CN101597648A - The screening method of meitogynogenetic diploid of pseudosciaena crocea - Google Patents
The screening method of meitogynogenetic diploid of pseudosciaena crocea Download PDFInfo
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Abstract
The screening method of meitogynogenetic diploid of pseudosciaena crocea.A kind of screening method of material cultivating process, parent DNA sample and record being preserved the meitogynogenetic diploid of pseudosciaena crocea that does not all have particular requirement is provided.Adopt phenol-chloroform-primary isoamyl alcohol method to extract the large yellow croaker genomic dna, dna profiling as the PCR reaction, adopt 5～10 to carry out pcr amplification and get the PCR reaction product with the closely linked microsatellite marker primer in kinetochore, behind denaturing polyacrylamide gel electrophoresis, show band with cma staining, if on a swimming lane 1 allelotrope is only arranged, then should on the detection locus, be homozygote by individuality; If on the swimming lane only is 2 allelotrope, then should on the detection locus, be heterozygote by individuality; All detect locus and be the individuality that isozygotys, select and remain as heterogeneous gynogenesis diploid; It is the individuality of heterozygosis that more than one locus is arranged, and mixes individual rejecting as big risk, will not select and remain.
Description
Technical field
The present invention relates to the screening method of the heterogeneous gynogenesis diploid of a kind of fish, especially relate to the screening method of the heterogeneous gynogenesis diploid of a kind of large yellow croaker (Pseudosciaena crocea).
Background technology
Heterogeneous gynogenesis diploid is meant the distinct species material that utilizes artificial induction's gynogenesis technology to cultivate, and promptly adopts the genetic inactivation sperm to start ovum and grows, and utilizes the genome doubling techniques to suppress the diploid that second meiotic division obtains again.Heterogeneously femalely authorize diploid and only contain the female parent gene group, do not have the genetic material of male parent, therefore the descendant who obtains all shows the maternal instinct proterties.Heterogeneous gynogenesis diploid has wide application prospect in aquatic living things genetic breeding and seedling production, comprising: set up pure lines fast, realize the complete female breed of mass-producing, genetic analysis, raising output; Comprehensive breeding, quickening breeding speed etc.
The induction method of meitogynogenetic diploid of pseudosciaena crocea is set up, and is applied to ([1] Wang Xiaoqing, Wang Zhiyong in the genetics research, Liu Xiaochun, Deng. large yellow croaker artificial induction gynogenesis offspring's microsatellite marker analysis [J]. heredity, 2006,28 (7): 831-837; [2] Xu Jianhe, You Feng, Wu Xiongfei, etc. the diplontic artificial induction of large yellow croaker gynogenesis [J]. ocean science, 2006,30 (12): 37-42; [3] Wang Dexiang, Su Yongquan, Wang Shifeng, etc. the allos sperm is induced the research [J] of large yellow croaker gynogenesis. hi-tech communication, 2006,16 (11): 1206-1210; [4] Xu J H, You F, Yan B L, et al.Effects of ultra-violetirradiation on sperm motility and diploid gynogenesis induction in large yellowcroaker (Pseudosciaena crocea) undergoing cold shock[J] .Aquac Int, 2007,15:371-382; [5] Li Y Y, Cai M Y, Wang Z Y, et al.Microsatellite-centromere mapping in the large yellow croaker (Pseudosciaena crocea) using induced gynogenetic diploid families[J] .Mar Biotech, 2008,10:83-90).But the heterogeneous gynogenesis diploid of artificial induction is mixed with common diploid sometimes.Be a small amount of sperm not by genetic inactivation, cause the ovum normal fertilization, produce amphimictic common diploid.For example, in the different gynogenesis family of two large yellow croakers that discoveries such as Wang Xiaoqing are analyzed, there is a family to be mixed with common diploid ([6] Wang Xiaoqing of 12.5%, Wang Zhiyong, Liu Xiaochun, Deng. the aflp analysis [J] of artificial gynogenesis large yellow croaker (Pseudosciaena crocea). Oceanologia et Limnologia Sinica, 2007,38 (1): 22-28).Heterogeneous gynogenesis diploid if be mixed with common diploid, will influence its using value in genetic research and seedling production as the germplasm materials of preciousness greatly, even the failure that causes the mass-producing unisexuality to be produced, and causes financial loss.Therefore, before being applied to seedling production and genetic breeding, screen, reject the common diploid that is mixed in wherein heterogeneous gynogenesis diploid as germplasm materials.
In " aflp analysis of artificial gynogenesis large yellow croaker (Pseudosciaena crocea) " literary composition that Wang Xiaoqing equals to deliver on " Oceanologia et Limnologia Sinica " the 38th volume 22-28 page or leaf in 2007, and in " large yellow croaker artificial induction gynogenesis offspring's microsatellite marker analysis " literary composition of on " heredity " the 28th volume 831-837 page or leaf, delivering in 2006, provide the method (reference [1] and [6]) that AFLP and microsatellite marker are differentiated gynogenesis diploid and common diploid of using respectively.Two kinds of discrimination methods all are based on the principle of paternity test, genotype and this genotype of father and mother of being about to the descendant compare, the individuality that does not contain the peculiar gene of male parent is judged to be heterogeneous gynogenesis diploid, and the individuality that contains the peculiar gene of male parent is judged to be common diploid.There is following problem in heterogeneous gynogenesis diploid screening method based on the paternity test principle:
1) the material cultivating process has particular requirement.Differentiate that from numerous and disorderly parent the difficulty and the workload that whether contain male parent gene all are huge.Simplify the paternity test workload, guarantee to identify accuracy, the method that the material require per stirpes is cultivated is grown seedlings and is cultivated.But in the actually operating, heterogeneous gynogenesis family cultivation difficulty is big, workload is big, expense is high.
2) preservation to sample and pedigree record requires high.Carry out paternity test and need preserve complete and identify parent DNA sample clearly, need well-documented history mating combination and descendant's pedigree simultaneously.In case sample or record are lost or mixed, just can't implement based on the discrimination method of paternity test principle.The complete sum that guarantees parent's dna sample and operating record is clear, must strengthen handling cost.
Summary of the invention
The object of the present invention is to provide a kind of screening method of material cultivating process, parent DNA sample and record being preserved the meitogynogenetic diploid of pseudosciaena crocea that does not all have particular requirement.
In heterogeneous gynogenesis diploid, with the homozygosity of the closely linked gene in kinetochore near 100%, and in common diploid colony the homozygosity of homologous genes (reference [5]: Li Y Y about 50% in theory, Cai M Y, Wang Z Y, et al.Microsatellite-centromere mapping in the large yellow croaker (Pseudosciaena crocea) usinginduced gynogenetic diploid families[J] .Mar Biotech, 2008,10:83-90.).Based on this result of study, the screening method of meitogynogenetic diploid of pseudosciaena crocea of the present invention may further comprise the steps:
1) extraction of template DNA: adopt phenol-chloroform-primary isoamyl alcohol method to extract the large yellow croaker genomic dna, as the required dna profiling of PCR reaction;
2) pcr amplification: pcr amplification is carried out in 5～10 of above-mentioned template DNA employings and the closely linked microsatellite marker primer in kinetochore respectively, get the PCR reaction product;
3) determine genotype: with step 2) gained PCR reaction product, carry out electrophoresis with denaturing polyacrylamide gel, after electrophoresis finishes, denaturing polyacrylamide gel is shown band with cma staining, according to showing the band result, if 1 allelotrope is only arranged on the swimming lane, then should on the detection locus, be homozygote by individuality; If on the swimming lane only be 2 allelotrope, then should on the detection locus, be heterozygote by individuality;
4) screening: all detect locus and be the individuality that isozygotys, select and remain as heterogeneous gynogenesis diploid; It is the individuality of heterozygosis that more than one locus is arranged, and mixes individual rejecting as big risk, will not select and remain.
Extracting the large yellow croaker genomic dna in phenol-chloroform described in the step 1)-primary isoamyl alcohol method is specially: clip waits to screen large yellow croaker tissue 10～20mg, place centrifuge tube, add pH 8.0STE damping fluid 600～1000 μ l and 20～40mg/ml Proteinase K, 10 μ l after shredding, place 50～60 ℃ of digestion 4～10h down; RNaseA4～6 μ the l that add 2～6mg/ml again, mixing are placed on 37～40 ℃ of reaction 1～2h down; Add saturated phenol 400～1000 μ l of pH 8.0Tris-again, the centrifugal 10min of 12000rpm moves on to supernatant liquor in another centrifuge tube behind the mixing, abandons lower floor; Add equal-volume phenol-chloroform-primary isoamyl alcohol (25: 24: 1), the centrifugal 10min of 12000rpm behind the mixing draws supernatant liquor in another centrifuge tube, abandons lower floor; Add equal-volume chloroform-primary isoamyl alcohol (24: 1), mixing repeatedly, the centrifugal 10min of 12000rpm moves on to supernatant liquor in another centrifuge tube, abandons lower floor; The dehydrated alcohol that adds 2.5 times of volumes places and precipitates more than the 10min on the trash ice; The centrifugal 5min of 12000rpm, precipitation is with the dissolving of 20 μ l ultrapure waters, as the required dna profiling of PCR reaction.
In step 2) in, described pcr amplification is specially: reaction system is: the extracting dna profiling 50～100ng of step 1) institute, primer 0.2 μ mol/L, Taq archaeal dna polymerase 1u, 10 * PCR reaction buffer, 2 μ l, the dNTPs 2.0 μ l of 2mmol/L add aseptic deionized water, make cumulative volume reach 20 μ l.
The one-tenth of PCR reaction buffer is grouped into and is preferably: 100mmol/L Tris-HCl, 100mmol/L KCl, 80mmol/L (NH
4)
2SO
4, 20mmol/L MgCl
2, 0.5%NP-40, pH 9.0.
In step 2) in, the PCR reaction conditions is preferably: pre-94 ℃ of 5min of sex change; 30～40 thermal cyclings (each cycling condition is: 94 ℃ of sex change 1min, 50～57 ℃ of annealing 30s, 72 ℃ of extension 45s); 72 ℃ are extended 10min;
In step 3), the composition of described denaturing polyacrylamide gel is preferably: the 60g acrylamide, and 3.2g methene acrylamide, 7mol urea, 10 * TBE 100ml, adding distil water is to 1L.
In step 3), the method for described denaturing polyacrylamide gel electrophoresis is preferably: at permanent power 60～90W electrophoresis 45～90min, electrophoretic buffer is 1 * TBE.
In step 3), described cma staining shows band and is specially: gel places fixedly 30min of 1% glacial acetic acid, after the water flushing one time, goes to the 1h that dyes in the staining fluid, goes in the developing solution and develops, and treats to take out after gel demonstrates band.
The composition of staining fluid is preferably: AgNO
31g, formaldehyde 1.5ml, 10mmol/L Na
2S
2O
30.2ml, add distilled water to 1L.
The composition of developing solution is preferably: NaOH 5g, formaldehyde 1.5ml adds distilled water to 1L.
Compare based on the heterogeneous gynogenesis screening method of paternity test principle with existing, the present invention preserves material cultivating process, parent DNA sample and pedigree record does not all have particular requirement, can simplify the meitogynogenetic diploid of pseudosciaena crocea cultivating technique, reduce and produce and handling cost.
Embodiment
Embodiment 1
1) extraction of template DNA
Get artificial induction's to be screened heterogeneous gynogenesis diploid large yellow croaker (inducing program) 30 tails and large yellow croaker 30 tails of cultivating by common production sequence referring to reference [5], dorsal fin 14～the 18mg of difference clip sample, place centrifuge tube, add pH 8.0STE damping fluid 600 μ l and 20mgml after shredding
-1Proteinase K 10 μ l place 50 ℃ of digestion 10h down; Add the RNase A4 μ l of 4mg/ml again, mixing is placed on 37 ℃ of following 1h; Add the saturated phenol 600 μ l of pH 8.0Tris-again, the centrifugal 10min of 12000rpm/min moves on to supernatant liquor in another centrifuge tube behind the mixing, abandons lower floor; Add the saturated phenol-chloroform-primary isoamyl alcohol of equal-volume Tris-(25: 24: 1), the centrifugal 10min of 12000rpm/min behind the mixing draws supernatant liquor in another centrifuge tube; Add equal-volume chloroform-primary isoamyl alcohol (24: 1), mixing repeatedly, the centrifugal 10min of 12000rpm/min moves on to supernatant liquor in another centrifuge tube, abandons lower floor; Add the dehydrated alcohol of 2.5 times of volumes, place and precipitate 10min on the trash ice; The centrifugal 5min of 12000rpm, precipitation is with the dissolving of 20 μ l ultrapure waters, as the required dna profiling of PCR reaction.
2) pcr amplification
Template DNA adopts the primer as table 1 to carry out pcr amplification respectively.
Table 1
The pcr amplification of LYC0004.The PCR reaction system is: primer LYC00080.2 μ mol/L, and step 1 an extracting dna profiling 50ng, Taq archaeal dna polymerase 1u, [one-tenth is grouped into 10 * PCR reaction buffer: 100mmol/LTris-HCl, 100mmol/LKCl, 80mmol/L (NH
4)
2SO
4, 20mmol/L MgCl
2, 0.5%NP-40pH 9.0] and 2 μ l, the dNTPs2.0 μ l of 2mmol/L adds aseptic deionized water, makes cumulative volume reach 20 μ l.The PCR reaction conditions is: pre-95 ℃ of 5min of sex change; 30 thermal cyclings (condition is: 94 ℃ of sex change 1min, 55 ℃ of annealing 30s, 72 ℃ of extension 45s); 72 ℃ are extended 10min.
The amplification of LYC0008, LYC0011, LYC0015, LYC0021, LYC0022, LYC0025, LYC0027, LYC0032 and LYC0033.With the amplification of LYC0004, only the annealing of microsatellite marker primer and thermal cycling is changed, referring to table 2.
3) determine genotype
Above-mentioned gained PCR reaction product adopts 6% denaturing polyacrylamide gel (6% acrylamide, 0.32% methene acrylamide, 7mol/L urea, 1 * TBE) electrophoresis, electrophoresis 45min under permanent power 80W.After electrophoresis finished, gel placed fixedly 30min of 1% glacial acetic acid; After the water flushing one time, go to staining fluid (AgNO
31g, formaldehyde 1.5ml, 10mmol/LNa
2S
2O
30.2ml, add distilled water to 1L) and middle dyeing 1h; Going to develops in the developing solution (NaOH 5g, formaldehyde 1.5ml add distilled water to 1L) to gel again demonstrates band.According to showing the band result, should individuality be homozygote promptly only on the swimming lane detecting on the locus by 1 allelotrope, should individuality be heterozygote promptly only on the swimming lane detecting on the locus by 2 allelotrope.
4) result
30 tails are waited to screen in artificial induction's the heterogeneous gynogenesis diploid large yellow croaker, have 12 tails all to isozygoty on 10 locus such as LYC0004, LYC0008, LYC0011, LYC0015, LYC0021, LYC0022, LYC0025, LYC0027, LYC0032 and LYC0033.This 12 tail fish is gynogenesis diploid through AFLP paternity test and ploidy analysis conclusive evidence.
The large yellow croaker that 30 tails are cultivated by common production sequence, not detecting at 10 locus such as LYC0004, LYC0008, LYC0011, LYC0015, LYC0021, LYC0022, LYC0025, LYC0027, LYC0032 and LYC0033 is homozygotic individuality simultaneously.
Embodiment 2
1) extraction of template DNA
Press embodiment 1 described method and extract the required dna profiling of PCR reaction.
2) pcr amplification
By embodiment 1 described method steps 2) carry out pcr amplification, amplimer is changed to 5 pairs, is respectively LYC0008, LYC0011, LYC0022, LYC0025 and LYC0033, and sequence and annealing temperature are referring to table 1.
3) determine genotype
Determine idiotype to be measured by the described method of embodiment 1 step 3).
4) result
30 tails are waited to screen in artificial induction's the heterogeneous gynogenesis diploid large yellow croaker, and it all is homozygote at 5 locus such as LYC0008, LYC0011, LYC0022, LYC0025 and LYC0033 that 22 tails are arranged.This 22 tail fish is gynogenesis diploid through AFLP paternity test and ploidy analysis conclusive evidence.
The large yellow croaker that 30 tails are cultivated by common production sequence, not detecting at LYC0008, LYC0011, LYC0022, LYC0025 and 5 locus of LYC0033 is homozygotic individuality simultaneously.
Sequence table
Embodiment 1: template DNA carries out the primer of pcr amplification:
Claims (9)
1. the screening method of meitogynogenetic diploid of pseudosciaena crocea is characterized in that may further comprise the steps:
1) extraction of template DNA: adopt phenol-chloroform-primary isoamyl alcohol method to extract the large yellow croaker genomic dna, as the required dna profiling of PCR reaction;
2) pcr amplification: pcr amplification is carried out in 5～10 of above-mentioned template DNA employings and the closely linked microsatellite marker primer in kinetochore respectively, get the PCR reaction product;
3) determine genotype: with step 2) gained PCR reaction product, carry out electrophoresis with denaturing polyacrylamide gel, after electrophoresis finishes, denaturing polyacrylamide gel is shown band with cma staining, according to showing the band result, if 1 allelotrope is only arranged on the swimming lane, then should on the detection locus, be homozygote by individuality; If on the swimming lane only be 2 allelotrope, then should on the detection locus, be heterozygote by individuality;
4) screening: all detect locus and be the individuality that isozygotys, select and remain as heterogeneous gynogenesis diploid; It is the individuality of heterozygosis that more than one locus is arranged, and mixes individual rejecting as big risk, will not select and remain.
2. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1, it is characterized in that extracting the large yellow croaker genomic dna in phenol-chloroform described in the step 1)-primary isoamyl alcohol method is specially: clip waits to screen large yellow croaker sample tissue 10～20mg, place centrifuge tube, add pH 8.0STE damping fluid 600～1000 μ l and 20～40mg/ml Proteinase K, 10 μ l after shredding, place 50～60 ℃ of digestion 4～10h down; Add 2～6mgml again
-1RNaseA4～6 μ l, mixing are placed on 37～40 ℃ of reaction 1～2h down; Add saturated phenol 400～1000 μ l of pH 8.0Tris-again, the centrifugal 10min of 12000rpm moves on to supernatant liquor in another centrifuge tube behind the mixing, abandons lower floor; Add the saturated phenol-chloroform-primary isoamyl alcohol of equal-volume Tris-(25: 24: 1), the centrifugal 10min of 12000rpm behind the mixing draws supernatant liquor in another centrifuge tube; Add equal-volume chloroform-primary isoamyl alcohol (24: 1), mixing repeatedly, the centrifugal 10min of 12000rpm moves on to supernatant liquor in another centrifuge tube, abandons lower floor; Add the dehydrated alcohol of 2.5 times of volumes, place and precipitate 10min on the trash ice; The centrifugal 5min of 12000rpm, precipitation is with the dissolving of 20 μ l ultrapure waters, as the required dna profiling of PCR reaction.
3. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1, it is characterized in that step 2) in, the reaction system of described pcr amplification is: primer 0.2 μ mol/L, the extracting dna profiling 50～100ng of step 1) institute, Taq archaeal dna polymerase 1u, 10 * PCR reaction buffer, 2 μ l, the dNTPs 2.0 μ l of 2mmol/L, add aseptic deionized water, make cumulative volume reach 20 μ l.
4. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1 is characterized in that in step 2) in, the reaction conditions of pcr amplification is: pre-94 ℃ of 5min of sex change; 30～40 thermal cyclings, each cycling condition is: 94 ℃ of sex change 1min, 50～57 ℃ of annealing 30s, 72 ℃ are extended 45s; 72 ℃ are extended 10min.
5. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1 is characterized in that in step 2) in, the composition of the reaction buffer of described pcr amplification consists of: 100mmol/L Tris-HCl, 100mmol/L KCl, 80mmol/L (NH
4)
2SO
4, 20mmol/L MgCl
2, 0.5%NP-40pH 9.0.
6. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1 is characterized in that in step 3), the consisting of of described denaturing polyacrylamide gel: the 60g acrylamide, 3.2g methene acrylamide, 7mol urea, 10 * TBE100ml, adding distil water is to 1L.
7. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1, it is characterized in that in step 3), described denaturing polyacrylamide gel carries out electrophoretic method: at permanent power 60～90W electrophoresis 45～90min, electrophoretic buffer is 1 * TBE.
8. the screening method of meitogynogenetic diploid of pseudosciaena crocea according to claim 1 is characterized in that in step 3), and the method that described cma staining shows band is specially: gel places fixedly 30min of 1% glacial acetic acid; After the water flushing one time, go to the 1h that dyes in the staining fluid; Go in the developing solution again and develop, treat to take out after gel demonstrates band.
9. as the screening method of meitogynogenetic diploid of pseudosciaena crocea as described in the claim 9, it is characterized in that in step 3) described cma staining shows consisting of with used staining fluid: AgNO
31g, formaldehyde 1.5ml, 10mmol/LNa
2S
2O
30.2ml, add distilled water to 1L; Described cma staining shows consisting of with used developing solution: NaOH 5g, formaldehyde 1.5ml adds distilled water to 1L.
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| CN107345934A (en) * | 2016-05-07 | 2017-11-14 | 中国海洋大学 | A kind of Fast silver-staining of long oyster microsatellite polyacrylamide gel electrophoresis |
| CN111321231A (en) * | 2020-03-14 | 2020-06-23 | 甘肃省水产研究所 | Method for molecularly identifying gynogenesis progeny of rainbow trout |
| CN111394478A (en) * | 2020-04-27 | 2020-07-10 | 西安理工大学 | PCR (polymerase chain reaction) microsatellite primer and method for paternity test of large yellow croaker by using same |
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2009
- 2009-06-26 CN CN2009101120828A patent/CN101597648B/en not_active Expired - Fee Related
Cited By (7)
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| CN107345934A (en) * | 2016-05-07 | 2017-11-14 | 中国海洋大学 | A kind of Fast silver-staining of long oyster microsatellite polyacrylamide gel electrophoresis |
| CN106191292A (en) * | 2016-08-18 | 2016-12-07 | 宁德市富发水产有限公司 | A kind of method cultivating Carnis Pseudosciaenae seed and the screening molecular marker used thereof |
| CN106191292B (en) * | 2016-08-18 | 2019-07-16 | 宁德市富发水产有限公司 | A kind of method for cultivating Larimichthys crocea seed and its used screening molecular labeling |
| CN111321231A (en) * | 2020-03-14 | 2020-06-23 | 甘肃省水产研究所 | Method for molecularly identifying gynogenesis progeny of rainbow trout |
| CN111321231B (en) * | 2020-03-14 | 2023-07-21 | 甘肃省水产研究所 | Method for molecular identification of rainbow trout gynogenesis offspring |
| CN111394478A (en) * | 2020-04-27 | 2020-07-10 | 西安理工大学 | PCR (polymerase chain reaction) microsatellite primer and method for paternity test of large yellow croaker by using same |
| CN111394478B (en) * | 2020-04-27 | 2022-06-17 | 西安理工大学 | PCR (polymerase chain reaction) microsatellite primer and method for paternity test of large yellow croaker by using same |
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