CN113215271B - SNP molecular marker related to weight and shank length of broiler chicken and application thereof - Google Patents
SNP molecular marker related to weight and shank length of broiler chicken and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker related to the weight and shin length of broiler chickens and application thereof. The SNP molecular marker is located at 135355388 th nucleotide of galGal6 edition chicken genome No.1 chromosome, corresponds to a mutation site g.880C > T in a chicken TMEM182 gene sequence, and has the genotypes of CC, TCA and TT. Wherein, the weight of the CC genotype individual is heavier than that of the TC and TT genotype individual, and the shank of the CC genotype individual is longer than that of the TC and TT genotype individual. The SNP molecular marker, namely the mutation site g.880C > T in the intron 2 sequence of the chicken TMEM182 gene is related to the body weight and the shank length, is a new molecular marker, and the shank length of the broiler chicken is selected at early stage by determining the genotype of the SNP site of the chicken, so that the production cost can be saved and the genetic progress can be accelerated.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a broiler shin length character related SNP molecular marker and application thereof.
Background
The consumer market determines the breeding direction of chickens to a large extent. Local broiler chickens in China are rich in resources, and most local broiler chickens have excellent properties such as strong stress resistance, weak nesting property and the like. With the improvement of living standard and the change of diet concept, people have higher requirements on the meat quality of the broiler chickens. The local variety is popular with consumers due to excellent meat quality and unique flavor, has larger market demand, but has the defects of slow growth, low material consumption ratio and the like compared with the foreign fast large broiler, thereby seriously restricting the large-scale production and industrialized application of the local chicken variety in China.
In addition, the chicken feet are rich in nutrients such as collagen, phospholipid, copper and the like, have the effects of reducing blood fat and blood pressure, promoting the development and repair of various tissue systems of a body and the like, are unique in flavor and rich in cooking modes, and are popular with consumers (particularly the masses in southern areas and eastern coastal areas). However, since most foreign poultry varieties are white feather fast-growing type, chicken feet are large, collagen is abundant, and the appearance is neat and bright, most of the chicken feet supplied in large and medium-sized wholesale markets are imported goods and are favored by domestic dealers and some consumers, the domestic chicken feet are small, and the shank length is one of the important factors influencing the purchase of the chicken feet by the consumers.
However, in the traditional breeding mode, a crossbreeding mode is adopted, parents are crossbred to form different genetic diversity, and filial generations with better shin length are obtained by screening the filial generations. However, the breeding process is slow and takes a lot of time. The breeding result is complex and unpredictable, and a large amount of seed selection and production work is required.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to the weight and the shin length of broiler chickens and application thereof, so as to solve one or more technical problems in the prior art and provide at least one beneficial selection or creation condition.
The invention provides an SNP molecular marker of broiler chicken in a first aspect. The SNP molecular marker is located at 135355388 th nucleotide of galGal6 version chicken genome No.1 chromosome, corresponds to a mutation site g.880C > T in a chicken TMEM182 gene intron 2 sequence, and has the genotypes of CC, TC and TT.
The second aspect of the present invention provides a primer pair for specifically amplifying the above-mentioned SNP molecular marker, preferably an upstream primer PCR-F and a downstream primer PCR-R, specifically:
the upstream primer PCR-F:5 'ACAAGACCGACCTTCA-3' (SEQ ID No. 1),
downstream primer PCR-R:5 'ACACCATTCCCACCT-3' (SEQ ID No. 2).
The third aspect of the invention provides application of the SNP molecular marker in broiler weight and shin length trait related breeding.
The specific application comprises early selection of the broiler weight and the shin length by using the genotype of the SNP molecular marker.
In some embodiments of the invention, the method comprises the steps of:
1) Extracting DNA of the broiler chickens to be detected;
2) Taking the DNA of the broiler to be detected as a template, and performing PCR amplification by using an upstream primer and a downstream primer with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 respectively to obtain a PCR product containing a chicken TMEM182 gene fragment;
3) Detecting the genotype of the mutation site g.880C > T of the chicken TMEM182 gene fragment;
4) And (4) early selecting the weight and the shank length of the broiler to be detected based on the genotype detected in the step 3), wherein the weight of a CC genotype individual is heavier than that of a TC genotype individual and that of a TT genotype individual, and the shank length of the CC genotype individual is longer than that of the TC genotype individual and that of the TT genotype individual.
In some application embodiments of the invention, the nucleotide sequence of the PCR product obtained by the detection in step 2) is shown as SEQ ID No. 3. In this embodiment, the length of the PCR product is 762bp, and the SNP molecular marker is located at the 439 th base of the PCR product.
Compared with the prior art, the invention has the following beneficial effects:
the SNP molecular marker, namely the mutation site g.880C > T in the intron 2 sequence of the chicken TMEM182 gene has correlation with the characters of weight and shin length, is a new molecular marker, and the broiler shin length character is early selected by determining the genotype of the chicken SNP site, so that the production cost can be saved, the genetic progress can be accelerated, the SNP molecular marker is better applied to broiler breeding, and has great economic application value and scientific research value.
Drawings
FIG. 1 is a graph showing the results of genotyping in the examples of the present invention.
Detailed Description
The present invention is specifically described below with reference to examples in order to facilitate understanding of the present invention by those skilled in the art. It should be particularly noted that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, as non-essential improvements and modifications to the invention may occur to those skilled in the art, which fall within the scope of the invention as defined by the appended claims. Meanwhile, the raw materials mentioned below are not specified and are all commercial products; the process steps or preparation methods not mentioned in detail are all process steps or preparation methods known to the person skilled in the art.
Examples
The broiler to be tested is a full-sib F2 resource group of apricot blossom chickens and recessive white rock chickens, wherein the recessive white rock chickens are fast large broiler chickens, and the apricot blossom chickens are local broiler chickens in Guangdong China. The chicken flocks are raised in a flat breeding mode and fed with corn-soybean meal type feed meeting the international formula standard. Then, the body weight and the shin length of the broiler chickens at each week of 1-17 weeks of age were measured. Supinal venous bleeding was performed before slaughter, and the collected blood was stored in a centrifuge tube containing 2% of EDTA anticoagulant for blood DNA extraction.
1. Extracting blood DNA of broiler chicken to be tested from F2 generation of resource group holomorphic family of apricot blossom chicken and recessive white rock chicken
Genomic DNA of all individuals
The DNA from blood was extracted by the Plant Mini Kit (Qiagen, hilden, CA; cat # 69104) Kit instructions, diluted to 50 ng/. Mu.L after quality and concentration testing, and stored at 4 ℃ until use.
2. SNP detection and DNA mixed pool sequencing
1. Designing primers and detecting specificity:
the sequence of TMEM182 gene was found from https:// www.ncbi.nlm.nih.gov/to design a primer pair, the information of the primer pair is shown in Table 1 (primer sequence 5'→ 3'), the sequence of the designed primer pair was synthesized by Guangzhou Biochemical company, and the blood DNA was amplified by PCR using the primer pair to test the specificity of the primer, and the result is shown in FIG. 1.
TABLE 1 primer information for SNP screening of TMEM182
2. DNA mixed pool sequencing:
from the total extracted DNA samples, 30 DNA samples were randomly selected to construct a pool, and every three samples (about 0.33. Mu.L each) were mixed into one mixed sample for a total of 10. The mixed pool sample is subjected to PCR amplification, the information of the used primer pair is shown in Table 1, and the reaction system is shown in Table 2.
TABLE 2 PCR reaction System
PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 50s, and 36 cycles; stretching for 10min at 72 ℃; storing at 4 ℃. And (3) sending the obtained PCR product to a biological company for sequencing, and detecting the sequencing result to obtain the mutation site g.880C > T in the intron 2 sequence of the chicken TMEM182 gene, wherein the sequencing result is analyzed as shown in figure 1.
3. Determining the genotype of the TMEM182 mutation site of the sample to be tested
PCR products of target fragments of the chicken TMEM182 gene are obtained by carrying out PCR specific amplification on all chicken blood DNAs to be detected in the example, and the information of the used primer pairs is shown in Table 1. After DNA sequencing, the genotype of each sequencing result was analyzed. In this example, 301 individuals were successfully typed, and then each genotype was statistically analyzed to obtain the genotype frequency and allele frequency at that site. The gene and genotype frequency statistical structure of the SNP mutation site is shown in the following table 3. Wherein:
(1) Genotype frequency refers to the ratio of the number of individuals of a certain genotype in a population to the total number of genotypes:
genotype frequency = total number of certain genotypes/total number of the population × 100%;
(2) The gene frequency refers to the ratio of a certain gene in a population to all genes at the same site:
gene frequency = number of genes/total number of genes at the same locus in a population × 100%.
As can be seen from the structure in Table 3, the CC genotype is the dominant genotype of the broiler group to be tested.
TABLE 3 Gene, genotype frequency and genetic diversity of TMEM182 Gene mutation site g.880C > T
4. Correlation analysis of TMEM182 mutation site and broiler weight and shank length traits
Statistical analysis is carried out by adopting the GLM process of the SAS statistical analysis software package, and the genotype of the tested chicken flock and the length of the broiler shin are subjected to correlation analysis. The mathematical model is as follows:
Yijkl＝μ+Gi+Dj+Hk+eijkl
y is the growth trait observation, μ is the growth trait population mean, G is the genotype effect, D is the pedigree effect, H is the batch effect, and e is the random error effect.
The correlation analysis results are shown in table 4 below. It can be shown from the table that the mutation site is significantly (P < 0.05) or extremely significantly (P < 0.01) associated with the body weight of broilers at 42, 49, 63 and 119 days and the length of shins at 42, 49, 63, 77 and 105 days. Wherein the weight of the CC genotype individual at each day age is obviously heavier than that of the TC and TT genotypes, and the shin length of the CC genotype individual at each day age is obviously longer than that of the TC and TT genotypes. The SNP locus can be used as an auxiliary selection and molecular genetic breeding marker for improving the weight and the shank length of the broiler.
TABLE 4 correlation analysis of mutation site g.880C > T of TMEM182 gene with weight and length of shin of broiler chicken
Note: the different lower case letters of the shoulder marks on the same row indicate that the difference is obvious (P < 0.05), the different upper case letters of the shoulder marks on the same row indicate that the difference is extremely obvious (P < 0.01), and the same letters of the shoulder marks on the same row indicate that the difference is not obvious.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> SNP molecular marker related to weight and shank length of broiler chicken and application thereof
<130> 2021
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> DNA
<213> Artificial sequence
<400> 1
acaagacgac cttcaa 16
<210> 2
<211> 15
<212> DNA
<213> Artificial sequence
<400> 2
acaccattcc cacct 15
<210> 3
<211> 762
<212> DNA
<213> TMEM182
<400> 3
acaagacgac cttcaaaacc tcttgtaaac agctgtaatc aagtctaaaa ttgattcatc 60
ttttatgata ctacttttgc aataagacat actttcaaag gaactataac tgtatagaca 120
ggctatagct aacctacgtt cagacaggct gtgtcactgt gatagtgtta ttttttacct 180
aaatatctta atttcacttg aacttgattc tttcagctgt ttgaaaaata gtatgataaa 240
ggctttcatt tttattttta cttttcatag acagagaagg ccacttttca tcatgaaggt 300
ttcttctgga ggtgctggtt tagtggaaat gtaagagagc ataacaccag catgtggaag 360
ttctggtaca gtaagtaata cctttctttt tatgttatct acaacagttg gatctcaaag 420
ctgtggagct ttttttagcg cttatgaagc attgaatttc atttggtgtc ttcagtgatc 480
aggttcttaa tctatcttca cattcaaaag cgacactttt aaaatctgta ctaaaggata 540
tcccaggaca aaatcatcca tgtttaacta tcagcttact ctttcagtag tagcctgtca 600
tcttaactga gagtccattt ttgtcaagac agtctcttgc aatgttgtta atggattgtt 660
catttccata aagcagggaa aaagagcttc atggttagga tggtccataa gtttttgcct 720
gcagtttctc cagctagttc tgtgagaagg tgggaatggt gt 762
Claims (1)
1. The application of a primer pair for specifically amplifying broiler SNP molecular markers in broiler weight and shin length related breeding is characterized in that the nucleotide sequences of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No.2, the broiler is F2 generation broiler hybridized by apricot chicken and recessive white rock, the SNP molecular markers are located at the 135355388 th nucleotide of the chromosome 1 of a galGal6 version chicken genome and correspond to the mutation site g.880C > T in the chicken TMEM182 gene sequence, and the genotypes of the SNP molecular markers are CC, TC and TT; and early selecting the weight and the shank length of the broiler to be detected according to the genotype of the SNP molecular marker, wherein the weight of a CC genotype individual is heavier than that of a TC genotype individual and that of a TT genotype individual, and the shank length of the CC genotype individual is longer than that of the TC genotype individual and that of the TT genotype individual.
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| CN114622020B (en) * | 2022-03-30 | 2022-09-27 | 华南农业大学 | KLHL31 gene molecular marker related to chicken growth traits and application thereof |
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| WO2009144809A1 (en) * | 2008-05-30 | 2009-12-03 | 独立行政法人 農業・食品産業技術総合研究機構 | Method of detecting gene mutation associated with chicken eggshell strength |
| CN106086229B (en) * | 2016-08-26 | 2019-08-16 | 广东省农业科学院动物科学研究所 | The relevant molecular labeling of chicken growth traits and its discrimination method and application |
| CN107385059B (en) * | 2017-08-11 | 2020-08-07 | 华南农业大学 | Molecular marker related to growth traits of broiler chickens and application thereof |
| CN111910008B (en) * | 2020-08-21 | 2022-05-31 | 云南农业大学 | Molecular marker related to chicken growth and development and application thereof |
| CN112501318B (en) * | 2020-12-30 | 2022-07-12 | 华南农业大学 | Chicken growth trait related TMEM18 gene molecular marker and application |
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| CN105441560A (en) * | 2015-12-29 | 2016-03-30 | 云南农业大学 | Chicken body size trait breeding molecular marker IGF-1R (insulin-like growth factor-1 receptor) gene and application thereof |
| CN112063725A (en) * | 2020-09-14 | 2020-12-11 | 华南农业大学 | Chicken shin length and shin diameter related AGO3 gene molecular marker and application |
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