CN103725790A - Molecular marker relevant to growth of fenneropenaeus chinensis and application of molecular marker - Google Patents
Molecular marker relevant to growth of fenneropenaeus chinensis and application of molecular marker Download PDFInfo
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Abstract
The invention aims to provide a molecular marker relevant to growth of fenneropenaeus chinensis and an application of the molecular marker. The molecular marker is a SNP (Single Nucleotide Polymorphism) locus in a FaMeT gene of the fenneropenaeus chinensis, which is relevant to the growth trait of the fenneropenaeus chinensis. The SNP locus is the 364th site of the FaMeT gene with a nucleotide sequence shown in SEQ ID NO:1, wherein the basic group of the SNP locus is G or T. The SNP locus is obtained by screening the GG-type fenneropenaeus chinensis; the weight and the head cuirass width of the SNP locus are both higher than those of a GT genotype individual. Thus, the GG-type individual can be preferentially selected as a breeding parent according to the SNP locus during the growth breeding selection process of the fenneropenaeus chinensis, thereby improving the efficiency of breeding selection.
Description
Technical field
The invention belongs to molecule marker Biotechnology in Genetic Breeding field, be specifically related to a kind of and molecule marker and application Chinese prawn growth correlation.
Background technology
Chinese prawn (Fenneropenaeu chinensis) is the Main economic shrimp in the yellow Bohai Sea, culture zone mainly concentrates on the coastal zone along the Huanghai Sea and the Bohai Sea of northern China, as the marine treasure having long enjoyed a good reputation, at home and abroad market has higher reputation, is subject to human consumer's favor.Nineteen ninety once accounted for 70% of China's prawn culturing output.But after 1993, due to the impact of germplasm, disease and environmental factors, cultured output sharply declines, 10% left and right of current production rate Jin Zhan China prawn output.Cultivate the basis that growth is quick, economic characters are good, improved seeds strong stress resistance have become the sustainable development of guarantee Culture of Penaeus Chinensis industry.
Seed selection is most important, one of the most effective means of Chinese prawn breed improvement at present, and the blending of traditional breeding technology and modern biotechnology advances animals and plants breeding the reach of science from depth and broadness.Molecular marking supplementary breeding is to utilize modern molecular biology technique from gene level, to accelerate the method for seed selection, can greatly shorten seed selection from generation to generation, reduces costs, and greatly improves Breeding Efficiency and benefit, has been subject to increasing attention.
Utilizing molecule marker to carry out the location of important economical trait, is the important foundation research of molecular breeding.SNPs is the abbreviation of Single Nucleotide Polymorphisms, i.e. single nucleotide polymorphism, and it refers to the polymorphism causing due to the replacement of single core thuja acid (A, T, C, G) in genomic dna sequence.In vegeto-animal genome, exist a large amount of SNPs, in any known or unknown gene or near all may find quantity SNPs not etc., it is the most extensive and stable point mutation that distributes in genome, for large-scale research, it is more reliable than microsatellite marker and other tumor-necrosis factor glycoproteins.
Most of vegeto-animal growths, quality, resistance etc. are all quantitative characters, and quantitative character is not only subject to minor-polygene control, but also is subject to the impact of one or several major gene.Key-gene is relative minor-polygene, and it refers to individual gene or the site that can produce to quantitative character huge effect.By association analysis, identify the relation of a certain colony internal object proterties and genetic marker or candidate gene, find out the candidate function SNP site associated with quantitative character.
Nowadays, this part is operated in the breeding researches of the herding class animals such as ox, sheep, pig, chicken extensively carries out, utilize major gene sequence polymorphism to carry out molecular mark, cultivate growth and accelerate, the new variety that production performance improves obtain the attention of breeding circle day by day.For example, Zhou(2005) and Sadeghi etc. (2008) adopt PCR-RFLP method to analyze respectively the polymorphism of Holstein bull growth hormone gene and STAT5A gene, base mutation and milk production trait that discovery lays respectively at growth hormone gene the 3rd intron and is arranged in STAT5A the 7th exon are significant correlation (P<0.01, P<0.082); Zhao Wen waits clearly (2008) to adopt PCR-SSCP to analyze seed goose growth hormone gene polymorphism, thinks that the base mutation of the 3rd intron and early livewerght gain and Carcass Traits exist cognation.But in Chinese prawn, also lack the report with growth traits mark of correlation.
Summary of the invention
The object of this invention is to provide a kind of and molecule marker and application Chinese prawn growth correlation, the i.e. SNP site relevant to Chinese prawn growth traits of in Chinese prawn FaMeT gene, this SNP site can be used for carrying out marker assisted selection and the application of Chinese prawn growth traits.
The molecule marker of of the present invention and Chinese prawn growth correlation, is the 364th of the nucleotides sequence FaMeT gene of classifying SEQ ID NO:1 as, and its base is G or T.
Molecule marker of the present invention is for the auxiliary genetic breeding of mark of Chinese prawn growth traits;
For detection of the primer of above-mentioned mark, be G364 and 364CP; Or T364 and 364CP; Concrete primer information is as follows:
G364：GCTACAGCACTGGCTTAGG（SEQ?ID?NO:2）
T364：GCTACAGCACTGGCTTAGT（SEQ?ID?NO:3）
364CP：ACATGGGTGTGGTCTCCTCAGG（SEQ?ID?NO:4）
The screening method of the molecule marker of Chinese prawn growth correlation of the present invention, comprises following step:
1) utilize amplimer P1(SEQ ID NO:5) and P2(SEQ ID NO:6) from Chinese prawn genomic dna, increasing obtains the fragment that length is 716bp;
2) amplified fragments obtaining from different Chinese prawns is checked order, filter out base mutation site;
3) for each mutational site design AS-PCR genotyping primer, adopt polyacrylamide gel electrophoresis, individuality in Chinese prawn cultured population at sunshine is carried out to genotype detection, utilize the generalized linear model GLM of SPSS13.0 software to carry out method of least squares estimation to the relation between the growth traits values such as Chinese prawn body weight, body length, body length, uromere length, periproct length, body width and genotype.
4) adopt the difference of body weight and morphological characters between method of least squares and multiple comparison analyse colony at sunshine different genotype, final determine and prawn body weight, carapace width between there is the SNP site of significant correlation (P < 0.05).
The SNP site of the present invention's screening is the Chinese prawn of GG type, its body weight and carapace width are all significantly higher than GT genotype individuality, in the growth Breeding Process of Chinese prawn, and can be for this site, the preferential individuality of GG type of selecting is as breeding parent, to improve the efficiency of seed selection.
Accompanying drawing explanation
Fig. 1: obtained the agarose electrophoresis figure of the FaMeT Gene Partial fragment that length is 716bp in the present invention by primer P1 and P2, M is Marker.
Fig. 2: detect genotypic electrophoresis result by specific gene type serotype specific primer in the present invention.Swimming lane 1-2,3-4 are respectively AA, the AG genotype in 135A/G site; 5-6,7-8 are respectively GG, the TT genotype in 364G/T site; 9-10,11-12,13-14 are respectively AA, AG, the GG genotype in 680A/G site.
Embodiment
Below in conjunction with example, method of the present invention is described further.The experimental technique of unreceipted actual conditions in the following example, conventionally condition routinely, condition described in the < < molecular cloning experiment guide > > writing as J. Pehanorm Brooker (Sambrook) etc., or the condition of advising according to manufacturer operation.Those skill in the art related can understand better and grasp the present invention by embodiment.
One, Chinese prawn FaMeT gene cDNA sequence pcr amplification and polymorphic detection
1. Chinese prawn FaMeT gene DNA sequence pcr amplification
Utilize Chinese prawn FaMeT gene mRNA in NCBI (GenBank Accession No.:JQ315119) sequence, design specific amplification primer P1 and P2, take 30 tail Chinese prawn genomic dnas as template, at Taq archaeal dna polymerase, buffer environment, Mg
2+, dNTPs exist situation under, utilize primer P1 and P2 to increase under PCR condition, product purification and order-checking, obtain length be 716bp fragment.
Described primer sequence is as follows:
Upstream primer P1:CCGATGAGAACAAGCAGTA(SEQ ID NO:5)
Downstream primer P2:ATCGCTGTCCTGGTATCC(SEQ ID NO:6)
The condition of described pcr amplification is: 20 μ L reaction systems, comprising: 50ng/ μ L DNA profiling 1 μ L; 10 × PCR Buffer2 μ L; The each 0.8 μ L of upstream and downstream primer of primer P1, the P2 of 10 μ mol/L; 1.6 μ LdNTP(2.5mM each), 0.2 μ L(5U/ μ L) Taq DNA polymerase; Ultrapure water 15.6 μ L.
Described pcr amplification reaction program is as follows:
95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 60s, carry out altogether 32 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.
2. sequence reclaims, connects, transforms and order-checking
PCR product reclaims and adopts miniprep dna to reclaim test kit, purchased from Shanghai, gives birth to work:
By agarose gel electrophoresis, as far as possible target DNA fragment and other fragments are separated, with clean knife blade, by cutting containing the sepharose piece of target DNA fragment, put into 1.5ml centrifuge tube and weigh; According to the weight of blob of viscose and concentration, the ratio that adds 300-600ul in every 100mg agarose adds Buffer B2; Centrifuge tube is placed in to 50 ℃ of water-bath 5-10min, or mix, until blob of viscose melts completely; The solution having dissolved is all moved into adsorption column, and the centrifugal 30s of 8000g, outwells the liquid in collection tube, and adsorption column is put into same collection tube; In adsorption column, add 500ul Wash Solution, the centrifugal 30s of 9000g, outwells the liquid in collection tube, and adsorption column is put into same collection tube; Repeat above-mentioned steps once; Suction attached column and collection tube are put into whizzer, the centrifugal 1min of 9000g; In adsorption film central authorities, add 15-40ulElution Buffer, the standing 1-2min of room temperature, the centrifugal 1min of 9000g, is placed in-20 ℃ of preservations by the DNA solution of gained.
Ligation: purified pcr product is connected with pMD18-T carrier (purchased from precious biotechnology (Dalian) company limited), ligation cumulative volume is 5 μ L, comprising Solution I2.5 μ L, pMD18-T0.5 μ L, purified pcr product 2 μ L, 4 ℃ are spent the night.
The preparation of competent cell: mono-colony inoculation of DH5 α of picking is in 2ml LB from 37 ℃ of fresh flat boards of having cultivated 16-20h, in 37 ℃ of shaking culture 3h, switching 1ml bacterium liquid is in the saline bottle that contains 30ml LB, continuation is at 37 ℃ of about 4h of shaking culture, when OD600 reaches 0.3-0.4, saline bottle is taken out from shaking table, the cooling 10-15min of ice bath, then bacterium liquid is proceeded in centrifuge tube in 4 ℃ of centrifugal 10min of 4000rpm, discard nutrient solution, by the resuspended precipitation of CaCl2 of the 0.1mol/l of 10ml ice precooling, ice bath 30min, repeat 4 ℃ of centrifugal 10min of 4000rpm, by the resuspended precipitation of CaCl2 of the 0.1mol/l of 4ml ice precooling, putting 4 ℃ saves backup.
Transform: under sterile state, get 100-120 μ L competent cell in 1.5ml centrifuge tube, the connection product of 5 μ L is added and mixed, on ice, place 30min, 42 ℃ of heat shock 90s, do not shake centrifuge tube therebetween, take out rear ice bath 3-4min, add the LB liquid nutrient medium of 400 μ L antibiotic-frees, 37 ℃ of shaking culture 45min.Get 100 μ L and coat 4h in advance and be coated with on the agar plate of isopropylthio-β-D-galactoside (IPTG) and X-gal, be inverted cultivation after keeping flat 1h for 37 ℃.
Cloning and sequencing is single positive colony of picking for order-checking, and sequencing is completed by Beijing AudioCodes biotechnology limited liability company, and each gene fragment is at least surveyed two clones.In theory, the nucleotides sequence of amplified fragments is classified SEQ ID NO:1 as.
3. Chinese prawn FaMeT gene base mutation site is detected
By the FaMeT sequencing fragment being obtained by 30 tail Chinese prawns, the sequence obtaining is carried out to sequence alignment, search single nucleotide mutation site.In 30 tail Chinese prawn samples, find altogether 3 base mutation sites, lay respectively at the 135th A/G of sequence SEQ ID NO:1, corresponding amino acid is by Ile-Val; 364 G/T, corresponding amino acid is by Gly-Val; 680 A/G, the amino acid of coding does not change.
Two, SNP serotype specific primer design
According to the above results, design genotyping primer, as probe, to detect the population material of larger quantity, and carries out association analysis with Chinese prawn growth traits.The SNP site obtaining according to order-checking, design AS-PCR(allele-specific polymerase chain reaction, allele specific pcr) primer.While being used for detecting SNP site, for two allelotrope, design respectively detection primer.Detect 3 ' of (somatotype) primer and hold last base generally corresponding respectively with the allelotrope of SNP, thereby the band of amplification is corresponding with corresponding allelotrope, reaches the object that SNP detects.In allele specific amplification reaction, at 3 ' end of pcr amplification primer, introduce and do not mate base, can part reduce the efficiency of amplification, but can optionally make goal gene matrix section effectively be increased.Based on this principle, detect each FaMeT gene mutation site two articles of Auele Specific Primers 3 ' hold third from the bottom or the 4th all introduced one and do not mated base, the specificity of primer extension is improved greatly, reduced the false positive that detects FaMeT transgenation with allele-specific PCR.According to different tests situation, AS-PCR can be divided into single tube and two-tube two kinds of methods, and this research adopts two-tube AS-PCR: three primers of each site design, two ASP respectively from the complementation of different SNP template, to expanding (table 1), polyacrylamide gel electrophoresis detects PCR product with a CP.In addition, in whole PCR reactions, increased the fragment of a 110bp of FaMeT gene as internal reference, in order to the integrity of judgement sample DNA and the quality of PCR simultaneously.Serotype specific primer for the design of each mutational site is as follows:
Related primer sequence in table 1:AS-PCR
Pcr amplification condition:
PCR reaction cumulative volume 10 μ L, wherein Chinese prawn genomic dna template 1 μ L, distilled water 7.1 μ L, 10 × buffer1 μ L, the each 0.3 μ L of 10mM forward primer and reverse primer, dNTP0.2 μ L, Taq enzyme 0.1 μ L (5U/ μ L).PCR reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 15s, circulate 32 times, and 72 ℃ are extended 5min.
Three, the genotype detection of the Chinese prawn FaMeT of colony at sunshine gene and with the association analysis of Chinese prawn growth traits
Utilize the allele-specific round pcr of setting up to carry out pcr amplification to 100 parts of genomic dnas of Chinese prawn colony at sunshine.The PCR product of getting 5 μ L mixes with 1 μ L sample-loading buffer, is splined on 5% non-denaturing polyacrylamide gel, the about 2h of the permanent power electrophoresis of 80W.After electrophoresis finishes, add in 0.1% silver nitrate solution, jog 10min on shaking table, then uses distilled water wash gel 2 times, adds nitrite ion (NaOH of 5g, the Na of 0.1g
2cO
3, add 0.5mL formaldehyde, be settled to 250mL), jog 15min on shaking table, then uses distilled water wash gel 2 times, the preservation of taking pictures.
Electrophoresis detection result: wherein 135A/G can detect AA, two kinds of genotype of AG; 364G/T can detect GG, two kinds of genotype of GT; 680A/G can detect AA, AG and tri-kinds of genotype of GG.
Gene and genotype frequency are calculated according to Hardy-Weinberg equilibrium law,
wherein d=e-o is the poor of expected value and observed value.Wherein p, q represent that, to the gene frequency on locating point, P, Q represent homozygous genotype frequency, and H represents the genotype frequency of heterozygote.Utilize the generalized linear model GLM of SPSS13.0 software to carry out method of least squares estimation to the relation between each character value and genotype, P < 0.05 thinks that there were significant differences.χ
2assay shows that the genotype frequency in five SNP sites is all in Hardy-Weinberg equilibrium state (P > 0.05) (table 2).
Table 2: the genotype in different SNP site and gene frequency (n=100)
Between employing method of least squares and multiple comparison analyse colony at sunshine different genotype, the difference of body weight and morphological characters, the results are shown in Table 3.Statistical result showed, between the each genotype of site 364G/T and Chinese prawn body weight, carapace width, there is significant correlation (P < 0.05), the individuality of genotype GG will be significantly higher than GT genotype individuality, not remarkable (P > 0.05) of difference between all the other each genotype in SNP site and Chinese prawn growth traits.Think thus, the molecule marker that Chinese prawn growth traits is selected can be served as in the SNPs site that adopts primer 364G and 364T to detect.
Table 3: the impact of the different polymorphic sites in five SNP sites on growth traits
Note: have same letter to represent not remarkable (P > 0.05) of difference in same column data shoulder mark, represent significant difference (P < 0.05) without same letter.
The present invention is directed to growth traits and carry out SNP screening, the 364GG genotype individuality screening shows obvious growth vigor in body weight and carapace width index, therefore, can be in the Breeding Process of Chinese prawn, the preferential individuality of GG type of selecting is as breeding parent, molecular marker assisted selection is combined with conventional genetic breeding seed selection mode, is expected to greatly improve the efficiency of seed selection.
Claims (5)
1. with the SNP site of Chinese prawn growth correlation, it is characterized in that, described SNP site is positioned at nucleotides sequence classifies the 364th of gene of SEQ ID NO:1 as, and its base is G or T.
2. the application of SNP claimed in claim 1 site in the auxiliary genetic breeding of mark of Chinese prawn growth traits.
3. for detection of the primer in SNP claimed in claim 1 site, it is characterized in that, the nucleotides sequence of described primer is classified SEQ ID NO:2 and SEQ ID NO:4 as; Or SEQ ID NO:3 and SEQ ID NO:4.
4. the screening method in SNP claimed in claim 1 site, comprises following step:
1) utilize amplimer P1 and P2 to increase from Chinese prawn genomic dna and obtain the fragment that length is 716bp;
2) amplified fragments obtaining from different Chinese prawns is checked order, filter out base mutation site;
3) for each mutational site design AS-PCR genotyping primer, adopt polyacrylamide gel electrophoresis, individuality in Chinese prawn cultured population at sunshine is carried out to genotype detection, utilize the generalized linear model GLM of SPSS13.0 software to carry out method of least squares estimation to the relation between the growth traits values such as Chinese prawn body weight, body length, body length, uromere length, periproct length, body width and genotype.
4) adopt the difference of body weight and morphological characters between method of least squares and multiple comparison analyse colony at sunshine different genotype, final determine and prawn body weight, carapace width between there is the SNP site of significant correlation (P < 0.05).
5. screening method as claimed in claim 4, is characterized in that the nucleotides sequence of described amplimer P1 is classified SEQ ID NO:5 as, and the nucleotides sequence of amplimer P2 is classified SEQ ID NO:6 as.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878004A (en) * | 2015-06-18 | 2015-09-02 | 广东海洋大学 | SNP marking detection associated with growth trait of haliotis diversicolor and its application |
| CN106834439A (en) * | 2016-12-26 | 2017-06-13 | 中国科学院海洋研究所 | A kind of related molecular labeling of Growth of Litopenaeus vannamei and its application |
| CN108184736A (en) * | 2018-03-06 | 2018-06-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of live body sampling without damage method of Procambius clarkii and application |
| CN110684776A (en) * | 2019-09-12 | 2020-01-14 | 中国水产科学研究院南海水产研究所 | Penaeus monodon Na +/K +/2 Cl-co-transporter NKCC gene and application thereof |
| CN111926020A (en) * | 2020-07-24 | 2020-11-13 | 中国科学院海洋研究所 | Two prawn growth related genes and application thereof in genetic breeding |
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2014
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878004A (en) * | 2015-06-18 | 2015-09-02 | 广东海洋大学 | SNP marking detection associated with growth trait of haliotis diversicolor and its application |
| CN104878004B (en) * | 2015-06-18 | 2018-11-16 | 广东海洋大学 | One kind SNP marker detection associated with haliotis diversicolor Reeve growth traits and its application |
| CN106834439A (en) * | 2016-12-26 | 2017-06-13 | 中国科学院海洋研究所 | A kind of related molecular labeling of Growth of Litopenaeus vannamei and its application |
| CN108184736A (en) * | 2018-03-06 | 2018-06-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of live body sampling without damage method of Procambius clarkii and application |
| CN110684776A (en) * | 2019-09-12 | 2020-01-14 | 中国水产科学研究院南海水产研究所 | Penaeus monodon Na +/K +/2 Cl-co-transporter NKCC gene and application thereof |
| CN110684776B (en) * | 2019-09-12 | 2020-10-27 | 中国水产科学研究院南海水产研究所 | Penaeus monodon Na+/K+/2Cl-Cotransporter NKCC gene and application thereof |
| CN111926020A (en) * | 2020-07-24 | 2020-11-13 | 中国科学院海洋研究所 | Two prawn growth related genes and application thereof in genetic breeding |
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Application publication date: 20140416 |