CN104328116A - SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof - Google Patents
SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof Download PDFInfo
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- CN104328116A CN104328116A CN201410623763.1A CN201410623763A CN104328116A CN 104328116 A CN104328116 A CN 104328116A CN 201410623763 A CN201410623763 A CN 201410623763A CN 104328116 A CN104328116 A CN 104328116A
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Abstract
The invention relates to an SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus. The SNP locus is 47th/48th site (the base is AA or A/DEL), 65th site (the base is T or C), 185th site (the base is C or T), 260th site (the base is C or T), 305th site (the base is T or A), 600th site (the base is T or C) or 815th site (the base is C or T) of HSP70 gene with the nucleic acid sequence of SEQ ID NO:1. The SNP locus is used for screening the heat-resistant individual/family of Paralichthys olivaceus. The heat-resistant family is screened by a molecular marker process by using the heat shock protein gene HSP70; the hereditary characters of all the families can be evaluated on the molecular level, and the mutant site of the genome can be clearly identified; and the relevance between the mutant site and heat resistance is analyzed by combination of the heating experiment to perform reasonable judgment on the heat-resistant molecular mechanism. Compared with the traditional breeding method, the method provided by the invention has the advantages of higher purposiveness and lower cost, and can obviously shorten the breeding time.
Description
Technical field
The invention belongs to the triage techniques field of the heat-resisting family of fish in aquaculture, be specifically related to a kind of SNP site relevant to lefteye flounder thermotolerance and application thereof.
Background technology
Lefteye flounder (Paralichthysolivaceus) is commonly called as tooth sheet, step, is cold warm nature ground fish, is the important marine fish of China.The optimum temperuture of lefteye flounder larvae cultivation is 17-20 DEG C, and the optimal temperature of adult fish growth is 14-23 DEG C, and optimum temperuture is 21 DEG C.Lefteye flounder is being low to moderate 1 DEG C, can of short durationly survive up under 33 DEG C of water temperature conditions.Compare with prelarva, the heat-resisting ability of adult fish can significantly decline, and more than 23 DEG C, food ration can reduce, and more than 25 DEG C stop growing, and being in 27 DEG C for a long time can cause mortality.Growth rate and the metabolic rate of Temperature on Fish have great effect, and in recent years, along with Global warming, seawater temperature progressively rises, and have a strong impact on the speed of growth and the surviving rate of lefteye flounder, reduce culture benefit, add aquaculture cost.Meanwhile, with the rising of seawater temperature, the cultivation scope of lefteye flounder also can reduce.Therefore, the active demand that the lefteye flounder strain with better thermotolerance is lefteye flounder breeding for stress tolerance is filtered out.There is the shortcoming of seed selection overlong time, seed selection high cost in traditional selection.Therefore, the screening method setting up the heat-resisting family of a kind of lefteye flounder is fast and effectively needed.
In research in fish are heat-resisting, mostly concentrate on genetic parameter assessment aspect, such as Perry etc. (2005) are to the research of rainbow trout high temperature resistance.The development of adjoint molecular biology and sequencing technologies, single nucleotide polymorphism (SNP) gets the nod in breeding field as molecule marker of new generation, in fish, researchist has screened a series of SNPs relevant with proterties, such as growth, disease-resistant relevant SNP marker.In research in flounder sole is heat-resisting, mainly concentrate on the screening of heat-resisting relevant micro-satellite aspect, seldom have the report that heat-resisting related SNP is screened.
Summary of the invention
The object of this invention is to provide a kind of SNP site relevant to lefteye flounder thermotolerance and the application in the heat-resisting Genealogy screening of lefteye flounder thereof, thus make up the deficiencies in the prior art.
First the present invention provides a kind of SNP site relevant to lefteye flounder thermotolerance, the 47/48th of described SNP site to be nucleotide sequence the be HSP70 gene of SEQ ID NO:1, and its base is AA or A/DEL; 65th, its base is T or C; 185th, its base is C or T; 260th, its base is C or T; 305th, its base is T or A; 600th, its base is T or C; 815th, its base is C or T.
Above-mentioned SNP site is used for the screening of lefteye flounder thermotolerance individuality/family;
The present invention also provides a kind of method of screening in lefteye flounder thermotolerance individuality/family, is to survey with pcr amplification primer quality testing the SNP site that the present invention screens;
For detecting the primer of above-mentioned SNP site, its upstream and downstream sequence is respectively;
HSP70fw1：5′-CGGGGATGAGTTTCAGACGA-3′；(SEQ ID NO:2)
HSP70rv1：5′-AGGAAAAGTGAACAAAGCAG-3′(SEQ ID NO:3)。
Present method selects heat shock protein gene HSP70, heat-resisting family is screened by the method for molecule marker, assess the hereditary property of each family on a molecular scale, its genomic mutational site can be identified clearly, and in conjunction with Heating Experiment, analyze mutational site and heat-resisting dependency, rational judgement is made to heat-resisting molecular mechanism, stronger relative to traditional selection purpose, cost is lower, can obviously shorten the seed selection time.
Embodiment
The present invention's homologous clone and RACE technology obtain lefteye flounder HSP70 gene, and this gene DNA total length 2646bp (SEQ ID NO:1), have an intron in 5 ' UTR district, length is 454bp.This gene cDNA total length is 2192bp, and wherein ORF length is 1923bp, 641 amino acid of encoding.Comprising 3 distinctive regions of HSP70 protein family (amino-acid sequence No.11-18IDLGTTYS, No.199-212IFDLGGGTFDVSIL and No.336-350IVLVGGSTRIPKIQK).
To be tested by chronic lethal and to the heavy sequencing technologies of lefteye flounder HSP70 gene, in conjunction with genotype frequency and the Holstein Cattle correlation analysis in each site, filter out 7 to heat-resisting relevant SNP site.This site can be used for screening and the marker assisted selection of the heat-resisting family of lefteye flounder.
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
Lefteye flounder HSP70 gene cDNA in the present invention and DNA sequence dna clone comprise the following steps:
A) extraction of lefteye flounder heart total serum IgE and the extraction of muscle DNA;
B) synthesis of cDNA first chain;
C) acquisition of goal gene full length cDNA sequence and DNA sequence dna.
Concrete operations are as follows:
A) extraction of lefteye flounder total serum IgE.From lefteye flounder heart, total serum IgE is extracted according to Trizol (Invitrogen, CA, USA) method; Extraction phenol/the chloroform of muscle DNA.
B) synthesis of cDNA first chain.With 2 μ g total serum IgE for template, add 1 μ l, 20 μMs of Oligo d (T) respectively, DEPC water complements to 13 μ l, mixing wink from.70 DEG C of insulation 10min, are placed in immediately on ice, add successively: 5 μ l 5 × M-MLV Buffer, 5 μ ldNTP (2.5mM), 1 μ lRNasin (40U/ μ l), 1 μ l M-MLV (200U/ μ l); Mix centrifugal after, 42 DEG C, 90min.Save backup in-20 DEG C after packing.
C) acquisition of goal gene full length cDNA sequence.Design primer according to the fragment (AB010871) in GenBank, utilize Clontech company SMART RACE cDNA Amplification Kit to carry out 5 ' RACE and 3 ' RACE and increase.Design 5 ' RACE primer: 5 '-GTTCTTGGCAGCATCTCCAATGAGCCTCTC-3 '.3 ' RACE primer: 5 '-CGAGTATGAGCACCAGCAGAAGGAAC-3 ', utilizes 3 ' RACE primer and 5 ' RACE primer and adapter-primer NUP (5' – AAGCAGTGGTATCAACGCAGAGT – 3') to carry out the amplification of 3 ' end and 5 ' end respectively.PCR primer detects through 1.5% agarose gel electrophoresis, reclaims test kit and carries out PCR primer recovery and purifying, connect with pMD-18T carrier, by the product conversion of connection to intestinal bacteria Trans5 α with glue.After bacterium colony PCR detects, each picking 3 positive colonies send Hua Da gene sequencing.CDNA total length is obtained after splicing.According to the cDNA sequence design primer HSP70fw15 '-CGGGGATGAGTTTCAGACGA-3 ' obtained; HSP70rv15 '-AGGAAAAGTGAACAAAGCAG-3 ' is that template carries out pcr amplification with DNA, and PCR primer, through reclaiming and purifying, connection, conversion, bacterium colony PCR etc., is selected 3 positive colonies and sent Hua Da gene sequencing, obtain this gene DNA total length.
3 ' and 5 ' RLM-RACE, utilize 50 μ L reaction systems:
Increase PCR response procedures used: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30sec, 72 DEG C of 3min, 5 circulations; 94 DEG C of sex change 30sec, 70 DEG C of annealing 30sec, 72 DEG C extend 3min, 5 circulations; 94 DEG C of sex change 30sec, 65 DEG C of annealing 30sec, 72 DEG C extend 3min, 28 circulations; 72 DEG C extend 10min.Check order to after the sequence assembly of cDNA, the sequencing result of multiple clone is consistent.This gene cDNA total length 2192bp, wherein ORF length is 1923bp, 641 amino acid of encoding.Comprising 3 distinctive regions of HSP70 protein family (amino-acid sequence No.11-18IDLGTTYS, No.199-212IFDLGGGTFDVSIL and No.336-350IVLVGGSTRIPKIQK).This gene DNA total length 2646bp, has an intron in 5 ' UTR district, and length is 454bp.
With the screening of heat-resisting associated SNP positions in embodiment 2 lefteye flounder HSP70 gene
Comprise the following steps with the screening of Holstein Cattle associated SNP positions and correlation analysis thereof in lefteye flounder HSP70 gene:
A) structure of lefteye flounder family
B) lefteye flounder temperature tolerance experiment
C) acquisition of relatively heat-resisting and thermo-labile trait population
D) screening of SNP site and correlation analysis
Concrete operation step is as follows:
A) structure of lefteye flounder family
The first-generation colony of the tool growth good character selected in family of learning from else's experience, as parent, selects not have akin individuality to carry out random combine.Be that 2:1 sets up family half sibs with female-male proportion; Be that 1:1 sets up family full-sibs with female-male proportion.Each family cultivates when culture condition is consistent.
B) lefteye flounder temperature tolerance experiment
After family cultivates 4 months, 17 familys that growth conditions is good, date of conception is close are chosen from the family full-sibs built and family half sibs, each family random selecting 35 tail individuality is tested, and raises together with the lefteye flounder fluorochrome label of each family chosen.
After raising together with 48h, carry out the experiment of temperature chronic lethal.Water temperature during starting experimental is 22 DEG C, and when lethal experiment, temperature rise rate is 1 DEG C/day, is warmed up to 31.3 ± 0.5 DEG C (determining that now the lefteye flounder colony death time continues longer according to preliminary experiment).From intensification, a few days ago average every 6h observes once, and remove dead individuality in time, the qualification of lefteye flounder death indicates using gill cover stop motion as death.After temperature is elevated to assigned temperature, every 2h observes once, removes dead individuals, and records family numbering, the tolerance time (calculating from being heated to top temperature) of dead individuals, and sample retention is to-80 DEG C.C) acquisition of relatively heat-resisting and thermo-labile shape colony
The experiment of lefteye flounder temperature tolerance continue for 15 days in fatal temperature, after the 11st day, have no dead individuals.Statistical study is carried out to dead individuals, assesses the difference of the thermotolerance between each family.The results of analysis of variance is in table 1.Analytical results shows, and between family, poor heat resistance heteropole significantly (P<0.01).According to the death time of each family individuality, multiple comparisons is carried out to each family and each family individual death time, the results are shown in Table 2, obtain relatively heat-resisting family (1,5,9,2,11,19,36) and relative thermo-labile family (13,14,27,38,40,42).Choose 30 relatively heat-resisting (wherein 2,11,19, No. 36 each familys of family choose 6 individualities that heat-resistant time is the longest, and 1,5, No. 9 each family of family chooses 2 individualities that heat-resistant time is the longest) heat labile lefteye flounder individualities relative to 30 (13,14,27,38,40,42 each familys choose 5 individualities dead at first).
Table 1: the difference table of the thermotolerance between each family
Table 2: the multiple comparisons table of each family and each family individual death time
D) screening of SNP site and correlation analysis
DNA extraction is carried out to the relatively heat-resisting and thermo-labile colony filtered out, primer HSP70fw1 and HSP70rv1 is utilized to carry out pcr amplification, reaction system is as follows: dNTP (2.5mM) 2 μ L, Buffer 2.5 μ L, template 10ng, the each 0.5 μ L of forward and reverse primer (10mM), all the other use ddH
2o mends 25 μ L.Reaction conditions is as follows: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, and 55-60 DEG C of annealing 1min, 72 DEG C extend 30s, and 35 circulations are carried out in reaction.HSP70 amplification and sequence of resurveying are carried out to each individuality, filters out 24 SNP site (being numbered 1-24) altogether.In these 24 sites, there are 23 same sense mutations, 1 nonsynonymous mutation (Ser/Thr).Chi-square test analyzes display, has 7 sites to show significant difference (P<0.01) between relatively heat-resisting and thermo-labile colony.Site 1 is the 47/48th, and its base is AA or A/DEL; Site 2 is the 65th, and its base is T or C; Site 3 is the 185th, and its base is C or T; Site 4 is the 260th, and its base is C or T; Site 5 is the 305th, and its base is T or A; Site 6 is the 600th, and its base is T or C; Site 7 is the 815th, and its base is C or T.Linkage analysis and haplotype analysis are carried out to 7 significant SNP site relevant to Holstein Cattle, finds that 13 and No. 15 SNP have significant linkage relationship.Haplotype analysis shows, nonrefractory TAGGAG and heat-resisting (DELT) GAATA significant correlation (P<0.005).
Use present method can analyze the frequency of the HSP70 haplotype of different family, assess its thermotolerance, avoid heat-resistant experiment to cause a large amount of individual death.When carrying out heat-resisting Genealogy screening, the fin ray can cutting family individuality carries out DNA extraction and pcr amplification, determines its haplotype on a molecular scale, avoids blindly screening.
Embodiment 3, the SNPs application in the heat-resisting individuality/family of screening
1, the screening of heat resistant type parent population
Fin ray is cut to parent individual to be screened, carries out DNA extraction, by primer HSP70fw1 and HSP70rv1 amplification parent HSP70 gene order, and check order, detect the SNP site genotype of HSP70 gene, pick out and meet the parent population that genotype is heat resistant type.
2, the structure of family
The parent population utilizing genotype to be heat resistant type builds full sibs and family half sibs, builds family as a control group with not screening parent population.
3, thermotolerance detects
After family cultivates 4 months, SNP detection and haplotype analysis are carried out to heat resistant type parent population and the F1 generation of not screening parent population, and temperature tolerance experiment is carried out to F1 generation individuality.Result shows, is significantly higher than the F1 generation individuality (P<0.05) not screening parent population in the F1 generation individuality of heat resistant type parent population to the genotype frequency of heat-resisting relevant SNP.Temperature tolerance experiment shows, the offspring of heat resistant type parent population is also significantly higher than the offspring (P<0.05) not screening parent population to the tolerance of temperature.Result shows, by the parent of the primer screening of SNP of the present invention, its offspring's thermotolerance of breeding is significantly higher than the control group do not screened.
Claims (4)
1. a SNP site relevant to lefteye flounder thermotolerance, is characterized in that, the 47/48th of described SNP site to be sequence the be HSP70 gene of SEQ ID NO:1, and its base is AA or A/DEL; 65th, its base is T or C; 185th, its base is C or T; 260th, its base is C or T; 305th, its base is T or A; 600th, its base is T or C; 815th, its base is C or T.
2. the application of SNP site according to claim 1 in the screening of lefteye flounder thermotolerance individuality/family.
3. screen the method in lefteye flounder thermotolerance individuality/family, it is characterized in that, described method surveys SNP site according to claim 1 with pcr amplification primer quality testing.
4. method as claimed in claim 3, it is characterized in that, the sequence of described primer is SEQ ID NO:2 and SEQ ID NO:3.
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| CN105950727A (en) * | 2016-05-19 | 2016-09-21 | 烟台大学 | Paralichthys olivaceus genotype detection primer and method based on microsatellite marker Paraoliva-1 |
| WO2020104503A1 (en) | 2018-11-22 | 2020-05-28 | Universiteit Gent | A single nucleotide polymorphism in the heat shock protein 70 constitutive gene for selecting heat-tolerant organisms |
| WO2021119980A1 (en) * | 2019-12-17 | 2021-06-24 | 中国水产科学研究院黄海水产研究所 | Gene chip for disease resistance breeding of olive flounder and application thereof |
| KR102384513B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105713974A (en) * | 2016-03-24 | 2016-06-29 | 中国水产科学研究院北戴河中心实验站 | SNP locus related to paralichthys olivaceus quantitative character and screening method and application thereof |
| CN105713974B (en) * | 2016-03-24 | 2019-04-19 | 中国水产科学研究院北戴河中心实验站 | One kind SNP marker relevant to lefteye flounder quantitative character, its screening technique and application |
| CN105950727A (en) * | 2016-05-19 | 2016-09-21 | 烟台大学 | Paralichthys olivaceus genotype detection primer and method based on microsatellite marker Paraoliva-1 |
| WO2020104503A1 (en) | 2018-11-22 | 2020-05-28 | Universiteit Gent | A single nucleotide polymorphism in the heat shock protein 70 constitutive gene for selecting heat-tolerant organisms |
| WO2021119980A1 (en) * | 2019-12-17 | 2021-06-24 | 中国水产科学研究院黄海水产研究所 | Gene chip for disease resistance breeding of olive flounder and application thereof |
| KR102384513B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
| KR102384515B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
| KR102384516B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
| KR102384512B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
| KR102384514B1 (en) * | 2021-09-01 | 2022-04-12 | 제주대학교 산학협력단 | SNP markers for predicting thermal tolerance of Paralichthys olivaceus and use thereof |
| WO2023033457A1 (en) * | 2021-09-01 | 2023-03-09 | 제주대학교 산학협력단 | Snp marker for predicting high water temperature tolerance of flatfish, and use thereof |
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