CN115992254A - Reagent for detecting genotype of SNP locus correlated with Leizhou black duck egg laying character - Google Patents
Reagent for detecting genotype of SNP locus correlated with Leizhou black duck egg laying character Download PDFInfo
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Abstract
The present application is a divisional application of an inventive patent application with the application number 2020115303638 and the name of "a reagent for detecting the genotype of SNP locus correlated with the egg laying property of black ducks in Rauzhou". The invention discloses a reagent for detecting genotype of SNP locus correlated with the egg laying character of black duck in Rauzhou. The invention discovers 7 SNP loci of the Leizhou duck HAL gene, the genotypes of the SNP loci are obviously related to the egg-laying properties, especially the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and individuals with the genotype of the SNP locus 6 being AA can be used as optimal selection of individuals with low open-time age, low open-time weight and high egg-laying amount for screening Leizhou black duck populations. The method provides a theoretical basis for molecular marker assisted breeding of the black ducks in Rauzhou and establishment of dominant high-yield populations.
Description
The present application is a divisional application of an inventive patent application with the application number 2020115303638 and the name of "a reagent for detecting the genotype of SNP locus correlated with the egg laying property of black ducks in Rauzhou".
Technical Field
The invention relates to the technical field of molecular breeding, in particular to a reagent for detecting genotypes of SNP loci correlated with the egg laying properties of black ducks in Rauzhou.
Background
China is a large poultry farming country, the annual poultry product yield is the leading world, and the poultry egg yield and the poultry meat yield in 2018 are obtained by current statistics and respectively located in the first world and the second world. But the local variety resources of domestic poultry are rich, but the fertility of the local variety is lower, the investment of farmers is increased, the output is reduced, and the problem of local variety resource loss is caused. The Guangdong province Guangxi region has various local poultry varieties, wherein the Razhou black ducks are local varieties mainly distributed in the Razhou peninsula, belong to egg-meat dual-purpose black feather ducks, are small in size and delicious in meat quality, have the advantages of high laying rate, coarse feeding resistance, strong disease resistance, strong environmental adaptability and the like, are widely favored by local people, and are less in related researches at present. The method for obtaining the laying rate of the black ducks in the Lei state is 27-41 weeks old and the laying rate in the peak period is more than 90% by using the egg laying curve fitting method is studied; the weight of the Lei Zhou black duck eggs is higher than that of other local varieties, such as a Jinyun sheldrake, a Huai nan sheldrake, a white duck, a county sheldrake and the like; the Leizhou black duck meat and eggs are rich in value, various microelements, especially Fe element required by human body, and high in nutrition content.
Chinese patent 201810410892.0 relates to a molecular marker related to egg laying performance of laying ducks and application thereof in breeding, and discloses a molecular marker which is obviously related to the egg laying amount of ducks at day-old, 34 week-old and 72 week-old. However, since the egg-laying traits are quantitative traits, the quantitative traits are often controlled by a plurality of genes, and the egg-laying traits can be judged more accurately by finding more molecular markers affecting the related traits.
histidine-Lyase (Histidine Ammonia-Lyase, HAL), also known as histidine enzyme (Histidase) or histidine deaminase, is a member of the family of carbon-nitrogen cleaving enzymes that catalyzes the first reaction of histidine catabolism, i.e. catalyzes the deamination of histidine to urocanic acid. Previous studies have shown that mutations in the HAL gene are associated with histidine, a risk of non-melanoma skin cancer and a incidence of coronary heart disease. The role of HAL genes in egg production performance has not been reported.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a reagent for detecting the genotype of SNP loci correlated with the egg laying characteristics of black ducks in Lei Zhou.
The first object of the invention is to provide a reagent for detecting the genotype of SNP locus correlated with the egg laying character of black ducks in Rauzhou.
The second object of the invention is to provide the application of the reagent in preparing a kit for evaluating the egg laying performance of the black ducks in Rauzhou.
A third object of the invention is to provide the use of said agent for evaluating the egg laying performance of black ducks in the state of rayleigh.
The fourth object of the invention is to provide a kit for evaluating the egg laying performance of the black ducks in Lei Zhou.
A fifth object of the invention is to provide the use of one or both of said reagents, or said kits, for evaluating the egg laying performance of black ducks in the state of rayleigh.
In order to achieve the above object, the present invention is realized by the following means:
the invention discovers that 7 SNP sites in the introns 1 and 2 of the HAL gene of the Rauzhou duck are respectively SNP site 1, SNP site 2, SNP site 3, SNP site 4, SNP site 5, SNP site 6 and/or SNP site 7:
wherein SNP site 1 is located at 288 th base of the first intron and C is mutated into T (I1-288C > T), the open yield weight of the sample of CC genotype is obviously higher than that of the sample of TT genotype, and the 300d egg yield of the sample of CC genotype is obviously lower than that of the sample of TT genotype;
SNP site 3 is located at 438 th base of the first intron, A is mutated to G (I1-438A > G), and the open birth date of the sample of AA genotype is extremely lower than that of the sample of AG genotype;
It was further found that individuals with genotype of SNP site 3 as AG, genotype of SNP site 4 as CA, genotype of SNP site 5 as TA, and genotype of SNP site 6 as AG were significantly older than individuals with genotype of SNP site 3 as AA, genotype of SNP site 4 as CC, genotype of SNP site 5 as TT, and genotype of SNP site 6 as AA, and individuals with genotype of SNP site 3 as AA, genotype of SNP site 4 as CC, genotype of SNP site 5 as TA, and genotype of SNP site 6 as AG;
the genotype of SNP locus 3 is AG, the genotype of SNP locus 4 is CA, the genotype of SNP locus 5 is TA, the genotype of SNP locus 6 is AG, the genotype of SNP locus 3 is AA, the genotype of SNP locus 4 is CC, the genotype of SNP locus 5 is TT, the individual with genotype of SNP locus 6 is AG has a significantly higher split weight than the individual with genotype of SNP locus 3 is AA, the genotype of SNP locus 4 is CC, the genotype of SNP locus 5 is TT, and the genotype of SNP locus 6 is AA;
the genotype of SNP locus 3 is AA, the genotype of SNP locus 4 is CC, the genotype of SNP locus 5 is TT, and the 300d egg yield of individuals with genotype of SNP locus 6 being AA is obviously higher than that of individuals with genotype of SNP locus 3 being AA, genotype of SNP locus 4 being CC, genotype of SNP locus 5 being TA, and genotype of SNP locus 6 being AG.
Therefore, the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the individuals with the genotype of the SNP locus 6 being AA can be used as the optimal selection of individuals with low open-time, low open-time weight and high egg yield for the Leizhou black duck population screening.
Therefore, the present invention claims a reagent for detecting genotype of SNP site having correlation with the Lei Zhou black duck egg laying trait, which is used for detecting genotype of SNP site 1, SNP site 2, SNP site 3, SNP site 4, SNP site 5, SNP site 6 and/or SNP site 7;
the SNP locus 1 is positioned at 288 th base of a first intron of the HAL gene, the open yield weight of a sample of the CC genotype is obviously higher than that of a sample of the TT genotype, and the 300d egg yield of the sample of the CC genotype is obviously lower than that of the sample of the TT genotype;
the SNP locus 2 is positioned at 438 th base of the first intron of the HAL gene, and the 300d egg yield of a sample of the TT genotype is obviously higher than that of a sample of the GG genotype;
the SNP locus 3 is positioned at the 438 th base of the first intron of the HAL gene, and the date of birth of a sample of the AA genotype is extremely lower than that of a sample of the AG genotype;
the SNP locus 4 is positioned at the 16 th base of the second intron of the HAL gene, and the date of birth of a sample of the AA genotype is obviously lower than that of a sample of the AC genotype;
the SNP locus 5 is positioned AT the 123 th base of the second intron of the HAL gene, the open date of a sample of the TT genotype is obviously lower than that of a sample of the AT genotype, and the 300d egg yield of the sample of the TT genotype is obviously higher than that of the sample of the AT genotype;
the SNP locus 6 is positioned at 141 th base of a second intron of the HAL gene, the open date of a sample of the AA genotype is obviously lower than that of a sample of the GG genotype, the open date of a sample of the AG genotype is obviously higher than that of a sample of the GG genotype, and the 300d egg laying number of a sample of the AG genotype is obviously lower than that of a sample of the GG genotype;
the SNP locus 7 is positioned at the 168 th base of the second intron of the HAL gene, the open date of the sample of the GG genotype is extremely lower than that of the AA type, the open date of the sample of the AG genotype is remarkably higher than that of the GG type, and the 300d egg yield of the sample of the AG genotype is remarkably lower than that of the GG type.
Preferably, SNP site 3, SNP site 4, SNP site 5 and SNP site 6 are detected.
The genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the individual with the genotype of the SNP locus 6 is AG, the date of birth is obviously higher than that of the individual with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TT, the genotype of the SNP locus 6 being AA, the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG;
the genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the genotype of the SNP locus 6 is AG, the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, the individual split weight of the genotype of the SNP locus 6 is AG is obviously higher than that of the individual split weight of the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the genotype of the SNP locus 6 is AA;
the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the 300d egg yield of individuals with the genotype of the SNP locus 6 being AA is obviously higher than that of individuals with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG.
More preferably, the reagent is a primer.
Even more preferably, the primer nucleotide sequence is shown in SEQ ID NO. 1-2.
SEQ ID NO.1：5’-GAATGGCACCTGAGTTAGCA-3’；
SEQ ID NO.2：5’-CCTGTTCTGGTTCTCGGTATTTGCT-3’。
The invention also claims the application of any reagent in preparing a kit for evaluating the egg laying performance of the black ducks in Lei Zhou.
The invention also claims the application of any one of the reagents in evaluating the egg laying performance of the black ducks in Lei Zhou.
Further, the invention also claims an evaluation of the egg laying performance of the black ducks in Lei Zhou, comprising the reagent.
Preferably, the reagent is a primer with a nucleotide sequence shown as SEQ ID NO. 1-2.
Most preferably, the kit for evaluating the egg laying performance of the black ducks in Rauzhou comprises a primer with a nucleotide sequence shown as SEQ ID NO. 1-2 and 2 xGreen Taq Mix;
the using method is as follows:
1. extracting sample DNA;
2. PCR amplification
PCR reaction system 20. Mu.L: 2 XGreen Taq Mix 10. Mu.L, each of the upstream/downstream primers (nucleotide sequences shown as SEQ ID NOS.1-2) 0.4. Mu.L, template 1. Mu.L, ddH 2 O 8.2μL；
PCR reaction procedure: thermal denaturation at 95℃for 5min, deformation at 95℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min,35 cycles, elongation at 72℃for 5min, and preservation at 4 ℃.
3. Sequencing of PCR products
Electrophoresis, recovery and sequencing of 1% agarose gel;
interpretation of the results:
The open-yield weight of the sample of the SNP locus 1 and the CC genotype is obviously higher than that of the sample of the TT genotype, and the 300d egg yield of the sample of the CC genotype is obviously lower than that of the sample of the TT genotype;
the 300d egg yield of the sample of the SNP locus 2, TT genotype is obviously higher than that of the sample of GG genotype;
the SNP locus 3, the age of the sample of the AA genotype at the birth date is extremely lower than that of the sample of the AG genotype;
the SNP locus 4, the age of the open birth of a sample of the AA genotype is obviously lower than that of a sample of the AC genotype;
the SNP locus 5, the open date age of the sample of the TT genotype is obviously lower than that of the sample of the AT genotype, and the 300d egg laying number of the sample of the TT genotype is obviously higher than that of the sample of the AT genotype;
the SNP locus 6, the open-time-of-production age of the sample of the AA genotype is obviously lower than that of the sample of the GG genotype, the open-time-of-production weight of the sample of the AG genotype is obviously higher than that of the sample of the GG genotype, and the 300d egg laying number of the sample of the AG genotype is obviously lower than that of the sample of the GG genotype;
the SNP locus 7, the open-birth age of the sample of the AG genotype is extremely lower than that of the AA type, the open-birth weight of the sample of the AG genotype is remarkably higher than that of the GG type, and the 300d egg yield of the sample of the AG genotype is remarkably lower than that of the GG type.
The genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the individual with the genotype of the SNP locus 6 is AG, the date of birth is obviously higher than that of the individual with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TT, the genotype of the SNP locus 6 being AA, the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG;
the genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the genotype of the SNP locus 6 is AG, the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, the individual split weight of the genotype of the SNP locus 6 is AG is obviously higher than that of the individual split weight of the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the genotype of the SNP locus 6 is AA;
the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the 300d egg yield of individuals with the genotype of the SNP locus 6 being AA is obviously higher than that of individuals with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG.
The application of the reagent or one or two of the evaluation of the egg laying performance of the Lei Zhou black ducks in the evaluation of the egg laying performance of the Lei Zhou black ducks also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers 7 SNP loci of the Leizhou duck HAL gene, the genotypes of the SNP loci are obviously related to the egg-laying properties, especially the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and individuals with the genotype of the SNP locus 6 being AA can be used as optimal selection of individuals with low open-time age, low open-time weight and high egg-laying amount for screening Leizhou black duck populations. The method provides a theoretical basis for molecular marker assisted breeding of the black ducks in Rauzhou and establishment of dominant high-yield populations.
Drawings
FIG. 1 is an electrophoretogram of the amplification product of the HAL gene of the Lei Zhou duck; lanes 1-4 are PCR amplified products from different individuals, respectively.
FIG. 2 is a sequencing map of 3 genotypes of SNP locus 1 of a HAL gene; FIG. I is a genotype CT individual; FIG. II is a genotype CC individual; FIG. III is a genotype TT individual.
FIG. 3 is a sequencing map of 3 genotypes of SNP locus 2 of a HAL gene; FIG. I is an individual with genotype TG; FIG. II is a person with genotype GG; FIG. III is a genotype TT individual.
FIG. 4 is a sequencing map of 3 genotypes of SNP locus 3 of a HAL gene; FIG. I is an individual with genotype AG; FIG. II is an individual with genotype AA; FIG. III shows individuals with genotype GG.
FIG. 5 is a sequencing map of 3 genotypes of SNP locus 4 of a HAL gene; FIG. I is an individual with genotype CA; FIG. II is a genotype CC individual; FIG. III is an individual with genotype AA
FIG. 6 is a sequencing map of 3 genotypes of SNP locus 5 of a HAL gene; FIG. I is an individual with genotype AT; FIG. II is a person with genotype TT; FIG. III is an individual with genotype AA.
FIG. 7 is a sequencing map of 3 genotypes of SNP locus 6 of a HAL gene; FIG. I is an individual with genotype GA; FIG. II is an individual with genotype AA; FIG. III shows individuals with genotype GG.
FIG. 8 is a sequencing map of 3 genotypes of SNP locus 7 of a HAL gene; FIG. I is an individual with genotype AG; FIG. II is an individual with genotype AA; FIG. III shows individuals with genotype GG.
FIG. 9 shows the SNPs locus linkage reaction of the HAL gene of dairy cow population.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The main reagent comprises: whole genome blood DNA extraction kit, purchased from Tiangen Biochemical technology (Beijing) Co., ltd; agarose, available from Boglin biosciences.
The main instrument is as follows: PCR instruments were purchased from Hangzhou Langmuir scientific instruments Co., ltd; DYY-6C electrophoresis apparatus was purchased from Beijing six biotechnology Co., ltd; a gel imaging system; the heaven-mei electronic balance analytical balance FA1204B was purchased from Shanghai precision.
Example 1 construction of a DNA Mixed pool PCR amplification
1. Experimental method
(1) Test animals
The test selects F4 generation black duck basic group from a certain farm in Zhanjiang city. Taking 93 black ducks in the state of 300d, taking 2mL of blood from the jugular vein, collecting the blood by using a heparin anticoagulation tube, and storing the blood at-20 ℃ for standby to extract blood genome DNA.
(2) Blood genomic DNA extraction
Extracting blood DNA by adopting a blood genome DNA extraction kit of the radix angelicae sinensis, and referring to a kit instruction, detecting the concentration and purity of the DNA by using a concentration analyzer, and preserving at-20 ℃ for later use.
(3) DNA pool construction
And randomly selecting 30-40 DNA samples to mix in equal quantity to prepare the DNA mixed pool sample.
(4) Primer design
Primer design of HAL gene was performed using Primer 5.0 software according to HAL genome sequence (number (ENSAPLG 00000003775.2)) provided by Ensemble database, and the primers were sent to the Probiotechnological engineering (Shanghai) Co., ltd for synthesis:
an upstream primer: 5'-GAATGGCACCTGAGTTAGCA-3' (SEQ IN NO. 1);
a downstream primer: 5'-CCTGTTCTGGTTCTCGGTATTTGCT-3' (SEQ ID NO. 2).
(5) Target gene PCR amplification and verification
PCR reaction system 20. Mu.L: 2 XGreen Taq Mix 10. Mu.L, each of the upstream/downstream primers (nucleotide sequences shown as SEQ ID NOS.1-2) 0.4. Mu.L, template 1. Mu.L, ddH 2 O 8.2μL；
PCR reaction procedure: thermal denaturation at 95℃for 5min, deformation at 95℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min,35 cycles, elongation at 72℃for 5min, and preservation at 4 ℃.
After electrophoresis verification, 1% agarose gel was sent to Shanghai Biotechnology Co.Ltd for sequencing.
2. Experimental results
The PCR amplified HAL gene product of the black duck is detected by 1% gel electrophoresis, and the result is shown in figure 1, and the PCR product has good amplification specificity, no impurity band and clear band, and can be directly used for sequencing identification.
7 SNP sites were found in the HAL gene introns 1 and 2, wherein SNP site 1, SNP site 2 and SNP site 3 were all located in the HAL gene intron 1, SNP site 4, SNP site 5, SNP site 6 and SNP site 7 were all located in the HAL gene intron 2, each genotype sequencing result of 7 SNPs is shown in FIGS. 2 to 8,
specifically, SNP locus 1 is positioned at 250 bases of the 5' end of the amplified product, and a C/T single nucleotide polymorphism exists;
EXAMPLE 2 population genetic characterization of 7 SNP loci of HAL Gene
1. Experimental method
Sequence alignment is carried out by using SeqMan software, SNP loci and genotypes of each individual are counted, and the gene frequency, genotype frequency, genetic heterozygosity (H), effective allele factor (Ne), polymorphism Information Content (PIC) and chi-square test value chi of the allele of each SNP locus are calculated 2 。
2. Experimental results
As a result, as shown in Table 1, the dominant genotypes at SNP locus 1 to SNP locus 7 were CT, TG, AA, CC, AT, GA and AG, respectively, and the dominant alleles were C, T, A, C, T, A and G, respectively, each having a frequency of more than 0.50; the gene heterozygosity (H) and Polymorphism Information Content (PIC) of 7 SNPs are all in the range of 0.25-0.5, which shows that the polymorphism of the population belongs to moderate polymorphism, and the X2 test shows that 7 SNPs are in Hardy-temperature-Berger balance (P > 0.05) in the Lei-Zhou black duck population.
TABLE 1 population genetic Properties of 7 SNPs of HAL Gene
EXAMPLE 3 analysis of correlation of SNPs of HAL Gene with egg laying Properties of Rauzhou black ducks
1. Experimental method
Multiple comparison analysis in single factor variance of SPSS 22.0 software was used to test for differences in significance of the association between egg laying traits and genotypes and haplotypes of black ducks in the state of rales, the results were expressed as "mean ± standard error", when P <0.05 indicated differences were significant, and when P <0.01 indicated differences were extremely significant.
2. Experimental results
The results are shown in Table 2. Thus, it was found that individuals of the CC genotype at SNP locus 1 had significantly higher open body weight than those of the TT genotype, while individuals of the CC genotype had significantly lower 300d egg count than those of the TT genotype (P < 0.05); the 300d egg yield of individuals of the TT genotype at SNP site 2 was significantly increased relative to individuals of the GG genotype (P < 0.05); individuals with the AA genotype at SNP locus 3 had very significantly lower age at birth than individuals with the AG genotype (P < 0.01); individuals with an AA size at SNP site 4 with significantly lower birth age than the AC genotype (P < 0.01); likewise, the open age AT date of individuals of the TT genotype AT SNP locus 5 was significantly reduced relative to individuals of the AT genotype, while the 300d egg count of individuals of the TT genotype was significantly higher than those of individuals of the AT genotype (P < 0.05); individuals with the AA genotype of SNP locus 6 have significantly lower open age than those with the GG genotype (P < 0.05), individuals with the AG genotype have significantly higher open weight than those with the GG genotype (P < 0.01), and individuals with the AG genotype have significantly lower 300d egg count than those with the GG genotype (P < 0.05); similarly, individuals of the GG genotype at SNP locus 7 had significantly lower open-term ages than those of the AA genotype (P < 0.01), individuals of the AG genotype had significantly higher open-term weights than those of the GG genotype (P < 0.05), and individuals of the AG genotype had significantly lower 300d egg numbers than those of the GG genotype (P < 0.05). 7 SNPs had no significant effect on the weight of eggs laid (P > 0.05).
TABLE 2 analysis of correlation of 7 SNPs from HAL Gene with egg laying Performance of Leisha ducks
From the results, except the SNP locus 1 and the SNP locus 2, the rest SNPs loci are obviously related to the open birth date of the black duck in Rauzhou, and the rest 5 SNPs loci can be presumed to be molecular marker loci of the open birth date character of the black duck in Rauzhou; SNP locus 1, SNP locus 6 and SNP locus 7, these 3 loci are all related with black duck of Rauzhou and open the weight of producing notably, this indicates these 3 SNPs may have important guiding effects on improving the weight of opening the production; except for SNP site 3 and SNP site 4, the rest sites are obviously related to the egg yield of the Rauzhou black duck 300d, which shows that the rest sites can be used as important markers for improving the egg yield of the Rauzhou black duck.
Example 4 7 SNP loci analysis of egg laying Performance of Rauzhou Duck
1. Experimental method
Linkage analysis was performed on 7 SNP sites using the biploview 4.2 software.
2. Experimental results
As a result, as shown in FIG. 9 and Table 3, it was found that 4 sites of SNP site 3, SNP site 4, SNP site 5 and SNP site 6 were strongly linked, and that 4 sites constituted a domain, and 10 different haplotypes were combined, wherein only 7 haplotypes were biologically statistically significant (the number of individuals was greater than 3) in H1H1 (AA-CC-TT-AA), H1H2 (AG-CA-TA-AG), H1H3 (AA-CC-TA-AG), H1H4 (AA-CC-TT-AG), H2H2 (GG-AA-AA-GG), H2H3 (AG-CA-AA-GG) and H3H4 (AA-CC-TA-GG), respectively.
The correlation of 7 haplotypes with the egg laying performance of the black ducks in Rauzhou was analyzed using the SPSS 22.0 single-factor variance and the results are shown in Table 4. From this, the age of the H1H2 haplotype in terms of open birth is significantly higher than that of the H1H1, H1H3 haplotypes (P < 0.05); the open product body weight of H1H2 and H1H4 haplotypes was significantly higher than that of H1 haplotypes (P < 0.05); while the 300d egg yield of the H1 haplotype was significantly higher than that of the H1H3 haplotype (P < 0.05). The remaining haplotypes have no obvious difference (P > 0.05) from the egg laying performance of the Rauzhou black ducks.
TABLE 3 haplotype frequencies of HAL genes
TABLE 4 analysis of correlation of HAL Gene haplotypes with egg laying Performance of Leizhou ducks
Note that: the same index labeled with completely different letters indicates significant differences (P < 0.05); the unnamed ones indicate that the difference is not significant (P > 0.05).
Example 5A kit for evaluating egg laying Performance of Rauzhou black ducks
1. Composition of the composition
The nucleotide sequence is shown as a primer shown as SEQ ID NO. 1-2, and 2 xGreen Taq Mix.
2. Application method
1. Extracting sample DNA;
2. PCR amplification
PCR reaction system 20. Mu.L: 2 XGreen Taq Mix 10. Mu.L, each of the upstream/downstream primers (nucleotide sequences shown as SEQ ID NOS.1-2) 0.4. Mu.L, template 1. Mu.L, ddH 2 O 8.2μL；
PCR reaction procedure: thermal denaturation at 95℃for 5min, deformation at 95℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min,35 cycles, elongation at 72℃for 5min, and preservation at 4 ℃.
3. Sequencing of PCR products
1% agarose gel was subjected to electrophoresis, recovered and sent to sequencing company for sequencing.
3. Interpretation of results
The open-yield weight of the sample of the SNP locus 1 and the CC genotype is obviously higher than that of the sample of the TT genotype, and the 300d egg yield of the sample of the CC genotype is obviously lower than that of the sample of the TT genotype;
the 300d egg yield of the sample of the SNP locus 2, TT genotype is obviously higher than that of the sample of GG genotype;
the SNP locus 3, the age of the sample of the AA genotype at the birth date is extremely lower than that of the sample of the AG genotype;
the SNP locus 4, the age of the open birth of a sample of the AA genotype is obviously lower than that of a sample of the AC genotype;
the SNP locus 5, the open date age of the sample of the TT genotype is obviously lower than that of the sample of the AT genotype, and the 300d egg laying number of the sample of the TT genotype is obviously higher than that of the sample of the AT genotype;
the SNP locus 6, the open-time-of-production age of the sample of the AA genotype is obviously lower than that of the sample of the GG genotype, the open-time-of-production weight of the sample of the AG genotype is obviously higher than that of the sample of the GG genotype, and the 300d egg laying number of the sample of the AG genotype is obviously lower than that of the sample of the GG genotype;
the SNP locus 7, the open-birth age of the sample of the AG genotype is extremely lower than that of the AA type, the open-birth weight of the sample of the AG genotype is remarkably higher than that of the GG type, and the 300d egg yield of the sample of the AG genotype is remarkably lower than that of the GG type.
The genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the individual with the genotype of the SNP locus 6 is AG, the date of birth is obviously higher than that of the individual with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TT, the genotype of the SNP locus 6 being AA, the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG;
the genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the genotype of the SNP locus 6 is AG, the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, the individual split weight of the genotype of the SNP locus 6 is AG is obviously higher than that of the individual split weight of the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the genotype of the SNP locus 6 is AA;
the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the 300d egg yield of individuals with the genotype of the SNP locus 6 being AA is obviously higher than that of individuals with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. A reagent for detecting genotype of SNP locus correlated with the laying character of black duck in Rauzhou, characterized in that the reagent is used for detecting genotype of SNP locus 1, SNP locus 2, SNP locus 3, SNP locus 4, SNP locus 5, SNP locus 6 and/or SNP locus 7;
the SNP locus 1 is positioned at 288 th base of a first intron of the HAL gene, the open yield weight of a sample of the CC genotype is obviously higher than that of a sample of the TT genotype, and the 300d egg yield number of the sample of the CC genotype is obviously lower than that of the sample of the TT genotype;
the SNP locus 2 is positioned at 373 bases of the first intron of the HAL gene, and the 300d egg yield of a sample of the TT genotype is obviously higher than that of a sample of the GG genotype;
the SNP locus 3 is positioned at the 438 th base of the first intron of the HAL gene, and the date of birth of a sample of the AA genotype is extremely lower than that of a sample of the AG genotype;
the SNP locus 4 is positioned at the 16 th base of the second intron of the HAL gene, and the date of birth of a sample of the AA genotype is obviously lower than that of a sample of the AC genotype;
the SNP locus 5 is positioned AT the 123 th base of the second intron of the HAL gene, the open date of a sample of the TT genotype is obviously lower than that of a sample of the AT genotype, and the 300d egg yield of the sample of the TT genotype is obviously high;
the SNP locus 7 is positioned at the 168 th base of the second intron of the HAL gene, the open date of the sample of the GG genotype is extremely lower than that of the AA type, the open date of the sample of the AG genotype is remarkably higher than that of the GG type, and the 300d egg yield of the sample of the AG genotype is remarkably lower than that of the GG type.
2. The reagent according to claim 1, wherein SNP site 3, SNP site 4, SNP site 5 and SNP site 6 are detected,
the genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the individual with the genotype of the SNP locus 6 is AG, the date of birth is obviously higher than that of the individual with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TT, the genotype of the SNP locus 6 being AA, the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG;
the genotype of the SNP locus 3 is AG, the genotype of the SNP locus 4 is CA, the genotype of the SNP locus 5 is TA, the genotype of the SNP locus 6 is AG, the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, the individual split weight of the genotype of the SNP locus 6 is AG is obviously higher than that of the individual split weight of the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the genotype of the SNP locus 6 is AA;
the genotype of the SNP locus 3 is AA, the genotype of the SNP locus 4 is CC, the genotype of the SNP locus 5 is TT, and the 300d egg yield of individuals with the genotype of the SNP locus 6 being AA is obviously higher than that of individuals with the genotype of the SNP locus 3 being AA, the genotype of the SNP locus 4 being CC, the genotype of the SNP locus 5 being TA, and the genotype of the SNP locus 6 being AG.
3. The reagent according to claim 1 or 2, wherein the reagent is a primer.
4. A reagent according to claim 3, wherein the primer nucleotide sequence is shown in SEQ ID No. 1-2.
5. Use of the reagent according to any one of claims 1 to 4 for preparing a kit for evaluating the egg laying performance of black ducks in the state of Rauzhou.
6. Use of the agent of any one of claims 1 to 4 for evaluating the egg laying performance of a black duck in the state of raddea.
7. A kit for evaluating the egg laying performance of a black duck in the state of rayleigh, comprising the reagent of claim 1.
8. The kit according to claim 7, wherein the reagent is a primer with a nucleotide sequence shown as SEQ ID NO. 1-2.
9. Use of one or both of the reagents of claim 1, or the kit of claim 8, for evaluating the egg laying performance of a black duck in the state of rex.
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| CN106755326B (en) * | 2016-11-28 | 2019-10-11 | 湖北省农业科学院畜牧兽医研究所 | One kind molecular genetic marker relevant to duck egg-laying deseription and application |
| CN108546764B (en) * | 2018-05-02 | 2021-12-24 | 浙江省农业科学院 | Molecular marker related to egg laying performance of laying ducks and application of molecular marker in breeding |
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