CN116377082B - Application of sheep LCORL gene single nucleotide polymorphism marker in growth trait selection - Google Patents
Application of sheep LCORL gene single nucleotide polymorphism marker in growth trait selection Download PDFInfo
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Abstract
The invention discloses application of sheep LCORL gene single nucleotide polymorphism markers in growth trait selection. The LCORL gene single nucleotide polymorphism marker is positioned at 38069494 of sheep chromosome 6. Taking Hu sheep as an example, individuals with the genotype of AA at this site have a faster growth rate than individuals with genotype of GA or GG. The LCORL gene single nucleotide polymorphism marker related to sheep growth traits is determined, and can be used for quickly establishing sheep populations with excellent growth traits, so that the method is used for early selection of sheep growth traits and accelerating the auxiliary selection breeding process of sheep molecular markers.
Description
Technical Field
The invention belongs to the field of biotechnology and livestock molecular breeding, and relates to a Single Nucleotide Polymorphism (SNP) marker affecting sheep growth traits and application thereof.
Background
Growth traits are important economic traits of sheep, and are important points of attention for genetic improvement of sheep. Sheep growth traits include body weight, body size, growth rate, etc. Growth traits are often affected by both genetic and environmental factors. In production practice, the growth traits of livestock can be effectively improved through genetic improvement. Thus, the search for candidate genes or effective QTLs associated with growth traits is an important approach to improving sheep growth traits and productivity.
A single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to a variation of a single nucleotide in the genome of an individual, which in turn results in a polymorphic change in the sequence of the entire locus where it is located. At present, the third generation genetic markers represented by SNP are rapidly developed in the booster animal industry, and compared with the first generation genetic markers represented by restriction fragment length polymorphism and the second generation genetic markers represented by microsatellite polymorphism, the third generation genetic markers represented by SNP have the characteristics of wide distribution, large quantity and the like, and are more suitable for researching genetic traits.
KASP (Kompetitive Allele-SPECIFIC PCR), i.e.competitive allele-specific PCR. According to the specificity matching of the primer terminal base, the SNP locus is typed by utilizing the Touch-down PCR and the fluorescence quenching probe and combining the emitted fluorescence signal. Specifically, KASP requires primer design inside the amplified target DNA sequence near the label: for co-dominant markers, since there are two target sequences, three primers, including two site-specific primers and one universal primer, were designed accordingly. The 3' end of the site-specific primer is SNP site, and is respectively combined with two universal quenching probes FAM and HEX in the KASP amplification process, so that fluorescent signals are emitted, and finally genotyping is carried out according to the fluorescent signals. The KASP technology can accurately detect SNPs widely existing in genome DNA, is a high-throughput, low-cost and low-error-rate SNP typing technology, and can avoid gel electrophoresis in a laboratory, thereby realizing automatic and platform operation.
LCORL (LIGAND DEPENDENT nuclear receptor compressor like) gene is located on sheep chromosome 6, also known as ligand-dependent nuclear receptor inhibitor. The LCORL gene is associated with arginine metabolism, which acts on C-terminal binding protein 1 (VINAYAGAM ET al.2011), affecting gene expression and cell cycle. LCORL genes encode a transcription factor that interacts with ubiquitin C, while polyubiquitin-encoding genes have been shown to be involved in multiple cellular biological processes (Kimura et al 2010), affecting livestock body weight and body size traits, and have been used in prior studies as candidate genes for affecting growth and development of horses and cattle (Han Yujiao et al 2016). Although literature reports that "LCORL is strongly selected among the Sichuan black goat flock" (Guo Guzhong, et al, sichuan black goat genome inbred coefficient and selection signal analysis, sichuan university of agriculture, university of Sichuan agricultural journal, 2022,40 (05): 656-663.DOI: 10.16036/j.issn.1000-2650.202209126), SNP markers applied to sheep for breeding with the characteristics of growth rate, weight and body size are not seen at present.
Disclosure of Invention
The invention aims to provide an application of a single nucleotide polymorphism marker of a sheep LCORL gene in growth trait selection.
In order to achieve the above purpose, the invention adopts the following technical scheme:
Use of a sheep LCORL gene single nucleotide polymorphism marker located at position 38069494 of chromosome 6 of a sheep reference genome (ARS-ui_ Ramb _v2.0) in sheep molecular marker-assisted selection breeding.
Preferably, the single nucleotide polymorphism marker is of the AA genotype.
A KASP primer for detecting a single nucleotide polymorphism marker of sheep LCORL gene, the KASP primer comprising a universal primer and a wild type site-specific primer having a FAM adaptor, and a mutant site-specific primer having a 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 a and G, respectively, located at the single nucleotide polymorphism locus 38069494 of chromosome 6 of the sheep reference genome (ARS-ui_ Ramb _v2.0), the universal primer being downstream primer (FAM adaptor added to wild type site-specific primer exhibits red fluorescence in KASP detection, HEX adaptor added to mutant site-specific primer exhibits blue fluorescence in KASP detection).
Preferably, the KASP primer specifically comprises:
upstream primer 1-FAM:
5'-gaaggtgaccaagttcatgctTCTGGCAACTAAGATAAAAATCACAAA-3' (FAM joint lower case letter)
Upstream primer 2-HEX:
5'-gaaggtcggagtcaacggattTCTGGCAACTAAGATAAAAATCACAAG-3' (lower case letter HEX joint)
A downstream primer:
5’-TGGTAAGTATTGAGCTCTGGATAATGT-3’。
A method for detecting single nucleotide polymorphism of sheep LCORL genes comprises the following steps:
The genome DNA of sheep to be detected is used as a template, partial fragments containing a single nucleotide polymorphism site of a LCORL gene are amplified through the KASP primer, and then the genotype of the single nucleotide polymorphism site is identified through detection of fluorescence, wherein the single nucleotide polymorphism site of the LCORL gene is 38069494 th site of a sheep reference genome (ARS-UI_ Ramb _v2.0) 6 th chromosome.
Preferably, 3 genotypes of the LCORL gene single nucleotide polymorphism sites (see mutation site of a/G at base 101 of the nucleotide sequence shown in seq id No. 1) are determined based on the discrimination of fluorescence: AA is red, GA is green, GG is blue.
Preferably, the sheep is a Hu sheep.
The detection method of the single nucleotide polymorphism of the sheep LCORL gene is applied to sheep molecular marker assisted selective breeding.
Preferably, the AA genotype of the LCORL gene single nucleotide polymorphism site is a genetic marker for improving growth traits.
Preferably, the growth trait is any one or more of growth rate (e.g., daily gain), body weight, and body size trait (e.g., body height, body length).
The beneficial effects of the invention are as follows:
The invention discovers and identifies the SNP locus of LCORL gene related to sheep growth characters (namely 38069494 locus of chromosome 6 of sheep reference genome) through nucleotide mutation detection analysis of sheep LCORL gene, and provides a molecular marker (genetic marker) and a method for identifying sheep growth characters, which can be used for rapidly establishing sheep populations with excellent growth characters, thereby providing genetic sample basis for sheep variety improvement, being beneficial to shortening variety cultivation period, accelerating variety cultivation process and improving seed selection accuracy.
Drawings
FIG. 1 is a general technical flow chart for detecting single nucleotide polymorphism of LCORL gene of sheep.
FIG. 2 is an electrophoresis chart of genomic DNA quality control of a Hu sheep sample; in the figure: 1. 8 and 16 are sample numbers, and M is Marker.
FIG. 3 shows the result of KASP typing at position 38069494 of chromosome 6 (AA, GA, GG genotype) of Hu sheep sample; 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 invention only and are not intended to limit the scope of the invention.
Identification of growth trait related single nucleotide polymorphism markers on sheep chromosome 6
1. Development of nucleotide mutation detection analysis for sheep LCORL Gene
Referring to fig. 1, haplotype analysis was performed on the obtained sheep resequencing data, and it was found that sheep having different growth traits had different haplotypes in the LCORL gene region of chromosome 6, and there was a significant difference in allele frequencies of different types of sheep (meat sheep, dairy sheep, chinese breeds) at 38069494 of chromosome 6, as described below.
The re-sequencing results of up to 266 sheep of 9 varieties including 25 SAFOKE sheep, 28 DuPoisson sheep, 31 non-horn Tasat sheep, 54 Dongfen sheep, 34 Babylonic sheep, 22 Kazakhstan sheep, 10 Euler Tibetan sheep, 5 Sunits sheep and 57 Hu sheep were obtained in the experiment (the sources of specific samples are shown in Table 1). Sheep of these breeds can be divided into three types according to their growth rates, respectively: meat sheep (SAFOKE sheep, duPong sheep, non-horned Taosite sheep), milk and meat sheep (Dongfen sheep) and Chinese local sheep (Babuk sheep, kazakhstan sheep, euler Tibetan sheep, sunit sheep, hu sheep). Wherein the growth speed of the meat sheep is higher than that of the dairy-meat sheep, and the growth speed of the dairy-meat sheep is higher than that of the sheep of the local variety in China.
TABLE 1 sample information Table
The re-sequencing data of 266 sheep of the 9 varieties were subjected to haplotype analysis, and it was found that three types of sheep (meat sheep, milk-meat sheep and China sheep) exhibited three different types of haplotypes in the LCORL gene region. The gene frequencies of the SNP loci of the LCORL gene regions of each sample were calculated by VCFtools software, and it was found that there was a significant difference in the gene frequencies of the 38069494 th chromosome of the sheep reference genome ARS-UI_ Ramb _v2.0 version 6 of three types (meat sheep, milk-meat sheep, chinese local variety sheep) of sheep (Table 2).
TABLE 2 statistics of genetic polymorphisms at the LCORL gene mutation sites (38069494 on chromosome 6) in different sheep breeds
Referring to Table 2, the number 38069494 of the chromosome 6 of the meat sheep was a reference base A, and the number of Chinese sheep was more mutant base G, and the gene frequency of the number 38069494 of the chromosome 6 was intermediate between that of meat sheep and Chinese sheep.
Accordingly, the 38069494 th position of chromosome 6 can be used as a candidate single nucleotide polymorphism site for predicting sheep growth performance.
(II) selection of sheep individuals with growth trait advantage by using sheep chromosome 6 single nucleotide polymorphism marker
The invention detects polymorphism at 38069494 th position of chromosome 6 of sheep sample by KASP typing method, and reveals that the locus has molecular marker which can be used as auxiliary selection for sheep breeding, and the specific description is as follows.
(1) Collecting blood samples
And (3) selecting Hu sheep as a test material. The Hu sheep are all from the river sheep factory in elm of Shaanxi province, and the blood sample collection time is 2022 and 6 months.
(2) Extraction of genomic DNA
DNA is extracted from a Hu sheep blood sample, and the DNA sample is extracted by adopting a genome DNA kit (TIANamp Genomic DNAKit) produced by Beijing Tiangen Biochemical technology Co., ltd.) according to the operation instruction provided by the kit.
(3) Sample DNA quality
The concentration of the extracted genome DNA is measured by using a Thermo Scientific Nanodrop 2000C ultra-micro spectrophotometer, and the result shows that the concentration of the sample is above 100 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) Primer design
The primer for PCR amplification was designed using the partial fragment of LCORL gene at position 38069494 of sheep reference genome ARS-UI_ Ramb _v2.0 version 6 chromosome as shown in SEQ ID No.1 as a template, and software such as Primer5.0:
Upstream primer F1:5'-TCTGGCAACTAAGATAAAAATCACAAA-3' A
The upstream primer F2:5'-TCTGGCAACTAAGATAAAAATCACAAG-3' A
The downstream primer R:5'-TGGTAAGTATTGAGCTCTGGATAATGT-3'.
Different KASP linkers, namely FAM linkers (shown as SEQ ID. NO. 5) and HEX linkers (shown as SEQ ID. NO. 6) were added to the 5' ends of the two upstream primers (F1 and F2), respectively, and the sequence of the upstream primer 1-FAM after the FAM linker and the sequence of the upstream primer 2-HEX after the HEX linker were as follows:
upstream primer 1-FAM:
5’-gaaggtgaccaagttcatgctTCTGGCAACTAAGATAAAAATCACAAA-3’
upstream primer 2-HEX:
5’-gaaggtcggagtcaacggattTCTGGCAACTAAGATAAAAATCACAAG-3’
The designed upstream primers 1-FAM and 2-HEX were sent to Kang Pusen agricultural technologies Co., ltd together with the universal primer (i.e., the downstream primer R).
(5) KASP amplification
The extracted genome DNA is used as a template, and the synthesized upstream primer 1-FAM, 2-HEX and a universal primer are used as an experimental primer group to carry out PCR amplification, wherein 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, 2X KASP MASTER mix 5. Mu.L, and KASP ASSAY
Mix (upstream primer 1-FAM concentration 12. Mu.M, upstream primer 2-HEX concentration 12. Mu.M, general primer concentration 30. Mu.M)
0.14μL。
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 and renaturation at 55℃for 60s, 26 cycles were repeated.
(6) Genotyping
Genotyping was performed using an ABI 7900HT real-time fluorescent quantitative PCR detection system, and the genotype at position 38069494 of chromosome 6 was determined from the analysis results, and the genotyping results are shown in fig. 3.
(7) Genotype and trait association analysis
The genotype at 38069494 th position of chromosome 6 of 536 Hu sheep was tested and the data of weight, height, body length, chest circumference, birth weight, daily gain, etc. of each individual at 2 months of age were determined. The genotype and the phenotype are subjected to association analysis, and the following least squares model is established:
Yilkm＝μ+Genotypei+Pk+Tl+Combinationm+εilkm
Wherein Y ilkm is the observed value of the trait, μ is the overall mean, genotype i is the genotype effect, P k is the field effect, T l
For the number of tires effect, combination m is a combined effect, epsilon ilkm is a random error, assuming epsilon ilkm is independent of each other, obeying N (0,
Σ 2) distribution.
The KASP genotyping results showed 73 AA genotypes, 282 GA genotypes,
There are 181 GG genotypes. The results of the relationship between genotypes and traits are shown in Table 3.
TABLE 3 analysis of the association of polymorphism at the mutation site (38069494 on chromosome 6) of Hu sheep LCORL with phenotype
Note that: the same column of data shoulder marks with different lower case letters indicate that the difference is significant (P < 0.05), and the same letter or non-standard letters indicate that the difference is not significant (P > 0.05);
daily gain is statistically from birth to 2 months of age
The results showed (table 3) that LCORL gene mutation sites (38069494 on chromosome 6) were significantly related to february age body weight, height, body length and daily gain of hu sheep: age of february of AA genotype individuals high body,
The body length and daily gain data are significantly higher for individuals of the GA genotype and GG genotype. Therefore, the sheep population with high growth performance (faster growth speed, better body weight and body size property) can be effectively established by screening out individuals with AA genotype.
In a word, the invention provides a method for detecting sheep growth performance on DNA level by utilizing SNP markers related to sheep growth traits and combining KASP typing technology through genetic information analysis of sheep LCORL genes, the method is not limited by sheep age and the like, can be used for more effectively and comprehensively identifying sheep growth performance in early breeding of sheep growth traits, is beneficial to overcoming the defects of long period, low efficiency and the like of traditional breeding, provides reference for rapidly establishing sheep population with high growth performance, improves pertinence and accuracy of a breeding selection process of sheep growth performance, accelerates the breeding selection process of sheep, and promotes cultivation improvement of sheep.
Claims (5)
1. The application of sheep LCORL gene single nucleotide polymorphism markers in sheep growth trait molecular marker assisted selection breeding is characterized in that: the single nucleotide polymorphism marker is positioned at 38069494 th chromosome 6 of sheep reference genome;
The sheep reference genome is in the version ARS-ui_ Ramb _v2.0;
The 38069494 th reference base of the chromosome 6 is A, and the mutant base is G;
The sheep is Hu sheep; the growth character is any one or more of weight, height, length and daily gain; the growth traits of AA genotype individuals are significantly higher in body weight, height, body length and daily gain than those of GA genotype and GG genotype.
2. The use according to claim 1, characterized in that: a KASP primer for detecting a single nucleotide polymorphism marker of sheep LCORL gene, the KASP primer comprising a wild-type site-specific primer having a FAM adaptor, a mutant site-specific primer having a HEX adaptor, and a universal primer, the wild-type site-specific primer and the mutant site-specific primer being competitive upstream primers having terminal bases identical to alleles a and G, respectively, of a single nucleotide polymorphism site located at 38069494 position of a sheep reference genome, chromosome 6, and the universal primer being a downstream primer;
The sheep reference genome is in the version ARS-ui_ Ramb _v2.0;
the single nucleotide polymorphism marker is AA genotype;
The sheep is Hu sheep; individuals of the AA genotype have significantly higher body weight, height, body length and daily gain than those of the GA genotype and GG genotype.
3. The use according to claim 2, characterized in that: the KASP primer specifically comprises the following components:
upstream primer 1-FAM:
5’-gaaggtgaccaagttcatgctTCTGGCAACTAAGATAAAAATCACAAA-3’
upstream primer 2-HEX:
5’-gaaggtcggagtcaacggattTCTGGCAACTAAGATAAAAATCACAAG-3’
A downstream primer:
5’-TGGTAAGTATTGAGCTCTGGATAATGT-3’。
4. The application of a detection method of single nucleotide polymorphism of sheep LCORL gene in sheep growth trait molecular marker assisted selective breeding is characterized in that: the method for detecting the single nucleotide polymorphism of the sheep LCORL gene comprises the following steps: amplifying partial fragments containing LCORL gene single nucleotide polymorphism sites by taking genome DNA of sheep to be detected as a template through KASP primers, and then identifying the genotype of the single nucleotide polymorphism sites through detection of fluorescence, wherein the LCORL gene single nucleotide polymorphism sites are 38069494 th bit of chromosome 6 of sheep reference genome;
The sheep reference genome is in the version ARS-ui_ Ramb _v2.0;
The 38069494 th reference base of the chromosome 6 is A, and the mutant base is G;
The sheep is Hu sheep; the growth character is any one or more of weight, height, length and daily gain; the growth traits of AA genotype individuals are significantly higher in body weight, height, body length and daily gain than those of GA genotype and GG genotype.
5. The use according to claim 4, characterized in that: the KASP primer comprises a wild type site-specific primer with FAM joint, a mutant site-specific primer with HEX joint and a universal primer, wherein the wild type site-specific primer and the mutant site-specific primer are competitive upstream primers with terminal bases respectively conforming to alleles A and G of a single nucleotide polymorphism site positioned at 38069494 th position of a sheep reference genome chromosome 6, the universal primer is a downstream primer, and 3 genotypes of the single nucleotide polymorphism site of the LCORL genes can be determined according to the differentiation of fluorescence: AA is red, GA is green, GG is blue.
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| Molecular Characterization and Expression of SPP1, LAP3 and LCORL and Their Association with Growth Traits in Sheep;Yongfu La等;Genes;20190814;第10卷;第616页 * |
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