CN117867133A - Application of PDGFD gene upstream SNP marker in sheep variety tail type selection - Google Patents
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Abstract
The invention discloses an application of an upstream SNP marker of a PDGFD gene in selection of sheep breeder breeds and breeds. The SNP marker is positioned at 3732399bp of chromosome 15 on the upstream of the sheep PDGFD gene, the mutation of the SNP locus is base substitution of G/A, the mutation affects the tail type of sheep individuals, genotype AA is obviously related to the fat tail characters of the individuals, and genotype GG is obviously related to the lean tail characters of the individuals. The identification of the invention obtains the SNP mark related to the tail type of sheep variety, which is beneficial to quickening the breeding of lean tail sheep.
Description
Technical Field
The invention belongs to the field of biotechnology and livestock breeding, and relates to parting detection of a Single Nucleotide Polymorphism (SNP) locus on the upstream of a sheep PDGFD gene and application thereof in sheep tail type character marker assisted selection and molecular breeding.
Background
Ancestors of sheep (Ovis aries) are thin-tailed sheep, which after domestication, gradually develop sheep breeds with different tail phenotype (i.e. tail type) patterns through long-term natural selection and artificial selection in order to adapt to different environments. As the human diet tends to "lean and anaerobic", the economic value of sheep tail fat gradually decreases. In addition, as the fat of the fat tail sheep is excessively high, the feed conversion rate is reduced, and the feeding cost is increased due to the large consumption of the feed; the mating and delivery are difficult due to the fat tail, and the intensive cultivation of sheep is restricted.
31 of 42 local varieties recorded in Chinese livestock and poultry genetic resources Zhi-Yangzhi (2011) belong to sheep with fat tails, and particularly sheep breeding provinces such as inner Mongolia, xinjiang, shandong, gansu, henan and the like are mainly sheep with fat tails. For the main variety types of local sheep and mutton sheep breeding, the improvement of 'tail thinning' of the fat tail is realized, the production performance of the variety is improved, and the method becomes the key point of sheep breeding work.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to a polymorphism in the DNA sequence that results from a single nucleotide base variation at the genomic DNA level. With the intensive research of SNP, SNP markers are widely used as third generation genetic markers in breeding technologies such as marker-assisted selection. After candidate genes are determined, the sheep population which not only maintains the excellent character of the variety but also meets the tail type improvement requirement from the fat tail to the lean tail can be established by utilizing the selected individuals through directly selecting the lean tail character of the fat tail sheep or the filial generation thereof at the molecular level, so that the method is very beneficial to relieving the limitation of the intensive breeding of the fat tail sheep variety.
The sheep PDGFD (Platelet-derived growth factor D) gene is located on chromosome 15. PDGFD, also known as platelet derived growth factor, is an alkaline protein stored in platelet alpha particles. It has been shown that PDGF-beta signaling, a PDGFD gene receptor, inhibits fat precursor cell differentiation by reducing two major regulatory factors for fat differentiation (PPARgamma, C/EBPalpha), i.e., PDGFD genes are associated with animal fat deposition. The research on the influence of the PDGFD gene on the tail fat deposition of the sheep indicates that the PDGFD gene is one of key candidate genes of sheep tail size characteristics. However, the problems to be solved are: the lack of molecular markers which can be used for rapidly establishing stable inheritance of lean-tail sheep populations with a simple and easy detection procedure.
Disclosure of Invention
The invention aims to provide an application of an upstream SNP marker of a PDGFD gene in tail type selection of sheep breeds. The SNP marker provided by the invention is not limited by sheep age, sex and the like, and can be used for assisting in screening sheep individuals with lean tail characters, thereby promoting the breeding improvement of local sheep and mutton sheep.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an application of a single nucleotide polymorphism site on the upstream of a sheep PDGFD gene in sheep molecular marker assisted selective breeding, wherein the single nucleotide polymorphism site on the upstream of the PDGFD gene is positioned at 3732399bp of chromosome 15 of a sheep reference genome (the version of the sheep reference genome is ARS-UI_Ramb_v2.0), and the polymorphism of the single nucleotide polymorphism site is G/A.
Preferably, the polymorphism of the single nucleotide polymorphic site upstream of the PDGFD gene affects the tail form of sheep individuals.
Preferably, the GG genotype of the single nucleotide polymorphism site at the upstream of the PDGFD gene is a molecular marker (specifically, a single nucleotide polymorphism marker, namely, SNP marker) related to sheep lean tail traits.
A KASP primer for detecting a single nucleotide polymorphism site upstream of sheep PDGFD gene, the KASP primer comprising a universal primer, a wild type site-specific primer having FAM adaptor and a mutant site-specific primer having HEX adaptor, the wild type site-specific primer and the mutant site-specific primer being competitive upstream primers having 3' terminal bases identical to alleles G and a of single nucleotide polymorphism site located at 3732399bp of chromosome 15 of sheep reference genome (ARS-ui_ramb_v2.0), respectively, the universal primer being downstream primer (FAM adaptor added to the wild type site-specific primer shows red fluorescence in KASP detection, and HEX adaptor added to the mutant site-specific primer shows blue fluorescence in KASP detection).
Preferably, the KASP primer specifically comprises:
upstream primer 1-FAM:5'-gaaggtgaccaagttcatgctTTGTCTAGGAGGACTTACAAATCG-3' (FAM joint lower case letter)
Upstream primer 2-HEX:5'-gaaggtcggagtcaacggattTTGTCTAGGAGGACTTACAAATCA-3' (HEX joint lower case letter)
A downstream primer: 5'-TTGACTTCCAACTCAAGGTCTTCT-3'.
A method for detecting a single nucleotide polymorphism upstream of a PDGFD gene in sheep, the method comprising the steps of:
the genome DNA of the sheep individual to be detected is used as a template, partial fragments containing the single nucleotide polymorphism site at the upstream of the PDGFD gene are amplified through the KASP primer, and then the genotype of the sheep individual to be detected at the single nucleotide polymorphism site is identified through fluorescence detection, wherein the single nucleotide polymorphism site at the upstream of the PDGFD gene is positioned at 3732399bp of chromosome 15 of sheep reference genome (ARS-UI_Ramb_v2.0).
Preferably, the 3 genotypes of the single nucleotide polymorphism sites upstream of the PDGFD gene are determined from the discrimination of the detected fluorescence: GG is red, AG is green, AA is blue.
Preferably, the sheep is selected from any one of a fat tail sheep (e.g. Bonga sheep, kido sheep, kefis sheep, aletai sheep, bashbia sheep, billow sheep, hu sheep, large tail han sheep) or a hybrid sheep of the fat tail sheep and a lean tail sheep (e.g. dorifen sheep, merino sheep, euler Tibetan sheep, grassland Tibetan sheep, safuke sheep, yunnan sheep).
Preferably, the detection method can detect the molecular marker related to sheep lean tail character based on KASP technology, and specifically comprises the following steps:
(1) Extracting genome DNA of sheep individuals to be detected;
(2) Performing PCR amplification by using the extracted genome DNA as a template and using designed specific primers (namely the three KASP primers);
(3) Genotyping after amplification is finished, and analyzing the genotype of the sheep individual to be detected (by using two upstream primers with different fluorescent joints and one universal primer, introducing different fluorescent signals into different amplification products, and judging the genotype by detecting the fluorescent signals);
(4) Judging the tail type of the sheep individual to be tested (specifically judging the fat tail or lean tail character) according to different genotypes: when the genotype at 3732399bp of chromosome 15 is GG, sheep individuals can show lean tails; sheep individuals will appear to be fat tails when the genotype at 3732399bp of chromosome 15 is AA.
The method for detecting the single nucleotide polymorphism of the sheep PDGFD gene upstream is applied to sheep molecular marker assisted selective breeding.
Preferably, the different genotypes of the single nucleotide polymorphism sites upstream of the PDGFD gene are significantly related to sheep tail type (such as tail root width and tail root perimeter), wherein the GG genotype is a SNP marker related to sheep lean tail trait, and the other homozygous genotype, i.e., AA genotype, is a SNP marker related to sheep fat tail trait.
The beneficial effects of the invention are as follows:
the invention discovers and identifies the upstream SNP locus of the PDGFD gene (namely 3732399 locus of chromosome 15 of sheep reference genome ARS-UI_Ramb_v2.0) obviously related to sheep tail type through the detection analysis of single nucleotide mutation on the upstream of the sheep PDGFD gene, and provides a detection method for identifying molecular markers related to sheep thin tail traits, and the sheep population with stable inheritance formed by thin tail trait individuals can be quickly established through the detection of sheep with thin tail traits, so that genetic sample basis is provided for sheep variety improvement, the variety cultivation period is shortened, the variety cultivation process is accelerated, and the accuracy of seed selection is also improved.
Furthermore, the SNP locus (namely 3732399 locus of sheep reference genome ARS-UI_Ramb_v2.0 chromosome 15) identified by the invention has SNP markers related to tail part thin-tail character in the hybridization population of local sheep varieties (for example, hu sheep and the like are taken as parents to be improved), which is beneficial to accelerating the breeding of local thin-tail sheep.
Drawings
FIG. 1 is a general technical flowchart of SNP detection in an embodiment of the invention.
FIG. 2 is an electrophoretogram of sample genomic DNA quality control; in the figure: 1.8 and 16 are sample numbers, and M is Marker.
FIG. 3 is a graph showing the result of KASP typing at position 3732399 of chromosome 15 (genotypes GG, AG, AA) of a offspring sample of a hybridization between Hu sheep and Dongfolisheng sheep; in the figure: NTC is no template control.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. It should be noted that the following examples are given for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.
(one) mining single nucleotide polymorphism sites related to sheep tail type on sheep chromosome 15
According to haplotype analysis of the sheep resequencing dataset, it is found that sheep breeds mainly comprising feverfew and sheep breeds mainly comprising leptin have different haplotypes in the upstream region of PDGFD gene of chromosome 15, and there is a significant allele frequency difference at the 3732399bp isosite of chromosome 15. The specific description is as follows.
The results of resequencing of the 14 breeds including 11 Bonga sheep, 10 Kido sheep, 13 Kefis sheep, 32 alentai sheep, 19 bash-kibe sheep, 26 multi-wave sheep, 45 hu sheep, 10 large tail han sheep, 54 dorifen sheep, 10 merino sheep, 14 euler Tibetan sheep, 10 grassland Tibetan sheep, 12 safoke sheep, 40 Yunnan sheep were obtained in the experiment (both the eastern fei sheep and the euler Tibetan sheep were collected at the national university of littoral in Shanxi of salt-Yang in 2021, and the rest of the sample resequencing data were publicly available in NCBI SRA databases, see Table 1). The 14 breeds of sheep can be divided into two types according to tail types, namely: fat tail sheep (including Bonga sheep, kido sheep, kefis sheep, aletai sheep, balsh sheep, doe sheep, hu sheep, large tailed han sheep) and lean tail sheep (including dorifen sheep, merino sheep, euler Tibetan sheep, grassland Tibetan sheep, safoke sheep, and Yunnan sheep).
Haplotype analysis was performed on the resequenced data of 306 sheep of the 14 breeds, and it was found that the haplotypes of the fat tail sheep and the lean tail sheep were significantly different in the region upstream of the PDGFD gene of chromosome 15 (sheep reference genome sequence: ARS-UI_Ramb_v2.0). The allele frequencies of the 14 varieties at each SNP site in the haplotype region were calculated by using VCFtools software, and obvious differences in the allele frequencies of the fat tail sheep and the lean tail sheep at the SNP sites were found, wherein the allele frequencies at the 3732399bp site of chromosome 15 are shown in Table 1.
TABLE 1 statistics of genetic polymorphisms at mutation sites upstream of PDGFD genes in different sheep breeds
Referring to Table 1, the fat tail sheep were most mutated base A at 3732399bp of chromosome 15, while the thin tail sheep were most reference base G. Accordingly, position 3732399 (i.e., chr15: 3732399) of chromosome 15, version 15 of the sheep reference genome ARS-UI_Ramb_v2.0, was taken as one of the candidate single nucleotide polymorphism sites for predicting the tail-type trait of sheep. Other candidate sites also include chr15:3769805, chr15:3809084, chr15:3855473.
(II) early selection of sheep individuals with lean tail trait Using sheep chromosome 15 Single nucleotide polymorphism markers
The invention uses KASP typing method to detect the polymorphism of the 3732399 th (chr 15: 3732399) waiting selection site of sheep sample chromosome 15 (see figure 1), and identifies the molecular marker of the candidate site which can be used as sheep breeding auxiliary selection. The specific description is as follows.
(1) Collecting blood samples
And selecting a hybrid offspring group of the Hu sheep and the Dongfolisheng sheep as a test sample, and collecting 8mL of anterior vena cava blood. All samples were from the sheep factory in the city of elm in Shaanxi, and the blood sample collection time was 2022 and 6 months.
(2) Extraction of genomic DNA
DNA was extracted from the sampled blood, and the DNA samples were all extracted using a genomic DNA kit (TIANamp Genomic DNA Kit) from Beijing Tiangen Biochemical technology Co., ltd. And the extraction was performed according to the instructions provided by the kit.
(3) Sample DNA quality
The concentration of the extracted genome DNA is measured by using a NanoDrop 2000C ultramicro spectrophotometer of Thermo Scientific company, and the result shows that the concentration of the sample is above 50 ng/mu L and the 260/280 value is between 1.8 and 2.0; the electrophoresis result shows that the genome DNA strip is clear and free of impurities (figure 2), which shows that the extracted genome DNA has good integrity and meets the subsequent test standard.
(4) Site-directed amplification (for example chr15: 3732399)
The PCR amplification primers were designed using software such as Primer5.0 using a partial fragment upstream of the 3732399 th PDGFD gene containing the sheep reference genome ARS-UI_Ramb_v2.0 version 15 chromosome as a template (see nucleotide sequence of G/A mutation site at 101 th base shown in Table 2, SEQ. ID. NO. 1), and the amplification primer sequences are shown in Table 2:
TABLE 2 design of amplification primers (KASP primers) for upstream mutation sites of sheep PDGFD gene
PCR amplification is carried out by using the sheep genome DNA to be detected as a template and utilizing SNP specific primers (three primers in Table 2), and the specific reaction system and the reaction program are as follows:
the reaction system is as follows: 20 ng/. Mu.L of genomic DNA, 5. Mu.L of 2 XSP Master mix, and 0.14. Mu.L of KASP Assay mix (concentration of 1-FAM for the upstream primer, 12. Mu.M for the upstream primer, 30. Mu.M for the common downstream primer), were amplified by a 384-well PCR instrument;
the reaction procedure is: pre-denaturation at 94.0 ℃ for 15min; denaturation at 94.0℃for 20s, renaturation at 61.0℃for 60s,10 cycles (0.6℃decrease in renaturation temperature per cycle); denaturation at 94.0℃for 20s, renaturation at 55℃for 60s, 26 cycles were repeated and stored at 4 ℃.
(5) Genotyping
Genotyping (detecting fluorescent signals on the products and collecting signals to determine genotypes) the amplified products of step 4 by using an ABI 7900HT real-time fluorescent quantitative PCR detection system, determining the genotype of the 3732399 th chromosome 15 according to the analysis result, wherein the genotyping result is shown in FIG. 3 (genotypes of a plurality of samples can be detected simultaneously).
(6) Genotype and trait association analysis (for example chr15: 3732399)
The genotype at 3732399 of chromosome 15 of the offspring (F2 generation) of the hybridization between 538 dof sheep and hu sheep was examined in this experiment and the width of the tail root and circumference of the tail root of each individual at february age were measured as raw phenotype data. Correlating polymorphisms at 3732399 of chromosome 15 with phenotype in 538 test samples, a least squares model was established as follows:
Y ilkm ＝μ+Genotype i +P k +T l+ Combination m +ε ilkm
wherein Y is ilkm Is the observed value of the property, mu is the overall average, genotype i For genotypic effect, P k T is the field effect l For the number of fetuses effect, coding m Epsilon as a combined effect ilkm For random error, assume ε ilkm Independently of each other, obey N (0, sigma) 2 ) Distribution.
For the 3732399 th chromosome of version 15 of the sheep reference genome ARS-UI_Ramb_v2.0, the KASP typing results showed 310 AA genotype individuals, 166 AG genotype individuals, and 62 GG genotype individuals in 538 test samples. The results of the analysis of the relationship between genotypes and traits in these individuals are shown in Table 3.
TABLE 3 analysis of the association of polymorphism at the upstream mutation site of PDGFD gene with phenotype in sheep test samples
Note that: the same column data shoulder marks different lower case letters represent significant differences (P < 0.05), and the same letters represent insignificant differences (P > 0.05)
The results (table 3) show that position 3732399 of chromosome 15 of sheep reference genome ARS-ui_ramb_v2.0 version 15 is significantly related to the february age tail root width and tail root perimeter of sheep individuals: the width and circumference of the root of individuals of the GG genotype are significantly smaller than those of the AA genotype. Screening individuals with the GG genotype may thus be effective in establishing a lean tail sheep population (e.g., a genotype homozygous breeding ram population).
While other candidate sites, such as chr15:3769805, chr15:3809084, chr15:3855473, have no correlation (or a weaker correlation) with the february-age tail root width and tail root perimeter of sheep individuals.
In a word, the invention develops mutation detection analysis on sheep genome, and searches SNP loci influencing sheep tail traits by correlating with the traits. Finally, the invention provides a method for detecting sheep tail type on DNA level by utilizing a found SNP locus (chr 15: 3732399) which is positioned in the upstream region of the PDGFD gene of chromosome 15 of sheep and has obvious influence on tail root width and tail root circumference, the method is not limited by sheep age, sex and the like, can provide a method basis for improving sheep fat tail variety lean tail, and the SNP locus is positioned in the upstream haplotype region of PDGFD genes of chromosome 15 of fat tail sheep and lean tail sheep, thereby being beneficial to improving the accuracy of breeding lean tail sheep, shortening the cultivation period and accelerating the cultivation process.
Claims (10)
1. The application of the single nucleotide polymorphism site on the upstream of the sheep PDGFD gene in sheep molecular marker assisted selective breeding is characterized in that: the upstream single nucleotide polymorphism site of the PDGFD gene is positioned at 3732399bp of chromosome 15 of sheep reference genome, and the polymorphism of the single nucleotide polymorphism site is G/A.
2. The use according to claim 1, characterized in that: the polymorphism of the single nucleotide polymorphic site at the upstream of the PDGFD gene influences the tail type of sheep individuals.
3. The use according to claim 1, characterized in that: the GG genotype of the single nucleotide polymorphism site at the upstream of the PDGFD gene is a molecular marker related to sheep lean tail characters.
4. A KASP primer for detecting a single nucleotide polymorphism site upstream of a PDGFD gene in sheep, characterized in that: the KASP primer comprises a wild type site-specific primer, a mutant site-specific primer and a universal primer, wherein the wild type site-specific primer and the mutant site-specific primer are competitive upstream primers, the 3' -end base of which is respectively identical with alleles G and A of a single nucleotide polymorphism site positioned at 3732399bp of chromosome 15 of a sheep reference genome, and the universal primer is a downstream primer.
5. A KASP primer for detecting a single nucleotide polymorphism site upstream of sheep PDGFD gene according to claim 4, wherein: the KASP primer specifically comprises the following components:
upstream primer 1-FAM:
5’-gaaggtgaccaagttcatgctTTGTCTAGGAGGACTTACAAATCG-3’
upstream primer 2-HEX:
5’-gaaggtcggagtcaacggattTTGTCTAGGAGGACTTACAAATCA-3’
a downstream primer:
5’-TTGACTTCCAACTCAAGGTCTTCT-3’。
6. a method for detecting single nucleotide polymorphism of sheep PDGFD gene upstream is characterized in that: the method comprises the following steps:
the genome DNA of sheep to be detected is used as a template, a partial fragment containing the single nucleotide polymorphism site at the upstream of the PDGFD gene is amplified through a KASP primer, and then the genotype of the single nucleotide polymorphism site is identified through fluorescence detection, wherein the single nucleotide polymorphism site at the upstream of the PDGFD gene is positioned at 3732399bp of chromosome 15 of a sheep reference genome.
7. The method for detecting an upstream single nucleotide polymorphism of sheep PDGFD gene according to claim 6, wherein: the KASP primer comprises a universal primer, a wild type site-specific primer with FAM linker and a mutant site-specific primer with HEX linker, wherein the wild type site-specific primer and the mutant site-specific primer are competitive upstream primers with 3' terminal bases respectively identical to alleles G and A of a single nucleotide polymorphism site located at 3732399bp of chromosome 15 of sheep reference genome, and the universal primer is a downstream primer; determining 3 genotypes of the single nucleotide polymorphism sites upstream of the PDGFD gene according to the distinction of fluorescence: GG is red, AG is green, AA is blue.
8. The method for detecting an upstream single nucleotide polymorphism of sheep PDGFD gene according to claim 6, wherein: the sheep is selected from one variety of sheep selected from the group consisting of fat tail sheep, a hybrid sheep of fat tail sheep and lean tail sheep.
9. Use of the method for detecting single nucleotide polymorphism upstream of sheep PDGFD gene according to any one of claims 6-8 in sheep molecular marker-assisted selection breeding.
10. The use according to claim 9, characterized in that: the GG genotype of the single nucleotide polymorphism site at the upstream of the PDGFD gene is a molecular marker related to sheep lean tail characters.
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