CN105925698A - SNP primers for early screening strongylocentrotus intermedius fine variety and screening method - Google Patents
SNP primers for early screening strongylocentrotus intermedius fine variety and screening method Download PDFInfo
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Abstract
The invention discloses SNP primers for early screening a strongylocentrotus intermedius fine variety and a screening method. The screening method comprises the following steps: selecting a strongylocentrotus intermedius second-generation male individual (Mars symbol) self-bred in Japan and a female individual (Venus symbol) bred for more than five generations in Longwangtang, Lvshun, China as male and female parents to crossbreed an F1 generation; establishing a mapping population containing 194 F1-generation individuals and two parents; cultivating in the same environment, extracting DNA, sequencing and taking other technical measures to find genetic markers of strongylocentrotus intermedius for the first time and design the relevant primers, wherein the primers can be used for early screening the strongylocentrotus intermedius fine variety. Compared with the prior art, the screening method has the advantages as follows: by the screening method, not only can manpower and material resources be saved and the seed selection be reduced, but also good genetic genes of the parents can be ensured, so that good progeny traits can be ensured.
Description
Technical field
The present invention relates to a kind of Strongylocentrotus intermedius Variety Selection primer and method, especially one can use manpower and material resources sparingly,
Reduce seed selection cost, the SNP primer guaranteeing the early screening Strongylocentrotus intermedius breeding that progeny character is good and screening technique.
Background technology
The traditional breeding method such as cross-breeding, selection and use, nothing is mostly used currently for the breeding of Strongylocentrotus intermedius
Opinion is cross-breeding or selection and use, is all with the good Hemicentrotus seu Strongylocentrotus of growth traits for standard Selection parent.Thus need
In Hemicentrotus seu Strongylocentrotus developmental process, the shell periodically measuring (such as every month etc.) Hemicentrotus seu Strongylocentrotus is high, shell footpath and the live body index such as heavily, carries out step by step with this
Screening, not only expends substantial amounts of manpower and materials, but also can there is the problem that selected parent gene are the most excellent, cause excellent
The filial generation seed quantum of output of good genotype is low.
Summary of the invention
The present invention is to solve the above-mentioned technical problem existing for prior art, it is provided that one can use manpower and material resources sparingly,
Reduce seed selection cost, the SNP primer guaranteeing the early screening Strongylocentrotus intermedius breeding that progeny character is good and screening technique.
The technical solution of the present invention is: the SNP primer of a kind of early screening Strongylocentrotus intermedius breeding, it is characterised in that
Described SNP primers DNA sequences is as follows:
Forward primer: 5'-CAGGTATCCTAAAGCAAT-3'；
Downstream primer: 5'-GTTTGTTGGTAAGCACTG-3'.
More than one state the method for SNP primer early screening Strongylocentrotus intermedius breeding, it is characterised in that in accordance with the following steps
Carry out:
A. prepared by DNA profiling:
Take Strongylocentrotus intermedius seedling pipe foot, extract genomic DNA；
B. PCR amplification target gene:
It is template with obtained DNA, carries out PCR reaction with described forward primer and downstream primer；
C. to PCR primer gene type:
With HRM 96 instrument, obtained PCR primer is carried out gene type, filter out containing following DNA sequence heterozygous genotypes
Strongylocentrotus intermedius seedling:
5'-CAGGTATCCTAAAGCAATTGTTCGTCCAGTGCTTACCAACAAAC-3'
CAGGTATCCTAAAGCAATTGTTCGTCCAGCGCTTACCAACAAAC
The present invention be selected Japan from the numerous secondary male of Strongylocentrotus intermedius (♂) and China the Dragon King pool, Lvshun breed five
It is Parent crossbreed F1 generation for above female individuals (♀), builds and comprise the individual mapping with 2 parents of 194 F1 generations
Colony cultivates in same environment, through extracting the technological means such as DNA, order-checking, is found that the heredity mark of Strongylocentrotus intermedius first
Remember and design relevant primer, can be used for early screening Strongylocentrotus intermedius breeding.Compared with prior art, manpower can not only be saved
Material resources, reduction seed selection cost, and can ensure that parent's gene are excellent, thus ensure that progeny character is good.
Accompanying drawing explanation
Fig. 1 is 4% agarose gel electrophoresis figure of embodiment of the present invention PCR primer.
Detailed description of the invention
One. the SNP marker screening process that growth traits is relevant:
1. high density genetic linkage maps builds
1.1 mapping populations build
Use and intend top-cross strategy, select Japan from the numerous secondary male of Strongylocentrotus intermedius (♂) with on the China Dragon King pool, Lvshun
The female individuals (♀) breeding five generations more than is Parent crossbreed F1 generation, builds and comprises 194 F1 generations individualities and 2 parents
Mapping population cultivate in same environment.
1.2 enzyme action schemes determine
Select purple ball Hemicentrotus seu Strongylocentrotus genome for carrying out electronics enzyme action prediction with reference to genome sequence, finally determine use RsaI+
HaeIII enzyme carries out enzyme action, and endonuclease bamhi length is at 414-464bp.
1.3 exploitation of SLAF label and screenings
Extracting tissue DNA with marine animal genome DNA extracting reagent kit, mapping population is carried out simplifying base by SLAF-seq technology
Because of group order-checking, the both-end Reads that order-checking produces is carried out sequence alignment by Blat software, according to sequence similarity more than 90%
Clustering, cluster sequence definition together is 1 SLAF label；It is polymorphic by the SLAF tag definition containing 2-4 bar tag
Property SLAF label.For ensureing genetic map quality, SLAF label is filtered, finally determines 150 individualities of upper figure labelling,
To can be used for 4678, the SLAF label of mapping, by calculating recombination fraction and MLOD value between label two-by-two, according to MLOD value, general marks
Label are divided into 21 linkage groups, finally determine upper figure labelling totally 4387.
1.4 genetic map construction
In units of linkage group, the linear arrangement of Marker in employing HighMap software analysis linkage group, between estimation Marker
The genetic distance, finally gives the genetic map that total map distance is 1907.21cM, mean chart away from 0.44cM, upper figure label integrity degree
99.75%。
2.QTL positions
The phenotypic number of 2.1 growth traitss is measured and is analyzed
Digital display electronic slide gauge (precision is 0.01mm) measures 150 individual shells high (TH), shell footpath (TD) of final upper figure；
Live body weight (BW) measured by electronic balance (precision is 0.01g).Utilize SPSS 16.0 software that phenotypic data is carried out normal distribution inspection
Test.
The QTL location of 2.2 growth traitss
The collection of illustrative plates and the growth traits phenotypic number that utilize 21 linkage groups of Strongylocentrotus intermedius carry out character association analysis.Pass through
Permutation inspection (1000 times) determines the significance level threshold value (LOD) of 95% confidence interval of each character；Use
Composite interval mapping (CIM) algorithm of RQTL software and Interval mapping (IM) algorithm of MapQTL software carry out shell height, shell footpath, work
3 growth traits location of body weight, navigate to 97 SLAF labels altogether.
3. the screening of polymorphism SNP marker, design of primers and typing
By 97 SLAF sequence labels and purple ball Hemicentrotus seu Strongylocentrotus genome alignment, similarity sets threshold value as 0.9, it is thus achieved that have gene note
15 the SLAF labels released, wherein find the SNP site 8 that can design small fragment primer.Utilize Primer Premier
5.0 softwares design small fragment primer near SNP site (makes Tm value control between 50 DEG C～60 DEG C, PCR primer a length of 40
～120bp, primer only comprises a SNP site, and does not comprise any insertion and deletion) carry out grads PCR amplification, the agar of 4%
Sugar gel electrophoresis (150V, 200mA, 20min) detects all primers, determines optimum annealing temperature.Through HRM testing goal fragment
Melting curve is single without miscellaneous peak, and is distributed in the primer between high/low temperature internal standard peak for gene type.
Utilize Light Scanner 96 analyser to garbled small fragment primer in mapping population (random choose
50 individualities) carry out gene type, it is thus achieved that 3 candidate SNP labellings relevant to Strongylocentrotus intermedius growth traits.
4. the checking of Strongylocentrotus intermedius growth traits related SNP labelling
Select Strongylocentrotus intermedius F5 for 3 familys (15 monthly age) of growth traits obvious difference, each family take at random 30 each and every one
Body, extraction tube foot DNA, measures the growth traits of familys to be checked, according to the method for above-mentioned third step to above-mentioned according to 2.1 methods
3 SNP marker obtained carry out gene type in 90 individualities of family, and by shell height, shell footpath, live weight character and SNP
The significance test of genotype (P< 0.05 is significant difference,P< 0.01 is that difference is the most notable), SNP-29 is marked at shell height, shell
Footpath, live on weight character, heterozygous genotypes all pole be noticeably greater than homozygous genotype (P＜ 0.01), it is shown in Table 1, i.e. heterozygous genotypes
For preponderant genotype.
The specifying information of SNP-29 is as follows:
The SNP primers DNA sequences of early screening Strongylocentrotus intermedius breeding is as follows:
Forward primer: 5'-CAGGTATCCTAAAGCAAT-3'；
Downstream primer: 5'-GTTTGTTGGTAAGCACTG-3'.
Affiliated linkage group numbering: LG20；
Affiliated SLAF bookmark name: 19255；
SNP type: T/C；
Product length: 44bp；
DNA sequence is as follows:
5'-CAGGTATCCTAAAGCAATTGTTCGTCCAGTGCTTACCAACAAAC-3'
CAGGTATCCTAAAGCAATTGTTCGTCCAGCGCTTACCAACAAAC
*
* the nucleotide variation i.e. SNP site of point is represented.
Related check between SNP-29 loci gene type and character such as table 1:
Related check between table 1 SNP-29 loci gene type and character
Note: the correlation analysis between different genotype and the various trait of same SNP site.Same a line of same SNP site
Right mark capitalization difference represent between genotype difference extremely notable (P＜ 0.01), contribution rate (P)= SS t / SS T ×100%。
Lower same.
Two. with SNP-29 labeled primer early screening Strongylocentrotus intermedius breeding
1. the selection of parent and cultivation
Select 2 familys that in 3 familys, growth traits is best to carry out colony to breed, be correlated with through Strongylocentrotus intermedius growth traits
The confirmatory experiment of SNP marker, screens in 2 familys the heterozygous genotypes containing SNP-29 labelling individual as breeding parent,
Parental number to these 2 familys is respectively 16 and 14 individualities, for the purpose of avoiding inbreeding, and female to these 2 familys
Hero carries out intersection and breeds, first filial generation numbered A group；16 and 14 are respectively randomly selected from identical two familys of laboratory cultures
Individuality, without the genotype detection of SNP-29 labelling, carries out intersection by male and female and breeds, first filial generation numbered matched group B.Put
Same breeding environment is cultivated to sexual maturity.
2. artificial breeding and growth traits phenotypic number are measured
Propagation method uses Strongylocentrotus intermedius healthy aquaculture technology.Adjust cultivation density according to sea urchin shell footpath size in good time, treat sea
When gallbladder grew to for 10 monthly age, from A, B two groups, 300 Hemicentrotus seu Strongylocentrotuss of each random choose measure shell height, shell footpath, weight character of living.
3. the detection of growth traits related SNP-29 labelling and assessment
Two groups of filial generations of A, B are screened in accordance with the following steps, and carries out gene type, result such as table 2
A. prepared by DNA profiling:
Take Strongylocentrotus intermedius seedling pipe foot, extract genomic DNA；
B. PCR amplification target gene:
It is template with obtained DNA, carries out PCR reaction with described forward primer and downstream primer；
Obtained PCR primer is carried out 4% agarose gel electrophoresis, obtains the display band of 44bp, result such as Fig. 1；
C. to PCR primer gene type:
With HRM 96 instrument, obtained PCR primer is carried out gene type, filter out containing following DNA sequence heterozygous genotypes
Strongylocentrotus intermedius seedling:
5'- CAGGTATCCTAAAGCAATTGTTCGTCCAGTGCTTACCAACAAAC-3'
CAGGTATCCTAAAGCAATTGTTCGTCCAGCGCTTACCAACAAAC
Table 2 is through the difference test of first filial generation that SNP-29 labelling instructs with matched group first filial generation
Result shows:
Parent first filial generation A group character value through the screening of SNP-29 labeled primer is noticeably greater than matched group B, and A group is containing SNP-
The number of individuals of 29 labelling heterozygous genotypes, also more than matched group B, illustrates that SNP-29 is marked in the A group of first filial generation polymerization effect more
Good, prove that the SNP-29 labelling relevant to growth traits is applicable to Strongylocentrotus intermedius seed selection in early days further.
Sequence table
<110>Dalian Ocean University
<120>the SNP primer of early screening Strongylocentrotus intermedius breeding and screening technique
<160> 4
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 1
CAGGTATCCTAAAGCAAT 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>primer
<400> 2
GTTTGTTGGTAAGCACTG 18
<210> 3
<211> 44
<212> DNA
<213>Strongylocentrotus intermedius
<400> 3
CAGGTATCCTAAAGCAATTGTTCGTCCAGTGCTTACCAACAAAC 44
<210> 4
<211> 44
<212> DNA
<213>Strongylocentrotus intermedius
<400> 4
CAGGTATCCTAAAGCAATTGTTCGTCCAGCGCTTACCAACAAAC 44。
Claims (2)
1. the SNP primer of an early screening Strongylocentrotus intermedius breeding, it is characterised in that described SNP primers DNA sequences is as follows:
Forward primer: 5'-CAGGTATCCTAAAGCAAT-3'；
Downstream primer: 5'-GTTTGTTGGTAAGCACTG-3'.
2. one kind with the method for SNP primer early screening Strongylocentrotus intermedius breeding described in claim 1, it is characterised in that according to such as
Lower step is carried out:
A. prepared by DNA profiling:
Take Strongylocentrotus intermedius seedling pipe foot, extract genomic DNA；
B. PCR amplification target gene:
It is template with obtained DNA, carries out PCR reaction with described forward primer and downstream primer；
C. to PCR primer gene type:
With HRM 96 instrument, obtained PCR primer is carried out gene type, filter out containing following DNA sequence heterozygous genotypes
Strongylocentrotus intermedius seedling:
5'-CAGGTATCCTAAAGCAATTGTTCGTCCAGTGCTTACCAACAAAC-3'
CAGGTATCCTAAAGCAATTGTTCGTCCAGCGCTTACCAACAAAC。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108411002A (en) * | 2018-05-22 | 2018-08-17 | 大连海洋大学 | PCR primer and discrimination method for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies |
| CN108660225A (en) * | 2018-07-18 | 2018-10-16 | 大连海洋大学 | Molecular mark primer and screening technique for Strongylocentrotus intermedius growth traits |
| CN109182540A (en) * | 2018-10-10 | 2019-01-11 | 大连海洋大学 | Screen the Strongylocentrotus intermedius primer and method with high-temperature stability |
| CN113718038A (en) * | 2021-07-26 | 2021-11-30 | 大连海洋大学 | Molecular marker assisted breeding primer group for fatty acid content of echinus intermedius and application |
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| CN102443637A (en) * | 2011-11-29 | 2012-05-09 | 大连海洋大学 | SSR marker screening method related to metamorphosis of sea urchins |
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| CN102443637A (en) * | 2011-11-29 | 2012-05-09 | 大连海洋大学 | SSR marker screening method related to metamorphosis of sea urchins |
Non-Patent Citations (3)
| Title |
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| ZUNCHUN ZHOU等: "High-Density Genetic Mapping with Interspecific Hybrids of Two Sea Urchins,Strongylocentrotus nudus and S. intermedius,by RAD Sequencing", 《PLOS ONE》 * |
| 初冠囡等: "中间球海胆生长分化相关的AFLP标记", 《中国水产科学》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108411002A (en) * | 2018-05-22 | 2018-08-17 | 大连海洋大学 | PCR primer and discrimination method for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies |
| CN108411002B (en) * | 2018-05-22 | 2021-07-13 | 大连海洋大学 | PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius |
| CN108660225A (en) * | 2018-07-18 | 2018-10-16 | 大连海洋大学 | Molecular mark primer and screening technique for Strongylocentrotus intermedius growth traits |
| CN108660225B (en) * | 2018-07-18 | 2021-06-15 | 大连海洋大学 | Molecular marker assisted breeding primer for growth traits of echinococcus intermedius and screening method |
| CN109182540A (en) * | 2018-10-10 | 2019-01-11 | 大连海洋大学 | Screen the Strongylocentrotus intermedius primer and method with high-temperature stability |
| CN109182540B (en) * | 2018-10-10 | 2021-06-15 | 大连海洋大学 | Primer and method for screening high-temperature-resistant strongylocentrotus intermedius |
| CN113718038A (en) * | 2021-07-26 | 2021-11-30 | 大连海洋大学 | Molecular marker assisted breeding primer group for fatty acid content of echinus intermedius and application |
| CN113718038B (en) * | 2021-07-26 | 2023-05-23 | 大连海洋大学 | Molecular marker assisted breeding primer group for fatty acid content of echinacea intermedius and application |
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