CN107022608B - SNP marker and application thereof - Google Patents
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Abstract
The invention discloses an SNP marker and application thereof. Wherein the SNP marker is SEQ ID NO: 1, the 501 th base C or T from the 5' end. The SNP marker is closely related to the growth speed of the epinephelus coioides, and can be effectively used for molecular marker assisted breeding of the epinephelus coioides.
Description
Technical Field
The present invention relates to SNP markers and their use. Specifically, the invention relates to an SNP marker related to the growth speed of Epinephelus coioides, a primer pair and a kit for detecting the SNP marker, application of the SNP marker, the primer pair and the kit in Epinephelus coioides breeding, and a method for detecting the growth speed of Epinephelus coioides.
Background
Grouper is a precious marine economic fish. In more than ten years, the artificial breeding technology of the groupers is relatively mature, the culture scale of the groupers is gradually enlarged, and the rapid development of the groupers industry has important significance for promoting the increase of fishermen, meeting the requirements of national aquatic products and optimizing the structure of the aquaculture industry. However, the germplasm degradation and the lack of excellent varieties are the major bottleneck of sustainable and healthy development of the grouper industry. Therefore, the cultivation of new excellent varieties of grouper becomes the key for the development of the grouper industry in China.
The traditional fish breeding method completely depends on phenotype, and has the insurmountable obstacles of long period, low efficiency and the like. Molecular breeding, namely molecular marker-assisted selective breeding, refers to the selection of breeding materials by using DNA molecular markers, the comprehensive improvement of important economic characters of breeding species, and is a breeding method organically combining traditional genetic breeding and modern molecular biology. The molecular breeding opens up a new way for fish breeding, and with the development of modern biotechnology, the function of molecular markers in fish breeding is increasingly prominent. In the breeding of grouper, people hope to achieve the goal of early seed selection and improving breeding accuracy through selection of DNA markers which are closely related to growth traits and closely linked with quantitative traits, thereby achieving greater genetic progress.
However, molecular markers related to growth traits that can be effectively used for grouper breeding at present are still to be explored.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide an SNP marker which is related to the growth characteristics of grouper and can be effectively used for grouper breeding and application thereof.
Among them, it should be noted that SNP (single nucleotide polymorphism) is a molecular genetic marker proposed by Lander, a scholarer of human genome research center of the american college of labor and technology, in 1996, and mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. SNPs exhibit polymorphisms involving only single base variations, including transitions, transversions, insertions, and deletions.
The invention also aims to provide an SNP marker for detecting the growth speed of the epinephelus coioides, a kit and application thereof.
The invention further aims to provide a method for detecting the growth speed of the epinephelus coioides.
The purpose of the invention can be realized by the following technical scheme:
an SNP marker related to the growth rate of epinephelus coioides, and the sequence of the SNP marker is shown as SEQ ID NO: 1, as shown in SEQ ID NO: 1 is C or T at the 501 st base from the 5' end. According to an embodiment of the invention, the SNP marker is SEQ ID NO: 1 (total length 1001bp) is represented by Y at the 501 st base from the 5' end, and Y represents C or T.
The growth rate of TT genotype individuals marked by the SNP markers is obviously higher than that of CC genotype individuals. The inventor finds that the body weight of the epinephelus coioides with the genotype homozygous TT at the locus is obviously higher than that of the epinephelus coioides with the genotype homozygous CC at the locus. Further, according to the embodiment of the present invention, the growth rate of the epinephelus coioides can be effectively determined by detecting the above-mentioned SNP of the epinephelus coioides, and specifically, as described above, the epinephelus coioides having the SNP site of homozygous TT has a significantly higher body weight than the epinephelus coioides having the genotype of homozygous CC, and for example, when the SNP site of homozygous TT is TT, the epinephelus coioides to be tested belongs to an individual having a high growth rate. Therefore, the inventor determines that the SNP marker of the invention is closely related to the growth rate of the epinephelus coioides and can be effectively used for molecular marker assisted breeding of the epinephelus coioides. And then the growth speed can be selected according to the actual breeding requirement to carry out early selection on the epinephelus coioides breeding material, the breeding efficiency and accuracy can be further effectively improved, and the genetic level of the epinephelus coioides breeding population is improved, so that the excellent variety of the epinephelus coioides can be accurately and efficiently bred. In addition, according to some embodiments of the invention, the SNP marker of the invention is utilized to carry out molecular marker assisted breeding of Epinephelus coioides, and the method has the advantages of early screening, time saving, low cost and high accuracy.
The invention also provides a primer pair for detecting the SNP marker. The primer pair has the sequence shown in SEQ ID NO: 2-3. Specifically, the sequences of the primer pairs of the present invention are as follows:
an upstream primer: 5'-CTCCTCTGCTGCTCAGCTTT-3' (SEQ ID NO: 2);
a downstream primer: 5'-CCGTACCTCCACTGATGTGA-3' (SEQ ID NO: 3).
The primer pair of the invention can effectively carry out PCR amplification on the segment of the SNP marker of the epinephelus coioides to be detected, which is related to the growth speed, thereby effectively realizing the detection of the SNP marker through sequencing, determining the genotype of the SNP marker locus of the epinephelus coioides to be detected, and further effectively determining the growth speed of the epinephelus coioides to be detected. Specifically, the growth speed of the epinephelus coioides with the genotype of homozygous TT at the SNP marker locus is obviously higher than that of the epinephelus coioides with the genotype of homozygous CC at the SNP marker locus, for example, when the genotype of the SNP marker locus is TT or CT, the epinephelus coioides to be detected can be determined to belong to an individual with high growth speed. Therefore, the primer pair for detecting the SNP marker can be effectively used for molecular marker assisted breeding of the epinephelus coioides, and further can assist in early-stage realization of breeding of excellent varieties of the epinephelus coioides in a short time, at low cost and with high accuracy.
The invention also provides a kit for detecting the SNP marker. The kit comprises the nucleotide sequence shown as SEQID NO: 2-3. According to the embodiment of the invention, the primer pair contained in the kit can effectively realize the polymorphism detection of the SNP marker related to the growth speed of the epinephelus coioides to be detected, determine the genotype of the SNP marker locus of the epinephelus coioides to be detected, and further effectively determine the growth speed of the epinephelus coioides to be detected. Specifically, the growth speed of the epinephelus coioides with the genotype of homozygous TT at the SNP marker locus is obviously higher than that of the epinephelus coioides with the genotype of homozygous CC at the SNP marker locus, for example, when the genotype of the SNP marker locus is TT, the epinephelus coioides to be detected can be determined to belong to an individual with high growth speed. Therefore, the kit for detecting the SNP marker can be effectively used for molecular marker assisted breeding of the epinephelus coioides, and further can assist in early breeding of excellent epinephelus coioides varieties in a short time, at low cost and with high accuracy.
The invention also provides application of the SNP marker, the primer pair or the kit in the breeding of the epinephelus coioides. As described above, the genotype of the SNP marker of the epinephelus coioides to be tested can be effectively determined by the reagent that can be used for detecting the SNP marker related to the growth rate of the epinephelus coioides of the present invention, for example, the primer set described above or the kit including the primer set, and further, the growth rate of the epinephelus coioides to be tested can be effectively determined based on the obtained genotype, thereby effectively assisting the breeding of the epinephelus coioides.
The invention also provides a method for detecting the growth speed of the epinephelus coioides. The method determines the growth speed of the epinephelus coioides to be detected by detecting the SNP marker of the epinephelus coioides to be detected. Specifically, the epinephelus coioides to be detected may be subjected to PCR amplification and sequencing by using a reagent which can be used for detecting the SNP marker related to the growth rate of the epinephelus coioides of the present invention, such as the aforementioned primer pair or a kit comprising the primer pair, so as to detect and determine the genotype of the SNP marker of the epinephelus coioides to be detected, and further, the growth rate of the epinephelus coioides to be detected can be effectively determined based on the obtained genotype. As mentioned above, the growth rate of the epinephelus coioides with the genotype of homozygous TT at the SNP marker locus is significantly higher than that of the epinephelus coioides with the genotype of homozygous CC at the SNP marker locus, for example, when the genotype of the SNP marker locus is TT, the epinephelus coioides to be detected belongs to an individual with high growth rate. Therefore, the method for detecting the growth speed of the epinephelus coioides can quickly, efficiently and accurately detect the growth speed of the epinephelus coioides, and further can be effectively used for molecular marker assisted breeding of the epinephelus coioides, so that the method can assist in early-stage realization of breeding of excellent epinephelus coioides varieties in a short time, at low cost and with high accuracy.
In addition, the method for detecting the growth rate of the epinephelus coioides according to the embodiment of the invention can also have the following additional technical characteristics:
according to an embodiment of the present invention, the method for detecting the SNP marker of the Epinephelus coioides to be detected is not particularly limited. SNP detection can be realized by technologies such as sequencing, single strand conformation polymorphism polymerase chain reaction (PCR-SSCP), restriction fragment length polymorphism polymerase chain reaction (PCR-restriction fragment length polymorphism, PCR-RFLP), time-of-flight mass spectrometry and the like. The sequencing is a detection technology with highest accuracy, strong flexibility, large flux and short detection period. Only one pair of primers is designed on both sides of the SNP locus to amplify a product of 200-1000bp, and then the genotype of the SNP locus can be directly detected by sequencing. Therefore, the method adopts a sequencing method to carry out SNP marker detection. According to some specific examples of the present invention, the method for determining the growth rate of the epinephelus coioides to be tested by detecting the SNP marker as described above further comprises the following steps: extracting the genome DNA of the epinephelus coioides to be detected; carrying out PCR amplification on the genomic DNA of the epinephelus coioides to be detected by using the primer pair so as to obtain a PCR amplification product; sequencing the PCR amplification product so as to obtain a sequencing result; determining the genotype of the SNP marker of the epinephelus coioides to be detected based on the sequencing result; and determining the growth speed of the epinephelus coioides to be detected based on the genotype of the SNP marker of the epinephelus coioides to be detected. Therefore, the efficiency of detecting the growth speed of the epinephelus coioides can be effectively improved.
According to the embodiment of the present invention, the method for extracting the genomic DNA of the epinephelus coioides to be tested is not particularly limited, and any known genomic DNA extraction method or kit may be used. According to some embodiments of the present invention, the genomic DNA of the Epinephelus coioides to be tested is extracted by a conventional phenol chloroform method. Therefore, the genomic DNA with good quality and high purity can be effectively obtained, and the subsequent steps can be conveniently carried out.
According to the embodiment of the present invention, the condition for performing PCR amplification on the genomic DNA of the epinephelus coioides to be detected is not particularly limited. According to some specific examples of the present invention, the amplification system of the PCR amplification is, in 25 μ l: 50-100 ng/. mu.l of template DNA 1. mu.l, 10 pmol/. mu.l of SEQ ID NO: 2-3, 1. mu.l of each of the upstream and downstream primers, 2.0. mu.l of 10mmol/L dNTPmix, 0.125. mu.l of 5U/. mu.l Taq DNA polymerase, 2.5. mu.l 10 XPCR reaction buffer, and the balance double distilled water; the reaction conditions for the PCR amplification are as follows: 5 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 53 ℃ for 30 seconds, 72 ℃ for 30 seconds; 5 minutes at 72 ℃. Therefore, the fragment of the SNP marker of the invention can be amplified rapidly, efficiently and accurately to obtain a target amplification product, which is convenient for the subsequent steps.
According to the embodiment of the present invention, the method for sequencing the PCR amplification product is not particularly limited as long as the sequence of the PCR amplification product, i.e., the fragment where the SNP marker is located, can be efficiently obtained. According to some specific examples of the present invention, the PCR amplification product may be sequenced using at least one selected from the group consisting of hipseq 2000, SOLiD, 454, and single molecule sequencing methods. Therefore, the sequencing result can be obtained quickly, efficiently and accurately in high flux.
According to the embodiment of the invention, based on the sequencing result, the genotype of the SNP marker of the epinephelus coioides to be detected as TT or CC can be effectively determined by comparing the reference genome sequences of the epinephelus coioides.
According to the embodiment of the invention, the growth rate of TT genotype individuals of the SNP marker is obviously higher than that of CC genotype individuals. That is, the SNP markers of the present invention described above are closely related to the growth rate of Epinephelus coioides. Therefore, the growth speed, namely the growth speed character of the epinephelus coioides to be detected can be accurately and effectively determined based on the determined genotype of the SNP marker of the epinephelus coioides to be detected, for example, when the genotype of the SNP locus is TT, the epinephelus coioides to be detected belongs to an individual with high growth speed. Furthermore, the method can be effectively used for the molecular marker assisted breeding of the epinephelus coioides, so that the method can assist in early breeding of the excellent variety of the epinephelus coioides in a short time, at low cost and with high accuracy.
The SNP marker related to the growth rate of Epinephelus coioides and the application thereof have the following advantages:
(1) the SNP marker provided by the invention is not limited by the age, sex and the like of the epinephelus coioides, can be used for early breeding of the epinephelus coioides, and can remarkably promote the breeding process of the epinephelus coioides;
(2) the method for detecting the 501 th SNP locus of the epinephelus coioides from the 5' end as shown in SEQ ID NO.1 is accurate, reliable and convenient to operate;
(3) the detection of the 501 th SNP locus from the 5' end of the epinephelus coioides as shown in SEQ ID NO.1 provides scientific basis for the marker-assisted selection of the growth traits of the epinephelus coioides.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
Unless otherwise defined, the terms used herein have the ordinary meanings as commonly understood in the art to which this invention belongs.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. The specific techniques or conditions are not specified in the examples and are generally performed according to conventional experimental conditions, such as the Molecular cloning handbook, Sambrook, et al (Sambrook J & Russell DW, Molecular cloning: analytical manual,2001), or according to the manufacturer's instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 acquisition of SNP marker associated with growth Rate of Epinephelus coioides
1.1 obtaining of Epinephelus coioides population
The adopted groups are Epinephelus coioides hatched in 11-10 th 2010 of a certain Epinephelus coioides cultivation farm in Hainan, the parents are 29 wild male fishes and 12 wild female fishes (captured in the south China sea area of Hainan san), and 15,000 fries are transferred to a net cage for continuous breeding in 12-10 th 2010 of the south China. 198 individuals are randomly selected from the net cage in 8.8.2011, and fin rays on the back fins of the fish bodies are cut and stored at the temperature of 95% ethanol-20 ℃ for extracting genome DNA.
1.2 extraction of genomic DNA from Epinephelus coioides
The test adopts a conventional phenol chloroform method to extract genome DNA in the fin ray of Epinephelus coioides, and comprises the following specific steps:
(1) taking 0.3-0.5 g of fin ray into a 1.5ml Eppendorf tube, shearing, and uncovering and drying on a super clean bench for 20 min;
(2) after ethanol is basically volatilized, washing the mixture for 1-2 times by using TE buffer solution (10mmol/ml Tris, 1mmol/ml EDTA, SDS5 percent and pH 8.0), adding 600 mu L of DNA extract (0.001mol/L Tris-Cl, 0.1mol/L EDTA, SDS5 percent and pH 8.0) and 3 mu L of proteinase k (200mg/ml), digesting the mixture for about 3 hours in a water bath at the temperature of 55 ℃, slightly shaking the centrifugal tube for 1 time every 10min for the first 30min, and digesting the liquid in the tube until the liquid in the tube is clear;
(3) add 600. mu.l of self-prepared phenol in chloroform (phenol: chloroform: isoamyl alcohol (V: V: V) ═ 25: 24: 1), invert the tube gently back and forth for 10min, and centrifuge at 12000r for 10 min. Extracting the upper water phase with the phenol chloroform with the same volume until no white precipitate exists between the water phase and the organic phase;
(4) extracting with chloroform for 1 time, taking out supernatant, adding 2 times volume of precooled absolute ethanol to precipitate DNA, reversing and mixing uniformly, standing at 4 ℃ for 30min, centrifuging at 12000r for 10min, washing the precipitate with 70% ethanol, centrifuging, drying, precipitating, and adding 50 μ l of sterile water to dissolve. Storing at 4 deg.C for use or storing at-20 deg.C for a long time.
1.3 construction of simplified genomic sequencing (RAD-seq) library and sequencing to obtain SNP (Single nucleotide polymorphism) markers related to the body weight of Epinephelus coioides
Based on a Hiseq2000 high-throughput sequencing platform, DNA samples of 198 individuals are sequenced by using an RAD simplified genome sequencing method, so that data volume of about 0.4G is generated, and the genome of the epinephelus coioides averagely covers 0.4X. Meanwhile, the 198 individuals were subjected to phenotypic identification of growth traits such as body weight. And (3) processing and screening the data by adopting PLINK software, and then carrying out GWAS analysis by using EMMAX software based on a mixed linear model to find out a SNP site which is obviously related to the body weight from 261,366 SNPs. The SNP site is located at the 501bp site of the sequence shown in SEQ ID NO.1, the site is represented by Y in the sequence shown in SEQ ID NO.1, and the base of the site is C or T. The weight of the epinephelus coioides with the genotype homozygous TT at the locus is obviously higher than that of the epinephelus coioides with the genotype homozygous CC at the locus.
Example 2 sequencing verification and application of SNP markers associated with growth rate of Epinephelus coioides
2.1 extraction of genomic DNA from the Fin line of Epinephelus coioides to be tested
The Epinephelus coioides to be detected is from the Epinephelus coioides population in example 1, 180 fish are randomly selected, and the genomic DNA is extracted according to the DNA extraction method described in example 1.
2.2 amplification of nucleotide fragments containing SNP sites
Taking the extracted genome DNA of each Epinephelus coioides to be detected as a template, and performing amplification reaction by using a forward primer F: 5'-CTCCTCTGCTGCTCAGCTTT-3' (SEQ ID NO: 2) and reverse primer R: 5'-CCGTACCTCCACTGATGTGA-3' (SEQ ID NO: 3), and amplifying the nucleotide fragment of the SNP marker to be detected. Wherein the PCR reaction system is calculated by 25 mul as: 50-100 ng/. mu.l template DNA 1. mu.l, 10 pmol/. mu.l each of primer F and R1. mu.l, 10mmol/L dNTP mix 2.0. mu.l, 5U/. mu.l Taq DNA polymerase 0.125. mu.l, 10 XPCR reaction buffer 2.5. mu.l, the balance double distilled water; the PCR reaction conditions are as follows: 5 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 53 ℃ for 30 seconds, 72 ℃ for 30 seconds; 5 minutes at 72 ℃.
2.3 sequencing and identification of SNP site genotypes
And (3) performing one-way sequencing on each PCR amplification product obtained in the step on an ABI3730 sequencer, and identifying the sequence represented by SEQ ID NO: 1 sequence at 501bp (i.e., the SNP marker of the present invention). The genotypes and weights of the SNP sites of 60 individuals of the Epinephelus coioides to be detected are shown in the following table 1.
TABLE 160 individuals genotypes of the SNP sites and body weights thereof
| Individual numbering | Body weight (g) | Genotype(s) | Individual numbering | Body weight (g) | Genotype(s) |
| s127 | 20 | CC | s77 | 72 | CC |
| s39 | 32 | CC | s101 | 74 | CC |
| s115 | 36 | CC | s116 | 74 | CC |
| s119 | 36 | CC | s18 | 74 | CC |
| s75 | 36 | CC | s109 | 78 | CC |
| s8 | 36 | CC | s138 | 80 | CC |
| s155 | 38 | CC | s181 | 80 | CC |
| s22 | 40 | CC | s98 | 80 | CC |
| s66 | 40 | CC | s11 | 82 | CC |
| s64 | 42 | CC | s182 | 90 | CC |
| s68 | 46 | CC | s129 | 92 | CC |
| s90 | 46 | CC | s172 | 92 | CC |
| s13 | 48 | CC | s197 | 92 | CC |
| s144 | 48 | CC | s73 | 100 | CC |
| s34 | 48 | CC | s71 | 104 | CC |
| s4 | 48 | CC | s135 | 114 | CC |
| s62 | 48 | CC | s189 | 118 | CC |
| s143 | 50 | CC | s85 | 132 | CC |
| s20 | 50 | CC | s27 | 156 | CC |
| s156 | 52 | CC | s95 | 170 | CC |
| s102 | 54 | CC | s2 | 146 | TT |
| s6 | 54 | CC | s26 | 150 | TT |
| s23 | 56 | CC | s187 | 180 | TT |
| s142 | 58 | CC | s174 | 180 | TT |
| s175 | 64 | CC | s179 | 186 | TT |
| s91 | 64 | CC | s185 | 190 | TT |
| s123 | 66 | CC | s165 | 210 | TT |
| s147 | 68 | CC | s188 | 228 | TT |
| s69 | 70 | CC | s72 | 262 | TT |
| s152 | 72 | CC | s163 | 262 | TT |
2.4 correlation analysis of SNP site genotype and growth Rate
Based on the results in Table 1, the correlation between the genotype of SNP site and growth rate was analyzed by linear simulation using the SAS9.0 software Mixed program, wherein the phenotype value was represented by the individual body weight in the analysis, and the model used was as follows:
Yijk＝μ+Gi+aj+eijk。
wherein, YijkIs the individual body weight value, mu population body weight mean value, GiIs a genotype effect vector, ajIs a micro-effective multi-gene vector, eijkIs a random residual effect vector.
The results of correlation analysis of the genotype of SNP sites with growth rate are shown in Table 2 below.
TABLE 2 genotype frequency at SNP site and correlation analysis with body weight
As can be seen from Table 2, the weight average of TT homozygous individuals is greater than the weight average of CC homozygous individuals.
The results of the correlation analysis shown in Table 2 indicate that the mean values of the body weights of individuals with the GT genotype and those with the TT genotype differ by a very significant level (P < 0.01). Further, it was confirmed that SEQ ID NO: the 501 th base G or T from the 5' end of the nucleotide sequence shown in 1 is obviously related to the growth speed of the epinephelus coioides and is an SNP marker related to the growth speed of the epinephelus coioides, and the growth speed of TT genotype individuals of the SNP marker is obviously higher than that of CC genotype individuals.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
SEQUENCE LISTING
<110> Zhenjianghua Dazai detection Co., Ltd
<120> SNP marker and use thereof
<130>2017
<160>3
<170>PatentIn version 3.3
<210>1
<211>1001
<212>DNA
<213> Epinephelus coioides
<221> nucleotide sequence of SNP marker related to growth rate of Epinephelus coioides
<400>1
gcacgcactt taatagacat caaacatcga cggtgcgata tagccccagg gattatattg 60
cagcctgttg tgcgactgcc ggctgcaggg ctctcactca atactagatc agattcaaaa 120
atcgatttcc ccttcagtca cctaagtcgt tttcatatca ggtatgactg atttgtacca 180
tgtctgagta tgattgcccc cccacctcca ctcctctgct gctcagcttt cacatttaat 240
gctgttccgt agctaagtac actttcatca tcatctacct gacatttttg ctacgtgtag 300
aaaagggcgc cgcagacata cactgctgct tcagggacta tcatctgctt ctggccagag 360
gaggagctct ctaccaccca ctgaagctct gacagcaggg tggagattgc agcgaagcct 420
gatctaagtc tgtccacagt gggctgcgct gccatctgat atagagactc ttgcataacc 480
agttaaaaac atcttgtagt yacattaaat agcagtccag ctttgtgtta tcgaacagtt 540
gctgttcata cttgatgtgc actgtaccag aactttacac aagaccacct tttcaagtgg 600
acttgggcaa gcatacttcc ttgtacactg gtacactggt tcacactaat caaacaaagt 660
ggactttgag gtcaagtgta ctcagatctg ggcccaggtc tctgatgtga aacccccatt 720
agagacaaaa gaatgagaaa ataggttgaa aaatgccagt ttcccattaa ataatgcatt 780
tcctccgatg acaacacaag aaattaagga gtcatggttt aaaccataag ttagcaagta 840
attcctgtct gatttaaata gctaatcgaa cacacagcac tttcacatca gtggaggtac 900
ggtaatgtca tttcattcac tctgggcttc attctcaaag gctgtgtcat aaccagtttc 960
aaactagcgc tttaacacca actatgtatt aaacttcctt t 1001
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<221> Forward primer F
<400>2
ctcctctgct gctcagcttt 20
<210>3
<211>20
<212>DNA
<213> Artificial sequence
<221> reverse primer R
<400>3
ccgtacctcc actgatgtga 20
Claims (6)
1. An SNP marker related to the growth rate of epinephelus coioides, wherein the sequence of the SNP marker is shown as SEQ ID NO: 1, as shown in SEQ ID NO: 1 is C or T at the 501 st base from the 5' end; the growth rate of TT genotype individuals marked by the SNP is obviously higher than that of CC genotype individuals.
2. A primer pair for detecting the SNP marker of claim 1, wherein the primer pair is set forth in seq id NO: 2 and SEQ ID NO: 3.
3. A kit for detecting the SNP marker according to claim 1, which comprises the primer set according to claim 2.
4. Use of the SNP marker according to claim 1, the primer pair according to claim 2 or the kit according to claim 3 for the selective breeding of Epinephelus coioides.
5. A method for detecting the growth rate of Epinephelus coioides, which is characterized in that the growth rate of the Epinephelus coioides to be detected is determined by detecting the SNP marker of claim 1 on the Epinephelus coioides to be detected.
6. The method according to claim 5, characterized in that it comprises in particular the steps of:
(1) extracting the genome DNA of the epinephelus coioides to be detected;
(2) performing PCR amplification on the genomic DNA of the epinephelus coioides to be detected by using the primer pair of claim 3 so as to obtain a PCR amplification product;
(3) sequencing the PCR amplification product so as to obtain a sequencing result;
(4) determining the genotype of the SNP marker of the epinephelus coioides to be detected based on the sequencing result; determining the growth speed of the epinephelus coioides to be detected based on the genotype of the SNP marker of the epinephelus coioides to be detected;
the growth rate of TT genotype individuals marked by the SNP is obviously higher than that of CC genotype individuals.
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| CN108411007B (en) * | 2018-05-25 | 2019-02-26 | 海南晨海水产有限公司 | SNP marker and its application |
| CN109852710B (en) * | 2019-04-11 | 2022-06-21 | 深圳华大海洋科技有限公司 | SNP marker related to ammonia tolerance of grouper and application thereof |
| CN110106281B (en) * | 2019-06-05 | 2023-03-31 | 四川省农业科学院生物技术核技术研究所 | Hexaploid I.trifida genome specific SNP molecular marker primer and application |
| CN112322759A (en) * | 2020-12-10 | 2021-02-05 | 镇江华大检测有限公司 | Detection method for identifying three kinds of cod based on high-throughput sequencing |
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| CN104419705B (en) * | 2013-09-10 | 2016-01-27 | 深圳华大基因研究院 | Snp marker and application thereof |
| CN104419706B (en) * | 2013-09-10 | 2016-01-27 | 深圳华大基因研究院 | Snp marker and application thereof |
| CN106086195A (en) * | 2016-06-28 | 2016-11-09 | 深圳华大基因研究院 | One SNP marker relevant to the cabrilla speed of growth and application thereof |
| CN106048028A (en) * | 2016-06-28 | 2016-10-26 | 深圳华大基因研究院 | Grouper-growth-rate-related SNP (single-nucleotide polymorphism) marker and application thereof |
| CN106048027A (en) * | 2016-06-28 | 2016-10-26 | 深圳华大基因研究院 | Grouper-growth-rate-related SNP (single-nucleotide polymorphism) marker and application thereof |
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