CN110951889A - Haplotype molecular marker related to chicken body weight and application thereof - Google Patents
Haplotype molecular marker related to chicken body weight and application thereof Download PDFInfo
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Abstract
The invention discloses a haplotype molecular marker related to chicken body weight and application thereof. The haplotype molecular marker consists of 6 SNP loci of SNP 1-SNP 6, wherein the 6 SNP loci are positioned at the tail end of No. 1 chromosome of chicken 169.195-169.209 Mb. The haplotype molecular marker is used for distinguishing high-weight chicken individuals from low-weight chicken individuals, and when the 6 SNPs form G, C, C, C, C, G homozygous haplotypes according to the genome sequence, the corresponding chicken has high-weight traits. The method for detecting the haplotype molecular marker is suitable for identifying Chinese and foreign chicken species and has wide range of application to chicken species. The method can be used for predicting the weight traits of the chickens in an early, rapid and low-cost manner, provides an effective means for molecular marker-assisted selective breeding of the chickens, has a wide application prospect in the aspect of chicken breed improvement, and can obtain excellent economic value.
Description
Technical Field
The invention relates to the field of molecular genetics, in particular to a haplotype molecular marker related to chicken weight traits and application thereof.
Background
Chickens are one of the most important agricultural livestock and poultry animals. At present, the records of poultry have 107 local chicken breeds in China, China has become the world with the most abundant poultry resources, and great contribution is made to the breeding and scientific research of poultry. Chicken and eggs provide important animal protein sources for human beings, and due to improvement of health concepts of people, the consumption demand of red meat represented by pig, cattle and sheep tends to be stable in recent years, while the consumption demand of white meat represented by chicken is steadily improved. In China, the consumption level of chicken is second to that of pork of the second largest meat animal, and the consumption of eggs per person is also increased year by year. Therefore, the method has great economic benefit for accelerating the genetic improvement of high-quality broiler chicken and laying hen varieties. Compared with the introduced white feather broiler variety, although local chicken species in China have distinct characteristics and excellent meat quality, and simultaneously have the advantages of low requirement on feeding environment, strong disease resistance, high survival rate and the like, the defects of slow growth, poor reproduction and the like exist due to no high-strength systematic breeding, and the low production efficiency brought by the method seriously restricts the large-scale production and the industrialized application of the local chicken species in China. Therefore, the method has multiple qualities and improves the production efficiency, and becomes an important problem in the high-quality yellow chicken breeding industry in China.
SNPs have been widely used as genetic markers in the research fields of gene mapping, cloning, genetic breeding, genetic diversity, and the like. However, the interference of factors such as genetic background differences, founder effects, multi-gene interaction effects, gene-environment interaction effects, incomplete selection/balanced selection effects, the limitation of the number of genetic markers, linkage disequilibrium and the like of different experimental groups still causes the lack of molecular markers with definite functions and obvious effects in the molecular breeding practice of the broiler chickens at present. Therefore, it is the current research focus to mine the general, effective and accurate molecular markers in the Chinese and foreign populations. Furthermore, if SNP molecular markers related to chicken weight traits can be found and the molecular mechanism of the site is finally analyzed, the genetic improvement of chicken is greatly promoted, and breakthrough progress is brought to the field of poultry breeding.
Disclosure of Invention
The invention aims to provide a haplotype molecular marker related to the weight traits of chickens and application thereof.
The invention has the following conception: and (3) increasing the recombination events among generations to assist in finely positioning candidate genome intervals related to the body weight by utilizing the chicken deep hybridization line constructed in the early stage, and accurately obtaining the high-density molecular marker by adopting a simplified genome sequencing technology. In addition, the method has the advantages that the method downloads the whole genome re-sequencing data of hundreds of chickens from an autonomous sequencing or database, avoids the influence of marker density and linkage disequilibrium on gene positioning in order to exhaust all types of mutations of the genome, verifies the whole genome association analysis result in a family, and obtains the general genetic markers related to growth traits of Chinese and foreign chicken varieties.
In order to achieve the purpose, the invention provides an SNP molecular marker related to the chicken weight trait, which is selected from at least one of ① - ⑥, is positioned in the range of 169.195-169.209 Mb of the tail end of No. 1 chromosome of chicken;
the SNP molecular marker ① comprises SNP1 as SNP locus, GGA1:169195899bp as SNP1 as genomic position, and T or G as basic group;
the SNP molecular marker ② comprises SNP2 as SNP locus, GGA1:169199539bp as SNP2 as genomic position, and T or C as basic group;
the SNP molecular marker ③ comprises SNP3 as SNP locus, GGA1:169202882bp as SNP3 as genomic position, and T or C as basic group;
the SNP molecular marker ④ comprises SNP4 as SNP locus, GGA1:169203717bp as SNP4 as genomic position, and T or C as basic group;
the SNP molecular marker ⑤ comprises SNP5 as SNP locus, GGA1:169208105bp as SNP5 as genomic position, and A or C as basic group;
the SNP molecular marker ⑥ comprises SNP6 as SNP locus, GGA1:169208133bp as SNP6 as genomic position, and A or G as basic group.
The above positional reference genome is the chicken Gallus _ Gallus-5.0 version genome (Gallus _ Gallus-5.0, INSDCAssembly GCA _ 000002315.3).
The rs numbers corresponding to SNP 1-SNP 6 are rs315589915, rs313571167, rs740211632, rs315809773, rs737654023 and rs740848201 respectively.
Further, the SNP molecular marker of the invention is selected from at least one of SEQ ID NO 1-6. Wherein, the base N in the SEQ ID NO. 1-6 sequences is T/G, T/C, T/C, T/C, A/C, A/G respectively.
In a second aspect, the invention provides a haplotype molecular marker related to the chicken weight trait, wherein the haplotype molecular marker consists of 6 SNP sites of the SNPs 1-6. The haplotype molecular marker is positioned near a chicken CAB39L gene (Ensembl database www.ensembl.org, ID: ENSGALG00000017005), and is specifically positioned at the chicken GGA1:169195899 bp-169208133 bp, and the total length is about 12.234 Kb.
When the haplotype molecular marker is G, C, C, C, C, G homozygous genotype (namely, two G, C, C, C, C, G haplotype and AA homozygous) according to SNP 1-SNP 6, the corresponding chicken has high weight character; when the haplotype molecular marker is G, C, C, C, C, G heterozygous genotype (namely, a G, C, C, C, C, G haplotype and Aa heterozygous exist), the corresponding chicken has the medium weight character; when the haplotype molecular marker does not have G, C, C, C, C, G genotype (aa homozygous), the corresponding chicken has low weight character.
In a third aspect, the present invention provides primers for amplifying said SNP molecular markers, the primer sequences are shown in Table 1.
TABLE 1 primers for amplifying respective SNP molecular markers
In a fourth aspect, the present invention provides a primer combination for amplifying the haplotype molecular markers, which consists of 6 pairs of primers for amplifying SNPs 1-6, respectively, and the primer sequences are shown in Table 1.
In a fifth aspect, the present invention provides a detection reagent or kit comprising the above primer or primer combination.
In a sixth aspect, the invention provides an application of the SNP molecular marker, or the haplotype molecular marker, or the primer, the primer combination, or the detection reagent or the kit in identifying and early screening the chicken varieties with the high weight traits.
In a seventh aspect, the invention provides a method for identifying or early predicting chicken weight traits by using the haplotype molecular markers, which comprises the following steps:
1) extracting the genome DNA of the chicken to be detected;
2) taking the genome DNA of the chicken to be detected as a template, and designing a primer combination for amplifying the haplotype molecular marker (table 1);
3) analyzing the PCR amplification product.
Wherein, the PCR reaction system is as follows: the dosage of 2 XPCR TaqMix is 12.5 mul which is half of the volume of a PCR amplification reaction system, 50ng of chicken genome DNA to be detected and 1 mul of each of 10 mul/l of forward primer and reverse primer, and finally dd H is used2O was supplemented to 25. mu.l. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; the following cycles were performed for 32: pre-denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 30 s; and (3) extending for 2min at 72 ℃ to obtain a PCR product.
Step 3) the PCR amplification product is analyzed by methods including but not limited to sequencing. Analyzing the six SNP loci, and reserving the individual when the six SNP loci form G, C, C, C, C, G homozygous haplotypes according to the genome sequence; the individual is eliminated when any other haplotype combination is composed.
In the eighth aspect, the invention provides a broiler chicken genetic improvement method, which comprises the steps of subculturing and breeding homozygous individuals of the haplotype molecular marker G, C, C, C, C, G genotype, and eliminating all other genotype individuals at the site.
In a ninth aspect, the invention provides the application of the SNP molecular marker, or the haplotype molecular marker, or the primer, the primer combination or the detection reagent or the kit in chicken molecular marker assisted breeding.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the haplotype molecular marker related to the chicken weight traits is used for distinguishing high-weight chicken individuals and low-weight chicken individuals, is suitable for identifying Chinese and foreign chicken breeds, and has a wide applicable chicken breed range. The method for detecting the haplotype molecular marker can predict the weight character of the chicken quickly and effectively at low cost in an early stage, provides an effective means for the molecular marker-assisted selective breeding of the chicken, has wide application prospect in the aspect of chicken variety improvement, and can obtain excellent economic value.
Drawings
FIG. 1 is a weight box plot of chickens 0-14 weeks old according to example 1 of the present invention.
FIG. 2 is a sequence peak diagram of chicken genotype determination in example 3 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The Huiyang bearded chicken, Lingnan yellow broiler chicken and deep hybrid line samples constructed by taking the two varieties as parents are from animal science research institute of agricultural science institute of Guangdong province, and specific construction methods of deep hybrid lines are described in Optimized double-digest genetic hybridization by sequencing (ddGBS) methods with high-sensitivity SNP markers and high genetic hybridization for chicken. Other varieties of chicken germplasm resources are all from poultry scientific research institute in Jiangsu province.
Example 1 acquisition of haplotype molecular markers associated with chicken weight traits and correlation analysis with weight traits
A Huiyang beard chicken and a fast and long-growing Lingnan yellow chicken are used as F0 generation parents to construct an F2 family, and the F2 generation later randomly mate to construct a deep hybrid line. About 600 chickens were selected for phenotyping at each week's age for the F9 generation of the deep hybrid line. F9 individuals for phenotype determination were hatched in two batches, mixed-bred before 40 days of age, and raised in a single cage after 40 days of age. Body weight was measured every 2 weeks. The distribution of the weight phenotype data of chickens at each week of age was characterized using a boxplot (fig. 1): the box plot arranges a set of data from small to large, showing the lower edge, lower quartile Q3, median, upper quartile Q1, and upper edge. Using Q3+1.5IQR (four-quadrant distance) and Q1-1.5IQR as upper and lower abnormal value critical points, and eliminating phenotype records beyond the lower critical point; if the phenotype record exceeding the upper critical point is a continuous phenotype, the record of different weeks of age is referred to, if the phenotype of different weeks of age is continuously at an abnormally high value, the record of the individual is kept, and the phenotype records exceeding the upper critical point except the above condition are eliminated. The chicken wing vein blood is tested and used for extracting genome DNA. And (3) constructing a double-enzyme-digestion simplified genome sequencing library by using EcoRI-MseI, and sequencing by using a Nextseq500 sequencer, wherein the average sequencing depth is 10 x. The simplified genome sequencing data were analyzed by using the Tassel software and the F9 generation whole genome SNP marker typing results were obtained.
A linear mixed model of GCTA software is adopted for analysis, and the model formula is as follows: y-a + bx + g + e, where y is the phenotype data, a is the estimated mean of the phenotype data, b is the additive effect (fixed effect), g is the random effect, e is the residual, and x is the genotype data. The weight traits of different weeks of age take birth weight, sex and batch as covariates; .
And (3) performing whole genome re-sequencing on parents of two varieties of the F0 generation, establishing a Library by adopting a TruSeq NanoDNA LT Library Prep kit of Illumina company, and performing whole genome sequencing on double ends of 151bp (PE 151+8+151 mode) of a Hiseq X Ten sequencing platform. Mean sequencing depth 10X. The whole genome re-sequencing data is mainly used for variation (SNP and InDel) analysis, the mainly adopted comparison software is BWA, and the mainly adopted variation identification software is GATK. The genome referenced during the alignment was the chicken Gallus _ Gallus-version 5.0 genome (Gallus _ Gallus-5.0, INSDC Assembly GCA _ 000002315.3). After the F0 generation genome-wide marker is obtained, a site with the allele frequency difference of more than or equal to 0.95 between two varieties is screened as a candidate site.
The whole genome association analysis result of the F9 generation obtains QTL (quantitative trait loci) intervals which are obviously related to growth traits such as chicken weight and the like at 168.6-171.7Mb of the tail end of the chicken chromosome I. The chromosome origin in this interval of each individual of the F9 generation was determined using a homologously homomapped approach, i.e., based on the locus where the allele frequencies in the two parental lines differ by more than 0.95. A total of four recombination events were detected in the 168.6-171.7Mb interval, which was divided into 5 block units, and correlation analysis showed significant correlation between chromosomes of different parental origin and the body weight phenotype (P < 0.05) only in the 168.6-169.8 Mb interval. Within the candidate interval, Sequenom flight mass spectrometry (sites with significant allele frequency difference (delta AF ≧ 0.75)) is performed on candidate sites mined in the F0 generation sequencing, which is added to all samples of the F9 generation again, so as to detect the degree of association of the sites with the target phenotype in the F9 generation.
Among the candidate sites, 6 SNP sites which are significantly related to the body weight of the chicken at the age of 6-10 weeks are finally obtained, the 6 SNP sites are all located near the CAB39L gene, specifically, the position, rs number, additive effect and variance of the genome are shown in the table 2, and the significantly related P values are shown in the table.
TABLE 26 information about SNP sites
The whole genome haploid typing and the genotype filling are carried out by adopting Beagle software, the Beagle software utilizes a hidden Markov model, based on the linkage disequilibrium principle, an algorithm uses gtgl (combining GT, GL and PL information in a VCF file), a parameter inpute is set to True, a reference genome is the same as a genome adopted in comparison, and chromosome division is carried out for genotype filling and haplotype construction.
The haplotype analysis of the six loci at F9 shows that the average body weight of an individual (AA) with homozygous G, C, C, C, C, G genotype is 1246 +/-196.23 g, the average body weight of an individual (AA) with heterozygous G, C, C, C, C, G genotype is 1181 +/-184.93 g, the average body weight of an individual (AA) without G, C, C, C, C, G genotype is 1131 +/-181.42 g, and the phenotypes of the individuals with the three genotypes have significant differences (P < 0.05).
Example 2 validation of haplotype molecular markers by different populations
Three commercial breeds were collected for 75 total broilers as high-weight chicken group, six local Chinese breeds were collected for 144 total broilers as low-weight chicken group, and the two groups had great differences in weight phenotype at seven weeks (Table 3). And (3) collecting blood samples from the 219 samples, extracting genome DNA, establishing a Library by adopting a TruSeq Nano DNA LT Library Prep kit of Illumina company, and sequencing the whole genome of a double-end 151bp (PE 151+8+151 mode) of a Hiseq X Ten sequencing platform. Mean sequencing depth 10X. The whole genome re-sequencing data is mainly used for variation (SNP and InDel) analysis, the mainly adopted comparison software is BWA, and the mainly adopted variation identification software is GATK. The genome referenced during the alignment was the chicken Gallus _ Gallus-version 5.0 genome (Gallus _ Gallus-5.0, INSDC Assembly GCA _ 000002315.3). The whole genome haploid typing and the genotype filling are carried out by adopting Beagle software, the Beagle software utilizes a hidden Markov model, based on the linkage disequilibrium principle, an algorithm uses gtgl (combining GT, GL and PL information in a VCF file), a parameter inpute is set to True, a reference genome is the same as a genome adopted in comparison, and chromosome division is carried out for genotype filling and haplotype construction.
TABLE 3219 sample correlation information
As can be seen from Table 3, all the chickens of the high body weight variety have a high frequency of the G, C, C, C, C, G haplotype after being manually selected, while the chickens of the low body weight variety have a very low frequency of the G, C, C, C, C, G haplotype.
EXAMPLE 3 establishment of haplotype molecular marker detection method and Breeding method for increasing body weight by using marker for auxiliary selection
Randomly selecting 300 chickens to be detected, collecting blood by veins, extracting genome DNA by a conventional phenol-imitation method, dissolving the extracted genome DNA in TE buffer solution, and then diluting the solution to the concentration of 50 ng/mu l. And (3) adopting the primers and the PCR reaction in the table 1 to amplify, sequencing by a direct sequencing method, and typing six SNP sites of the haplotype. And (3) carrying out allele sequencing on the amplification product by adopting a direct sequencing method.
Performing PCR amplification by using chicken genome DNA as a template and using the primer pair shown in the table 1 to obtain five groups of PCR products (rs737654023 and rs740848201 are close in labeled position, and detection is completed by using a pair of primers); sequencing the five groups of PCR products, analyzing the six SNP loci, and reserving the individual when the six SNP loci form G, C, C, C, C, G homozygous haplotypes according to the genome sequence; when any other haplotype combination is formed, the individual is eliminated. The PCR reaction system is as follows: the dosage of 2 XPCR RTaqmix is half of the volume of the PCR amplification reaction system, namely 12.5 mul, 50ng of chicken genome DNA to be detected, the amounts of 10 mul/l of forward primer and reverse primer are respectively 1 mul, and finally dd H is used2O was supplemented to 25. mu.l. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; the following cycles were performed for 32: pre-denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 30 s; and (3) extending for 2min at 72 ℃ to obtain a PCR product. The PCR products were sequenced, and the genotypes were judged by sequencing the peak patterns (FIG. 2), with the sequencing primers underlined in Table 4. Wherein, after PCR is carried out on the three sites of rs740211632, rs737654023 and rs740848201, the used sequencing primer is a reverse sequencing primer, and therefore, the results shown by the sequencing peak diagrams corresponding to the three sites are the base sequences of the complementary chains of the reference genome.
TABLE 4 sequencing primers for each SNP molecular marker
Six loci are selected and remained according to the genotyping result, and the healthy individuals with homozygous G, C, C, C, C, G genotypes are formed by haplotypes. Reserving seeds according to the quantity that the ratio of male to female is not less than 1:3, and establishing new family breeding seeds in the egg laying peak period.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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<211>864
<212>DNA
<213> Chicken (Gallus Gallus)
<400>2
acagtgaaga cccctgacag gttctggctt tggacactgg gaaaaaaaat tactcaccac 60
atgacatgaa tggtaatatc taaatataca ggttgtatca ggtcaaatat agtgatggaa 120
atcttcttgg caaacatcac ttgtcggcac atctttcaat gcaatctgac agctccttta 180
ttactcagcg tacagatcaa ttgttacact tgctggaaat agccaagaaa tgtcagcttt 240
ctcgtgataa tggtggatta catatctgct ctctttctac agaaataatt tattttttct 300
cctttgtgtg tttttttgtt tgtttgggtt ttggtttgtt tttttttgtt tttccagaat 360
gcaatcccaa attaacaagg aggcaaaacc acctatatac taaattaacc agggtctctg 420
atccaaggag ctggaactgt aggcctgcta gtggaaatat gacagaaaag tcttgaaacc 480
acttccttaa aaccagcaaa gatcataagg acatcaaatt ttgtctgaca ttatcgtcaa 540
actggagtgt ccatctaagc accagtgaaa agaaaaggct tatttcatga gtgcagaatc 600
ctgtgagaag gtcgtgtacc aagagtcaga ccactttaac ttnccatgac cacagcaaga 660
cagtccctac tagctgtcag catgggatga aattactttc acaagcacaa gaacttctac 720
tgaaatattt cagcacacaa agacctgaat ccaaacagct gcatattagc caagtaacta 780
aagacataaa tataagctat ttaatatctt actgtttatg tgctatgtga ctcctgtatt 840
tgaaagagaa cgctgctaga aggc 864
<210>3
<211>812
<212>DNA
<213> Chicken (Gallus Gallus)
<400>3
agagaaagcc gatacagcca tggctccctg ctgaaaaatg aaaaagtcct aacaaagagc 60
tgaaggttac atgttgctag gatcactaca gttgctggaa aagaattttc attccatgct 120
atttcagttt ctccatattc agtaacttcc agtgctctta agaagtcata tataatgtct 180
taccaacact taagcgcaac tgaactgtat agttgcaata aaacattaca gctaaatttg 240
ttcgtgtcca ttttttcaca tgctcttgtg ctagattgac aagggggggc ctatggaaaa 300
tctgtcagtc caacagctat cactcagttg gctccatatt tcaaaactca gttatgcagt 360
attggtcttt ggtcatacac tgggagggct tcccttccct tcccttccct tcccttccct 420
tcccttccct tcccttccct tcccttccct ctcccttccc ttccctctcc cttccctctc 480
ccttccctct ccccctctgg aagtaaaaag cacctgctta ttcagcactt tctttcatct 540
aaattacggt ctgttggagc aagaaaagca agaaacaaac atcactaata tcaatggatt 600
ttttataaaa agcttatgct gtaatatcat ctaaagacgt catttcacaa ttaaattgct 660
ctaggaangt atggctgcca atctattgtc tagtatcatt cagtctttgt ctgggtattg 720
tattgtagca aatagtatac ttatcttctt cagtgccaaa ctcatatatt agaattaagc 780
tcagccagcc agcacactgc tggaaacctg aa 812
<210>4
<211>547
<212>DNA
<213> Chicken (Gallus Gallus)
<400>4
taacaaggga gggctgagtg aagcagtaag aacagaacca cttcactgtc tgacaatgcc 60
aaatttaagt gtatgcagga ttgtggctta cctgatcctt ctccataaag ctttttcatt 120
gtttcctaca gcngtaatca aatttttctt agcatactta aaaatgtgtt aacttccagt 180
tttatttgac agtggttatt gcatctatac ataataatta cttcagtttt gctacatgca 240
tcattttgat gggctttgtt atgtcaggtg caactctaag tgaggcactc actgctttgt 300
gtttgtactc acctatcaat caccaggcat cacagtgaag gcttgcaact tactctacgc 360
tttgatttaa aaaacacaag gcaaaacaac aacaaaaaaa gaagctctgg tacaacatat 420
aatacaactc ctagtagtgg gaacaccgca agttcactgt atctagaaat ttgtctgcta 480
catcttcatt taattaaaat attttgtgca cgtagtttga tctgcatgca aacccacaaa 540
caaagcc 547
<210>5
<211>490
<212>DNA
<213> Chicken (Gallus Gallus)
<400>5
gagcacctca gaccagacta ggcccgacac agagctctga ggcagccctt ttcccctccc 60
ccccattgcc aagatggcgg gacagcacga acacggcgac gcccccaaaa cacccggccc 120
caaactcgcc ccgcccaccc aagatggcgg acacaccgcc ttcctctcgg ctccgccccc 180
agccccctct ctctccctgc ggccacccgg cccccttctc gctctcccgc tcgttcccat 240
tggccagcga cgcgcgcacc gcgaccccag ccgcagccaa tagcaacaga ggcactcgca 300
cnacctgccg ggagcggccc gcctcctcca cgcagctatt cgctcgggcg gcccggcccc 360
gccttgcgca tgcgcagaag gtgcaacgcg aggtaacgaa gcctagctcg gcgcatgcgc 420
atagctgttg gtgcttggca ccccctggcg ggcggtgacg gcgcagcagc tgactgaggt 480
aacgctgtgt 490
<210>6
<211>490
<212>DNA
<213> Chicken (Gallus Gallus)
<400>6
gagcacctca gaccagacta ggcccgacac agagctctga ggcagccctt ttcccctccc 60
ccccattgcc aagatggcgg gacagcacga acacggcgac gcccccaaaa cacccggccc 120
caaactcgcc ccgcccaccc aagatggcgg acacaccgcc ttcctctcgg ctccgccccc 180
agccccctct ctctccctgc ggccacccgg cccccttctc gctctcccgc tcgttcccat 240
tggccagcga cgcgcgcacc gcgaccccag ccgcagccaa tagcaacaga ggcactcgca 300
caacctgccg ggagcggccc gcctcctccn cgcagctatt cgctcgggcg gcccggcccc 360
gccttgcgca tgcgcagaag gtgcaacgcg aggtaacgaa gcctagctcg gcgcatgcgc 420
atagctgttg gtgcttggca ccccctggcg ggcggtgacg gcgcagcagc tgactgaggt 480
aacgctgtgt 490
Claims (8)
1. The haplotype molecular marker related to the chicken weight trait is characterized in that the haplotype molecular marker consists of 6 SNP loci of SNP 1-SNP 6;
wherein, the genomic position of the SNP1 is GGA1:169195899bp, and the basic group is T or G;
the genomic position of SNP2 is GGA1:169199539bp, and the basic group is T or C;
the genomic position of SNP3 is GGA1:169202882bp, and the basic group is T or C;
the genomic position of SNP4 is GGA1:169203717bp, and the basic group is T or C;
the genomic position of SNP5 is GGA1:169208105bp, and the basic group is A or C;
the genomic position of SNP6 is GGA1:169208133bp, and the basic group is A or G.
When the haplotype molecular marker is G, C, C, C, C, G homozygous genotype according to SNP 1-SNP 6, the corresponding chicken has high weight character; when the haplotype molecular marker is G, C, C, C, C, G heterozygous genotype, the corresponding chicken has medium weight character; when the haplotype molecular marker does not exist in G, C, C, C, C, G genotype, the corresponding chicken has a low weight character.
2. The primer combination for amplifying the haplotype molecular markers of claim 1, which consists of 6 pairs of primers for amplifying SNPs 1-6, respectively, and has the following sequences:
3. a detection reagent or kit comprising the primer combination of claim 2.
4. Use of the haplotype molecular marker of claim 1, the primer combination of claim 2 or the detection reagent or kit of claim 3 for identifying and early screening chicken breeds with high weight traits.
5. The method for identifying or early predicting the chicken weight traits by using the haplotype molecular markers as claimed in claim 1, which comprises the following steps:
1) extracting the genome DNA of the chicken to be detected;
2) using the genome DNA of the chicken to be detected as a template, designing a primer combination for amplifying the haplotype molecular marker in the claim 1, and carrying out PCR amplification reaction;
3) analyzing the PCR amplification product.
6. The method according to claim 5, wherein the combination of primers used in step 2) is the same as that described in claim 2.
7. The method according to claim 5 or 6, wherein the PCR reaction conditions in step 2) are as follows: pre-denaturation at 94 ℃ for 2 min; pre-denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 2 min.
8. Use of the haplotype molecular marker of claim 1, the primer combination of claim 2 or the detection reagent or kit of claim 3 in molecular marker assisted breeding of chicken.
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| CN112609009B (en) * | 2021-01-15 | 2022-07-05 | 广西大学 | SNP molecular marker combination related to weight and body size of Guangxi three-yellow chicken based on whole genome sequencing screening and application |
| CN112885408A (en) * | 2021-02-22 | 2021-06-01 | 中国农业大学 | Method and device for detecting SNP marker locus based on low-depth sequencing |
| CN113005205A (en) * | 2021-04-23 | 2021-06-22 | 江苏省家禽科学研究所 | Haploid marker for identifying variety of Henan fighting chicken based on sex chromosome and application thereof |
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