CN105969882B - One kind haplotype SNP marker relevant to channel catfish fast-growth and its detection method and application - Google Patents
One kind haplotype SNP marker relevant to channel catfish fast-growth and its detection method and application Download PDFInfo
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Abstract
The invention patent belongs to aquatic livestock molecular mark field, and in particular to one kind haplotype SNP marker relevant to channel catfish fast-growth and its detection method and application.The SNP marker is obtained from channel catfish PRL gene cloning, and the nucleotide sequence of the molecular labeling is as shown in sequence table SEQ ID NO:1 and SEQ ID NO:2.Application of the above-mentioned molecular labeling in channel catfish growth traits Molecular Detection is also disclosed in the present invention.Screening-gene type is respectively the individual of AA, CC, TT, CC, AA, i.e., haplotype combination is the individual of H3/H3 (ACTCA/ACTCA) as breeding parent.Method of the invention, it is easy to operate, detection speed is fast, greatly reduces the blindness of channel catfish parental line selection, it can be quickly obtained that the speed of growth is fast and the channel catfish parent of inheritance stability.
Description
Technical field
The invention patent belongs to aquatic livestock molecular mark field, and in particular to a kind of fast with channel catfish
The long relevant haplotype SNP marker of fast-growing and its detection method and application.
Background technique
Channel catfish (Ictalures punctatus) originates in the U.S., is a kind of important economic species of aquatic products,
Introducing China in 1984 has the characteristics that meat is fine and smooth, suitable temperature range is wide, the speed of growth is fast, disease resistance is strong.However, with
The fast development of China's channel catfish aquaculture industry, it is uneven to have had already appeared apparent decreased growth, body-color diversity and specification
Equal germplasm decay.Therefore, the channel catfish new varieties (being) that growth is fast, economic characters are good, resistance is strong are cultivated to have become
For current channel catfish industry sustainable development major issue urgently to be resolved.
Prolactin (Prolactin, PRL) is that the single chain polypeptide class for being synthesized and being secreted by vertebrate gene expression swashs
Element has different physiological roles, relates generally to osmotic adjustment, growth and development, immune, endocrine and metabolic regulation etc..Stimulating milk secretion
Element and growth hormone (Growth hormone, GH) and somatomammotropin (Somatolactin, SL) have similar structure and
Function, newest molecular evolution is research shows that GH gene, PRL gene and SL gene are likely to by ancient GH gene through polygenes
Copy and then very long evolutionary process are evolved.PRL gene has been used as the candidate gene of growth traits, SNPs and
The association analysis of SSR polymorphism and growth traits has had more report in poultry, herding and aquatic livestock.Tilapia mossambica
One site SSR of the gene promoter area PRL1 grows in different salinity level to it significant related.On the perch PRL gene of Asia
The growth traits such as one SNP site (c.264+269T > C) and its weight, body be long are significant related, and genotype is the body of CC individual
Quality and the long conspicuousness of body are greater than the individual that genotype is CT and TT.Therefore, in channel catfish find identification PRL gene with
The associated SNPs of growth traits and haplotype molecular labeling be fast to the breeding speed of growth and the channel catfish parent of inheritance stability
Originally have great importance.
Summary of the invention
The object of the present invention is to provide a kind of haplotype SNP marker relevant to channel catfish fast-growth and its
Using, that is, utilize channel catfish PRL gene a haplotype SNP marker relevant to channel catfish growth traits
Carry out the screening and identification of fast-growth parent.
The invention patent is adopted the technical scheme that:
The present invention provides a kind of haplotype SNP marker relevant to channel catfish fast-growth character, described
The nucleotide sequence of haplotype SNP marker is as shown in SEQ ID NO.1 and SEQ ID NO:2, in SEQ ID NO.1 sequence
The the 296th, 362,623 base at be respectively present A/T, C/T, C/T 3 mutation；The 94th of SEQ ID NO.2 sequence the
Be respectively present at 788 bases C/T and A/G 2 mutation.
It is a kind of for detecting the primer of above-mentioned haplotype SNP marker, which includes PRLP5~13；The PRLP5
~13 DNA sequence dna is as shown in NO:7~15 SEQ ID.
It is a kind of for detecting the kit of above-mentioned haplotype SNP marker, which includes above-mentioned primer PRLP5
~13.
Haplotype SNP marker, primer or kit of the invention is in quick screening and identification growth channel catfish
Application in parent.
The screening of fast-growth channel catfish candidate parent a kind of and identification method include the following steps: to detect spot
Sequence shown in SEQ ID NO:1 and SEQ ID NO:2 in Cha Wei Channel-catfish core selective breeding group, to every tail in core selective breeding group
Each SNP site of channel catfish candidate parent carries out parting, and selection has haplotype combination H3/H3 (ACTCA/ACTCA)
Individual as breeding parent.
Specifically comprise the following steps:
(1) the clip channel catfish fin ray sample to be measured from core selective breeding group extracts genomic DNA.
(2) it to extract obtained genomic DNA as template, is carried out using primer PRLP5/PRLP6, PRLP7/PRLP8
SNP site PCR amplification, PRLP5/PRLP6 for expand site g.296A > T, g.362C > T and g.623C > T, PRLP7/PRLP8
For expand site g.94C > T and g.788A > G, obtain first PCR product.Primer PRLP5, PRLP6, PRLP7 and PRLP8's
DNA sequence dna is as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
(3) after purification by amplification obtains in step (2) PCR product, respectively using primer PRLP9, PRLP10,
It after PRLP11, PRLP12 and PRLP13 carry out extension, is tested and analyzed, is determined on the automatic genetic analysis systems of ABI
The genotype of each SNP site of candidate parent to be measured.The DNA sequence of primer PRLP9, PRLP10, PRLP11, PRLP12 and PRLP13
Column are as shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
(4) haplotype combination is H3/H3 (ACTCA/ by the haplotype combination for analyzing every tail candidate blob Cha Wei Channel-catfish parent
ACTCA Juvenile stage), which comes out, reserves seed for planting in case subsequent breeding.
SNP site PCR amplification uses instant UtraTaq enzyme PCR kit (Shanghai Jierui Biology Engineering Co., Ltd),
Reaction system are as follows:
Amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, anneal 30s, 72 DEG C of extension 60s, 30 circulations；72
DEG C extend 10min.
SNPs parting uses ABI SNaPshot Multiplex PCR kit (ABI, the U.S.), and reaction system is 6 μ L:
Reaction condition are as follows: 96 DEG C of initial denaturation 1min；96 DEG C of denaturation 10s, 52 DEG C of annealing 5s, 60 DEG C of extension 30s, totally 30 are followed
Ring.
1U FastAP is added after reaction, 37 DEG C of 60min, 85 DEG C of 15min are purified.With GeneScan-120Liz
Size Standard is internal standard, and 1 μ L product is taken to mix with the Hi-Di (GS-120Liz:Hi-Di=1:200) that 9 μ L contain Liz,
95 DEG C of denaturation 3min go up sequenator immediately after ice bath 3min.Detection and analysis are automatically hereditary using ABI PRISM 3730XI type
Analysis system (ABI, the U.S.), and parting is carried out using Genemapper v4.1 software.
There are 3 kinds of genotype through multiple Snapshot method parting, each site in 5 sites SNPs.Utilize SHEsis
Software analyzes the haplotype type and frequency of this 5 SNPs generation, and R software carries out the association analysis between genotype and character.Knot
Fruit shows, in 5 SNPs, g.623C>T and g.788A>G have a significant impact (P<0.05) to measured growth traits, 5
The haplotype of site reconstruct, different haplotype combinations all have a significant impact (P < 0.05) to measured growth traits, wherein
The weight and body long value of H3/H3 (ACTCA/ACTCA) haplotype combination are maximum.Retaining haplotype combination in actual production is
The channel catfish parent of H3/H3 (ACTCA/ACTCA) can obtain the speed of growth fastly and the channel catfish product of inheritance stability
System.This method has that purpose is strong, easy to operate, detection speed is fast compared to traditional selection, easy to spread etc. excellent
Point.
Method of the invention greatly reduces the blindness of channel catfish parental line selection, shortens the breeding time limit, can be fast
Speed obtains the speed of growth fastly and the channel catfish parent of inheritance stability.
Detailed description of the invention
Fig. 1 is the location of PRL gene schematic diagram and 5 sites SNPs
Wherein, primer pair PRLP5/PRLP6 amplify SNP site g.296A > T, g.362C > T and g.623C > T, primer pair
PRLP7/PRLP8 amplify SNP site g.94C > T and g.788A > G, except SNP site g.788A > G, remaining SNP site is in
Include subregion.
Specific embodiment
Embodiment 1
1.1 experimental animal
Channel catfish sample standard deviation used in this experiment is from the proving ground Jiangsu Fresh Water Aquatic Product Inst. Lu Kou.
1997-2004 from the U.S. introduce Texas (1997) group, Arkansas (1999) group, Mississippi (2001) group,
Arkansas (2003) group and 405 tail of Arkansas (2004) group establish breeding basic population, carry out G0 generation using basic population
Family building.In June, 2013 constructs G1 for family, in order to reduce the shadow that environment grows channel catfish for family using G0
It rings, seed rearing is carried out according to same standard, i.e., uniform changes water speed rate, feeding volume, cultivation density, oxygenation capacity and water temperature.
Until scanning record living individuals number when family mean age in days is 520d acquires the tail fin tissue of every tail fish, the leaching of 95% alcohol
Bubble, -20 DEG C of preservations.
In the present invention, the channel catfish growth traits specifically includes that weight and weight.
1.2 design of primers
According to the channel catfish PRL complete genome sequence (AF267990.1) of GenBank warehouse publication, Primer is used
5 software design two of Premier to primer PRLP1/PRLP2, PRLP3/PRLP4, amplified production size be respectively 1762bp and
1776bp。
PRLP1:5’-TGTTGGATTGGTCTGGAGTGG-3’(SEQ ID NO:3)
PRLP2:5’-CGTCTACTCTCCACCTGCTTC-3’(SEQ ID NO:4)
PRLP3:5’-GGATCACGTTTCCGTTCTCTCT-3’(SEQ ID NO:5)
PRLP4:5’-AGAAGGTTCCTCCCAGCCATA-3’(SEQ ID NO:6)
1.3PCR amplification
PCR amplification uses instant UtraTaq enzyme PCR kit (Shanghai Jierui Biology Engineering Co., Ltd), reactant
System are as follows:
Amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, anneal 30s, 72 DEG C of extension 2min, 30 circulations；
72 DEG C of extension 10min.1% agarose gel electrophoresis detects PCR product, and gel imager observes electrophoresis result.
1.4 sequencing
Ultraviolet lamp incision glue purification recycles PCR product, and the PCR product of each pair of primer is respectively chosen 12 samples, sent to Shanghai
Sheng Gong bioengineering limited liability company is directly sequenced using ABI 3730XL sequenator (ABI, the U.S.).It is soft using chromas
Part observation sequencing peak figure carries out DNA sequence dna comparison in conjunction with ClustalX software to judge the base type of SNP site.
1.5SNPs parting
The site SNPs PCR amplification is carried out using primer PRLP5/PRLP6, PRLP7/PRLP8, PRLP5/PRLP6 is for expanding
Increase site g.296A > T, g.362C > T and g.623C > T, PRLP7/PRLP8 for expand site g.94C > T and g.788A > G, obtain
To first PCR product.
PRLP5:5 '-TATCAGGGTGGCGAAAGACAG-3 ' (SEQ ID NO:7),
PRLP6:5'-GATGCTCTTCACGATGCTCCT-3'(SEQ ID NO:8)；
PRLP7:5 '-CACCTAATGTCAGTGAGCGAGTA-3 ' (SEQ ID NO:9),
PRLP8:5’-GAAGGTTCCTCCCAGCCATAC-3’(SEQ ID NO:10)。
Respectively using primer PRLP9, PRLP10, PRLP11, PRLP12 and PRLP13 carry out extension after, ABI from
It is tested and analyzed on dynamic genetic analysis systems, determines the genotype in each site of candidate parent to be measured.
PRLP9:5 '-TTTTTTACAGAGCTGTGCTTTATCCTCG-3 ' (SEQ ID NO:11),
PRLP10:5 '-TTTTACATTCCTACTTGTGAACAGTA-3 ' (SEQ ID NO:12),
PRLP11:5 '-CGCGAGACGTGGCGAGGAGACG-3 ' (SEQ ID NO:13),
PRLP12:5 '-TGTACCACCATGCTGCCACCAT-3 ' (SEQ ID NO:14),
PRLP13:5’-ACAGAGCTGTGCTTTATCCTCG-3’(SEQ ID NO:15)。
The site SNPs PCR amplification uses instant UtraTaq enzyme PCR kit (the limited public affairs of Shanghai JaRa bioengineering
Department), reaction system are as follows:
Amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, anneal 30s, 72 DEG C of extension 60s, 30 circulations；72
DEG C extend 10min.
SNPs parting uses ABI SNaPshot Multiplex PCR kit (ABI, the U.S.), and reaction system is 6 μ L:
Reaction condition are as follows: 96 DEG C of initial denaturation 1min；96 DEG C of denaturation 10s, 52 DEG C of annealing 5s, 60 DEG C of extension 30s, totally 30 are followed
Ring.
1U FastAP is added after reaction, 37 DEG C of 60min, 85 DEG C of 15min are purified.With GeneScan-120Liz
Size Standard is internal standard, and 1 μ L product is taken to mix with the Hi-Di (GS-120Liz:Hi-Di=1:200) that 9 μ L contain Liz,
95 DEG C of denaturation 3min go up sequenator immediately after ice bath 3min.Detection and analysis are automatically hereditary using ABI PRISM 3730XI type
Analysis system (ABI, the U.S.), and parting is carried out using Genemapper v4.1 software.
1.6 haplotype building and association analysis
The haplotype type and frequency of this 5 SNPs generation are analyzed using SHEsis software, R software carries out genotype and property
Association analysis between shape is calculated using generalized linear model (GLM).Concrete model: Yij=μ+Gi+eij, in formula: Yij
For the individual observation of i-th of some character, j-th of label；μ is the average value (i.e. population mean) of all individuals of experimental observation；
Gi is the effect value of i-th of label；Eij is the random residual effect corresponding to observed value.
1.7 interpretation of result
Sequence alignment result is shown on PRL gene and detects 5 SNPs altogether, respectively g.296A > T, g.362C > T,
G.623C > T, g.94C > T and g.788A > G, are respectively positioned on noncoding region.Wherein g.296A > T, g.362C > T and the equal position g.623C > T
In intron 2, g.94C > T is located at the 4th introne, and g.788A > G is located at 3 ' end flanking sequences, as shown in Fig. 1.This 5
There are 3 kinds of genotype through multiple Snapshot method parting, each site in the site SNPs.5 sites SNPs reconstruct to form 7 kinds
Haplotype generates 10 kinds of haplotype combinations.Association analysis is the results show that different haplotype combinations have significant shadow to growth traits
It rings (P < 0.05), wherein the weight of H3/H3 (ACTCA/ACTCA) haplotype combination and body long value are maximum.Therefore, PRL gene
5 SNPs constitute haplotype molecular labeling can apply in channel catfish molecular marking supplementary breeding.
The association analysis of table 1 channel catfish PRL gene haplotype combination and growth traits
Note: different lowercase letter indication differences are significant (P < 0.05), different capitalizations indicate difference it is extremely significant (P <
0.01)。
Claims (7)
1. a kind of haplotype SNP marker relevant to channel catfish fast-growth, it is characterised in that: the haplotype
The nucleotide sequence of SNP marker is as shown in SEQ ID NO:1；It is punished in the 296th, 362,623 base of the sequence
Not there are A/T, C/T, C/T 3 mutation.
2. a kind of haplotype SNP marker relevant to channel catfish fast-growth, it is characterised in that: the haplotype
The nucleotide sequence of SNP marker is as shown in SEQ ID NO:2；It is deposited respectively at the 94th and 788 of sequence base
It is mutated in C/T and A/G 2.
3. a kind of for detecting the primer of the as claimed in claim 1 or 22 haplotype SNP markers, it is characterised in that: described draws
Object includes PRLP5 ~ 13；The DNA sequence dna of PRLP5 ~ 13 is as shown in NO:7 ~ 15 SEQ ID.
4. a kind of for detecting the kit of the as claimed in claim 1 or 22 haplotype SNP markers, it is characterised in that: include power
Benefit require 3 described in primer.
5. described in haplotype SNP marker of any of claims 1 or 2, primer as claimed in claim 3 or claim 4
Application of the kit in quick screening and identification growth channel catfish parent.
6. a kind of screening technique of fast-growth channel catfish parent, characterized by the following steps: detection spot fork
Sequence shown in SEQ ID NO:1 and SEQ ID NO:2 in Wei Channel-catfish core selective breeding group selects haplotype combination ACTCA/
The individual of ACTCA is as breeding parent.
7. screening technique according to claim 6, characterized by the following steps:
(1) the clip channel catfish fin ray sample to be measured from core selective breeding group extracts genomic DNA；
(2) to extract obtained genomic DNA as template, SNP are carried out using primer PRLP5/PRLP6, PRLP7/PRLP8
Point PCR amplification, PRLP5/PRLP6 are used for expanding site g.296 A > T, g.362 C > T and g.623 C > T, PRLP7/PRLP8
In amplification site g.94 C > T and g.788 A > G, first PCR product is obtained；
(3) after purification by amplification obtains in step (2) first PCR product, respectively using primer PRLP9, PRLP10,
It after PRLP11, PRLP12 and PRLP13 carry out extension, is tested and analyzed, is determined on the automatic genetic analysis systems of ABI
The genotype of each SNP site of candidate parent to be measured；
(4) haplotype combination of every tail candidate blob Cha Wei Channel-catfish parent, the parent for being ACTCA/ACTCA by haplotype combination are analyzed
It chooses and reserves seed for planting in case subsequent breeding；
The DNA sequence dna of primer PRLP5, PRLP6, PRLP7 and PRLP8 of the SNP site PCR amplification such as SEQ ID NO:7,
Shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10；
The DNA sequence dna of primer PRLP9, PRLP10, PRLP11, PRLP12 and PRLP13 of the extension such as SEQ ID NO:
11, SEQ ID NO:12, SEQ ID NO:13, shown in SEQ ID NO:14 and SEQ ID NO:15.
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| CN107502663B (en) * | 2017-09-19 | 2020-07-07 | 江苏省淡水水产研究所 | Channel catfish microsatellite family identification method |
| CN113215284B (en) * | 2021-06-18 | 2023-08-08 | 西南大学 | SNP (Single nucleotide polymorphism) marker related to growth rate of leiocassis longirostris and application thereof |
| CN113186307B (en) * | 2021-06-21 | 2023-08-01 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method and application based on channel catfish male specific gene zbtb38-Y |
| CN113293218B (en) * | 2021-06-21 | 2024-02-06 | 江苏省淡水水产研究所 | SNP molecular marker for selecting weight gain character of channel catfish and application |
| CN113789392B (en) * | 2021-10-09 | 2024-02-02 | 江苏省淡水水产研究所 | SNP (Single nucleotide polymorphism) marker related to growth of channel catfish and application thereof |
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| CN103409516A (en) * | 2013-07-24 | 2013-11-27 | 罗志萍 | Identification method of channel catfish species |
| CN104073486A (en) * | 2014-06-23 | 2014-10-01 | 中国水产科学研究院珠江水产研究所 | SNP site related to rapid growth of largemouth black bass as well as identification method and application thereof |
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| CN103409516A (en) * | 2013-07-24 | 2013-11-27 | 罗志萍 | Identification method of channel catfish species |
| CN104073486A (en) * | 2014-06-23 | 2014-10-01 | 中国水产科学研究院珠江水产研究所 | SNP site related to rapid growth of largemouth black bass as well as identification method and application thereof |
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