CN117487931A - Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof - Google Patents
Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker related to the low-oxygen-tolerance trait of sillago sihama, which is one or more of the following 5 SNP molecular markers, and the SNP molecular markers are respectively positioned in SEQ ID NO:1, 583 th, 611, 629, 633 and 937 th base from 5' end, 583 th base is T or C, 611 st base is A or G, 629 th base is T or A, 633 st base is T or A, 937 th base is A or G, and the hypoxia tolerance of individuals with genotypes CT, AG, AT, AT and AA is significantly stronger than that of other genotypes, primers and kits for amplifying SNP molecular markers and methods for detecting hypoxia tolerance of Lepium to be obtained by using the primers, and application of the SNP molecular markers, the primers, the kits or the detection methods in identifying or breeding the hypoxia tolerance-trait Lepioglita varieties.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof.
Background
Sillago sihama belonging to the order Perferae, the genus Sillago, which is a tropical India-western Pacific shallow sea fish, is widely distributed in coastal areas of China. The fish meat is tender, delicious in taste, rich in unsaturated fatty acid content, high in nutritive value, and very popular with consumers, and has important economic value. The sillago sihama is sensitive to environmental stress, so that the cultivation of the strain with excellent hypoxia tolerance is a necessary condition for guaranteeing sustainable development of the sillago sihama breeding industry.
The subject group of the inventor breaks through the full artificial breeding technology of the Sillago sihama at early stage, realizes full artificial large-scale seedling raising of the Sillago sihama, and lays a foundation for the next step of fine variety breeding of the Sillago sihama. However, the lack of directional breeding of the Sillago sihama cultured offspring seed has the character degradation risks of slow growth speed, reduced stress resistance and the like caused by inbreeding, so that the healthy development of the aquaculture industry is severely limited, and development of molecular genetic breeding research and analysis of important economic character related genetic mechanisms are urgently needed. In recent years, the academy has been researched in aspects of anoxia and salinity tolerance of the sillago sihama, population genetics, artificial propagation, genomics and the like, but has not been reported in molecular marker development and related application.
Molecular marker assisted selective breeding is a recently developed fish genetic breeding method combining traditional genetic breeding and modern molecular biology, and aims at improving important economic characters of breeding species, and a breeding method for selecting breeding materials by using DNA molecular markers. With the development of molecular biotechnology, molecular marker assisted breeding is increasingly important in fish breeding work, opens up a new way for fish breeding, and shows unique superiority.
The single nucleic acid polymorphism (Single nucleotide polymorphisms, SNPs) marker has the advantages of high polymorphism, genetic stability, convenient detection and the like as a third-generation molecular marker, can be used for researching the association between genotype and phenotype, and is widely applied to the research field of animal and plant molecular breeding.
At present, reports of SNP molecular markers of related genes of the low oxygen resistance properties of Sillago sihama are not seen. The SNP molecular marker can be used for analyzing the hypoxia tolerance of the sillago sihama, carrying out genetic improvement on the sillago sihama, improving the cultivation efficiency and adaptability of the sillago sihama, increasing the economic value and ecological value of the sillago sihama, revealing the genetic mechanism and molecular regulation network of the sillago sihama, and providing a new view and thinking for understanding the adaptation mechanism of animals to the anoxic environment.
Disclosure of Invention
The invention aims to provide a SNP molecular marker related to the hypoxia tolerance of the sillago sihama, and the SNP molecular marker is obviously related to the survival rate of the sillago sihama under hypoxia stress.
The invention also aims at providing a primer for amplifying the SNP molecular marker, a kit comprising the primer and a method for detecting the hypoxia tolerance of the sillago album by using the primer.
The final object of the invention is to provide the application of the SNP molecular marker, the primer, the kit or the detection method in the identification or breeding of the low-oxygen-resistant character of the variety of the Siraia SSO.
The first object of the present invention can be achieved by the following technical means: the Sillago sihama hypoxia tolerance character related SNP molecular markers are one or more of the following 5 SNP molecular markers, and the 5 SNP molecular markers are respectively positioned in the sequence shown in SEQ ID NO:1, wherein the base at 583 position is T or C, the base at 611 position is A or G, the base at 629 position is T or A, the base at 633 position is T or A, and the base at 937 position is A or G.
According to the invention, SNP loci of SsmGST3b genes of Sillago sihama are taken as research targets, and 5 SNP loci (g.583T > C; g.611A > G; g.629T > A; g.633T > A; g.937A > G) located in an intron region of the SsmGST3b genes are found to be obviously related to the hypoxia tolerance characteristics of the Sillago sihama, so that the 5 SNP molecular markers are obtained through screening.
Further experiments show that in the identification of SNP loci of the SsmGST3b gene of the Sillago sihama:
the hypoxia tolerance character of a CT genotype individual with the temperature of 583T > C is obviously higher than that of a TT genotype individual;
the hypoxia tolerance character of AG genotype individuals of g.611A > G is obviously higher than that of AA genotype individuals;
the hypoxia tolerance character of the AT genotype individuals with g.629T > A is obviously higher than that of the AT genotype individuals with TT genotype;
the hypoxia tolerance character of AT genotype individuals with g.633T > A is obviously higher than that of TT genotype individuals;
the hypoxia tolerance of the AA genotype individuals of g.937A > G is obviously higher than that of the AG genotype individuals.
The hypoxia tolerance of each genotype at 5 SNP sites located in the intronic region of the SsmGST3b gene was significantly different (P < 0.05).
Namely, the hypoxia tolerance of CT genotype individuals marked by SNP molecules AT 583 of the invention is obviously higher than those of TT genotype individuals, the hypoxia tolerance of AG genotype individuals marked by SNP molecules AT 611 is obviously higher than those of AA genotype individuals, the hypoxia tolerance of AT genotype individuals marked by SNP molecules AT 629 is obviously higher than those of TT genotype individuals, the hypoxia tolerance of AT genotype individuals marked by SNP molecules AT 633 is obviously higher than those of TT genotype individuals, and the hypoxia tolerance of AA genotype individuals marked by SNP molecules AT 937 is obviously higher than those of AG genotype individuals.
The hypoxia tolerance property is that the hypoxia tolerance property can survive in a low dissolved oxygen environment, for example, the dissolved oxygen concentration ranges from a choking point dissolved oxygen concentration (about 1.5 mg/L) to a normal dissolved oxygen concentration (about 7 mg/L).
The second object of the present invention can be achieved by the following technical means: a primer for amplifying the SNP molecular marker, wherein the forward primer SsMGST3b-F and the reverse primer SsMGST3b-R are primers, and the base sequence of the forward primer SsMGST3b-F is as shown in SEQ ID NO:2, the base sequence of the reverse primer SsMGST3b-R is shown as SEQ ID NO: 3.
Specifically, the nucleotide sequences of the forward primer SsMGST3b-F and the reverse primer SsMGST3b-R are as follows:
forward primer SsMGST3b-F:5'-ACAGCGACAAGGAGCAAGTATTCAA-3' (shown as SEQ ID NO: 2);
the reverse primer SsmGST3 b-R5'-CAGTGGCAGATGAGAAGGTGGATG-3' (shown as SEQ ID NO: 3).
The invention also provides a kit for detecting the SNP molecular marker related to the hypoxia tolerance property of the sillago sihama, and the kit comprises the primer.
The invention further provides a method for detecting the hypoxia tolerance of the sillago sihama, which comprises the following steps:
(S1) extracting genomic DNA of tail fins of the sillago album to be detected;
(S2) carrying out PCR amplification on the genomic DNA of the multi-scale tail fin to be detected by adopting the primer to obtain a PCR amplification product;
(S3) sequencing the PCR amplification product, and determining the genotype of the SNP molecular marker based on the sequencing result;
(S4) determining the hypoxia tolerance of the to-be-detected sillago sihama through genotypes.
In the method for detecting the hypoxia tolerance of the sillago sihama:
preferably, the PCR reaction system used in the PCR amplification in the step (S2) is 40. Mu.L, comprising:PCR Supermix 20. Mu.L, 10mM forward and reverse primers each 2. Mu.L, 40 ng/. Mu.L DNA template 2. Mu.L, ddH 2 O 14μL。
Preferably, in the PCR amplification in the step (S2), the PCR reaction program is used for 35 cycles in total, the pre-denaturation at 94℃for 5min before the cycle, each cycle comprising denaturation at 94℃for 30sec, annealing at 60℃for 30sec, extension at 72℃for 30sec, and extension at 72℃for 10min after the cycle is completed.
Preferably, determining the hypoxia tolerance of the to-be-tested sillago sihama by genotype in the step (S4), wherein the hypoxia tolerance of the CT genotype individual of g.583t > C is significantly higher than that of the TT genotype individual; the hypoxia tolerance character of AG genotype individuals of g.611A > G is obviously higher than that of AA genotype individuals; the hypoxia tolerance character of the AT genotype individuals with g.629T > A is obviously higher than that of the AT genotype individuals with TT genotype; the hypoxia tolerance character of AT genotype individuals with g.633T > A is obviously higher than that of TT genotype individuals; the hypoxia tolerance of the AA genotype individuals of g.937A > G is obviously higher than that of the AG genotype individuals.
The hypoxia tolerance of each genotype at 5 SNP sites located in the intronic region of the SsmGST3b gene was significantly different (P < 0.05).
According to the method for detecting the hypoxia tolerance property of the sillago sihama, the hypoxia tolerance of the sillago sihama to be detected is determined by detecting the SNP molecular marker.
The last object of the invention can be achieved by the following technical scheme: the SNP molecular marker, the primer, the kit or the detection method are applied to the identification or breeding of the low-oxygen-resistant character variety of the silvery-grass.
In general, 5 SNP loci which are obviously related to the hypoxia tolerance trait of the sillago sihama and a pair of primers for amplifying the SNP loci are discovered by screening the hypoxia tolerance trait of the sillago sihama (the base sequences are respectively shown as SEQ ID NO:2 and SEQ ID NO: 3). The SNP loci are respectively positioned at positions 583, 611, 629, 633 and 937 of the nucleotide sequence of the SsmGST3b gene (as shown in SEQ NO: 1) from the 5' end, and the SNP loci are expressed as g.583T > C; g.611A > G; 629t > a; g.633T > A and g.937A > G. The SNP locus disclosed by the invention is obviously related to the survival rate of the sillago sihama under the hypoxia stress, the hypoxia tolerance of individuals with genotypes CT, AG, AT, AT and AA in the SNP locus is obviously stronger than those of individuals with other genotypes, and the SNP locus can be used for carrying out molecular marker assisted breeding and genetic improvement of the sillago sihama, improving the hypoxia tolerance of the sillago sihama and increasing the cultivation benefit.
The beneficial effects of the invention are as follows:
(1) According to the invention, SNP loci of SsmGST3b genes of Sillago sihama are taken as research targets, and 5 SNP loci (g.583T > C; g.611A > G; g.629T > A; g.633T > A; g.937A > G) located in an intron region of the SsmGST3b genes are found to be obviously related to the hypoxia tolerance property of Sillago sihama;
(2) Wherein the hypoxia tolerance of the CT genotype individuals of g.583T > C is significantly higher than that of TT genotype individuals (P < 0.05); the hypoxia tolerance of AG genotype individuals of g.611A > G is significantly higher than that of AA genotype individuals (P < 0.05); the hypoxia tolerance of AT genotype individuals with g.629T > A is significantly higher than those with TT genotype (P < 0.05); the hypoxia tolerance of AT genotype individuals with g.633T > A is significantly higher than those with TT genotype (P < 0.05); the hypoxia tolerance of the AA genotype individuals of g.937A > G is obviously higher than that of the AG genotype individuals (P < 0.05);
(3) The SNP locus disclosed by the invention is obviously related to the survival rate of the sillago sihama under the hypoxia stress, the hypoxia tolerance of individuals with genotypes CT, AG, AT, AT and AA in the SNP locus is obviously stronger than those of individuals with other genotypes, and the SNP locus can be used for carrying out molecular marker assisted breeding and genetic improvement of the sillago sihama, improving the hypoxia tolerance of the sillago sihama and increasing the cultivation benefit;
(4) In the genetic breeding research process of Sillago sihama with hypoxia tolerance as a breeding index, individuals with g.583T > C locus CT, g.611A > G locus AG, g.629T > A locus AT, g.633T > A locus AT and g.937A > G locus AA can be preferentially selected as breeding parents, and the genetic breeding research process has important guiding significance for breeding new varieties with excellent hypoxia tolerance of Sillago sihama.
Drawings
FIG. 1 is a diagram of sequencing peaks of the PCR amplification product using SsMGST3B-F primer in example 1, sequencing using forward primer SsMGST3B-F, A shows g.583T > C site position, B shows g.611A > G site position, C shows g.629T > A and g.633T > A site position, and D shows g.937A > G site position.
Detailed Description
The following detailed description of the present invention is given with reference to the accompanying drawings and specific examples, which are given for illustrative purposes only and are not to be construed as limiting the scope of the present invention. The reagents or materials used in the examples, unless otherwise specified, were all commercially available. Unless otherwise indicated, all laboratory instruments used are laboratory conventional.
Example 1
The screening and verifying method for the SNP molecular markers related to the low-oxygen-tolerance traits of Sillago sihama provided by the embodiment comprises the following steps:
(S1) acquisition of Sillago sihama population
The test was performed at the east sea island breeding base of the university of ocean, guangdong. Before the experiment starts, sexually mature male and female individuals with good body forms are selected as parents, and F1 isotactic cell families are constructed through one-to-one propagation. Under this condition, the seedling raising field is set in a concrete pool (5.8mX4.8mX1.8m) at 27+ -0.5deg.C, pH 7.2+ -0.6 and dissolved oxygen 7.1+ -0.5 mg/L. After one year of breeding, 109-12 month old sillago sihama is randomly selected for hypoxia stress treatment, and hypoxia tolerance character (min) phenotype data are measured and recorded.
(S2) subjecting Sillago sihama to hypoxia stress treatment
The nitrogen is injected into the water body to reduce the content of dissolved oxygen, so that the dissolved oxygen level is kept at 1.5mg/L (concentration of dissolved oxygen at choking point) to reach a low-oxygen environment, and the time from the start of low-oxygen treatment to the generation of floating head phenomenon of each fish is used as low-oxygen resistant phenotype character data. And according to the recorded time of the floating head of the experimental fish, collecting 109 individuals from the initial floating head to the last floating head of the fish for extracting genome DNA.
(S3) extracting Sillago sihama DNA to be detected
The fin tissues of 109 individuals are cut, and sample genome DNA is extracted according to a CTAB method, and the specific operation steps are as follows:
(1) The fish on a fasting day were totally anesthetized with MS-222 at a concentration of 100 mg/L. For subsequent DNA extraction, shearing fin bars of each individual, and freezing and preserving the fin bar samples in liquid nitrogen;
(2) DNA separation is carried out on a sample by using a CTAB technology, the sample is properly ground under the condition of liquid nitrogen, about 0.1g of liquid is conveyed into a pre-cooled centrifuge tube, and CTAB and beta-mercaptoethanol solution are added;
(3) The mixed sample was placed in a 65 ℃ water bath for about 1 hour, ensuring uniform stirring in the water bath, and then the sample was transferred to a centrifuge for centrifugation at 12,000rpm for 10min;
(4) Adding a mixed solution of phenol, chloroform and isopropanol, uniformly mixing, centrifuging at 12,000rpm for 10min, and shifting the phase of the upper water into a new centrifuge tube;
(5) Adding a mixed solution of chloroform and isopropanol, uniformly mixing, centrifuging at 12,000rpm for 10min, and shifting the phase of the upper water into a new centrifuge tube;
(6) Precipitating and washing the sample with isopropanol and absolute ethyl alcohol, centrifuging, and discarding the supernatant;
(7) Repeating the above steps, centrifuging briefly, drying with ethanol, and adding appropriate ddH 2 O, placing at-20deg.C and preserving;
(8) Finally, the integrity of the DNA sample is detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA sample are detected by a NanoDrop2000 micro-spectrophotometer.
(S4) PCR amplification of the fragment of interest
The SsmGST3b gene of Sillago sihama (shown as SEQ ID NO: 1) is obviously and differentially expressed under hypoxia stress, belongs to glutathione S-transferase (GST) gene superfamily, and plays an important role in the response of fish to environmental stress.
The nucleotide sequence of the SsmGST3b gene of Sillago sihama (shown as SEQ ID NO: 1) is as follows:
primers were designed from the nucleotide sequence of the SsMGST3b gene of Sillago sihama using Primer6, and the primers include forward Primer SsMGST3b-F and reverse Primer SsMGST3b-R, as follows:
the forward primer SsMGST3b-F:5'-ACAGCGACAAGGAGCAAGTATTCAA-3' (shown as SEQ ID NO: 2);
the reverse primer SsmGST3 b-R5'-CAGTGGCAGATGAGAAGGTGGATG-3' (shown as SEQ ID NO: 3).
Using the DNA of the sillago sihama extracted in the step (S3) as a template, usingThe DNA polymerase carries out PCR amplification on the target fragment (769 bp), and the specific primer PCR amplification on the target fragment comprises the following steps: 4, wherein the SNP molecular marker sequence is in brackets.
acagcgacaaggagcaagtattcaactgcatccagagagcacaccagaacaccctggaggtgtaccctcagtggcttgttttccagaccattgcagctcttgtctacccggtatgtgcaatttatcca[t(g.583T>C)]acagtcataattaagtttgtgcgggat[a(g.611A>G)]cctacattcttttacaa[t(g.629T>A)]gtt[t(g.633T>A)]cttggacttattagcgcatatcaagtacaaatctcacctcacacactcaccgtgccgtgtgtgtgggaggggcatgcagcactcgggatcatgatctttgccatacattcacagataacagtcatacacaaaaaccactcactgtgaataagcatattttcaccccagtctgttgcagtgttgcatttcctcttgtgtaattctgtgtttctttgttcaccgtctcagttatcagcatcggtgctgggggctatttgggtgaccagcaggttttcctacgcctggggctattacacaggaggt[a(g.937A>G)]agactgacaatacattgacttaaaataataataatataaaatagaagcacttcatgggggccaaaagcacctaaactgagttcacctacacttctactagatttccacacatctgtattgaaaatttgatacatgtaaaacatagaacacctagggttgaatgtaattcaataaagtaaatgtgtgaaatggtttggggatatagtcatttgtgctggaggctgcagtgctttataatgcgcattgacttaaggtcatagttcatccaccttctcatctgc cactg(shown in SEQ ID NO: 4).
Wherein underlines indicate positions of the forward primer and the reverse primer (forward sequence, base sequence of SsmGST3b-R after reverse complementation), and brackets indicate SNP sites detected.
Wherein:
the PCR reaction system was 40. Mu.L:PCRSuperMix 20. Mu.L, 10mM forward and reverse primers each 2. Mu.L, 40 ng/. Mu.L of LDNA template 2. Mu.L, ddH 2 O14μL。
The PCR reaction procedure was performed for a total of 35 cycles, with pre-denaturation at 94℃for 5min before the cycle, each cycle consisting of denaturation at 94℃for 30sec, annealing at 60℃for 30sec, extension at 72℃for 30sec, and extension at 72℃for 10min after the cycle was completed.
And (3) carrying out electrophoresis detection on the amplified product by using 1% agarose gel, and carrying out sequencing reaction on the product after the detection is qualified.
(S5) sequencing the PCR amplified product to determine the genotype of the SNP site
Based on a Hiseq2000 high-throughput sequencing platform, the PCR amplification products of 109 individuals of the sillago sihama are subjected to bidirectional sequencing on an ABI3730XL sequencer, and based on the sequencing result, the SNP loci of the sillago sihama are subjected to genotyping.
FIG. 1 is a schematic diagram of a peak pattern of a PCR amplification product detected by capillary electrophoresis using SsMGST3B-F primer, and the forward primer SsMGST3B-F is used for sequencing, wherein the position of g.583T > C site is shown in FIG. 1A, the position of g.611A > G site is shown in FIG. 1B, the positions of g.629T > A and g.633T > A site are shown in FIG. 1C, and the position of g.937A > G site is shown in FIG. 1D.
Therefore, the embodiment obtains the SNP molecular marker related to the low-oxygen-resistant property of the sillago sihama, which comprises one or more of 5 SNP molecular markers, wherein the 5 SNP molecular markers are as follows: g.583T > C; g.611A > G; 629t > a; g.633T > A; g.937A > G.
(S6) correlation analysis of SNP locus genotype and low oxygen resistance property of the sillago sihama
The correlation analysis of genotypes and hypoxia tolerance quantitative traits of each SNP locus is detected by adopting single factor analysis of variance in a general linear model of SPSS26, and multiple comparison analysis is carried out on the SNP locus with obvious expression by adopting a Ducan method, wherein the analysis results are as follows:
TABLE 1 correlation of SNP loci of Sillago sihama SsMGST3b genes with hypoxia tolerance
The results show that: the 5 SNP loci (g.583T > C; g.611A > G; g.629T > A; g.633T > A; g.937A > G) on SsmGST3b gene are dominant genotypes CT, AG, AT, AT, AA, and the genotypes of the loci and the hypoxia tolerance traits are obviously different (P < 0.05), thus the method has statistical significance. The 5 SNP loci are obviously related to hypoxia tolerance in the process of cultivating the hypoxia tolerance character of the sillago sihama population.
Thus, the individual of the sillago sihama with the genotype CT, AG, AT, AT, AA has a significant difference between the average survival time in the water body of the dissolved oxygen below the choking point and the individual of the sillago sihama with the genotype CC or TT, GG or AA, TT or AA, GG or AG.
Therefore, the SNP molecular marker can be applied to screening of the low-oxygen-resistant sillago sihama colony, and individuals with the genotype CT, AG, AT, AT, AA of 5 SNP loci (g.583T > C; g.611A > G; g.629T > A; g.633T > A; g.937A > G) on the SsMGST3b gene can be used as breeding objects to cultivate the low-oxygen-resistant sillago sihama colony.
The invention can further develop a kit for detecting the SNP molecular marker, comprising the primer.
The SNP molecular marker is applied to the breeding process of the low-oxygen-resistant property of the sillago sihama, and particularly, in the breeding process of the sillago sihama, the genotype detection of the SNP locus is carried out on candidate populations of the sillago sihama, and then the genotypes of other loci related to the growth property, the disease resistance and the stress resistance are combined, so that the individual with the SNP genotype CT, AG, AT, AT, AA is preferentially selected as a parent of the multi-scale for breeding the low-oxygen-resistant property.
Therefore, the SNP molecular marker, the primer pair or the kit can be applied to the identification of the low-oxygen property of the sillago sihama and the selective breeding.
In addition, the application of the SNP molecular marker, the primer and the kit in evaluating the hypoxia tolerance of the Sillago sihama belongs to the protection scope of the invention.
The above embodiments are merely illustrative of the present invention, and the protective scope of the present invention is not limited to the above embodiments only. The object of the present invention can be achieved by a person skilled in the art based on the above disclosure, and any modifications and variations based on the concept of the present invention fall within the scope of the present invention, which is defined in the claims.
Claims (8)
1. A Sillago sihama hypoxia tolerance character related SNP molecular marker is characterized in that: the SNP molecular markers are one or more of the following 5 SNP molecular markers, and the 5 SNP molecular markers are respectively positioned in the sequences shown in SEQ ID NO:1 from 5 ， The ends are at positions 583, 611, 629, 633 and 937, wherein the base at position 583 is T or C, the base at position 611 is A or G, the base at position 629 is T or A, the base at position 633 is T or A, and the base at position 937 is A or G.
2. The molecular marker of the SNP related to the hypoxia tolerance trait of sillago sihama according to claim 1, wherein the molecular marker is characterized in that: the hypoxia tolerance of CT genotype individuals marked by SNP molecules AT 583 is obviously higher than that of TT genotype individuals, the hypoxia tolerance of AG genotype individuals marked by SNP molecules AT 611 is obviously higher than that of AA genotype individuals, the hypoxia tolerance of AT genotype individuals marked by SNP molecules AT 629 is obviously higher than that of TT genotype individuals, the hypoxia tolerance of AT genotype individuals marked by SNP molecules AT 633 is obviously higher than that of TT genotype individuals, and the hypoxia tolerance of AA genotype individuals marked by SNP molecules AT 937 is obviously higher than that of AG genotype individuals.
3. A primer for amplifying the SNP molecular marker of claim 1, characterized in that: the base sequences of the forward primer SsMGST3b-F and the reverse primer SsMGST3b-R are shown in SEQ ID NO:2, the base sequence of the reverse primer SsMGST3b-R is shown as SEQ ID NO: 3.
4. A kit for detecting SNP molecular markers related to low oxygen resistance properties of Sillago sihama is characterized in that: the kit comprises the primer of claim 3.
5. The method for detecting the low-oxygen-resistant property of Sillago sihama is characterized by comprising the following steps of:
(S1) extracting genomic DNA of tail fins of the sillago album to be detected;
(S2) performing PCR amplification of genomic DNA of the multi-scale tail fin to be detected using the primer set forth in claim 3, to obtain a PCR amplification product;
(S3) sequencing the PCR amplification product, and determining the genotype of the SNP molecular marker based on the sequencing result;
(S4) determining the hypoxia tolerance of the to-be-detected sillago sihama through genotypes.
6. The method for detecting the hypoxia tolerance of sillago sihama as claimed in claim 5, wherein the method comprises the following steps: the PCR reaction system used in the PCR amplification in the step (S2) was 40. Mu.L, and it comprises:PCR Supermix 20. Mu.L, 10mM forward and reverse primers each 2. Mu.L, 40 ng/. Mu.L DNA template 2. Mu.L, ddH 2 O 14μL。
7. The method for detecting the hypoxia tolerance of sillago sihama as claimed in claim 5, wherein the method comprises the following steps: in the PCR amplification in the step (S2), the PCR reaction program was used for a total of 35 cycles, the pre-denaturation at 94℃for 5min before the cycle, each cycle comprising denaturation at 94℃for 30sec, annealing at 60℃for 30sec, extension at 72℃for 30sec, and extension at 72℃for 10min after the cycle was completed.
8. Use of the SNP molecular marker of claim 1 or 2, the primer of claim 3, the kit of claim 4 or the detection method of any one of claims 5-7 for identifying or breeding low-oxygen-tolerance trait sillago sihama varieties.
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| CN114752683A (en) * | 2022-04-18 | 2022-07-15 | 广东海洋大学 | Method for constructing QTL (quantitative trait locus) related to sex characters of sillago sihama |
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| CN105146221A (en) * | 2015-10-21 | 2015-12-16 | 通威股份有限公司 | Compound feed for mixed culture of penaeus vannamei boone and silver sillago and preparation method of compound feed |
| CN114752683A (en) * | 2022-04-18 | 2022-07-15 | 广东海洋大学 | Method for constructing QTL (quantitative trait locus) related to sex characters of sillago sihama |
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