive.
Reproductive traits of sows are one of the important economic traits
in pigs. However, due to the low heritability of pigs, the conventional
selection of reproductive traits in sows proceeded slowly. With the rise and
rapid development of molecular biology technology, the combination of
conventional breeding and marker assisted selection (MAS) has been
developed, which can effectively speed up the selection progress of
selection in number of piglets born alive.
DKK2 belongs to DKK protein family and is a kind of secretory
glycoprotein. It plays a role by antagonizing WNT signaling pathway,
mainly including signal peptide sequence, coesterase folding and two
conserved domains rich in cysteine (Niehrs, 2006). The characterization of
SNPs associated with number of piglets born alive plays an important role
in marker assisted selection. Up to now, the reports related to the DKK2
gene as a molecular marker assisted selection are rare.
Any discussion of the prior art throughout the specification should in
no way be considered as an admission that such prior art is widely known
or forms part of common general knowledge in the field.
The object of the present invention is to overcome or ameliorate at
least one of the disadvantages of the prior art, or to provide a useful
alternative.
The invention relates to an application of a molecular marker assisted
selection in number of piglets born alive. The invention explored molecular
markers associated with the number of piglets born alive and provides a
new molecular marker for molecular marker assisted selection in number
of piglets born alive.
In one aspect, the present invention provides a molecular marker
associated with the number of piglets born alive, wherein the molecular
marker is located in the partial sequence of porcine DKK2 gene. The
nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is a mutation of A>G at 261 bp of the SEQ ID NO. 1, which showed AA, AG or GG genotypes, and the number of piglets born alive in
GG genotype is significantly lower than that of AA genotype and AG
genotype.
In another aspect, the present invention provides the use of the
molecular marker shown in SEQ ID NO. 1 assisted selection in number of
piglets born alive.
In another aspect, the present invention provides a pair of primers
comprising an upstream primer and a downstream primer for amplifying
the molecular markers defined above, the nucleotide sequence of the
upstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence
of the downstream primer is shown in SEQ ID NO. 3.
In another aspect, the present invention provides the use of the pair of
primers shown above assisted selection in number of piglets born alive.
In another aspect, the present invention provides a kit for detecting
SNP associated with number of piglets born alive, wherein the kit
comprises the pair of primers for amplifying the sequence of SEQ ID NO.
1, and the upstream primer is shown in SEQ ID NO. 2, the downstream
primer is shown in SEQ ID NO. 3, and the kit can be used in assisted
selection in number of piglets born alive.
In an embodiment, the kit also comprises 10 x buffer, dNTPs and
TaqDNA polymerase.
The invention explored molecular markers (8:g.114967878A>G)
associated with the number of piglets born alive in DKK2 gene, the
molecular markers located in the partial sequence of second intron of
porcine DKK2 gene, and the single nucleotide polymorphism can be used
as a molecular marker, and it provides a new molecular breeding marker
for marker assisted breeding of the number of piglets born alive.
In another aspect, the present invention provides use of a molecular
marker assisted selection in number of piglets born alive, wherein the
nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1,
and there is a mutation of A>G at 261 bp of the SEQ ID NO. 1, which
showed AA, AG or GG genotypes, and the number of piglets born alive in
GG genotype is significantly lower than that of AA genotype and AG
genotype.
In another aspect, the present invention provides use of a SNP detecting
kit assisted selection in number of piglets born alive, wherein the kit
comprises the pair of primers for amplifying the sequence of SEQ ID NO.
1, and the upstream primer is shown in SEQ ID NO. 2, the downstream
primer is shown in SEQ ID NO. 3.
Figure 1: The agarose gel electrophoresis map of the sequence of SEQ
ID NO. 1;
Figure 2: The Sanger sequencing results of 8:g.114967878A>G site
of porcine DKK2 gene, figure A is the AA genotype, figure B is the AG
genotype, figure C is the GG genotype.
Embodiment 1: Acquisition of SNP detection fragment of DKK2 gene
and establishment of the detection method of polymorphic sites
1. Extraction of porcine genomic DNA
The experimental pig variety of the invention is a new black pig
variety jointly cultivated by the Institute of Animal Science and Veterinary
Medicine, Hubei Academy of Agricultural Sciences and Hubei Tianzhili
High Quality Pig Breeding Co., Ltd. The porcine genomic DNA was
extracted by the animal tissue genomic DNA kit produced by Beijing
Qingke Xinye Biotechnology Co., Ltd according to the instructions.
2. Acquisition of SNP fragment of porcine DKK2 gene
(1) PCR amplification
Designed a pair of primers to amplify the fragment of a polymorphic
site according to the SNP marker sequence shown in SEQ ID NO. 1 of
DKK2 gene sequence (Gene ID: NC_010450.4), and the sequences of the
primers are shown as follows:
The upstream primer is: 5'ATAAGGAATGTGAAGTTGGGAGAT3'
(also shown in SEQ ID NO.2), and the downstream primer is: 5'
GCTTTGAGGAAGGCTAGGGA 3'(also shown in SEQ ID NO. 3).
PCR amplification was carried out by the genomic DNA of the new black
pig with the above primers. The PCR reaction system was 50 pL and it
comprised: template DNA 100 ng, 10xbuffer comprising Mg2 4 gL,
upstream primer 0.5 pM, downstream primer 0.5 pM, dNTPs 2.5 pM, 1U
TaqDNA polymerase.
The operation procedure of PCR comprises: pre-denaturation at 94°C
for 3min, denaturation at 94°C for 30s, annealing at 60°C for 30s,
extending at 72°C for 45s, 35 cycles; then extending at 72°C for 10min,
and maintaining at 4°C.
(2) Purification of PCR products
The PCR product was purified by Gel Extraction Kit of Sangon
Biotech (Shanghai) Co., Ltd, and the specific steps are shown in the
instructions of the kit.
3. Sanger sequencing for detection of molecular markers
The recovered PCR products were sent to Beijing AuGCT
Biotechnology Co., Ltd. for Sanger sequencing.
Embodiment 2: Polymorphism distribution detection of the molecular
marker in the new black pig
In this embodiment, the polymorphism of the 8: g.114967878A>G
located in the second intron of porcine DKK2 gene was detected in the new
black pigs, and the detection results are shown in Table 1.
Table Genotype frequency and allele frequency of 8:g.114967878A>G
site of DKK2 gene in new black pigs
Polymorphic Genotype Allele frequency Chi-square test Variety Number loci AA AG GG A G X1 P value
8:g.1149678 The new 417 332 83 2 0.90 0.10 1.77 0.18 78A>G black pigs
The results in Table 1 showed that: in the new black pig, the
8:g.114967878A>G site of DKK2 gene showed three genotypes: AA, AG
or GG, and the frequency of A allele was 0.9, which was the dominant
allele. Chi-square test showed that the 8:g.114967878A>G site in the new
black pig obeyed Hardy-Weinberg equilibrium (P>0.05).
Embodiment 3: Correlation analysis between molecular markers of
the invention and the number of piglets born alive and its application
In order to determine the correlation between 8:g.114967878A>G site
of DKK2 gene and the difference of the number of piglets born alive, the
method established in embodiment 1 was used to detect the polymorphism,
and the correlation between the different genotypes of 8:g.114967878A>G
site of DKK2 gene and total number of piglets born and number of piglets
born alive was analyzed. The GLM program of SAS software (SAS
Institute Inc, Version 9.1) was used to analyze the variance of different SNP
genotypes, and apply the significant test. The model used was as follows:
Yij=p+Gi+Fj+eij
Wherein, Yij was the phenotypic value of traits, p was the average
value, Gi was the genotype effect (comprising the additive gene effect and
the dominant effect; in the additive gene effect, 1, 0 and -1 represent AA,
AG and GG genotypes respectively; in the dominant effect, 1, -1 and 1
represent AA, AG and GG genotypes respectively); Fj was the
comprehensive effect of pig farm, and the eij was the random error.
The correlation analysis between different genotypes and total
number of piglets born and number of piglets born alive was carried out in
new black pig, and the statistical analysis results are shown in Table 2.
Table2 Correlation analysis results between 8:g.114967878A>G site of
DKK2 gene and the number of piglets born alive
Genotype Value of effect Traits AA AG GG additive gene dominant effect effect Number 332 83 2
total number of 11.83+0.17 11.66+0.33 8.50+2.13 0.08+0.19 1.62+1.07 piglets born
number of piglets 10 . 8 1±0. 15b 10 .8 4 ±0. 2 9b 7.00±1.90a -0.02+0.16 1.91+0.95* born alive Notes: a and b indicate significant difference(P<0.05), * indicate significant difference(P<0.05)
The table 2 showed that in 8:g.114967878A>G site, the number of
piglets born alive in GG genotype was significantly lower than that of AA
genotype and AG genotype (P<0.05), and the dominant effect reached to
significant level (P<0.05), but the SNP site had no significant correlation
with the total number of piglets born in the new black pigs.
Throughout the description and claims of the specification, the word
"comprise" and variations of the word, such as "comprising" and
"comprises", is not intended to exclude other additives, components,
integers or steps.
The above embodiments are only preferred embodiments of the
invention and do not limit the invention. Any modification, equivalent
replacement, improvement, etc. made within the spirit and principles of
the invention shall be included in the protection scope of the invention.
Claims (4)
1. Use of a molecular marker assisted selection in number of piglets
born alive, wherein the nucleotide sequence of the molecular marker is
shown in SEQ ID NO. 1, and there is a mutation of A>G at 261 bp of the
SEQ ID NO. 1, which showed AA, AG or GG genotypes, and the number
of piglets born alive in GG genotype is significantly lower than that of AA
genotype and AG genotype.
2. The use according to claim 1, wherein a pair of primers comprising
an upstream primer and a downstream primer for amplifying the molecular
markers defined in claim 1, the nucleotide sequence of the upstream primer
is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream
primer is shown in SEQ ID NO. 3.
3. Use of a SNP detecting kit assisted selection in number of piglets
born alive, wherein the kit comprises the pair of primers for amplifying the
sequence of SEQ ID NO. 1, and the upstream primer is shown in SEQ ID
NO. 2, the downstream primer is shown in SEQ ID NO. 3.
4. The use according to claim 3, wherein the kit also comprises 10 x
buffer, dNTPs and TaqDNA polymerase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019113386718 | 2019-12-23 | ||
| CN201911338671.8A CN110846422B (en) | 2019-12-23 | 2019-12-23 | Molecular marker associated with pig number of live piglets and application thereof |
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| AU2020104202A4 true AU2020104202A4 (en) | 2021-03-11 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801946A (en) * | 2021-11-02 | 2021-12-17 | 吉林省农业科学院 | Molecular marker influencing sow reproductive performance, detection method and application thereof |
| CN114134236A (en) * | 2021-12-07 | 2022-03-04 | 中国农业科学院北京畜牧兽医研究所 | Application of reagent for detecting SNP molecular marker in goat RBP4 genotyping and/or goat molecular marker assisted breeding |
| CN117757959A (en) * | 2024-02-22 | 2024-03-26 | 海南省农业科学院三亚研究院(海南省实验动物研究中心) | SNP molecular marker related to sow dystocia traits and application thereof |
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| CN112011623B (en) * | 2020-08-25 | 2022-05-10 | 华中农业大学 | Molecular marker related to sow live litter size and effective live litter size traits |
| CN111996264B (en) * | 2020-09-17 | 2022-05-17 | 湖北省农业科学院畜牧兽医研究所 | Application of pig SNP molecular marker in pig breeding character screening and pig breeding |
| CN112126688B (en) * | 2020-09-17 | 2022-05-17 | 湖北省农业科学院畜牧兽医研究所 | Multiple allele molecular marker related to reproductive traits in pig OLR1 gene and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313156B (en) * | 2014-10-27 | 2016-06-29 | 湖北省农业科学院畜牧兽医研究所 | The genetic marker of pig number born character and application |
| CN105671172A (en) * | 2016-03-14 | 2016-06-15 | 江苏农林职业技术学院 | Molecular marker relevant with reproduction traits of pigs and detection method and application thereof |
-
2019
- 2019-12-23 CN CN201911338671.8A patent/CN110846422B/en active Active
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- 2020-12-21 AU AU2020104202A patent/AU2020104202A4/en not_active Ceased
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113801946A (en) * | 2021-11-02 | 2021-12-17 | 吉林省农业科学院 | Molecular marker influencing sow reproductive performance, detection method and application thereof |
| CN113801946B (en) * | 2021-11-02 | 2023-06-20 | 吉林省农业科学院 | Molecular marker affecting reproductive performance of sow, detection method and application thereof |
| CN114134236A (en) * | 2021-12-07 | 2022-03-04 | 中国农业科学院北京畜牧兽医研究所 | Application of reagent for detecting SNP molecular marker in goat RBP4 genotyping and/or goat molecular marker assisted breeding |
| CN114134236B (en) * | 2021-12-07 | 2023-11-21 | 中国农业科学院北京畜牧兽医研究所 | Application of reagent for detecting SNP molecular markers in goat RBP4 genotyping and/or goat molecular marker assisted breeding |
| CN117757959A (en) * | 2024-02-22 | 2024-03-26 | 海南省农业科学院三亚研究院(海南省实验动物研究中心) | SNP molecular marker related to sow dystocia traits and application thereof |
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| Publication number | Publication date |
|---|---|
| CN110846422A (en) | 2020-02-28 |
| CN110846422B (en) | 2020-12-25 |
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