CN114921570B - Molecular marker related to chicken high temperature tolerance and application thereof - Google Patents
Molecular marker related to chicken high temperature tolerance and application thereof Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention relates to a SNP molecular marker related to chicken high temperature tolerance, wherein the SNP molecular marker locus corresponds to the 23059382, 23062420 and 23062522 positions of the 9 th chromosome sense strand of a reference genome (Gallus 5.0) of chicken on NCBI, and the base at the 3 positions is G or A, C or T, G or A respectively. The 3 SNP molecular markers related to the high temperature tolerance of the chicken are utilized for auxiliary selection, so that the genetic progress is rapidly and accurately obtained; the invention also provides the sequences and the primers of the 3 SNP molecular markers, which can establish a rapid, efficient and accurate molecular marker assisted breeding selection technology and accelerate the breeding work of high-temperature tolerant chicken strains.
Description
Technical Field
The invention relates to the fields of genetic engineering and genetic breeding, in particular to a molecular marker related to chicken high temperature tolerance and application thereof.
Background
Global warming has resulted in extended times of high temperature seasons in many areas over the past decades, with increasing impact of high temperature stress on poultry health in an intensive farming environment. Because the body surface feathers of the poultry are thick, sweat glands are not formed on the whole body, metabolism is vigorous, the body temperature is high, and modern commercial poultry can generate more body temperature due to rapid metabolism, so that the poultry is particularly sensitive when suffering from high temperature, the growth speed of a light person is low, the death of a heavy person is high, and huge economic loss is caused to the poultry farming industry. It is counted that high temperatures cause economic losses to the poultry industry worldwide of up to $1.28 to $1.65 per year. How to ensure that the poultry has higher production performance and economic benefit in a high-temperature environment is a focus problem of current poultry cultivation. The current strategy of effective alleviation is also continuously developed in the production of poultry, including adopting measures such as ventilation and cooling, nutrition regulation and control and the like to cope with inadaptation in high-temperature environment, wherein the development of high-temperature resistant poultry strains through breeding means can also become one of effective and long-term measures.
The national science and technology department major research and development plan declaration guideline provides the aims of focusing and accelerating the breaking of the basic science of agricultural biology, improving the design and breeding capability and guaranteeing the national grain safety, takes the research main points of the important properties of agricultural biology and the environmental adaptability as guideline content for the first time, and highlights the high importance of the country on the biological and environmental adaptation. However, the improvement of the productivity of the poultry is necessarily required to improve the adaptability to the environment, especially to the high-temperature environment in the future. Thus, high temperature tolerance is considered as an environmental adaptation capability of poultry, and breeding of high temperature tolerant strains by genetic breeding means is one of the directions of contemporary poultry breeding. The effective molecular marker is utilized to select high temperature tolerance, so that the cultivation of chicken high temperature tolerance varieties or strains can be effectively promoted. Only a few studies report that the gene can be used as a potential genetic marker for chicken high temperature tolerance, mainly focuses on the development of known genes, and is lack of more potential molecular marker studies on chicken genome. Therefore, the SNP molecular marker for positioning high temperature tolerance can obtain faster genetic progress, and has important significance for promoting the process of breeding new high temperature tolerance chicken lines.
Disclosure of Invention
In order to solve the breeding problem of new high-temperature tolerance lines in production, the invention provides a SNP molecular genetic marker related to high-temperature tolerance, and the SNP marker is helpful for improving the high-temperature tolerance of poultry on genetic level, and can be used for molecular marker auxiliary selection of chickens.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a SNP molecular marker related to chicken high temperature tolerance, wherein the SNP molecular marker locus corresponds to the 23059382, 23062420 and 23062522 positions of the sense strand of chromosome 9 of a reference genome (Gallus 5.0) of chicken on NCBI, and the base at the 3 positions is G or A, C or T, G or A respectively.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.7, and the 126 th base is G or A.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.8, and the 120 th base is C or T.
Preferably, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.9, and the 96 th base is C or T.
In a second aspect, the invention provides a primer combination, which consists of an upstream primer with nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 and a downstream primer with nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6.
The third aspect of the invention provides a method for breeding high temperature tolerant chickens, comprising the following steps:
(1) Detecting genotypes of the SNP molecular marker loci in the genome DNA with a certain sample number;
(2) And selecting high-temperature resistant chickens based on genotypes of SNP loci, wherein the selected genotypes meet 3 loci of GG, CC and GG chickens respectively, and eliminating chickens with other genotypes.
Preferably, the step (1) uses the primer combination to detect genotypes of three SNP molecular markers.
Preferably, the number of samples in the step (1) is 100-1000.
The 3 SNP molecular markers related to the high temperature tolerance of the chicken are utilized for auxiliary selection, so that the genetic progress is rapidly and accurately obtained; the invention also provides the sequences and the primers of the 3 SNP molecular markers, which can establish a rapid, efficient and accurate molecular marker assisted breeding selection technology and accelerate the breeding work of high-temperature tolerant chicken strains.
Drawings
FIGS. 1a and 1b are schematic representations of selection signals on chicken genomes associated with high temperature tolerance.
FIG. 2 is a schematic diagram showing mutation frequencies of 3 SNP loci in 4 varieties in a selected signal region.
FIG. 3 is a genotype schematic of 3 SNP loci in 8 variety populations.
FIG. 4 is a diagram showing the sequence of 3 SNP loci sanger.
Detailed Description
In order to make the technical contents of the present invention more clearly understood, the following examples are specifically described.
Example 1
Selecting 4 local chickens growing in different environments in China, including 50 Wenchang chickens, dongxiang green-shell chickens, tibetan chickens and Lindian chickens, collecting 2mL blood samples from the wing veins in an EDTA-Na anticoagulant tube, and extracting genome DNA by using a phenol-chloroform method. Whole genome sequencing was performed based on Illumina Hiseq PE150 platform after DNA quality control. The adaptor sequence was omitted (> 10nt calibrated to the adaptor, allowing for. Ltoreq.10% mismatches), and low quality sequences including. Gtoreq.10% unrecognized nucleotides or > 50% and base sequencing quality < 5 were removed, removing PCR repeats that may occur during library construction. The remaining high quality paired sequences were matched using BWA (Burows-Wheeler Aligner) (version 0.7.8) software package with the command mem-t 4-k 32M to chicken reference genome gallus_gap-5.0; while the repeated sequences were removed by SAMtools (version 0.1.19) software package in order to reduce the mismatches generated by pre-sequencing PCR amplification. The obtained sequence was annotated with SNP and InDel using the ANNOVAR software package according to the gallus_gallous-5.0 genome.
Selection signal screening: the screening thinking is to compare and screen selected signals of Wenchang chickens (WCC) growing in a high-temperature area, green-shell laying hens (GSC) and Tibetan chickens (TBC) growing in a non-high-temperature area with those of Wenchang chickens (LDC) growing in a low-temperature area respectively, and the three groups of signals are combined and analyzed to remove two groups of negative control (non-high-temperature environment) selected signals of the green-shell laying hens and the Tibetan chickens from the Wenchang chickens selected signals so as to obtain high-temperature selected signals. The screening method is to locate the selected signal by a method based on the combination of population differentiation, a population genetic statistical method Fst based on the precondition of Hash balance, θpi ratio based on nucleotide diversity and Tajima' D (see figure 1), and analyze and screen SNP sites on the selected signal which are related to temperature adaptation (see figure 2). SNP analysis is carried out in the group by using a Bayesian method in a SAMtools software package, the genotype of each individual of 4 varieties is calculated (see figure 3), and the frequency of three locus genotypes GG, CC and GG of the chickens growing in the high-temperature areas is higher than that of the chickens growing in the temperate areas and the cold areas; further statistics of allele frequencies in the samples (see fig. 2 and table 1) revealed that 3 SNPs had lower mutation frequencies on Wenchang chickens, and that mutation frequencies from green-shelled layer chickens to meadow chickens were gradually increased, probably because domestication of chickens originated initially in southeast asia and had strong adaptability to high-temperature environments, 3 loci of chickens growing in high-temperature areas were closer to ancestors of chickens, and it was presumed that the several SNPs could play a role in high-temperature environment adaptation.
Table 1: mutation frequency of 3 SNP loci of 4 chicken breeds
Example 2
In order to further study whether the allele mutation frequency pattern of the 3 SNP loci is related to high temperature environmental adaptability, the experiments select other 4 local varieties of China including Minqing maojiao chicken (MQMC), guangxi Shenfeng chicken (LSFC), shandong Jining Bai Ri chicken (JNC) and Beijing oil chicken (BJYC) 100, and the fin veins collect 2mL blood samples in EDTA-Na anticoagulant tubes, and the genome DNA extraction is carried out by using a phenol-chloroform method. After DNA quality control, amplifying target fragments of 3 SNP loci related to high temperature tolerance, wherein the upstream and downstream primers for 3-segment sequence amplification are respectively as follows:
primer with base at 23059382 of chromosome 9:
upstream primer F: cagacagaagaaagagcgggtcag (SEQ ID NO. 1)
The downstream primer R: actcaccaacacaccaagcctttc (SEQ ID NO. 2)
Primer with base at 23062420 of chromosome 9:
upstream primer F: gcaggaaccatcgtgtacca (SEQ ID NO. 3)
The downstream primer R: tcacacagggaacccacaga (SEQ ID NO. 4)
Primer with base at 23062522 of chromosome 9:
upstream primer F: gtctgtgggttccctgtgtg (SEQ ID NO. 5)
The downstream primer R: tctgggagatggagattattggc (SEQ ID NO. 6)
And (3) PCR amplification: in this example, the reagents were obtained from Beijing Tiangen Biochemical technology Co., ltd, and primer synthesis and sequencing were performed by Shanghai Biotechnology Co., ltd. PCR amplification was performed using the other 4 varieties of chicken genomic DNAs obtained in the examples as templates, and the primers described above, respectively. The amplification system is as follows:
the PCR reaction procedure was as follows:
sequencing and identification of amplified sequences:
the amplified PCR product was sent to Sanger two-way sequencing by Shanghai Biotechnology Co., ltd. And comparing the obtained sequence with a reference genome of the chicken to obtain a corresponding SNP mutation site. The PCR amplification products were as follows:
SEQ ID NO.7
SEQ ID NO.8
SEQ ID NO.9
note that: the first bolded base sequence of the sequence is the position of the upstream and downstream primer sequences, the bolded base marked with underline in the sequence is the mutation site, the mutation base is in brackets and is the allelic mutation.
As can be seen from fig. 3, with the increase of the temperature of the growing places of the 4 local varieties of Beijing oil chickens (BJYC), jining Bai Er chickens (JNC), minqing maojiao chickens (MQMC), and longshengfu chickens (LSFC), the frequencies of the genotypes GG, CC and GG of the three sites gradually increased, which indicates that there is a strong correlation between the genotype frequencies of the local chickens of different varieties and the ambient temperature. Further analysis shows that the mutation frequency of 3 SNP loci of Dragon and phoenix chicken < Minqing maojiao chicken < Jining white ear chicken < Beijing oil chicken (see table 2, specific sequencing diagram see figure 4), namely the mutation frequency becomes lower with the increase of the temperature of the growing environment of the chicken, and the 3 loci are further considered to be closely related to high temperature tolerance.
TABLE 24 mutation frequencies of 3 SNP loci of chicken breeds
Example 3
In order to further explore whether the allele mutation frequencies of the 3 SNP loci are suitable for screening high-temperature tolerance groups, 240 broiler chickens of 50 days old are selected from broiler chickens A and broiler chickens B respectively, and all chickens are fed with basic daily ration and eat freely. This test was subjected to a cyclic heat stress test at the temperature set to: 20:00-00:00, 00:00-06:00 set to 28 ℃,06:00-10:00, 16:00-20:00 set to 32 ℃,10:00-16:00 set to 35 ℃. The test period was 2 weeks. On test day 14, the productivity was counted, and 3 primer sequences and PCR conditions in example 2 were collected for DNA extraction, and then transferred to sanger sequencing by PCR, and genotypes of 3 SNP sites in example 2 were detected.
As shown in table 3, the rectal temperature, respiratory rate and mortality of group B chickens were lower than those of group a, presumably the group B chickens were more resistant to high temperatures than the group a chickens; the sequencing analysis shows that the genotypes of GG, CC and GG of the B group chicken at 3 SNP loci are high in frequency, and the B group chicken population is supposed to have relatively low mortality rate due to better high temperature adaptability when the B group chicken population is in response to a high temperature environment, and meanwhile, the 3 SNP loci are further proved to have certain application value in the selection of high temperature tolerance populations or strain varieties.
TABLE 3 productivity of group 2 chickens and mutation frequencies of 3 SNP loci under heat stress environment
In this specification, the invention has been described with reference to specific embodiments thereof. It will be apparent, however, that various modifications and changes may be made without departing from the spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.
Sequence listing
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<120> molecular marker related to chicken high temperature tolerance and application thereof
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cagacagaag aaagagcggg tcagaatatt catagtaagt tgagctctgt cttgtgcctt 60
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atacagtagc attagaggag ggctttcctg ctcttactca gtgtcaataa ttccctgttc 180
atggactgtt ctgactagat ttgggacttc atataattta gaggtgaagt aagaaaggct 240
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gtctgtgggt tccctgtgtg aggtgcacag ctgtaatttc taaagaagca gtttggatag 60
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cagacagaag aaagagcggg tcagaatatt catagtaagt tgagctctgt cttgtgcctt 60
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atacaatagc attagaggag ggctttcctg ctcttactca gtgtcaataa ttccctgttc 180
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gcaggaacca tcgtgtacca aacactttac acccctcaat gaatttttaa ctttcagttc 60
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gtctgtgggt tccctgtgtg aggtgcacag ctgtaatttc taaagaagca gtttggatag 60
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tcctttggcc aataatctcc atctcccaga 150
Claims (4)
1. A primer combination of SNP molecular markers related to chicken high temperature tolerance, which is characterized by comprising an upstream primer of a nucleotide sequence shown as SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 and a downstream primer of a nucleotide sequence shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6;
the SNP molecular marker loci correspond to positions 23059382, 23062420 and 23062522 of the sense strand of the fifth version 9 chromosome of the reference genome of the chicken on NCBI, and the bases at the 3 positions are G or A, C or T, G or A respectively.
2. A method for breeding high temperature tolerant chickens, comprising the steps of:
(1) Detecting the genotype of the SNP molecular marker locus according to claim 1 in a sample amount of genomic DNA;
(2) And selecting high-temperature resistant chickens based on genotypes of SNP loci, wherein the selected genotypes meet 3 loci of GG, CC and GG chickens respectively, and eliminating chickens with other genotypes.
3. The method for breeding high temperature tolerant chicken according to claim 2, wherein the step (1) detects genotypes of three SNP molecular markers using the primer set as set forth in claim 1.
4. The method for breeding high temperature tolerant chicken according to claim 2, wherein the number of samples in the step (1) is 100 to 1000.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948832A (en) * | 2010-09-16 | 2011-01-19 | 华南农业大学 | Heat resistance associated molecular marker on chicken heat stress protein 70 gene and acquiring method and application thereof |
| CN102154266A (en) * | 2010-09-16 | 2011-08-17 | 华南农业大学 | Chicken heat resistance-associated molecular marker and identification method and use thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948832A (en) * | 2010-09-16 | 2011-01-19 | 华南农业大学 | Heat resistance associated molecular marker on chicken heat stress protein 70 gene and acquiring method and application thereof |
| CN102154266A (en) * | 2010-09-16 | 2011-08-17 | 华南农业大学 | Chicken heat resistance-associated molecular marker and identification method and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| Genetic polymorphisms in part of intron 7 and exon 8 of HSP90AA1 gene and its association with heat tolerance traits in two exotic layer chicken strains;Young I Irivboje等;《Trop Anim Health Prod》;第52卷(第3期);第969-977页 * |
| Whole genome analyses reveal novel genes associated with chicken adaptation to tropical and frigid environments;Shourong Shi等;《Journal of Advanced Research》;第47卷;第13-25页 * |
| 鸡Hsc70基因5′侧翼区SNP与耐热性状的相关分析;陈卓宇等;《中国兽医学报》;第32卷(第9期);第1339-1343页 * |
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