CN105969882A - Haplotype SNP molecular marker associated with rapid growth of Ictalures punctatus and detection method and application thereof - Google Patents
Haplotype SNP molecular marker associated with rapid growth of Ictalures punctatus and detection method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular marker-assisted breeding of aquatic animals, and particularly relates to a haplotype SNP molecular marker associated with rapid growth of an Ictalures punctatus and a detection method and application thereof. The SNP molecular marker is obtained by cloning a PRL (Prolactin) gene of the Ictalures punctatus; and the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1 and SEQ ID NO: 2. The invention further discloses application of the molecular marker to molecular detection of growth traits of the Ictalures punctatus. Individuals with genotypes of AA, CC, DD, CC and AA respectively, namely individuals with haplotype combinations of H3/H3(ACTCA/ACTCA), are screened as breeding parents. The detection method provided by the invention is simple in operation and high in detection speed; and by the detection method, the Ictalures punctatus parent screening blindness is greatly reduced and Ictalures punctatus parents having the characteristics of rapid growth and genetic stability can be quickly obtained.
Description
Technical field
Patent of the present invention belongs to aquatic animal molecular mark field, is specifically related to a kind of fast with channel catfish
Haplotype SNP marker that fast-growing length is relevant and detection method thereof and application.
Background technology
Channel catfish (Ictalures punctatus) originates in the U.S., is a kind of economic species of important Aquatic product,
Within 1984, introduce China, there is the features such as meat is fine and smooth, suitable temperature range is wide, fast growth, resistance against diseases are strong.But, along with
The fast development of China's channel catfish aquaculture industry, has occurred in that obvious decreased growth, body-color diversity and specification are uneven
Deng kind of a matter decay.Therefore, cultivation grows soon, economic characters channel catfish new varieties (being) good, strong stress resistance become
For the major issue that current channel catfish industry sustainable development is urgently to be resolved hurrily.
Prolactin antagonist (Prolactin, PRL) is that the single chain polypeptide class being synthesized by vertebrates gene expression and secreting swashs
Element, has different physiological roles, relates generally to Osmoregulation, growth promoter, immunity, endocrine and metabolic regulation etc..Lactogenic
Element and growth hormone (Growth hormone, GH) and somatomammotropin (Somatolactin, SL) have similar structure and
Function, up-to-date molecular evolution research shows that GH gene, PRL gene and SL gene are likely to by ancient GH gene through polygenes
Copy and the veryest long evolutionary process develop.PRL gene as the candidate gene of growth traits, its SNPs and
SSR polymorphism has had more report with the association analysis of growth traits in poultry, herding and aquatic animal.Tilapia
One SSR site of PRL1 gene promoter area grows significant correlation in different salinity level with it.On the perch PRL gene of Asia
One SNP site (c.264+269T > C) and the growth traits significant correlation such as its weight, body length, genotype is the body that CC is individual
Quality and the long significance of body are more than the individuality that genotype is CT and TT.Therefore, in channel catfish find identify PRL gene with
SNPs and haplotype molecular marker that growth traits is associated are close to the channel catfish of selection-breeding fast growth and inheritance stability
Originally have great importance.
Summary of the invention
It is an object of the invention to provide a kind of haplotype SNP marker relevant to channel catfish fast-growth and
Application, i.e. utilizes a haplotype SNP marker relevant to channel catfish growth traits of channel catfish PRL gene
Carry out screening and the qualification of fast-growth parent.
Patent of the present invention 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 base of the 296th, 362,623 at be respectively present A/T, C/T, C/T 3 sudden change；In SEQ ID NO.2 sequence the 94th
C/T and A/G 2 sudden change it is respectively present at base with 788.
A kind of primer for detecting above-mentioned haplotype SNP marker, this primer includes PRLP5～13；Described PRLP5～13
DNA sequence as shown in SEQ ID NO:7～15.
A kind of test kit for detecting above-mentioned haplotype SNP marker, this test kit comprise above-mentioned primer PRLP5～
13。
Haplotype SNP marker, primer or the test kit of the present invention is in rapid screening and identifies growth channel catfish
Application in parent.
The screening of a kind of fast-growth channel catfish candidate parent and authentication method, comprise the steps: to detect speckle
SEQ ID NO:1 in fork-tail core selective breeding colony and the sequence shown in SEQ ID NO:2, to tail every in core selective breeding colony
Each SNP site of channel catfish candidate parent carries out typing, selects have haplotype combination H3/H3 (ACTCA/ACTCA)
Individuality as selection-breeding parent.
Specifically include following steps:
(1) clip channel catfish to be measured fin ray sample from core selective breeding colony, extracts genomic DNA.
(2) to extract the genomic DNA that obtains as template, primer PRLP5/PRLP6, PRLP7/PRLP8 is utilized to carry out
SNP site PCR expands, and PRLP5/PRLP6 is used for expanding site g.296A > T, g.362C > T and g.623C > T, PRLP7/PRLP8
For expanding site g.94C > T and g.788A > G, obtain first PCR primer.Primer PRLP5, PRLP6, PRLP7 and PRLP8's
DNA sequence is as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
(3) PCR primer amplification in step (2) obtained is after purification, use respectively primer PRLP9, PRLP10,
After PRLP11, PRLP12 and PRLP13 carry out extension, the automatic genetic analysis systems of ABI carries out detection and analyzes, determine
The genotype of each SNP site of candidate parent to be measured.The DNA sequence of primer PRLP9, PRLP10, PRLP11, PRLP12 and PRLP13
Arrange 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) analyze the haplotype combination of every tail candidate blob fork-tail parent, be H3/H3 (ACTCA/ by haplotype combination
ACTCA) Juvenile stage is out reserved seed for planting in case follow-up breeding.
The amplification of SNP site PCR uses instant UtraTaq enzyme PCR kit (Shanghai Jierui Biology Engineering Co., Ltd),
Reaction system is:
Amplification condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, anneal 30s, and 72 DEG C extend 60s, 30 circulations；72
DEG C extend 10min.
SNPs typing uses ABI SNaPshot Multiplex PCR kit (ABI, the U.S.), and reaction system is 6 μ L:
Reaction condition is: 96 DEG C of denaturations 1min；96 DEG C of degeneration 10s, 52 DEG C of annealing 5s, 60 DEG C extend 30s, and totally 30 are followed
Ring.
Reaction adds 1U FastAP, 37 DEG C of 60min, 85 DEG C of 15min after terminating and is purified.With GeneScan-120Liz
Size Standard is internal standard, takes 1 μ L product and Hi-Di (GS-120Liz:Hi-Di=1:200) mixing that 9 μ L contain Liz,
95 DEG C of degeneration 3min, immediately upper sequenator after ice bath 3min.Detection and analysis use ABI PRISM 3730XI type the most hereditary
Analysis system (ABI, the U.S.), and utilize Genemapper v4.1 software to carry out typing.
All there are 3 kinds of genotype through multiple Snapshot method typing, each site in 5 SNPs sites.Utilize SHEsis
The haplotype kind of these 5 SNPs generations of software analysis and frequency, R software carries out the association analysis between genotype and character.Knot
Fruit display, in 5 SNPs, measured growth traits is had a significant impact (P<0.05) by g.623C>T and g.788A>G, 5
The haplotype of site reconstruct, measured growth traits is all had a significant impact (P < 0.05), wherein by different haplotype combination
The weight of H3/H3 (ACTCA/ACTCA) haplotype combination and body long value are maximum.Retaining haplotype combination in actual production is
The channel catfish parent of H3/H3 (ACTCA/ACTCA), just can obtain the channel catfish product of fast growth and inheritance stability
System.The method compared to traditional selection, have purposiveness strong, simple to operate, detection speed fast, it is easy to popularizations etc. are excellent
Point.
The method of the present invention, greatly reduces the blindness of channel catfish parental line selection, shortening the breeding cycle, can be fast
Speed obtains fast growth and the channel catfish parent of inheritance stability.
Accompanying drawing explanation
Fig. 1 is PRL gene schematic diagram and 5 SNPs site locations
Wherein, g.296A primer amplifies SNP site to PRLP5/PRLP6 > T, g.362C > T and g.623C > T, primer pair
G.94C PRLP7/PRLP8 amplifies SNP site > T and g.788A > G, except SNP site g.788A > G, remaining SNP site is in
Intron region.
Detailed description of the invention
Embodiment 1
1.1 laboratory animal
This experiment channel catfish sample standard deviation used comes from Jiangsu Fresh Water Aquatic Product Inst. Lu Kou proving ground.
1997-2004 from the U.S. introduce Texas (1997) colony, Arkansas (1999) colony, Mississippi (2001) colony,
Arkansas (2003) colony and Arkansas (2004) colony 405 tail set up breeding basic population, utilize basic population to carry out G0 generation
Family builds.Utilize in June, 2013 G0 for family structure G1 for family, in order to reduce the shadow that channel catfish is grown by environment
Ringing, seed rearing is carried out according to same standard, and the most homogeneous changes water speed, feeding volume, cultivation density, oxygenation capacity and water temperature.
Until scanning record living individuals numbering when family mean age in days is 520d, gather the tail fin tissue of every tail fish, 95% ethanol leaching
Bubble, 20 DEG C of preservations.
In the present invention, described channel catfish growth traits specifically includes that weight and body weight.
1.2 design of primers
Channel catfish PRL complete genome sequence (AF267990.1) according to GenBank warehouse publication, uses Primer
Premier 5 software design two 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 is:
Amplification condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, anneal 30s, and 72 DEG C extend 2min, 30 circulations；
72 DEG C extend 10min.1% agarose gel electrophoresis detection PCR primer, electrophoresis result observed by gel imaging instrument.
1.4 order-checking
Uviol lamp incision glue purification reclaims PCR primer, and the PCR primer of every pair of primer is chosen 12 samples, delivered to Shanghai
Sheng Gong biological engineering limited company, directly uses ABI 3730XL sequenator (ABI, the U.S.) order-checking.Use chromas soft
Part observes order-checking peak figure, carries out DNA sequence comparison to judge the base type of SNP site in conjunction with ClustalX software.
1.5SNPs typing
Utilizing primer PRLP5/PRLP6, PRLP7/PRLP8 to carry out SNPs site PCR amplification, PRLP5/PRLP6 is used for expanding
Increase site g.296A > T, g.362C > T and g.623C > T, PRLP7/PRLP8 is used for expanding site g.94C > T and g.788A > G, must
To first PCR primer.
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)。
After using primer PRLP9, PRLP10, PRLP11, PRLP12 and PRLP13 to carry out extension respectively, at ABI certainly
Carry out detection on dynamic genetic analysis systems to analyze, determine 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)。
PCR amplification in SNPs site uses instant UtraTaq enzyme PCR kit (the Shanghai limited public affairs of JaRa biological engineering
Department), reaction system is:
Amplification condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, anneal 30s, and 72 DEG C extend 60s, 30 circulations；72
DEG C extend 10min.
SNPs typing uses ABI SNaPshot Multiplex PCR kit (ABI, the U.S.), and reaction system is 6 μ L:
Reaction condition is: 96 DEG C of denaturations 1min；96 DEG C of degeneration 10s, 52 DEG C of annealing 5s, 60 DEG C extend 30s, and totally 30 are followed
Ring.
Reaction adds 1U FastAP, 37 DEG C of 60min, 85 DEG C of 15min after terminating and is purified.With GeneScan-120Liz
Size Standard is internal standard, takes 1 μ L product and Hi-Di (GS-120Liz:Hi-Di=1:200) mixing that 9 μ L contain Liz,
95 DEG C of degeneration 3min, immediately upper sequenator after ice bath 3min.Detection and analysis use ABI PRISM 3730XI type the most hereditary
Analysis system (ABI, the U.S.), and utilize Genemapper v4.1 software to carry out typing.
1.6 haplotypes build and association analysis
Utilizing haplotype kind and the frequency of these 5 SNPs generations of SHEsis software analysis, R software carries out genotype and property
Association analysis between shape, uses generalized linear model (GLM) to calculate.Concrete model: Yij=μ+Gi+eij, in formula: Yij
For certain character i-th labelling jth individuality observation；μ is the meansigma methods (i.e. population mean) of all individualities of experimental observation；
Gi is the effect value of i-th labelling；Eij is the random residual effect corresponding to observed value.
1.7 interpretation of result
Sequence alignment result shows and 5 SNPs detected on PRL gene altogether, is respectively the most g.296A > T, g.362C > T,
G.623C > T, g.94C > T and g.788A > G, is respectively positioned on noncoding region.The most g.296A > T, g.362C > T and g.623C > the equal position of T
In intron 2, g.94C > T is positioned at the 4th intron, and g.788A > G is positioned at 3 ' end flanking sequences, as shown in Figure 1.These 5
All there are 3 kinds of genotype through multiple Snapshot method typing, each site in SNPs site.5 SNPs site reconstruct form 7 kinds
Haplotype, produces 10 kinds of haplotype combination.Association analysis result shows, different haplotype combination all have notable shadow to growth traits
Ringing (P < 0.05), wherein weight and the body long value of H3/H3 (ACTCA/ACTCA) haplotype combination are maximum.Therefore, PRL gene
The haplotype molecular marker that constitutes of 5 SNPs can apply in channel catfish molecular marking supplementary breeding.
Table 1 channel catfish PRL gene haplotype combination and the association analysis of growth traits
Note: different lowercase alphabets show significant difference (P < 0.05), different capitalizations represent difference extremely notable (P <
0.01)。
Claims (9)
1. a haplotype SNP marker relevant to channel catfish fast-growth, it is characterised in that: described haplotype
The nucleotide sequence of SNP marker is as shown in SEQ ID NO:1；The base punishment of the 296th, 362,623 of this sequence
Not there is A/T, C/T, C/T3 sudden change.
2. a haplotype SNP marker relevant to channel catfish fast-growth, it is characterised in that: described haplotype
The nucleotide sequence of SNP marker is as shown in SEQ ID NO:2；Deposit respectively at the base of the 94th and 788 of this sequence
In C/T and A/G 2 sudden change.
3. one kind requires the primer of haplotype SNP marker described in 1 or 2 for test right, it is characterised in that: described draws
Thing includes PRLP5～13；The DNA sequence of described PRLP5～13 is as shown in SEQ ID NO:7～15.
4. one kind requires the test kit of haplotype SNP marker described in 1 or 2 for test right, it is characterised in that: comprise power
Profit requires the primer described in 3.
5. described in the haplotype SNP marker described in claim 1 or 2, the primer described in claim 3 or claim 4
Test kit rapid screening and identify growth channel catfish parent in application.
6. the screening technique of a fast-growth channel catfish parent, it is characterised in that: comprise the steps: to detect speckle fork
SEQ ID NO:1 in tail core selective breeding colony and the sequence shown in SEQ ID NO:2, select haplotype combination H3/H3
(ACTCA/ACTCA) individuality is as selection-breeding parent.
Screening technique the most according to claim 6, it is characterised in that: comprise the steps:
(1) clip channel catfish to be measured fin ray sample from core selective breeding colony, extracts genomic DNA；
(2) to extract the genomic DNA that obtains as template, primer PRLP5/PRLP6, PRLP7/PRLP8 is utilized to carry out SNP position
Point PCR amplification, PRLP5/PRLP6 is used for expanding site g.296A > T, g.362C > T and g.623C > T, PRLP7/PRLP8 is used for
Amplification site is g.94C > T and g.788A > G, obtain first PCR primer；
(3) first PCR primer amplification in step (2) obtained is after purification, use respectively primer PRLP9, PRLP10,
After PRLP11, PRLP12 and PRLP13 carry out extension, the automatic genetic analysis systems of ABI carries out detection and analyzes, determine
The genotype of each SNP site of candidate parent to be measured；
(4) analyze the haplotype combination of every tail candidate blob fork-tail parent, be H3/H3 (ACTCA/ by haplotype combination
ACTCA) Juvenile stage is out reserved seed for planting in case follow-up breeding.
Screening technique the most according to claim 7, it is characterised in that: described SNP site PCR amplification primer PRLP5,
The DNA sequence of PRLP6, PRLP7 and PRLP8 such as SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:
Shown in 10.
Screening technique the most according to claim 7, it is characterised in that: the primer PRLP9 of described extension, PRLP10,
The DNA sequence of PRLP11, PRLP12 and PRLP13 such as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
Shown in NO:14 and SEQ ID NO:15.
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| CN107502663A (en) * | 2017-09-19 | 2017-12-22 | 江苏省淡水水产研究所 | A kind of channel catfish microsatellite Parentage determination method |
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| CN113215284A (en) * | 2021-06-18 | 2021-08-06 | 西南大学 | SNP (Single nucleotide polymorphism) marker related to growth speed of leiocassis longirostris and application thereof |
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| CN113186307A (en) * | 2021-06-21 | 2021-07-30 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method based on channel catfish male specific gene zbtb38-Y and application |
| CN113293218A (en) * | 2021-06-21 | 2021-08-24 | 江苏省淡水水产研究所 | SNP molecular marker for selecting weight gain character of channel catfish and application |
| 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 |
| CN113789392A (en) * | 2021-10-09 | 2021-12-14 | 江苏省淡水水产研究所 | SNP marker related to growth of channel catfish and application thereof |
| 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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