WO2023033457A1 - 넙치의 고수온 내성 예측용 snp 마커 및 이의 용도 - Google Patents

넙치의 고수온 내성 예측용 snp 마커 및 이의 용도 Download PDF

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WO2023033457A1
WO2023033457A1 PCT/KR2022/012747 KR2022012747W WO2023033457A1 WO 2023033457 A1 WO2023033457 A1 WO 2023033457A1 KR 2022012747 W KR2022012747 W KR 2022012747W WO 2023033457 A1 WO2023033457 A1 WO 2023033457A1
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base
polynucleotide consisting
seq
polynucleotide
nucleotide sequence
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French (fr)
Korean (ko)
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이제희
이숙경
정태혁
스리팔 리야나게딜리파
무디얀세라게비라즈 우다얀타 헤라스
이지훈
김가은
김문관
고형범
원승환
오성립
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제주대학교 산학협력단
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Publication of WO2023033457A1 publication Critical patent/WO2023033457A1/ko

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

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  • the present disclosure relates to a SNP marker composition for predicting High Water Temperature Tolerance (HWTT) of flounder and a method for predicting high temperature tolerance of flounder using the same.
  • HWTT High Water Temperature Tolerance
  • seawater temperature is an important variable in aquaculture production in onland farms or cage farms using natural seawater. It is known that it can cause various problems. Therefore, a new breed that can withstand the high temperature period is needed, and in order to develop the breed, genetic selective breeding based on a method for selecting high temperature resistant halibut is required.
  • An object of the present disclosure is to provide a SNP marker composition for predicting high temperature resistance of flounder, a composition comprising an agent capable of detecting or amplifying it, a kit for predicting high temperature resistance of flounder including the composition, and a method for predicting high temperature resistance of flounder will be.
  • the present disclosure provides that the 36th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is G or A, and the polynucleotide consisting of 10 to 100 consecutive bases including the 36th base.
  • a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 wherein the 36
  • the present disclosure is a composition for predicting high temperature resistance of flounder, including an agent capable of detecting or amplifying a SNP marker for predicting high temperature resistance of flounder, wherein the SNP marker is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1
  • the 36th base of is G or A, and a polynucleotide consisting of 10 to 100 consecutive bases including the 36th base or a polynucleotide complementary thereto;
  • the agent may be a primer or probe capable of detecting or amplifying the SNP marker.
  • the present disclosure provides a kit for predicting high-temperature resistance of flatfish, including the composition for predicting high-temperature resistance of flounder.
  • the present disclosure provides (a) amplifying or detecting a polymorphic site of a SNP marker for predicting high temperature resistance of flounder from DNA of a sample isolated from flounder; and (b) determining the base of the polymorphic site amplified or detected in step (a), wherein the SNP marker in step (a) is 36 nucleotides of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the present disclosure is a kit for predicting high temperature tolerance of halibut; And in a method for predicting high-temperature tolerance of flatfish, it provides the use of the above-described SNP marker composition for predicting high-temperature tolerance of flatfish.
  • the SNP marker composition of the present disclosure can be used for genetic selection of halibut with high temperature tolerance.
  • Figure 1 is a graph showing the cumulative mortality according to the water temperature change in the high temperature tolerance experiment.
  • Figure 2 shows the GWAS analysis by type (survival status, survival days, survival time) for high temperature tolerance.
  • 3 to 32 show the phenotypic distribution of genotypes of 30 SNPs for prediction of high temperature tolerance.
  • the present disclosure is a single nucleotide polymorphism that can predict high-temperature tolerance through genotypic analysis and genome-wide association study (GWAS) using a 70K SNP chip for halibut for individuals selected from high-temperature exposure experiments ( SNP) was confirmed.
  • GWAS genotypic analysis and genome-wide association study
  • the present disclosure relates to a SNP marker composition for predicting high temperature tolerance of halibut.
  • the present disclosure discloses that the 36th base of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is G or A, and a polynucleotide consisting of 10 to 100 consecutive bases including the 36th base or a polynucleotide complementary thereto polynucleotide;
  • a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 wherein the 36th base is A or G, and a polynucleotide consisting of 10 to 100 consecutive bases including the 36th base, or a polynucleotide complementary thereto
  • high water temperature resistance refers to the property of enduring an increase in water temperature, and specifically, when a high water temperature of 29.0 ° C to a maximum of 33.7 ° C is maintained, resistance to growth slowdown or disease occurrence due to water temperature shock, weakening of immunity, decline in physiological function, etc. means to have
  • SNP marker in the present disclosure refers to a single base polymorphic allele base pair on a DNA sequence used to identify an individual or species. Since SNPs have a relatively high frequency, are stable, and are distributed throughout the genome, resulting in genetic diversity of individuals, SNP markers can serve as indicators of genetic proximity between individuals. SNP markers usually include phenotypic changes accompanying single nucleotide polymorphisms, but may not in some cases. In the case of the SNP marker of the present disclosure, it may represent a difference in phenotype of an individual such as amino acid sequence variation or high temperature tolerance of halibut.
  • the term "individual” of the present disclosure means a halibut (flatfish), which is a target for confirming high-temperature resistance, and by analyzing the genotype of the SNP marker using a sample obtained from the flounder (flatfish), the high-temperature resistant flatfish (flatfish) can be identified.
  • a 70K SNP chip prepared after searching for SNPs in the mother genome by whole genome re-sequencing of the flounder mother generation was used.
  • the search was conducted by narrowing the scope to SNPs associated with high temperature tolerance.
  • effective SNPs highly related to high temperature tolerance were found on chromosomes 18 and 19.
  • the phenotypic distribution of the genotypes of the SNPs was analyzed to determine 30 SNP information, and the reliability and accuracy of the SNP markers of the present disclosure were confirmed to confirm that high-temperature tolerance of flounder could be predicted.
  • a polynucleotide consisting of 10 to 100 contiguous bases comprising the nucleotide sequence of SEQ ID NO: 1 of the present disclosure or a polynucleotide complementary thereto to 10 to 100 contiguous bases comprising the nucleotide sequence of SEQ ID NO: 30 It suggests that halibut containing at least one selected from the group consisting of a polynucleotide consisting of or a polynucleotide complementary thereto may exhibit high-temperature resistance traits, and such a base sequence was identified for the first time by the present inventors.
  • the present disclosure provides a composition for predicting high-temperature tolerance of flounder, including an agent capable of detecting or amplifying a SNP marker for predicting high-temperature resistance of flounder.
  • agent capable of detecting or amplifying a SNP marker refers to a composition capable of predicting high-temperature resistant flounder by confirming the polymorphic region of the gene as described above through detection or amplification, preferably the SNP A primer set or probe capable of specifically detecting or amplifying a polynucleotide of a marker.
  • the primers used to amplify the SNP markers are prepared in the form of template-directed DNA under suitable conditions (eg, four different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. It can be a single-stranded oligopolynucleotide that can serve as a starting point for synthesis.
  • the appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 polynucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template.
  • the primer sequence need not be perfectly complementary to the SNP marker, but must be sufficiently complementary to hybridize with the SNP marker.
  • primer in the present disclosure is a base sequence having a short free 3' terminal hydroxyl group, capable of forming a base pair with a complementary template, and starting for copying the template strand. It refers to a short sequence that functions as a point, and is mainly used in the form of a primer set that amplifies a specific section.
  • a primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. At this time, PCR conditions and lengths of sense and antisense primers may be modified based on those known in the art.
  • the term "probe” is DNA or RNA nucleotide sequences of various lengths labeled with radioactive or fluorescent labels, and is composed of single-stranded nucleotide sequences and is specific for single-stranded DNA or RNA having complementary sequences. It is characterized in that it binds and hybridizes with the target, and the target can be detected through a method of detecting the label signal of the hybridized probe.
  • the primers or probes of this disclosure can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods.
  • Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).
  • the present disclosure provides a kit for predicting high temperature resistance of flounder, including the composition for predicting high temperature resistance of flounder.
  • the kit of the present disclosure may be a DNA chip kit, but is not limited thereto.
  • the DNA chip kit uses a chip attached with a polynucleotide sequence containing a SNP marker, which is a marker for predicting high temperature resistance of flounder, or an oligopolynucleotide containing a complementary nucleotide sequence thereof, to specifically hybridize and bind to target DNA Characterized in that, it is possible to predict high-temperature tolerant halibut using the change in hybridization level according to the base variation of the SNP.
  • a SNP marker which is a marker for predicting high temperature resistance of flounder
  • an oligopolynucleotide containing a complementary nucleotide sequence thereof to specifically hybridize and bind to target DNA Characterized in that, it is possible to predict high-temperature tolerant halibut using the change in hybridization level according to the base variation of the SNP.
  • the DNA chip kit for predicting high temperature resistance of flounder is selected from the group consisting of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 30 At least one polynucleotide or a polynucleotide consisting of 10 to 100 bases including its 36th base and an oligopolynucleotide complementary to a marker are attached to a chip, and target DNA is reacted with the chip to determine the genotype through hybridization. It may be a kit for predicting high temperature tolerance of halibut, which is a kit for determining.
  • step (a) amplifying or detecting a polymorphic site of the SNP marker for predicting high temperature resistance of claim 1 from the DNA of a flounder sample; and (b) determining the base of the polymorphic site amplified or detected in step (a).
  • any method known to those skilled in the art may be used for amplifying the polynucleotide from the DNA sample in step (a). For example, it can be obtained by amplifying a target nucleic acid through PCR and purifying it. Other techniques such as ligase chain reaction (LCR), transcription amplification, and self-maintained sequence replication and nucleic acid-based sequence amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • NASBA self-maintained sequence replication and nucleic acid-based sequence amplification
  • Step (a) may be amplifying or detecting a base using a primer or a probe.
  • the method for determining the base of the amplified SNP marker region in step (b) includes sequencing analysis, microarray hybridization, allele specific PCR, and dynamic allele hybridization technique.
  • allele-specific hybridization DASH
  • PCR extension analysis PCR-SSCP
  • PCR-RFLP analysis HRM analysis or TaqMan techniques
  • SNPlex platform Applied Biosystems
  • mass spectrometry eg Sequenom's MassARRAY system
  • mini-sequencing mini-sequencing
  • Bio-Plex systems BioRad
  • CEQ and SNPstream systems Beckman
  • Molecular Inversion Probe array technology eg, Affymetrix GeneChip
  • BeadArray Technologies eg, Illumina GoldenGate and Infinium assays).
  • One or more alleles in the SNP marker can be identified by the above methods or other methods available to those skilled in the art.
  • Example 1 High-density SNP 70K chip for halibut
  • MS loci loci Individuals selected from 1,291 flounder populations prepared based on the allele and correlation analysis results of 11 microsatellite (MS) loci loci were selected based on QC results for genomic DNA, group, mating status, Considering gender, 100 fish were finally selected from the flounder population for whole genome re-sequencing analysis.
  • MS microsatellite
  • the Axiom TM my Design TM SNP chip (50 to 90K) platform was selected.
  • the secured high-quality SNPs are classified into tiers according to their importance, and 154,964 SNPs are classified according to 1) genotype rate for 100 animals, 2) LD block, 3) repeat, and 4) MAF range.
  • the population structure of 100 halibut was analyzed based on the selected 155K SNPs, a pattern similar to the results of the analysis with the 11 MS markers used previously was found, indicating that the collected flounder population was different according to the collection area (group). It is assumed that the specificity is strong and genetic fixation within the population is judged to be high.
  • 71,364 SNPs were finally determined considering the SNP chip (50 to 90K) platform. have been selected The selected SNPs were evenly distributed across 24 chromosomes, and 70% of the distances between SNPs were within 5,000 bp, and most of them were identified as less than 10,000 bp.
  • a 70K SNP chip for halibut in the form of a total of 384 wells/plate was manufactured so that 71,364 SNPs were planted in one chip.
  • the flounder used as the experimental fish was a flounder group produced according to the breeding guidelines of Jeju National University from a flounder mother group collected at the Jeju Maritime and Fisheries Research Institute. Specifically, the nF0 generation was mass-produced from 354 sexually mature mother candidates (126 females, 288 males) through natural spawning and natural fertilization, and reared to a size of 20 to 30 cm. 400 animals per tank were stocked in 2 m-sized tanks and allowed to acclimate for 3 days at 19.4°C water temperature. At this time, the water tank used a circulating filter system that can control the water temperature with a boiler. A total of 769 animals were used in the high-temperature exposure experiment by removing objects showing abnormal symptoms during the acclimatization period. Feed was fed twice a day until the water temperature was 30°C, and feeding was stopped after the first death.
  • the water temperature was adjusted so that it could rise by about 1 ° C per day from the temperature of the fish to 30 ° C, and it was adjusted so that it could rise by about 0.5 ° C per day to 32.5 ° C, which is known as the critical temperature of halibut.
  • Water temperature and dissolved oxygen were measured at regular intervals to ensure that oxygen saturation was maintained at 90% or more.
  • the dead individuals were collected at regular intervals from the time of the first death, and the size, weight, and external symptoms were checked, and the tail fins were collected and stored frozen.
  • Genomic DNA was extracted from 50 mg of the caudal fin of the experimental fish using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Germany).
  • a microarray was performed on 763 samples that passed gDNA quality control (QC) using a 70K SNP chip designed and manufactured for halibut in Example 1.
  • the average total length of 769 experimental fish was 25.42 ⁇ 1.63 cm, the average width was 8.84 ⁇ 0.65 cm, and the average body weight was 159.1 ⁇ 29.9 g.
  • the water temperature at which the first death occurred after the temperature increase was 30.7 ° C, and the water temperature was raised and maintained from 30.7 ° C to a maximum of 32.7 ° C for 4 days from the first death.
  • the water temperature was lowered from dawn on the 4th day after the first death, and the surviving individuals were classified as survivors.
  • the total number of dead fish was 538, which was calculated as a cumulative mortality rate of 69.96%, and the number of surviving fish was confirmed as 231 (Table 2).
  • the survival analysis according to high temperature exposure was 1) survival (survival), 2) number of survival days (DPC_Day), which is how many days have elapsed since the first death, and 3) survival time (DPC_Time, which is how many hours have passed since the first death) ), the score was given and applied to the analysis.
  • GWAS Genome-Wide Association Study
  • the heritability ( h 2 ) according to survival was estimated to be 0.620, and specific SNPs related thereto were identified on chromosomes 18 and 19.
  • the heritability according to DPC_Day was estimated to be 0.753, and specific SNPs related thereto were identified on chromosomes 18 and 19, similar to the results of survival GWAS.
  • SNPs were selected for prediction of high temperature resistant individuals, and the nucleotide sequence of the selected SNPs and the genes related to each SNP were selected. Information is as shown in Table 6.
  • the 30 SNPs were determined as high temperature resistant genotypes based on the following analysis results.
  • Table 7 and FIGS. 3 to 32 show information on each SNP associated with high temperature resistance and average and standard deviation of survival days and survival time for each genotype and graphs.
  • the number of survival days (DPC_Day) was assigned a value of 1 to 8 as many as the number of survival days for each individual, and the survival time (DPC_Time) was the date of occurrence of the first dead individual after converting the death date of each individual into a serial number using Excel. 1 was performed.
  • DPC_Day The number of survival days (DPC_Day) was assigned a value of 1 to 8 as many as the number of survival days for each individual
  • the survival time (DPC_Time) was the date of occurrence of the first dead individual after converting the death date of each individual into a serial number using Excel. 1 was performed.
  • the survival and genotype of each individual the number of surviving or dead individuals for each genotype was confirmed, and based on this, it was shown in
  • G. (215) mean ⁇ standard deviation number of days to live 6.71 ⁇ 2.36 3.85 ⁇ 2.63 2.18 ⁇ 1.84 survival time 10.38 ⁇ 4.42 5.14 ⁇ 4.72 2.60 ⁇ 2.98 14 19 4476416 AX-419310954 C Variation (Number of Populations) CC (139) CT (376) TT (207) mean ⁇ standard deviation number of days to live 6.61 ⁇ 2.42 4.03 ⁇ 2.68 2.05 ⁇ 1.71 survival time 10.18 ⁇ 4.53 5.44 ⁇ 4.83 2.44 ⁇ 2.76 15 19 4230401 AX-419194853 G Variation (Number of Populations) AA (27) AG (334) G. G.
  • G. (39) mean ⁇ standard deviation number of days to live 4.97 ⁇ 2.88 3.02 ⁇ 2.46 2.03 ⁇ 1.77 survival time 7.18 ⁇ 5.25 3.91 ⁇ 4.21 2.37 ⁇ 2.83 21 18 11576382 AX-419304244 T Variation (Number of Populations) G. G.
  • G. (282) mean ⁇ standard deviation number of days to live 2.68 ⁇ 2.30 3.04 ⁇ 2.45 5.45 ⁇ 2.81 survival time 3.43 ⁇ 3.91 3.92 ⁇ 4.20 8.02 ⁇ 5.19 30 19 4781952 AX-419194922 T Variation (Number of Populations)
  • G. G. (334) TG (338) TT (48) mean ⁇ standard deviation number of days to live 2.42 ⁇ 2.03 5.09 ⁇ 2.80 6.42 ⁇ 2.53 survival time 2.94 ⁇ 3.33 7.34 ⁇ 5.20 9.80 ⁇ 4.75
  • Table 8 shows the heritability estimated from the GWAS analysis for high temperature tolerance through genetic parameter analysis.
  • Heritability associated with high temperature tolerance phenotypic trait heritability Heritability ( h 2 ) P-value Significant to select major related chromosomes Survival 0.620 8.47 x 10 -7 Significant 18 and 19 Days to surviveDPC_Day 0.753 8.47 x 10 -7 Significant 18 and 19 survival timeDPC_Time 0.710 8.47 x 10 -7 Significant 18 and 19
  • the single nucleotide polymorphism (SNP) marker composition for predicting high temperature resistance can induce the development and continuous production of high temperature resistant varieties, which can be of great help to flounder farming.

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PCT/KR2022/012747 2021-09-01 2022-08-25 넙치의 고수온 내성 예측용 snp 마커 및 이의 용도 WO2023033457A1 (ko)

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CN104328116A (zh) * 2014-11-06 2015-02-04 中国海洋大学 一种与牙鲆耐热性相关的snp位点及其应用
CN105624318A (zh) * 2016-03-24 2016-06-01 中国水产科学研究院北戴河中心实验站 一种与牙鲆生长性状相关的snp位点、其筛选方法及应用
CN105713974A (zh) * 2016-03-24 2016-06-29 中国水产科学研究院北戴河中心实验站 一种与牙鲆数量性状相关的snp位点、其筛选方法及应用
CN109897905A (zh) * 2019-04-26 2019-06-18 上海海洋大学 一种与牙鲆体色异常关联的snp分子标记及其应用
KR102384512B1 (ko) * 2021-09-01 2022-04-12 제주대학교 산학협력단 넙치의 고수온 내성 예측용 snp 마커 및 이의 용도

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KR102080120B1 (ko) 2019-11-15 2020-02-21 대한민국 전복 고수온 내성 형질 예측용 바이오마커 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328116A (zh) * 2014-11-06 2015-02-04 中国海洋大学 一种与牙鲆耐热性相关的snp位点及其应用
CN105624318A (zh) * 2016-03-24 2016-06-01 中国水产科学研究院北戴河中心实验站 一种与牙鲆生长性状相关的snp位点、其筛选方法及应用
CN105713974A (zh) * 2016-03-24 2016-06-29 中国水产科学研究院北戴河中心实验站 一种与牙鲆数量性状相关的snp位点、其筛选方法及应用
CN109897905A (zh) * 2019-04-26 2019-06-18 上海海洋大学 一种与牙鲆体色异常关联的snp分子标记及其应用
KR102384512B1 (ko) * 2021-09-01 2022-04-12 제주대학교 산학협력단 넙치의 고수온 내성 예측용 snp 마커 및 이의 용도

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