CN113337616B - Molecular marker highly related to cockscomb and application thereof - Google Patents

Molecular marker highly related to cockscomb and application thereof Download PDF

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CN113337616B
CN113337616B CN202110718095.0A CN202110718095A CN113337616B CN 113337616 B CN113337616 B CN 113337616B CN 202110718095 A CN202110718095 A CN 202110718095A CN 113337616 B CN113337616 B CN 113337616B
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CN113337616A (en
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马荆鄂
周敏
朱学农
谭玉文
许继国
杨艳北
李金�
徐淘
邓方琪
何凡
李景浩
文正亚
饶友生
王樟凤
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Abstract

The invention discloses a molecular marker highly related to cockscomb and application thereof, belonging to the technical field of biological genes. The molecular marker nucleotide sequence is shown as SEQ ID NO: 1, SEQ ID NO: 1, a base mutation of A/G is arranged at the g.1061 position of the SNP site of the sequence shown in the specification, and different genotypes of AA, AG and GG appear. The invention also discloses a primer pair for detecting the molecular marker, a kit containing the molecular marker and a method for detecting the molecular marker, and the molecular marker can be used for typing local breeder cocks so as to provide theoretical basis and reference data for breeding of reproductive traits and molecular assisted breeding work of the local breeder cocks.

Description

Molecular marker highly related to cockscomb and application thereof
Technical Field
The invention relates to the technical field of biological genes, in particular to a molecular marker highly related to cockscomb and application thereof.
Background
In the modern poultry industry, a plurality of researchers and breeders all realize that breeding cocks play an important role in production level, particularly influence the fertility rate and the hatchability of hatching eggs and further influence the economic benefit of breeding hens. Besides the qualified henhouse environment and strict feeding management, the breeding of the reproductive traits is also a very important factor for obtaining higher breeding value of the breeding cocks. The second characteristics of the appearance of the cocks, such as cockscomb, meat drop and the like, are regulated by androgen, have different degrees of correlation with each other, can indirectly reflect the degree of masculinization development of the cocks, and can be used as breeding indexes of reproductive traits. The height of the crown is measured by a caliper rule from the basal part of the cockscomb to the highest crown tooth, and the height of the crown is one of important indexes of the second sexual characteristics of the poultry and is closely related to the total quality of the testis. The testicular character can be indirectly selected by breeding the crown height character.
The Ningdu yellow chicken is originally named as 'Ningdu Sanhuang chicken', is a high-quality yellow-feather broiler, is a famous local broiler variety in Jiangxi province, is a valuable poultry variety resource, and is listed in 'Yangxi province livestock and poultry variety book' published in 1999. The Ningdu yellow chicken also has the characteristics of short and small individual and fresh and tender meat, and indexes such as protein, total amino acid, chicken fiber quantity per unit area and the like are superior to those of a plurality of domestic broiler varieties, so that the Ningdu yellow chicken has larger market demand. However, no report of screening Ningdu yellow chicken through the highly-related properties of cockscomb exists at present.
Disclosure of Invention
The invention aims to provide a molecular marker highly related to cockscombs and application thereof, so as to solve the problems in the prior art, and the molecular marker can be used for typing local breed cocks through the correlation with the cockscombs, so as to provide theoretical basis and reference data for breeding of reproductive traits and molecular-assisted breeding.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker highly related to cockscomb, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1 is shown.
Preferably, SEQ ID NO: 1, a base mutation of A/G is arranged at the g.1061 position of the SNP site of the sequence shown in the specification, and different genotypes of AA, AG and GG appear.
The invention also provides a primer pair for detecting the molecular marker highly related to the cockscomb, which comprises the following components:
an upstream primer F: 5'-CGAGCACCCCCATCCATTT-3', respectively;
a downstream primer R: 5'-TTTCCTCACTCTTTCCCGGC-3' is added.
The invention also provides a kit comprising the primer pair.
The invention also provides a method for detecting the molecular marker highly related to the cockscomb, which comprises the following steps:
step 1: extracting the genome DNA of the chicken to be detected;
step 2: carrying out PCR amplification by using a primer pair to obtain an amplification product;
and step 3: sequencing the amplification product obtained in the step 2 to obtain the genotype of the molecular marker;
and 4, step 4: correlation with rooster comb height was analyzed by genotype, and rooster comb height for AG or GG genotype individuals was higher than for AA genotype individuals.
Preferably, in step 2, the PCR amplification reaction system comprises: DNA 1-2. mu.L, 2 XPCR mix 7.5. mu.L, upstream primerAnd downstream primers 1. mu.L each, H 2 And O is supplemented to 15 mu L.
Preferably, the PCR amplification reaction procedure in step 2 is as follows: 3min at 95 ℃; 94 ℃ for 15s, 55 ℃ for 15s, 72 ℃ for 30s, 20 cycles; 3min at 72 ℃.
The invention also provides application of the molecular marker, the primer pair or the kit in detecting the high correlation traits of the cockscomb.
The invention also provides the application of the molecular marker, the primer pair and the kit in breeding local variety cock breeders.
The invention discloses the following technical effects:
the invention provides a molecular marker highly related to cockscombs, which detects a site by using a GH gene g.1061 site, analyzes the correlation between the polymorphism of the site and the height of cockscombs, and finds that an allele G of the site is favorable for the high development of the cockscombs through experiments. The method provides theoretical basis and reference data for local breeding cock breeding and molecular marker-assisted selection through the height of the cockscomb. Compared with the traditional phenotypic data breeding, the detection method constructed by the molecular marker has the advantages of low cost, simple and convenient operation, accurate result, simplicity and practicability, strong repeatability and capability of being carried out in a common laboratory.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the results of typing of the target site in example 2.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 method for detecting molecular markers highly correlated with rooster combs
Typing the corresponding mutation sites by adopting a PCR method, and selecting breeding cocks with different cockscomb heights according to the genotypes of the target sites of the blood of the detected cocks. The method comprises the following specific steps:
step 1: sample DNA preparation.
(1) 339 cock blood of 16 weeks old is collected, and DNA of blood is extracted by phenol-chloroform extraction method.
The specific steps for extracting genomic DNA by phenol-chloroform extraction (Ospor F et al, 1998) are as follows:
putting 30 mu L of whole blood into a 1.5mL centrifuge tube, respectively adding 470 mu L of 1 xSET buffer solution, 12.5 mu L of 20% SDS and 6 mu L of 10mg/mL proteinase K, uniformly mixing, and putting in a water bath at 55 ℃ for overnight;
② taking out the sample to a 1.5mL centrifuge tube, adding 500 μ L saturated phenol, shaking gently for 20min, centrifuging at 10,000rpm for 10 min;
thirdly, taking the supernatant, adding 500 mu L of saturated phenol again, shaking gently for 20min, and centrifuging at 10,000rpm for 10 min;
fourthly, taking the supernatant, adding 500 mu L chloroform-isoamylol (23: 1) to shake for 20min, and centrifuging for 10min at 10,000 rpm;
fifthly, taking the supernatant, adding 1mL of ice absolute ethyl alcohol (-20 ℃), swinging back and forth to precipitate DNA, centrifuging at 10,000rpm for 10min, and pouring out the ethyl alcohol;
sixthly, washing the DNA once by using 1mL of 75% ethanol, pouring off the ethanol, and drying in a drying oven at 50 ℃;
seventhly, after the DNA is completely dried, 300 mu L of sterilized double distilled water is added for dissolving, and the DNA is dissolved in a water bath kettle at the temperature of 50 ℃ overnight;
storing in a refrigerator at-20 ℃ for later use.
(2) DNA quality detection and dilution: a DNA sample with qualified quality is taken and diluted to 100 ng/. mu.L, and is stored at the temperature of minus 20 ℃ for standby.
Step 2: PCR amplification
The synthesized primers were dissolved in 1 XTE to a concentration of 10pmol, and the primers in one set were added together, mixed well and centrifuged. The PCR amplification primers are shown below:
the upstream primer F (SEQ ID NO:2) is CGAGCACCCCCATCCATTT;
the downstream primer R (SEQ ID NO:3) is TTTCCTCACTCTTTCCCGGC.
The PCR reaction system is shown in Table 1:
TABLE 1 PCR reaction System
Figure BDA0003135781600000041
The prepared PCR master tube is subpackaged into 200 mu L PCR, centrifuged, 2ul DNA sample is added into each hole, centrifuged and loaded into a PCR instrument.
The PCR reaction procedure is shown in Table 2.
TABLE 2 PCR reaction procedure
Figure BDA0003135781600000042
Figure BDA0003135781600000051
The amplification sequence is as follows SEQ ID NO: 1, and the following components:
CACGAGCACCCCCATCCATTTTAAACAGACCCCCAGCTATATAAGGGGTGTCT CACCTGTTATCATCACCTGGATGAAAGGAGGAAACGTTCAAGCAACACCTGAGC AACTCTCCCGGCAGGAATGGCTCCAGGTACTTTGCTTTATCTCAGTTCTAATGGGT GTTCCAATGCTGCTGCATGCTTTGGGTGATGGGATACGATGGTGGGGTGTGCTGTG GTGGGCTGACACACGCAGAGCCGGCTCTGAACTAAAATGTGGCAACTTACAGAT CAGTGACAAAGGATCTCCTTCCCTACAGTGCAACTTCAAACCATGAGCTGACTCA GGTAACCCTGAGCCTAACCTTGACAGGGGGCAGGAATGAGCTGCAGAATACGAA GAACAAGGCAAACCAGAGTTGTAATGGTGATTGCTATCATACGTGTTGCCAGGGA TATTAAAACTCAGTTCCAAGGCTTTAAAACGGAGATCAGGATGATGTCTAATCAG ACTCATAGAATGGCCCGGGTTGAAAAGCACTTCAAAGATCACCTAATTTCAACCC CTTGCACTTGTCCAAGTCCTGCAGGCTCCAGGGCATTCCTCCACTGAAGTTAAAC CCTACTGAGATTAACTTTTGTAAGCGGACACTCATGTG【A/G】 GCTGGATGTCGAGGGTTAATAACCTTCAGGCTTGACAGTGACCTCCAGATCCTAC AGGTGTGTCCCAGAGAGCCACAGCGCAGGTAATGCAGCCACTTCTCACCCCAGT GAAGGCAGACAGTGCCATGGCAGCAGCACGGTGCAAATAGGCTCAGCTGAGCTG TTCCCAGTCCTCACCCACTGCTCTCCACCCTGTTGGCTCACGCGCCAAAGAGTGT ACCGTGCTCTGCTCAGGAAGGTGAAACCTACCAAAAAACATCAAAAAACATGAG CACGTTAGGGGAAAATAAAGGGACGGACCCACATGGAGCAACATGTGGGCTTGT GTGGAGGGCTGCACTCACAGGTGGACACAACCCTGAGCCCCAGGTGCCACCAAA GGACGGGTAACCCCTCTCCTCTCCGCTCTGCTGTAGGCTCGTGGTTTTCTCCTCTC CTCATCGCTGTGGTCACGCTGGGACTGCCGCAGGAAGCTGCTGCCACCTTCCCTG CCATGCCCCTCTCCAACCTGTTTGCCAACGCTGTGCTGAGGGCTCAGCACCTCCA CCTCCTGGCTGCCGAGACATATAAAGAGTTCGTAAGTGTTGGCCATCTCCTCATTA GCTTGATGCCTCCAGGACCGTGCCACGGCTTCCCACCCTGCATAAATTCCTGCAA GCAGAGACTTTTCAGCTATCGGTGCCTCCTGAGGAGGAGAAAGGGTCTTAATAGG GTAGGAGAGGTGATGACCGACCAGCCTGTCCTCCCCACAGCTCTCTGCCTCCTCC TGCAAGGAGTCACCTCCTCCTGTCCATGTGTTCTGTGCTCACCTCAACCCTTCAGT GAGAGCAATCTCTAAGACCAGTAGGTGTTGTGCCAACACGTGGGCTCTGCTTTCC CACCTGCCAGTCCTGCACAGGGATGCACATCATGTCCCACGTTTCCTTCTTGCAA AGAGCAACCTGCCGGGAAAGAGTGAGGAAAGAGTCCGTGCTCTTCTCTTATCAC AC
note: the target locus is in the 'interior'.
(4) Sequencing typing
And (4) sending the PCR product obtained by amplification to a biological company for sequencing. And reading an experimental result by using a SeqMan module of DNAStar software, and checking the target site genotype.
And step 3: trait association analysis
Adopting SAS 9.0GLM program to carry out statistical analysis on the different genotypes of the GH gene locus g.1061 and the high character of the cockscomb, including the correlation of 4-week-old crown height (mm), 6-week-old crown height (mm), 8-week-old crown height (mm), 10-week-old crown height (mm), 12-week-old crown height (mm), 14-week-old crown height (mm) and 16-week-old crown height (mm). The result shows that the site has obvious character difference with the crown height from 4 weeks to 16 weeks, the height of the cockscomb of GG genotype is extremely higher than that of AA type individuals in 4 weeks to 10 weeks, and the specific result is shown in the following table 3:
TABLE 3 relationship between different genotypes of GH gene locus g.1061 and different ages of cockscomb height traits
Figure BDA0003135781600000061
Example 2 use of molecular markers highly associated with rooster combs in selecting breeder cocks of different comb heights
Selecting breeding cocks with different cockscomb heights through GH gene locus g.1061 genotype.
1. Sample collection and preparation
(1) Hatching 400 Ningdu yellow-breed eggs, and identifying male and female eggs on the day of birth;
(2) blood was collected from male individuals.
DNA extraction
Genomic DNA was extracted using a phenol-chloroform extraction method as described in example 1 (Ospor F et al, 1998).
3. Typing of the site of interest
PCR amplification was performed using the primers, reaction system and reaction program shown in example 1 to obtain an amplification product.
4. Sequencing typing
And (3) sending the obtained PCR amplification product to a biological company for sequencing, and performing sequence sequencing by adopting a downstream primer F. And reading an experimental result by using a SeqMan module of DNAStar software, and checking the target site genotype.
Peak images were reviewed using chromas software. The typing results were 3 as shown in FIG. 1:
5. and (6) judging the result.
According to the typing result, the individuals with the genotypes AA, AG and GG are respectively labeled, the individuals are raised to 16 weeks old, the heights of cockscombs at 4 weeks old, 6 weeks old, 8 weeks old, 10 weeks old, 12 weeks old, 14 weeks old and 16 weeks old are measured and recorded, correlation analysis is carried out, and experimental data show that the heights of cockscombs of the individuals with the genotypes GG are extremely different from those of the individuals with the genotypes AA, and allele G is beneficial to the height traits of the cockscombs and is consistent with the prediction result.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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cacgagcacc cccatccatt ttaaacagac ccccagctat ataaggggtg tctcacctgt 60
tatcatcacc tggatgaaag gaggaaacgt tcaagcaaca cctgagcaac tctcccggca 120
ggaatggctc caggtacttt gctttatctc agttctaatg ggtgttccaa tgctgctgca 180
tgctttgggt gatgggatac gatggtgggg tgtgctgtgg tgggctgaca cacgcagagc 240
cggctctgaa ctaaaatgtg gcaacttaca gatcagtgac aaaggatctc cttccctaca 300
gtgcaacttc aaaccatgag ctgactcagg taaccctgag cctaaccttg acagggggca 360
ggaatgagct gcagaatacg aagaacaagg caaaccagag ttgtaatggt gattgctatc 420
atacgtgttg ccagggatat taaaactcag ttccaaggct ttaaaacgga gatcaggatg 480
atgtctaatc agactcatag aatggcccgg gttgaaaagc acttcaaaga tcacctaatt 540
tcaacccctt gcacttgtcc aagtcctgca ggctccaggg cattcctcca ctgaagttaa 600
accctactga gattaacttt tgtaagcgga cactcatgtg ngctggatgt cgagggttaa 660
taaccttcag gcttgacagt gacctccaga tcctacaggt gtgtcccaga gagccacagc 720
gcaggtaatg cagccacttc tcaccccagt gaaggcagac agtgccatgg cagcagcacg 780
gtgcaaatag gctcagctga gctgttccca gtcctcaccc actgctctcc accctgttgg 840
ctcacgcgcc aaagagtgta ccgtgctctg ctcaggaagg tgaaacctac caaaaaacat 900
caaaaaacat gagcacgtta ggggaaaata aagggacgga cccacatgga gcaacatgtg 960
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aggacgggta acccctctcc tctccgctct gctgtaggct cgtggttttc tcctctcctc 1080
atcgctgtgg tcacgctggg actgccgcag gaagctgctg ccaccttccc tgccatgccc 1140
ctctccaacc tgtttgccaa cgctgtgctg agggctcagc acctccacct cctggctgcc 1200
gagacatata aagagttcgt aagtgttggc catctcctca ttagcttgat gcctccagga 1260
ccgtgccacg gcttcccacc ctgcataaat tcctgcaagc agagactttt cagctatcgg 1320
tgcctcctga ggaggagaaa gggtcttaat agggtaggag aggtgatgac cgaccagcct 1380
gtcctcccca cagctctctg cctcctcctg caaggagtca cctcctcctg tccatgtgtt 1440
ctgtgctcac ctcaaccctt cagtgagagc aatctctaag accagtaggt gttgtgccaa 1500
cacgtgggct ctgctttccc acctgccagt cctgcacagg gatgcacatc atgtcccacg 1560
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Claims (6)

1. A molecular marker highly related to rooster combs of Ningdu yellow cocks, characterized in that the nucleotide sequence is as shown in SEQ ID NO: 1 is shown.
2. The molecular marker highly correlated to Ningdu yellow cock comb according to claim 1, characterized by the fact that the sequence of SEQ ID NO: 1 has a base mutation of A/G at the 641 th position, and has different genotypes of AA, AG and GG.
3. A method for detecting the molecular marker highly correlated to rooster combs of Ningdu yellow cock as claimed in claim 1, comprising the steps of:
step 1: extracting genome DNA of Ningdu yellow cock to be detected;
and 2, step: carrying out PCR amplification by using a primer pair to obtain an amplification product; the primer pair comprises: an upstream primer F: 5'-CGAGCACCCCCATCCATTT-3', respectively; a downstream primer R: 5'-TTTCCTCACTCTTTCCCGGC-3';
and step 3: sequencing the amplification product obtained in the step 2 to obtain the genotype of the molecular marker;
and 4, step 4: the height of the cockscomb of an individual with AG or GG genotype is higher than that of an individual with AA genotype.
4. The method of claim 3, wherein in step 2, the PCR amplification reaction system comprises: DNA 1-2. mu.L, 2 XPCR mix 7.5. mu.L, mixed primers 2. mu.L, H 2 And O is supplemented to 15 mu L.
5. The method of claim 3, wherein the PCR amplification reaction procedure in step 2 is as follows: 3min at 95 ℃; 15s at 94 ℃, 15s at 55 ℃, 30s at 72 ℃, 20 cycles; 3min at 72 ℃.
6. Use of a molecular marker according to claim 1 or 2 for breeding cocks of different rooster comb heights, wherein the breeding cocks are Ningdu yellow cocks.
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