CN115181804B - Molecular marker related to sheep growth traits, detection method and application thereof - Google Patents
Molecular marker related to sheep growth traits, detection method and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker related to sheep growth traits, a detection method and application thereof. The invention discovers that an A/T polymorphic site exists at the 558 th site of an amplified fragment by carrying out PCR amplification and sequence analysis on sheep ALB genes, further uses KASPar primer pair 1271 to detect the polymorphic site of sheep and establishes a least squares model, carries out correlation analysis on genotype and growth traits, and finally determines that the amplified ALB gene fragment can be used as a molecular marker related to average daily gain of sheep. The detection of the molecular marker can be used for selecting the sheep with the TT homozygous gene into the core group as the sheep so as to improve the growth characteristics of sheep and facilitate the increase of economic benefits.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an ALB gene fragment serving as a molecular marker affecting sheep growth traits, a detection method and application thereof.
Background
Albumin (ALB) is the most abundant circulating protein that acts as a carrier protein for a variety of endogenous molecules, including fatty acids, hormones, and metabolites. In humans, several studies have shown that ALB levels are inversely related to body fat percentage (Chang, xu, ferronte, & Krakoff, 2019), that a decrease in maternal serum ALB ratio in late pregnancy will affect infant birth weight of pregnant women, and that there is a positive correlation (Wada et al, 2021). Furthermore, one report showed that the duck ALB gene was strongly inversely related to FCR and RFI. It is not clear whether ALB is related to, or has some relationship with, sheep growth traits.
The sheep industry is an important component of modern animal husbandry, can be used as a raw material for producing wool spinning industry, and improves the economic income of people. With the widespread rise of the living standard of people, mutton is popular because of its low cholesterol, lean meat, high nutrition, tenderness (Hehua et al, 2020).
The invention discusses the relevance of different genotypes and sheep growth traits by sequencing and analyzing ALB genes, and aims to provide gene materials for improving the genetic improvement aspect of sheep growth traits and accelerate the cultivation process of high-quality mutton sheep.
Disclosure of Invention
In order to solve the technical problems, the invention provides a molecular marker related to sheep growth traits and application thereof. The molecular marker is amplified from sheep ALB gene, and the specific nucleotide sequence is shown as SEQ ID NO. 1. The DNA sequence of the sheep ALB gene is amplified and sequenced, polymorphic sites of the ALB gene are searched, the correlation between different genotypes and sheep growth characters is analyzed, a detection method of a molecular marker containing the polymorphic sites is established, and the molecular marker can be applied to the cultivation of novel high-quality mutton sheep new varieties.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a molecular marker related to sheep growth characteristics has a nucleotide sequence shown in SEQ ID NO.1, wherein W at 558bp represents A or T, and the A/T mutation at 558bp of the sequence causes the A/T polymorphism of sheep ALB gene at the site.
The results of genotype and trait association analysis showed that the ALB g.8699a > T mutation sites were significantly correlated with sheep Average Daily Gain (ADG) with prolonged assay cycles. Average daily gain of 80-100, 80-140 and 80-160 of sheep carrying TT genotype is better than that of sheep carrying TA or AA genotype (P < 0.05), so that TT is taken as dominant genotype.
Preferably, the primer pair for detecting the molecular marker comprises a sequence shown as SEQ ID NO.2 and SEQ ID NO. 3.
A KASPar primer pair for detecting the molecular marker comprises a forward primer 1 with a nucleotide sequence shown as SEQ ID NO.4, a forward primer 2 with a nucleotide sequence shown as SEQ ID NO.5 and a universal reverse primer C with a nucleotide sequence shown as SEQ ID NO. 6.
A kit for detecting the molecular marker, wherein the kit comprises a PCR primer pair or a KASPar primer pair for detecting the molecular marker.
A method for detecting molecular markers related to sheep growth traits, wherein the nucleotide sequence of the molecular markers is shown as SEQ ID NO.1, W at 558bp position represents A or T, the method comprises the steps of detecting sheep genome DNA by using the primer pair or the kit, and the specific detection method comprises the following steps:
a) Amplifying sheep genomic DNA using the primer pair, KASPar primer pair, or a kit comprising the primer pair;
b) Identifying the polymorphic site of the amplification product obtained in step a).
Wherein, in step b), the method of typing identification includes, but is not limited to, direct sequencing method, probe method, gene chip method, high resolution dissolution profile method.
The method for detecting the molecular marker related to sheep growth traits by using the primer pair comprises the following steps:
a) Extracting genome DNA from sheep blood as a sample, and performing high-throughput water bath PCR amplification by using primer pairs with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
b) After amplification, fluorescence signals were detected using BMG PHERAstar instrument and the typing results were checked.
The molecular marker and the polymorphic site, the primer pair or the kit thereof are applied to sheep growth trait detection, and the type of the polymorphic site is analyzed by detecting the molecular marker in genome DNA of sheep to be detected, so that the growth trait of sheep can be determined, and then the fast-growing sheep can be screened out.
The molecular marker and the polymorphism site, the primer pair or the kit are applied to sheep breeding, and the primer pair or the kit is used for amplifying and detecting genomic DNA of sheep to determine the genotype of ALB gene of a sample to be detected, so that a sheep variety with rapid growth can be bred from the molecular marker.
Finding mutation sites of genes, finding the relationship between genes and traits through association analysis between the genes and the traits is an important means for researching gene functions and is also a basis for marker-assisted selection.
The invention discovers that an A/T polymorphism site exists at the 558 th site of an amplified fragment by carrying out PCR amplification and sequencing on ALB genes of sheep representing a hu sheep variety, and determines a molecular marker related to sheep growth traits by detecting 1271 hu sheep polymorphism and an established least square model, and the molecular marker can be used for cultivating high-quality new mutton sheep varieties, provides an effective genetic engineering means for genetic improvement of sheep growth traits, and has great practical application value.
According to the invention, the detection is carried out on the molecular marker by designing the KASPar primer required by competitive allele specific PCR (KASP), the detection method does not need to synthesize a specific fluorescent probe aiming at each SNP locus, and all locus detection is finally carried out by using a universal fluorescent primer based on the unique ARM PCR principle, so that the cost of reagents is greatly reduced, the accuracy is higher, and a simple, convenient, accurate and low-cost operation method is provided for the detection of the molecular marker.
The invention has the beneficial effects that:
The invention provides a molecular marker related to sheep growth traits and a polymorphic site of A/T thereof, and the polymorphism genotype is measured to effectively identify whether the sheep is a fast-growing sheep or not, so that an effective detection means is provided for breeding the fast-growing sheep. The invention can be used for selecting the sheep with the retention gene TT homozygous as the breeding sheep to be used for breeding by detecting the molecular marker and the polymorphism locus, so as to improve the growth character of the sheep and be beneficial to improving the economic benefit of the breeding industry.
Drawings
FIG. 1 is a gel electrophoresis diagram of sheep ALB gene fragment used as a molecular marker in the present invention; wherein, lane M: DL 2000marker, lanes 1-10: ALB gene amplification results.
FIG. 2 shows the sequencing result of the mutation site of sheep ALB gene in the present invention.
FIG. 3 shows the typing result of sheep ALB gene g.8699A > T mutation site KASPAR SNP in the present invention; wherein, the red dot near the left represents TT genotype, the green dot near the middle represents TA genotype, and the blue dot near the right represents AA genotype.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the methods are of analytical purity or above.
EXAMPLE 1 amplification of ALB Gene
A pair of primers was designed using oligo7.0 software using sheep ALB gene DNA (GenBank accession number: NC-040257.1) as a template: forward and reverse primers, primer sequences were as follows:
Forward primer (SEQ ID No. 2): 5'-TTCATAGCAGGCATATTGGT-3' A
Reverse primer (SEQ ID NO. 3): 5'-TCTAGAGCATCTGCCACAA-3' A
(2) Amplification and sequencing of ALB Gene
The genomic DNA extracted from sheep whole blood cells was amplified by PCR as a DNA template in a total volume of 25. Mu.L, wherein the DNA template was 1. Mu.L, 2X PCR MASTER Mix was 12.4. Mu.L, the forward primer was 0.8. Mu.L (concentration was 10. Mu. Mol/L), the reverse primer was 0.8. Mu.L (concentration was 10. Mu. Mol/L), and ddH 2 O was 10. Mu.L. PCR amplification reaction conditions: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 54.5℃for 30s, extension at 72℃for 30s, cycling for 35 times, and extension at 72℃for 10min.
The PCR amplification reaction products were detected by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1, which shows that 603bp specific amplified fragment was obtained. Sequencing the amplified PCR fragment, wherein the specific nucleotide sequence of the amplified fragment is shown as SEQ ID NO.1, and a polymorphic site exists in the fragment, specifically W at the 558 th bp site is A or T, namely the amplified ALB gene fragment (SEQ ID NO. 1) has an A/T polymorphism at the 558 th bp site (see FIG. 2).
Wherein, SEQ ID NO.1:
TAGTTCTGTCTGTGCCTCTAGGCTTAGCAGCACGGGTTGAAAATCTTCATAGCAGGCATATTGGTACAAGTATGGTCTTAGAGCACAATGAATGTGTTCTGTCGACATAGTAAATGTCTGGGGAGGAAGCACAGGAATGTCACTTTACAGTGAACACTTTTCACCTACTTGCTTGTAGGCAGATCTTGCCAAGTACATATGTGATCATCAAGACGCACTCTCCAGTAAACTGAAGGAATGCTGTGATAAGCCTGTGTTGGAAAAATCCCACTGCATTGCTGAGGTAGATAAAGATGCCGTGCCTGAAAACCTGCCCCCATTAACTGCTGACTTTGCTGAAGATAAGGAGGTTTGCAAAAACTATCAGGAAGCAAAAGACGTCTTCCTGGGCTCGTAAGTGGATAAAAAATGATCCTTTCATACTTTTATATGATCTCAGAATTTAGGAAATTATTCTAGGCTTTCCTTTGGAATTGCTACAATTTTTCACGTTGCCTGCAATGTTTCTTTATTCTTTCCTCTTCTGGCCGTTAAACATTTTTGGAAAAATTGCTTTTWAAAAATTGTCATAAAATAATACATGCTTGTGGCAGATGCTCTAGA.
DNA sequence homology search identification:
The DNA sequence obtained after sequencing was compared for sequence homology with known physiological functional genes published in GenBank database by 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 to identify and obtain functional information of the DNA sequence. The search result shows that the homology of the sequence to the partial sequence of sheep ALB gene DNA (GenBank accession number: NC_ 040257.1) reaches 99%.
Example 2 establishment of genotyping assay
1) Primer sequence design
A KASPar primer pair was designed for the A/T polymorphic site of the amplified fragment of example 1, so as to be used for specific detection of the polymorphic site, and the nucleotide sequence of the designed KASPar primer pair was:
Forward primer A1 (SEQ ID No. 4) for detection AlleleX:
5’-GAAGGTGACCAAGTTCATGCTCCGTTAAACATTTTTGGAAAAA TTGCTTTTA-3’；
forward primer A2 (SEQ ID No. 5) for detection AlleleY:
5’-GAAGGTCGGAGTCAACGGATTCCGTTAAACATTTTTGGAAAAA TTGCTTTTT-3’；
Universal reverse primer C (SEQ ID No. 6): 5'-GCATCTGCCACAAGCATGTATT ATTTTATG-3'.
The primers are entrusted to be synthesized by Beijing Biotechnology, inc. Each set of primers in the KASPar primer pair was diluted to 10. Mu. Mol/L and was prepared as per primer A1: primer A2: the primer C is uniformly mixed for standby according to the volume ratio of 12:12:30.
2) DNA quality control
The extraction of genomic DNA from sheep whole blood can be performed using a DNA extraction kit. The quality detection of the extracted genome DNA is carried out, 1% agarose electrophoresis and Nanodrop2100 are adopted for detection respectively, and the qualified DNA requires: (1) Agarose electrophoresis showed a single DNA band without significant diffusion. (2) Nanodrop2100 detection a260/280 is between 1.8-2.0; a260/230 is between 1.8 and 2.0; there is no significant light absorption at 270 nm. And according to KASPar detection technology and genome size of LGC company in UK, the dosage of DNA is 10-20 ng/sample, and the diluted concentration of extracted genome DNA is 10-20 ng/mu L as DNA template for standby.
3) Genotyping
Firstly, a K-pette liquid-separating workstation is used for respectively adding 1.5uL of diluted DNA template to be detected (10-20 ng/. Mu.L) and blank control (No template control, NTC, sterilized water is adopted) into 384-hole reaction plates, and drying is carried out for 30min at 60 ℃ (a drying oven, LGC company) so that the DNA is changed into dry powder for standby.
Each of the above KASPar primer pairs was diluted to 10. Mu. Mol/L and was prepared as described for primer A1: a2: and C, uniformly mixing the mixture according to the volume ratio of 12:12:30 to obtain a primer mixture for later use.
And then adding a mixed solution of 1 XMaster Mix (1536 microwell plates, product number: part No. KBS-1016-011) and a primer into each reaction well by using a Meridian sample adding workstation under Kraken operation system, putting the microwell plates on a Kube heat sealing instrument and a Fusion laser membrane sealing instrument in sequence for membrane sealing after Mix split charging, and carrying out high-flux water bath PCR amplification by using Hydrocyler. The PCR reaction was performed in a high throughput water bath system Hydrocycler, with the following specific procedures:
pre-denaturation at 94 ℃ for 15 min;
94 ℃,20 seconds (denaturation) -61 ℃ -55 ℃,1 minute (renaturation & extension), 10 cycles of amplification in the touchdown order, 0.6 ℃ decrease per cycle;
amplification was continued for 26 cycles at 94℃for 20 seconds (denaturation) -55℃for 60 seconds.
After amplification, a BMG PHERAstar instrument is used for detecting fluorescent signals and checking the parting condition, and the specific result is shown in fig. 3. Each dot in the figure represents a piece of material to be tested, wherein the red dot near the left side indicates that the locus is homozygous for genotype "TT"; blue dots near the right indicate that the locus is homozygous genotype "AA"; the green dots near the middle indicate that the locus is heterozygous genotype "TA" or "AT"; the black dots indicate NTC (not shown in fig. 3), i.e., a blank.
4) Application of molecular marker in sheep growth trait correlation analysis
The polymorphism of 1271 Hu sheep is detected in the test, the genotype of the Hu sheep is determined, a least squares model is established as described below, and the correlation analysis of the genotype and the growth character is carried out.
Y ijk＝μ+Genotypei+Pj+Fk+Ml+εijkl wherein Y ijk is a phenotypic observation of growth traits, μ is the overall mean, genotype i is the genotype effect, P j is the batch effect, F k is the parent effect, M l is the maternal effect, ε ijkl is a random error, assuming ε ijkl is independent of each other, subject to N (0, σ 2) distribution.
Genotype test results showed 418 AA genotypes, 642 TA genotypes and 211 TT genotypes out of 1271 individuals. The results of genotype-trait association analysis are shown in Table 1, where ADG represents average daily gain in kg. ADG80-100 represents average daily gain of sheep 80-100 balance; ADG80-120 represents average daily gain of sheep 80-120 balance; ADG80-140 represents average daily gain of sheep 80-140 balance; ADG80-160 represents daily gain of sheep 80-160 balance; ADG80-180 indicates daily gain on a balance of sheep 80-180.
TABLE 1 ALB Gene polymorphism and growth trait association analysis of Hu sheep
Note that: p <0.05 indicates significant differences.
The results showed that with prolonged assay period, ALB g.8699a > T mutation sites were significantly correlated with average daily gain in hu sheep. The staged daily gain of sheep carrying the TT genotype was significantly better than that of sheep carrying the TA genotype (P < 0.05). From this, it was found that the T allele was the dominant allele. And selecting TT genotype for preserving the breed during breeding, and hybridizing the breeding sheep with other sheep by taking the TT genotype as a breeding sheep. In particular, artificial insemination is carried out by adopting the semen of the TT genotype stud ram, so that the breeding efficiency can be greatly improved, and the flock with the advantage of growth speed can be obtained.
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
gcatctgcca caagcatgta ttattttatg 30
Claims (9)
1. The application of a molecular marker related to sheep growth traits in sheep breeding is disclosed, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, W at 558bp represents A or T, and the mutation leads to the A/T polymorphism of the molecular marker; the method is characterized in that the staged daily gain of the sheep carrying the TT genotype is better than that of the sheep carrying the TA genotype.
2. Use of a PCR primer pair for detecting a molecular marker associated with sheep growth traits in sheep breeding, wherein the PCR primer pair comprises sequences of nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, wherein W at 558bp represents a or T, the mutation resulting in a/T polymorphism of the molecular marker; the method is characterized in that the staged daily gain of the sheep carrying the TT genotype is better than that of the sheep carrying the TA genotype.
3. Use of a KASPar primer pair for detecting a molecular marker associated with sheep growth traits in sheep breeding, wherein the KASPar primer pair comprises a forward primer 1 having a nucleotide sequence shown as SEQ ID No.4, a forward primer 2 having a nucleotide sequence shown as SEQ ID No.5 and a universal reverse primer C having a nucleotide sequence shown as SEQ ID No.6, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, wherein W at 558bp represents a or T, the mutation resulting in an a/T polymorphism of the molecular marker; the method is characterized in that the staged daily gain of the sheep carrying the TT genotype is better than that of the sheep carrying the TA genotype.
4. Use of a kit for detecting a molecular marker associated with sheep growth traits in sheep breeding, wherein the kit comprises a PCR primer pair having a nucleotide sequence shown as SEQ ID No.2-3 or a KASPar primer pair having a nucleotide sequence shown as SEQ ID No.4-6, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, wherein W at 558bp represents a or T, the mutation resulting in an a/T polymorphism of the molecular marker; the method is characterized in that the staged daily gain of the sheep carrying the TT genotype is better than that of the sheep carrying the TA genotype.
5. Use of a method for detecting a molecular marker associated with sheep growth traits in sheep breeding comprising the steps of:
a) Amplifying sheep genome DNA by using a PCR primer pair with a nucleotide sequence shown as SEQ ID NO.2-3 or a KASPar primer pair with a nucleotide sequence shown as SEQ ID NO.4-6, or a kit containing the PCR primer pair or the KASPar primer pair;
b) And c) carrying out typing identification on specific sites of A or T represented by W at 558bp of the nucleotide sequence of the amplified product obtained in the step a) as shown in SEQ ID NO.1, wherein the staged daily gain of sheep carrying the TT genotype is better than that of sheep carrying the TA genotype.
6. The use according to claim 5, wherein the typing method in step b) is a sequencing method, a fluorescent probe method, a gene chip method or a high resolution dissolution profile method.
7. The use according to claim 5, wherein the PCR amplification is performed using a KASPar primer pair having a nucleotide sequence shown in SEQ ID NO.4-6, and the typing result is determined by detecting a fluorescent signal after the amplification is completed.
8. Use of a molecular marker associated with a sheep growth trait, or a PCR primer pair for detecting a molecular marker associated with a sheep growth trait, a KASPar primer pair, or a kit comprising a PCR primer pair or KASPar primer pair for detecting a molecular marker associated with a sheep growth trait, or a method for detecting a molecular marker associated with a sheep growth trait in sheep growth trait detection, characterized in that the nucleotide sequence of the molecular marker is as shown in SEQ ID No.1, wherein W at 558bp in the sequence SEQ ID No.1 is a or T, the mutation resulting in an a/T polymorphism of the molecular marker, wherein the staged daily gain of sheep carrying the TT genotype is superior to sheep carrying the TA genotype;
the nucleotide sequence of the PCR primer pair is shown as SEQ ID NO. 2-3;
The nucleotide sequences of the KASPar primer pairs are shown in SEQ ID NO. 4-6.
9. Use according to any one of claims 1 to 7, characterized in that the breeding purpose is to pick out fast-growing sheep.
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| CN113913530B (en) * | 2021-07-08 | 2023-11-17 | 甘肃润牧生物工程有限责任公司 | Molecular marker related to sheep body height and application thereof |
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