CN105441536A - SNP markers for discriminating the sex in the olive flounder - Google Patents
SNP markers for discriminating the sex in the olive flounder Download PDFInfo
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Abstract
The present invention has a purpose of providing a marker for discriminating the sex of olive flounder fry before sex discrimination. Provided in the method is a single nucleotide polymorphism (SNP) marker which can generically discriminate sex of olive flounder fry by comparing and analyzing genome of male and female olive flounders.
Description
To the cross reference of related application
The right of priority that this application claims the korean patent application No.10-2014-0124525 that on September 18th, 2014 submits to and the ownership equity obtained from it, its content is by mentioning complete being incorporated to.
Background of invention
Present disclosure relates to the mark for gender discrimination, and relates more specifically to the mark for the gender discrimination in lefteye flounder (oliveflounder).
Growing sooner than male Japanese flounders due to female lefteye flounder (Paralichthysolivaceus) and have higher economic worth, female lefteye flounder is preferred relative to male Japanese flounders.Usually, sex ratio between male and female lefteye flounder in the lefteye flounder cultivated is almost 1:1, replace with female flounder the production cost that male Japanese flounders can make to reduce the lefteye flounder cultivated, and so greatly improve its productivity (productivity).But the routine techniques producing the female seed (femaleseed) of lefteye flounder is very difficult and complexity, and the mechanism of the sex measured in fish also greatly changes with species.Lefteye flounder congenital imparting genetic sex decision systems (it determines sex at birth) and environment sex decision systems (Dot Blots Sex of female lefteye flounder can be become male Japanese flounders by it during the sexual differentiation phase of being assisted by high water temperature or low water temperature) both.Therefore, if by male for vacation (pseudomale), (it is female at birth, but change into male by cultivating in high water temperature or low water temperature) and female breeding, then can produce hereditary female fingerling, so improve female per-cent when seed produces.
The open text No.2012-0027843 of Korean Patent discloses the method for the gender typing in lefteye flounder, and it can measure in early days via using the primer sets of the gender typing be used in lefteye flounder to compare aromatase enzyme gene expression amount.
Summary of the invention
But the technology described above is for measuring the property method for distinguishing after sexual differentiation, and not yet develops the property method for distinguishing before can measuring sexual differentiation.
Present disclosure is to solve various restriction, comprises above-described restriction, and in order to provide other mark of lefteye flounder seminality that can measure before sexual differentiation.But above-mentioned theme is only in order to illustrate object, and the scope of present disclosure is not limited thereto.
According to an exemplary embodiment, provide for measuring the DNA polynucleotide of the sex in lefteye flounder or the polynucleotide with its complementation, it is selected from lower group: i) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 523rd base of SEQIDNO:1 is C or T; Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:2, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:2 is A or T; Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:3, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 252nd base of SEQIDNO:3 is A or C; Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:4, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 329th base of SEQIDNO:4 is T or G; V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:5, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 108th base of SEQIDNO:5 is C or G; Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T; Vii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G; Viii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:8, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 367th base of SEQIDNO:8 is C or T; Ix) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T; X) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; And xi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
The embodiment exemplary according to another, provides polynucleotide, itself and above-mentioned polynucleotide or its complementary polynucleotide specific hybrid.
The embodiment exemplary according to another, provides the test kit for the gender typing in lefteye flounder, and it comprises above-mentioned polynucleotide, the primer pair of the above-mentioned polynucleotide that maybe can increase.
The embodiment exemplary according to another, provide the method for the gender typing in lefteye flounder, the method comprises: extract genomic dna from female and male Japanese flounders; The DNA extracted is used to comprise the polynucleotide of single nucleotide polymorphism (SNP) with the primer pair amplifies of the above-mentioned polynucleotide that can increase as template; And identify the genotype of polynucleotide.
The embodiment exemplary according to another, provide for measure morphology male in the male method of vacation, it comprises: by via the abdominal compression of adult lefteye flounder being measured to the existence of testis and lacking and select morphology male; From the male extraction genomic dna of morphology selected; Can the increase primer pair of polynucleotide described below and the genomic dna of extraction of use comprises the polynucleotide described below of single nucleotide polymorphism as template amplification; And identify the genotype of polynucleotide; For measuring the DNA polynucleotide of the sex in lefteye flounder or being selected from lower group with the polynucleotide of its complementation:
I) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 523rd base of SEQIDNO:1 is C or T;
Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T;
Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G;
Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T;
V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; With
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
Accompanying drawing is sketched
According to the following description adopted by reference to the accompanying drawings, exemplary embodiment can be understood in more detail, wherein:
The figure of Fig. 1 shows the SNP mark based on the genomic comparative analysis qualification by male and female lefteye flounder, 24 chromosomal collection of illustrative plates based on SNP, and wherein red rod indicates female SNP district, and blue rod indicates male SNP district;
The figure of Fig. 2 shows the electrophorogram (electropherogram) from the SNP#9 mark of female lefteye flounder;
The figure of Fig. 3 shows the electrophorogram of the SNP#10 mark from female lefteye flounder; With
The figure of Fig. 4 shows the electrophorogram of the SNP#11 mark from female lefteye flounder.
Embodiment
The definition of term:
As used in this article, term " lefteye flounder " refers to belong to Bothidae (FamilyParalichthyidae), the saltwater fish of flounder order (OrderPleuronectiformes), it has flat health, eye is gathered in left side, and also referred to as flatfish (flatfish).Lefteye flounder from the place of tide set to the deep-sea life with 1, the 000m degree of depth, and has from the long small-sized type of the body with about 5cm to many species of type with 80cm length.Tail fin obviously separates on dorsal fin and anal fin, and the body colour of eye location is brown or dead color, and the color of opposition side is white.The laying season of lefteye flounder is between February and July, and it can omit in southern area early.During laying season, lefteye flounder moves on to tide the husky mud/husky gravel or rocky zone (sandymud/sandygravelorrockyarea) lay eggs that horizontal plane 20 to 70m locates.
As used in this article, term " produces female diploid (gynogeneticdiploid) " and refers to that diploid is individual, it is only made up of the female genetic stocks after removing male inherited genetic factors completely, and can by the female individuality of product suppressing the release of second polar body preparation have high genetic purity.In addition, in gender typing's culture, female be homozygote (XX) time, by this mechanism induction all individualities become female.When the lefteye flounder that Korea S cultivates or Red sea porgy (redseabream) etc., the growth velocity of raun is very fast, and implements seed produces by this mechanism so at present.
As used in this article, term " false male (pseudomale) " refers to individual lefteye flounder, its in heredity as female birth, but can change into and have the male of testis by cultivating in high-temperature water or water at low temperature in the sexual differentiation phase (roughly before and after 60 days of hatching).Thus, by finding female parent (mother) (false male) (it is female in heredity, but is male on morphology), and with female breeding, hereditary female seed can be produced, and so can improve female per-cent during seed produces.
As used in this article, term " single nucleotide polymorphism (SNP) " refers to the nucleotide variation between colony or individual interior allelotrope in specific gene locus.
Detailed Description Of The Invention
According to an aspect of present disclosure, provide for measuring the DNA polynucleotide of the sex in lefteye flounder or the polynucleotide with its complementation, it is selected from lower group:
I) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 523rd base of SEQIDNO:1 is C or T;
Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:2, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:2 is A or T;
Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:3, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 252nd base of SEQIDNO:3 is A or C;
Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:4, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 329th base of SEQIDNO:4 is T or G;
V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:5, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 108th base of SEQIDNO:5 is C or G;
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T;
Vii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G;
Viii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:8, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 367th base of SEQIDNO:8 is C or T;
Ix) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T;
X) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; With
Xi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
According to another aspect of present disclosure, provide polynucleotide, itself and above-mentioned polynucleotide or its complementary polynucleotide specific hybrid.
In above-mentioned polynucleotide, can be the polynucleotide as allele-specific probe or allele-specific primers with the polynucleotide of polynucleotide or its complementary polynucleotide specific hybrid.
According to another aspect of present disclosure, provide the test kit for the gender typing in lefteye flounder, it comprises according to above-mentioned polynucleotide, the primer pair of the above-mentioned polynucleotide that maybe can increase.Test kit can be the test kit provided as the microarray containing polynucleotide.
According to another aspect of present disclosure, provide the method for the gender typing in lefteye flounder, the method comprises:
Genomic dna is extracted from female and lefteye flounder;
By using the DNA that extracts as template with the primer pair amplifies of polynucleotide comprising the polynucleotide of single nucleotide polymorphism (SNP); And
The genotype of qualification polynucleotide.
In the property method for distinguishing for measuring in lefteye flounder, can be to identify that the various methods of SNP implement the genotype of described qualification polynucleotide via previous report, described method comprises order-checking, the hybridisation assays using allele-specific microarray, ApoE gene, capillary electrophoresis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), the HLPC revised, mass spectrometry, restrictive fragment length polymerphism (RFLP) or TaqManSNP genotyping assay method.
Can by analyzing the high-flux sequence of lefteye flounder genomic dna and implementing order-checking by calling (variantcalling) via the variant of location (mapping).In enforcement variant calls, only qualification has the variant of the minimum mass numerical value (minimumqualityvalue) of 30 or higher, and the last male genome (it is previously for the order-checking completely of lefteye flounder) that uses is as standard gene group investigation variant.By the SNP mark of Analysis and Identification, there is the male display special-shaped (heterotype) of testis, and there is the female display homotype of ovum, so make it possible to the difference detection sex based on only single base.The female diploid of product due to the lefteye flounder used in present disclosure only has x chromosome, so female in heredity, and its genotype is homotype with regard to female genotypes, but also show that false male genotype is the same with female genotype has homotype.This is because false male Japanese flounders is female in heredity, although it shows as male on morphology.According to above, have been found that and malely in heredity, show abnormal shape, and the female homotype SNP genotype that shows in heredity, so realize genetic sex difference.The method for the gender typing in lefteye flounder available at present comprises the measurement of the sex steroid hormone in visual inspection, the histological observation of sexual gland, blood, the measurement of vitellin(Vt), CT measure, the measurement etc. of aromatase enzyme genetic expression.But, these methods only can use when lefteye flounder has certain size or ripe size usually, and according to the mensuration ability of examiner, also produce larger error, and it is killing and using sexual gland to apply after dissecting experimenter's lefteye flounder, so only available after the sexual differentiation of lefteye flounder.But usefully, the genetic sex that the measuring method of present disclosure may be used for before sexual differentiation childhood of seed measures.
In addition, method may further include for any one following situation, determines that the sex of lefteye flounder seed is male:
A kind of situation is wherein C with the 523rd base that base pair is answered of the nucleotide sequence based on SEQIDNO:1;
A kind of situation is wherein T with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:2;
A kind of situation is wherein A with the 252nd base that base pair is answered of the nucleotide sequence based on SEQIDNO:3;
A kind of situation is wherein T with the 329th base that base pair is answered of the nucleotide sequence based on SEQIDNO:4;
A kind of situation is wherein C with the 108th base that base pair is answered of the nucleotide sequence based on SEQIDNO:5;
A kind of situation is wherein T with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:6;
A kind of situation is wherein C with the 322nd base that base pair is answered of the nucleotide sequence based on SEQIDNO:7;
A kind of situation is wherein C with the 367th base that base pair is answered of the nucleotide sequence based on SEQIDNO:8;
A kind of situation is wherein A with the 253rd base that base pair is answered of the nucleotide sequence based on SEQIDNO:9;
A kind of situation is wherein G with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:10; With
A kind of situation is wherein G with the 249th base that base pair is answered of the nucleotide sequence based on SEQIDNO:11.
In addition, according to an aspect of present disclosure, provide for measure morphology male in the male method of vacation, it comprises:
By selecting morphology male via the existence and shortage that the abdominal compression of adult lefteye flounder are measured to testis;
From the male extraction genomic dna of morphology selected;
Can the increase primer pair of polynucleotide described below and the genomic dna of extraction of use comprises the polynucleotide described below of single nucleotide polymorphism as template amplification; And
Identify the genotype of polynucleotide described below;
Wherein polynucleotide are that it is selected from lower group for measuring the DNA polynucleotide of the sex in lefteye flounder or the polynucleotide with its complementation:
I) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 523rd base of SEQIDNO:1 is C or T;
Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T;
Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G;
Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T;
V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; With
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
In false male method, the genotype of polynucleotide described below can analyzed further for determining:
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:2, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:2 is A or T;
Viii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:3, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 252nd base of SEQIDNO:3 is A or C;
Ix) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:4, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 329th base of SEQIDNO:4 is T or G;
X) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:5, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 108th base of SEQIDNO:5 is C or G; With
Xi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:8, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 367th base of SEQIDNO:8 is C or T.
For determining in false male method, when the genotype of the single nucleotide polymorphism (SNP) of polynucleotide is homotypes, method may further include determines that lefteye flounder is false male.
By use according to an embodiment of present disclosure for measuring false male method, can measure false male, but its in heredity be female change into male, and using the male and female breeding as the vacation of hybrid strain, thus it is female only to generate 100% heredity, and so the method can be effective to the seed produces of lefteye flounder very much.
Hereinafter, present disclosure is described in more detail with reference to following examples.But present disclosure is not limited to these embodiments, and can embody in various forms different from each other.On the contrary, provide these embodiments, thus present disclosure can be thorough and completely, and scope of the present invention can be conveyed to those skilled in the art completely.
Embodiment 1: produce female diploid and the male generation of vacation
Female diploid is produced in order to generate according to the embodiment of present disclosure, seminal fluid is obtained from 2 – 3 age Red sea porgys male (fish farm from single) of maturation by abdominal compression, with woods lattice (ringer) solution dilution, and by irradiating 4,800erg/mMUV deactivation when stirring under UV irradiating unit.With the seminal fluid of lefteye flounder ovum artificial insemination Red sea porgy, and for haploid diploidization (2n), produce female diploid in the subzero treatment zygote of 0 to 2 DEG C to prepare, and cultivate at 5 tons of water pots.In order to be trained by female for product diploid male (namely false male), use the male hormone with 17 Alpha-Methyl testosterone (methyltesterone) (MT) of final concentration 10ppb and 100ppb.In three different water pots, distribute 2,000 tail lefteye flounder for each concentration, and the 33rd day after hatching starts HORMONE TREATMENT.After this, implement submergence two hours every days and continue 60 day time period, and the vacation of cultivating induction in 5 tons of water pots is male.
Embodiment 2: genome analysis
According to an embodiment of present disclosure, from male and female lefteye flounder isolation of genomic DNA, and analyze male and female gene group DNA by high-flux sequence.
Particularly, via the blood using the ultracentrifugation of CsCl-EtBr gradient from the male parent population of 1 tail (broodstock) and the female parent population of 14 tails (Good-broodstocksResearchCenter, NationalFisheriesResearch & DevelopmentInstitute), seminal fluid and ovum separate tissue high purity genomic dna.The DNA be separated is used to build the short section of reading library, and by using sequenator HiSeq2000 (Illumina, USA) of future generation to implement the high-flux sequence of lefteye flounder to the high-flux sequence of lefteye flounder.
The qualification of embodiment 3:SNP mark
Use the high-flux sequence analyzed according to embodiment of present disclosure, the genomic dna of the male and female lefteye flounder of comparative analysis.The present inventor, in order to identify the karyomit(e) relevant with lefteye flounder sex or mark, selects the SNP mark with high variation and reproducibility from molecular marker.The heritable variation of the difference of single base sequence (A, T, G, C) in show dna sequence due to SNP mark, be therefore investigation display male and female between the most suitable mark of mark of difference.
Particularly, use SOAPdenovo to assess the high-flux sequence analyzed in embodiment 2, and call via base and locate and implement variant and call.Use the ELAND program developed by IlluminaCompany to implement location, and use the Mpileup of Samtools to implement the qualification of SNP variant.In enforcement SNP calls, only identify that those have the variant of minimum mass numerical value 30 or higher, and in order to check the genotype in SNP district, using the single base sequence of Seqman red tape operation, then arranging.Based on canonical sequence, the sequence of male and female individuals containing primer in collating sequence, and qualification show male and female between the SNP of difference.Finally, by using male genome (it had previously been used as, and lefteye flounder is genomic to check order completely) as reference, BWA locating procedure is used to locate the male and female lefteye flounder experimenter analyzed above, finally screen variant, and based on the SNP mark identified to the collection of illustrative plates of each karyomit(e) preparation based on SNP.
Result discloses except 9,22 and No. 23 karyomit(e)s, and SNP is same distribution in male and female all regions.In No. 9 chromosomal situations, SNP distributes in female all regions, but have SNP be not present in male in many regions.Compared with other karyomit(e), these regions are high conservatives, and so they seem to have the gene for the vital effect of male performance.In 22 and No. 23 chromosomal situations (it shows contrary result), these regions seem to have the gene for the vital effect of female performance.Thus, determine that one or more sex genes involveds or mark can be present in 9,22 and No. 23 karyomit(e)s, and especially, infer that No. 9 karyomit(e)s (it is male specific) are sex chromosome (Fig. 1).
The genotypic analysis of embodiment 4:SNP mark
By the order-checking of an embodiment according to present disclosure, first select the display between the SNP mark that exists in No. 9 karyomit(e)s male and female between 55 SNP districts of difference in height.Male and the female lefteye flounder inspection that the genotype in 55 SNP districts uses each 6 tails to distinguish via phenotype or histological observation, and specify 17 kinds of SNP marks (its with regard to male and female between phenotype and genotype with regard to display consistency) as the material standed for of sex related SNP mark.In order to confirm the relation of these 17 kinds of SNP marks and lefteye flounder sex, just each kind of groups (female diploid of hereditary female product; In heredity, female but its phenotype is that male vacation is male; Female female parent (mother), and male female parent) analyze SNP genotype.
Particularly, female lefteye flounder (confirmed as by abdominal compression and there is the female of ovum) that 75 tails cultivate is analyzed and 40 tails produce the genotype of the SNP mark of female diploid lefteye flounder (in heredity female lefteye flounder).Further, the genotype analyzing SNP mark in vacation prepared by female diploid male (in heredity female but have the male of seminal fluid) is produced in 70 tail male Japanese flounders (confirmed as by abdominal compression and there is the male of seminal fluid) and 40 tail male hormone process.Use OLIGO5.0 program based on the information design primer of the base sequence containing SNP district, synthesized by GENOTECH, and for PCR reaction (see table 1).In addition, be separated male and female subject by using MagExtractor-genomic DNA purification kit from fin, produce the genomic dna of female diploid and false masculine subjects, and using 10 μ L reaction solns altogether to implement PCR reaction with the region of amplification containing SNP via PTC-220 thermal cycler, described reaction soln contains genomic dna 100ng, 0.2mMdNTP, 20mMTris-HCl (pH8.0), 100mMKCl, 1.5mMMgCl
2, the forward primer of each 2 μ L and reverse primer and 0.25UEX-taq polysaccharase.PCR condition was as follows: in 95 DEG C of denatured DNAs 5 minutes; Reach 30 seconds in 94 DEG C of denatured DNAs, in the annealing temperature indicated in Table 1 45 seconds, and extend 35 circulations of 45 seconds in 72 DEG C; 10 minutes are reached in the final DNA synthesis of 72 DEG C.Use the amplified reaction product that Acroprep96 orifice plate is separating obtained, and use implements two-way order-checking for the primer increased, and use ABI3130xl automatization sequencing analysis instrument to measure base sequence.
Therefore, confirm that 11 kinds of SNP marks between 17 kinds of SNP marks are that tight sex is correlated with.Female individuals is homotype (AA, CC, TT and GG) (see table 2) with the genotype major part of the 11 kinds of SNP marks produced in female diploid.Isotype ratios in female individuals accounts for 96.0% to 100%, and the isotype ratios produced in female diploid accounts for 100%.In addition, the genotype major part of 11 kinds of SNP marks in male is special-shaped (CT, AT, AC, GT, CG and AG), and special-shaped ratio accounts for 77.1% to 80.0% (see table 3).But the special-shaped ratio during the vacation of Dot Blots Sex is male is 0%, so all shows homotype.This has pointed out the vacation of Dot Blots Sex male is that heredity is female, and be so all shown as homotype, and when male, abnormal shape is not 100%, because lefteye flounder can pass through environmental factors (mainly high water temperature or low water temperature) during the sexual differentiation phase experience Dot Blots Sex, so the male of those display homotypes is that heredity is female at birth, and during the Dot Blots Sex phase, due to the impact of water temperature, Dot Blots Sex becomes male for it.Produce female diploid lefteye flounder and only have x chromosome, and be so that heredity is female, and its genotype is homotype, as when female genotypes, and when vacation is male, its genotype is shown as homotype, because they are that heredity is female, although false male is that morphology is male.According to aforementioned result, can find in heredity, the special-shaped SNP genotype of male display, and female display homotype SNP genotype.In addition, by using after hatching the female diploid of product of use and the male sample of vacation of Dot Blots Sex in this research of seed inspection of the 30th day, and so not think and have they carry out Dot Blots Sex possibility by environmental factors.
Gather the above results, can reach a conclusion when the genotype of 11 kinds of SNP is homotype (homozygotes), they are that heredity is female, and when they are special-shaped (heterozygotes), they are Genetic male.Particularly, in 11 kinds of SNP, when 6 kinds of SNP of SNP#1, #6, #7, #9, #10 and #11 are homotypes, all confirm as 100% heredity female, and show male 77.1% be special-shaped.In male, those male height showing homotype in 6 kinds of SNP may be that growing period Dot Blots Sex becomes male vacation male, and so only can determine the genetic sex of lefteye flounder to the genotypic analysis of single SNP, no matter it is male or female when its commitment.When other SNP, they be hereditary female time, the high per-cent of their display homotypes, and so can improve accuracy by combining with other SNP, and so can be effective to the early stage differentiation of the genetic sex of lefteye flounder.
In 11 kinds of SNP marks, confirm representative 3 kinds of SNP marks (SNP#9, #10 and #11) (Fig. 2 to 4) via electrophorogram.In addition, the analytical results about the primer designed and the genotypic information of SNP is hereafter shown in table 1,2 and 3 above.
[table 1]
[table 2]
* those sign with runic are special-shaped, and remainder is homotype.
* Gyno-2N: produce female diploid.
[table 3]
* those sign with runic are special-shaped, and remainder is homotype.
As described above, according to one of present disclosure exemplary embodiment, can by the effect using the gender typing's mark via the genomic comparative analysis exploitation of male and female lefteye flounder to realize the early sex mensuration in lefteye flounder seed.Certainly, the scope of present disclosure limits with this effect.
Although describe the SNP mark distinguished for sex with reference to specific embodiment, they are only exemplary objects.Therefore, it will be readily appreciated by those skilled in the art that and under the prerequisite of spirit and scope not departing from the present disclosure that appended claims limits, various modifications and variations can be carried out to it.Thus, the real technical protection scope of present disclosure should be determined by the technical conceive of appended claims.
Claims (11)
1., for determining the DNA polynucleotide of the sex in lefteye flounder (oliveflounder) or the polynucleotide with its complementation, it is selected from lower group:
I) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 523rd base of SEQIDNO:1 is C or T;
Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:2, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:2 is A or T;
Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:3, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 252nd base of SEQIDNO:3 is A or C;
Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:4, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 329th base of SEQIDNO:4 is T or G;
V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:5, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 108th base of SEQIDNO:5 is C or G;
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T;
Vii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G;
Viii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:8, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 367th base of SEQIDNO:8 is C or T;
Ix) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T;
X) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; With
Xi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
2. polynucleotide, the polynucleotide of itself and claim 1 or its complementary polynucleotide specific hybrid.
3. the polynucleotide of claim 2 are wherein allele-specific probe or allele-specific primers with the polynucleotide of described polynucleotide or its complementary polynucleotide specific hybrid.
4. for the test kit that the sex in lefteye flounder is determined, it comprises the polynucleotide any one of claims 1 to 3, the primer pair of the described polynucleotide that maybe can increase.
5. the test kit of claim 4, wherein said test kit provides with the microarray comprising described polynucleotide.
6., for the method that the sex in lefteye flounder is determined, the method comprises:
Genomic dna is extracted from female and male Japanese flounders;
The polynucleotide of single nucleotide polymorphism (SNP) are comprised as template and the primer pair amplifies of the polynucleotide of the claim 1 that can increase by using extract DNA; And
Identify the genotype of described polynucleotide.
7. the method for claim 6, wherein implements the genotype of described qualification polynucleotide via check order, the use hybridisation assays of allele-specific microarray, ApoE gene, capillary electrophoresis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), the HLPC revised, mass spectrometry, restrictive fragment length polymerphism (RFLP) or TaqManSNP genotyping assay method.
8. the method for claim 6, it comprises further for any one following situation, determines that the sex of lefteye flounder seed is male:
A kind of situation is wherein C with the 523rd base that base pair is answered of the nucleotide sequence based on SEQIDNO:1;
A kind of situation is wherein T with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:2;
A kind of situation is wherein A with the 252nd base that base pair is answered of the nucleotide sequence based on SEQIDNO:3;
A kind of situation is wherein T with the 329th base that base pair is answered of the nucleotide sequence based on SEQIDNO:4;
A kind of situation is wherein C with the 108th base that base pair is answered of the nucleotide sequence based on SEQIDNO:5;
A kind of situation is wherein T with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:6;
A kind of situation is wherein C with the 322nd base that base pair is answered of the nucleotide sequence based on SEQIDNO:7;
A kind of situation is wherein C with the 367th base that base pair is answered of the nucleotide sequence based on SEQIDNO:8;
A kind of situation is wherein A with the 253rd base that base pair is answered of the nucleotide sequence based on SEQIDNO:9;
A kind of situation is wherein G with the 150th base that base pair is answered of the nucleotide sequence based on SEQIDNO:10; With
A kind of situation is wherein G with the 249th base that base pair is answered of the nucleotide sequence based on SEQIDNO:11.
9. for determine morphology male in the male method of vacation, it comprises:
By via the abdominal compression determination testis to adult lefteye flounder existence and lack select morphology male;
From the male extraction genomic dna of morphology selected;
Can the increase primer pair of described polynucleotide and the genomic dna of extraction of use has the polynucleotide described below of single nucleotide polymorphism as template amplification; And
Identify the genotype of described polynucleotide;
Wherein said polynucleotide are that it is selected from lower group for determining the DNA polynucleotide of the sex in lefteye flounder or the polynucleotide with its complementation:
I) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:1, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 523rd base of SEQIDNO:1 is C or T;
Ii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:6, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:6 is A or T;
Iii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:7, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 322nd base of SEQIDNO:7 is C or G;
Iv) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:9, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 253rd base of SEQIDNO:9 is A or T;
V) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:10, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:10 is C or G; With
Vi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:11, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 249th base of SEQIDNO:11 is A or G.
10. the method for claim 9, it comprises analyzes following genotype further:
Vii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:2, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 150th base of SEQIDNO:2 is A or T;
Viii) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:3, and have comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, wherein the 252nd base of SEQIDNO:3 is A or C;
Ix) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:4, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 329th base of SEQIDNO:4 is T or G;
X) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:5, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 108th base of SEQIDNO:5 is C or G; With
Xi) polynucleotide, it is selected from the polynucleotide of the nucleotide sequence with SEQIDNO:8, and has comprise single nucleotide polymorphism (SNP) district 10 to 100 or more nucleic acid continuously, and wherein the 367th base of SEQIDNO:8 is C or T.
The method of 11. claims 9, determines when its genotype being included in the single nucleotide polymorphism (SNP) of described polynucleotide is further homotype that lefteye flounder is false male.
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| KR101770966B1 (en) | 2016-10-21 | 2017-08-24 | 대한민국 | Genetic marker and method for identifying the sex in the olive flounder |
| KR102000878B1 (en) | 2018-07-31 | 2019-07-18 | 주식회사 불루젠코리아 | Marker for male and female of fish identification and HRM-PCR analysis method |
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| KR101281529B1 (en) * | 2010-09-13 | 2013-08-01 | 임봉수 | Primer set for sex determination in flatfish and method for sex determination in flatfish using these primers |
| KR101418139B1 (en) * | 2011-04-29 | 2014-07-14 | 제주대학교 산학협력단 | Compisition and method for diagnosting sex of Pa-ralichthyidae |
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Non-Patent Citations (2)
| Title |
|---|
| WOO JIN KIM: "Development of Polymorphic Microsatellite Markers Suitable for Genetic Linkage Mapping of Olive Flounder Paralichthys olivaceus", 《FISHERIES AND AQUATIC SCIENCES》 * |
| 邵长伟: "半滑舌鳎性别决定的基因组学研究", 《中国博士学位论文全文数据库农业科技辑》 * |
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|---|---|---|---|---|
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