CN111793700B - SNP (Single nucleotide polymorphism) marker, detection primer pair, kit and application of deer antler yield trait - Google Patents
SNP (Single nucleotide polymorphism) marker, detection primer pair, kit and application of deer antler yield trait Download PDFInfo
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Abstract
The invention discloses SNP markers of deer antler yield traits, a detection primer pair, a kit and application, wherein the SNP markers comprise five SNP marker loci: s is S 1 、S 2 、S 3 、S 4 And S is equal to 5 The five corresponding detection primer pairs SEQ ID NO. 1-10, a method for detecting the deer antler yield traits, the application of the constructed primer pairs or SNP marker loci in breeding technology and the kit constructed by the primer pairs are provided; the beneficial effects of the invention are as follows: through systematic analysis, five SNP marker loci are selected to realize accurate detection, and meanwhile, the five SNP marker loci provide new materials for molecular breeding, provide basis for marker-assisted selection of high-yield traits of the deer antler, and realize multiple purposes of the SNP marker loci; the method for detecting the deer antler yield characteristics effectively relieves the problems in actual production and improves economic benefits; through the detection kit, the operation is simple, and the accuracy is high.
Description
Technical Field
The invention relates to the technical field of genetic biology, in particular to SNP markers, detection primer pairs, a kit and application of the deer antler yield trait.
Background
The pilose antler has irreplaceable functions in the field of Chinese medicinal materials by virtue of the magic medicinal value and the nourishing function. The deer is mainly raised in the world, but the deer industry science and technology is always in a platform period due to weak basic research, and has no major breakthrough, low breeding level and phenotype seed selection stage. The key technical problems affecting the deer yield cannot be effectively solved, the overall production benefit of the industry is low, and the stability and sustainable development of the deer industry are seriously affected.
Cultivation of high-yield spotted deer species of deer is always a pursued goal, the growth speed of deer with high yield is faster, and the weight of deer produced in the same growth period is larger. For a long time, people mainly select and breed the characters through the surface values of the traditional individuals or relatives, but the characters are influenced by external unstable factors such as environment and the like, so that the accuracy of the selection is directly caused, and the progress of the selection and breeding work of the high-quality and high-yield sika deer variety is hindered. Researches show that the genetic force and the repeated force of the heavy character of the double-yang, western-style and Changbai mountain spotted deer antler are respectively 0.53, 0.49, 0.36 and 0.67, 0.63 and 0.64, which belong to high genetic force and repeated force, and provide reliable theoretical and practical basis for the molecular marker auxiliary selection of the deer antler yield character.
At present, most of researches on the yield traits of the deer antler are related analysis of SNP markers and the weight of the deer antler, for example, SNP detection of growth hormone genes shows that among 3 genotypes generated by G-A mutation of the 2 nd intron, BB genotype individuals have differences between the deer antler yield of a fifth saw and AA genotype individuals; in addition, research shows that the estimated value of the antler production of CT genotype and CC genotype of the mutation of the growth hormone gene C139-T139 is obviously higher than that of TT genotype, the estimated value of the antler production of CC genotype of the mutation of the exon G629-C629 of the melatonin I type receptor subtype a gene is obviously higher than that of GC genotype, the estimated value of the antler production of the GC genotype is obviously higher than that of GG genotype, the estimated value of the antler production of CT genotype of the mutation of the exon C75-T75 of the androgen receptor gene 3 is higher than that of CC genotype, the antler production of 3 genotypes produced by the mutation of the intron G371T of the 7 of the transforming growth factor beta 1 gene is higher than that of other two genotypes, the allelic typing is carried out on the major histocompatibility complex gene of the sika deer, and the correlation research on the antler growth characters shows that the allelic DRB3 is obviously positively correlated with the yield of the Xylodes, and the DRB8 and DRB11 genes are obviously inversely correlated with the yield of the Xylodes. However, because different research methods, environmental factors and standard definition of research samples are inconsistent, the obtained SNP markers are not verified, and the experimental repeatability is poor, so that the deer antler yield traits have no definite and reliable molecular markers so far.
More importantly, the deer antler yield is a complex character, the variation of a single gene locus can be completely controlled, a series of loci are needed for regulation and control, and the variation of a plurality of gene loci can bring about little influence, and the effects of the gene loci have superposition effects. If the related site variation of the complex character is to be accurately positioned, more valuable molecular markers are obtained through deep analysis of large data such as deer genome, transcriptome and the like, and large-scale population verification analysis is carried out on the markers, so that the molecular marker sites obviously related to the deer yield character can be accurately obtained, and the aims of early selection of the deer yield character and improvement of breeding accuracy are fulfilled.
In conclusion, the development and application of SNP markers related to the deer yield traits have important significance for accelerating the breeding process of high-yield deer and improving the overall level and economic benefit of deer industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides SNP markers, detection primer pairs, kits and applications of the character of the yield of the antler so as to at least achieve the aim of accurately detecting the yield of the antler and provide a direct basis for the establishment of the accurate detection standard of the yield of the antler.
The aim of the invention is realized by the following technical scheme:
an SNP marker for deer antler yield traits, comprising five SNP marker loci: s is S 1 、S 2 、S 3 、S 4 And S is equal to 5 ；
The S is 1 To indicate that the SNP locus is positioned at 11137347bp of a red deer genome chromosome 8, and the nucleotide sequence of the SNP locus is C/T;
the S is 2 To indicate that the SNP locus is positioned at 28438186bp of a red deer genome chromosome 8, and the nucleotide sequence of the SNP locus is A/G;
the S is 3 To indicate that the SNP locus is positioned at 20928571bp of a No. 14 chromosome of a red deer genome, and the nucleotide sequence of the SNP locus is A/G;
the S is 4 To indicate that the SNP locus is positioned at 86027207bp of a red deer genome chromosome 15, the nucleotide sequence of the SNP locus is A/T;
the S is 5 To indicate that the SNP locus is located at 69698130bp of the No. 23 chromosome of the red deer genome, the nucleotide sequence of the SNP locus is C/G.
Preferably, in order to further achieve the purpose of accurate detection, the five SNP marker loci are obtained by performing systematic analysis on a red deer genome (Genbank accession number: PRJNA 564362) and a sika deer genome (Genbank accession number: PRJNA 541418); the system analysis comprises the following steps:
s1, taking the difference of the yields in the same deer growth period as a reference, and combining with the comparative analysis of a red deer genome (Genbank accession number: PRJNA 564362) and a sika deer genome (Genbank accession number: PRJNA 541418) to obtain a preliminary SNP marker locus;
s2, carrying out genome-wide association analysis and screening on the primary SNP marker loci to obtain secondary SNP marker loci related to the yield of the deer antler;
s3, carrying out gene annotation and co-expression network analysis on the secondary SNP marker loci to obtain candidate SNP marker loci;
s4, analyzing candidate SNP marker loci through a Massarray technology, and carrying out genotyping on sika deer individuals with different yields in the same deer growth period to obtain different genotypes;
s5, carrying out correlation analysis on the obtained genotype and the deer antler yield to obtain five SNP loci; through genome comparison analysis, whole genome association analysis, gene annotation and co-expression network analysis, massary ARRAY technical analysis and correlation analysis, five SNP marker loci related to deer yield traits are selected from a red deer genome and a sika deer genome, and further, the deer yield of two deer species can be accurately detected, so that the purpose of accurate detection is achieved. Meanwhile, five SNP marker loci provide new materials for molecular breeding and scientific basis for marker-assisted selection of high-yield traits of the deer antler.
Preferably, in order to further realize versatility, the DNA fragments where the five SNP marker loci are located can be amplified by primers with sequences shown in SEQ ID nos. 1 to 10, respectively, so that reverse inference or breeding design can be performed by using primer pairs, and the five SNP marker loci and the primer pairs designed by the SNP marker loci can be further used in breeding selection and deer antler yield detection, thereby realizing the purpose of multi-purposed SNP marker loci.
The invention also provides a primer pair for detecting SNP markers of the deer antler yield traits, and the primer pair sequences are shown in SEQ ID NO. 1-10:
1F：5’-GTAGACTCCGGGGGTTTGTG-3’(SEQ ID NO.1)，
1R：5’-AATTCTCCAGCAGTCCGGTG-3’(SEQ ID NO.2)；
2F：5’-AAGTGACTACTCCCTGTCCCA-3’(SEQ ID NO.3)，
2R：5’-CCAATCAACCCACCAACCCA-3’(SEQ ID NO.4)；
3F：5’-CCAATCAACCCACCAACCCA-3’(SEQ ID NO.5)，
3R：5’-TCCTAACCACTGGACCACCA-3’(SEQ ID NO.6)；
4F：5’-GTCGGGAAAATCCCCTGGAG-3’(SEQ ID NO.7)，
4R：5’-CCTTCGGTCCTAGGGTGAGA-3’(SEQ ID NO.8)；
5F：5’-TGGTGATGCCATCCAACCAT-3’(SEQ ID NO.9)，
5R:5'-AATGCTGGTGGGAAAATGGC-3' (SEQ ID NO. 10); amplifying a sample to be detected by using the primer pair, wherein if the amplified fragment meets three or more of a homozygote or a heterozygote containing a base T at 11137347bp of a red deer genome 8, a homozygote or a heterozygote containing a base C at 69698130bp of a red deer genome 23, a homozygote genotype containing a base A at 28438186bp of the red deer genome 8, a homozygote genotype containing a base G at 20928571bp of the red deer genome 14 and a homozygote genotype containing a base T at 86027207bp of the red deer genome 15, the corresponding deer is a dominant individual with a high-yield phenotype of the deer; the five SNP marker loci for the deer yield traits are defined firstly, and then five pairs of specific primer pairs are designed, so that a method for detecting the deer yield traits is established by utilizing the primer pairs, detected objects are not limited by the age of deer, early screening can be carried out from young deer and bred deer, the problems of long selection time and low accuracy of deer with high deer yield in actual production are effectively solved, the breeding cost is reduced, the economic benefit of deer breeding is improved, and the method plays an important role in individual selection and new variety cultivation of deer with high deer yield.
The invention also provides application of the SNP marker or the primer pair in breeding selection, and the primer pair or the SNP marker detects and screens dominant individual deer with high-yield phenotype of the deer through combining with specific primers for detecting other deer phenotypes.
The invention also provides application of the SNP marker or the primer pair in preparation of a kit or in a detection method, and the kit or the detection method is used for detecting the deer antler output of sika deer or red deer.
The invention further provides a method for detecting the deer antler yield traits, which comprises the following steps:
1) Extracting genomic DNA of sika deer or red deer to be detected;
2) Using the extracted genomic DNA as a template, using SEQ ID NO:1-10, performing PCR amplification to obtain an amplification product; the total amount of the amplification system was 50. Mu.l, including 2XPCR Master Mix 25. Mu.l, 10 pmol/. Mu.l forward and reverse primers each 0.5. Mu.l, 50 ng/. Mu.l template DNA 1. Mu.l, and 23. Mu.l sterile distilled water; the reaction conditions for amplification are: 94℃for 5min,94℃for 30s,60℃for 30s,72℃for 45s,30 cycles; 72 ℃ for 5min;
3) Sequencing the amplified product, respectively detecting the base at the positions of 11137347bp and 28438186bp of the red deer genome No. 8 chromosome and the position of 20928571bp of the red deer genome No. 14 chromosome, the position of 86027207bp of the red deer genome No. 15 chromosome and the position of 69698130bp of the red deer genome No. 23 chromosome, and judging the genotype;
4) Judging whether the deer to be detected is a dominant individual with the high-yield phenotype of the deer antler according to the determined genotype.
The invention also provides a detection kit for the deer antler yield traits, which comprises a primer pair shown in SEQ ID NO. 1-10, a PCR reaction buffer solution and a standard positive template; by utilizing the designed primer pair, a detection kit is constructed, so that the deer antler yield characteristics of deer can be detected rapidly, small-scale farmers and large-scale deer breeding enterprises can be adopted, the operation method is simple, the accuracy is high, and the method has high practical application value.
The beneficial effects of the invention are as follows:
1. through genome comparison analysis, whole genome association analysis, gene annotation and co-expression network analysis, massary ARRAY technical analysis and correlation analysis, five SNP marker loci related to deer yield traits are selected from a red deer genome and a red deer genome, and further, the deer yield of two deer species can be accurately detected, so that the aim of accurate detection is fulfilled, and simultaneously, the five SNP marker loci provide new materials for molecular breeding and scientific basis for marker-assisted selection of the deer high yield traits.
2. The five SNP marker loci and the primer pair designed by the SNP marker loci can be further utilized in breeding selection and deer antler yield detection by utilizing the primer pair to carry out reverse inference or breeding design, thereby realizing the purpose of multiple purposes of the SNP marker loci.
3. The five SNP marker loci for the deer yield traits are defined firstly, and then five pairs of specific primer pairs are designed, so that a method for detecting the deer yield traits is established by utilizing the primer pairs, detected objects are not limited by the age of deer, early screening can be carried out from young deer and bred deer, the problems of long selection time and low accuracy of deer with high deer yield in actual production are effectively solved, the breeding cost is reduced, the economic benefit of deer breeding is improved, and the method plays an important role in individual selection and new variety cultivation of deer with high deer yield.
4. By utilizing the designed primer pair, a detection kit is constructed, so that the deer antler yield characteristics of deer can be detected rapidly, small-scale farmers and large-scale deer breeding enterprises can be adopted, the operation method is simple, the accuracy is high, and the method has high practical application value.
Drawings
FIG. 1 is a schematic diagram showing the gene frequencies of different genotypes after genotyping according to the present invention,
wherein, the left and right areas of the straight line represent the gene frequency data corresponding to different genotypes, a) represent the marking site data of TT, CT and CC of the No. 8 chromosome of the red deer genome, b) represent the marking site data of AA, AG and GG of the No. 8 chromosome of the red deer, c) represent the marking site data of AA, AG and GG of the No. 14 chromosome of the red deer, d) represent the marking site data of TT, AT and AA of the No. 15 chromosome of the red deer, e) represent the marking site data of GG, CG and CC of the No. 23 chromosome of the red deer.
Detailed Description
The technical scheme of the present invention is described in further detail below, but the scope of the present invention is not limited to the following.
The technical means used in the examples are all conventional means known to the person skilled in the art, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the raw materials used are all commercially available according to the conditions recommended by the manufacturer's instructions.
Example 1 acquisition of SNP markers related to cornu Cervi Pantotrichum yield traits
Based on the high similarity of sika deer and sika deer genome sequences (genome sequencing data comparison rate > 99%), the sika deer genome (Genbank accession number:PRJNA 564362) and sika deer genome (Genbank accession number:PRJNA 541418) are subjected to systematic analysis, and the specific steps are as follows:
s1, taking the difference of the output in the same deer growth period as a reference, taking a high-quality chromosome level red deer genome (Genbank accession number: PRJNA 564362) as a reference genome, sequencing the body weight of 100 red deer individuals with obvious difference of the deer output, detecting the data of the red deer genome (Genbank accession number: PRJNA 541418), and comparing and analyzing with the reference genome to obtain 39,788,029 primary SNP marker loci;
s2, carrying out genome-wide association analysis on the obtained primary SNP marker loci, screening SNP marker loci related to the deer antler yield, and obtaining 1,182 secondary SNP marker loci related to the deer antler yield;
s3, carrying out gene annotation and co-expression network analysis on the obtained secondary SNP marker loci, screening and analyzing genotypes of the secondary SNP marker loci, and screening out 100 candidate SNP marker loci;
s4, carrying out genotyping on the obtained candidate SNP marker loci in 341 sika deer individuals with the same growing period of the deer antler and the deer antler yield record by using a nucleic acid mass spectrometry analysis system (Massarray) technology to obtain different genotypes, wherein the obtained candidate SNP marker loci are shown in figure 1;
s5, performing correlation analysis on the obtained genotype and the deer antler yield to obtain five SNP marker loci shown in Table 1.
TABLE 1 correlation analysis of genotypes at five SNP loci and deer antler yield traits
Note that: the corner mark A, B in Table 1 shows that the difference of the data of different deer antler yields is remarkable (P < 0.05)
As can be seen from Table 1, the five SNP sites are respectively identified as: s is S 1 、S 2 、S 3 、S 4 And S is equal to 5 ；
The S is 1 To indicate that the SNP locus is positioned at 11137347bp of a red deer genome chromosome 8, and the nucleotide sequence of the SNP locus is C/T;
the S is 2 To indicate that the SNP locus is positioned at 28438186bp of a red deer genome chromosome 8, and the nucleotide sequence of the SNP locus is A/G;
the S is 3 To indicate that the SNP locus is positioned at 20928571bp of a No. 14 chromosome of a red deer genome, and the nucleotide sequence of the SNP locus is A/G;
the S is 4 To indicate that the SNP locus is positioned at 86027207bp of a red deer genome chromosome 15, the nucleotide sequence of the SNP locus is A/T;
the S is 5 To indicate that the SNP locus is located at 69698130bp of the No. 23 chromosome of the red deer genomeThe nucleotide sequence is C/G.
Through genome comparison analysis, whole genome association analysis, gene annotation and co-expression network analysis, massary ARRAY technology analysis and correlation analysis of system analysis, five SNP marker loci related to deer yield traits are selected from a red deer genome and a red deer genome, and further, the deer yield of two deer species can be accurately detected, so that the purpose of accurate detection is realized. Meanwhile, five SNP marker loci provide new materials for molecular breeding and scientific basis for marker-assisted selection of high-yield traits of the deer antler.
Example 2 verification of deer individuals high in deer yield by five SNP markers
Designing a detection primer pair by combining Primer5.0 aiming at the DNA fragments corresponding to the five SNP marker loci obtained in the example 1, and finally obtaining the primer pair shown in SEQ ID NO. 1-10:
1F：5’-GTAGACTCCGGGGGTTTGTG-3’(SEQ ID NO.1)，
1R：5’-AATTCTCCAGCAGTCCGGTG-3’(SEQ ID NO.2)；
2F：5’-AAGTGACTACTCCCTGTCCCA-3’(SEQ ID NO.3)，
2R：5’-CCAATCAACCCACCAACCCA-3’(SEQ ID NO.4)；
3F：5’-CCAATCAACCCACCAACCCA-3’(SEQ ID NO.5)，
3R：5’-TCCTAACCACTGGACCACCA-3’(SEQ ID NO.6)；
4F：5’-GTCGGGAAAATCCCCTGGAG-3’(SEQ ID NO.7)，
4R：5’-CCTTCGGTCCTAGGGTGAGA-3’(SEQ ID NO.8)；
5F：5’-TGGTGATGCCATCCAACCAT-3’(SEQ ID NO.9)，
5R:5'-AATGCTGGTGGGAAAATGGC-3' (SEQ ID NO. 10); after the primer design is finished, the primer pair SEQ ID NO. 1-10 is used for carrying out PCR amplification and sequencing on genome DNA of 56 sika deer species deer (deer growth for 70 days and weight is more than 5 kg) with high deer yield and 125 sika deer species deer (deer growth for 70 days and weight is less than 4 kg) with clear deer phenotype record, wherein the total amount of an amplification system is 50 mu l, the amplification system comprises 2XPCR Master Mix 25 mu l,10 pmol/mu l forward and reverse primers respectively 0.5 mu l,50ng/ul template DNA 1 mu l and sterile distilled water 23 mu l; the amplification reaction conditions are shown in Table 2, the theoretical value of the primer annealing temperature is 60 ℃, the genotype analysis is carried out on the obtained amplification product, and the sika deer number which accords with the high-yield genotype of the deer antler is counted to obtain Table 3.
TABLE 2PCR amplification reaction conditions
TABLE 3 detection conditions of 56 high-production deer and 125 production deer using SNP marker loci
As shown in Table 3, when the genotypes of the high-yield species satisfying 3 or more SNP loci are identical, 88.5% of the high-yield species deer can be identified, and only 4.0% of the production deer can be identified, thus excluding 96% of the production deer. And when the high-yield genotypes of 2 SNP loci and more are consistent, 98.9% of high-yield deer can be identified, but 36.8% of high-yield deer can be identified, so that the consistent SNP marker loci are limited to 3 loci and more, thereby more accurately distinguishing the high-yield traits of the deer antler and improving the accuracy of judging the yield traits of the deer antler.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Sequence listing
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<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
tggtgatgcc atccaaccat 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
aatgctggtg ggaaaatggc 20
Claims (5)
- The application of SNP markers in detecting the deer antler yield traits is characterized in that: at least 3 SNP marker loci comprising five SNP marker loci S1, S2, S3, S4 and S5 respectively;s1 is 11137347bp of a red deer genome chromosome 8, which represents that the SNP locus is located, and the nucleotide sequence of the locus is C/T;s2 is 28438186bp of the SNP locus on a red deer genome chromosome 8, and the nucleotide sequence of the locus is A/G;s3 is 20928571bp of the SNP locus on the chromosome 14 of the red deer genome, and the nucleotide sequence of the locus is A/G;s4 is 86027207bp of the SNP locus on the chromosome 15 of the red deer genome, and the nucleotide sequence of the locus is A/T;s5 is 69698130bp of the SNP locus of the red deer genome chromosome 23, and the nucleotide sequence of the SNP locus is C/G.
- 2. The use of the SNP marker according to claim 1 for detecting the character of the production of deer antler, characterized in that: the five SNP marker loci are obtained by systematically analyzing a red deer genome and a sika deer genome.
- 3. The use of the SNP marker according to claim 2 for detecting the character of the production of deer antler, characterized in that: the system analysis comprises the following steps:s1, obtaining a preliminary SNP marker locus by taking the difference of the output in the same deer growing period as a reference and combining with the comparison analysis of a red deer genome and a sika deer genome;s2, carrying out genome-wide association analysis and screening on the primary SNP marker loci to obtain secondary SNP marker loci related to the yield of the deer antler;s3, carrying out gene annotation and co-expression network analysis on the secondary SNP marker loci to obtain candidate SNP marker loci;s4, analyzing candidate SNP marker loci through a Massarray technology, and carrying out genotyping on sika deer individuals with different yields in the same deer growth period to obtain different genotypes;s5, performing correlation analysis on the obtained genotype and the deer antler yield to obtain the five SNP loci.
- 4. The use of the SNP marker according to claim 1 for detecting the character of the production of deer antler, characterized in that: the DNA fragments with the five SNP marker loci can be respectively amplified by primers with sequences shown as SEQ ID NO. 1-10.
- 5. The method for detecting the deer antler yield traits is characterized by comprising the following steps: the method comprises the following steps:1) Extracting genomic DNA of sika deer or red deer to be detected;2) Using the extracted genomic DNA as a template, using SEQ ID NO:1-10, performing PCR amplification to obtain an amplification product;3) Sequencing the amplified product, respectively detecting the base at the positions of 11137347bp and 28438186bp of the red deer genome No. 8 chromosome and the position of 20928571bp of the red deer genome No. 14 chromosome, the position of 86027207bp of the red deer genome No. 15 chromosome and the position of 69698130bp of the red deer genome No. 23 chromosome, and judging the genotype;4) Judging whether the deer to be detected is a dominant individual with the high-yield phenotype of the deer antler according to the determined genotype.
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