CN106086216B - A kind of and associated SNP marker of pteria martensii growth traits and primer and application - Google Patents
A kind of and associated SNP marker of pteria martensii growth traits and primer and application Download PDFInfo
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Abstract
The invention discloses a kind of and associated SNP marker of pteria martensii growth traits and primer and applications.Detection primer includes: ASSN1184FI, ASSN1184RI, ASSN1184FO, ASSN1184RO.Utilize the detection primer of SNP marker associated with pteria martensii growth traits of the invention, it can amplify and the associated SNP genotype locus of pteria martensii growth traits, growth vigor individual can be intuitively filtered out further according to PCR amplification electropherogram and carries out breeding as standby parent, realize molecular mark.
Description
Technical field:
The invention belongs to aquatic livestock heredity and technical field of molecular marker-assisted breeding, and in particular to a kind of and geneva pearl
The associated SNP marker of female shellfish growth traits and primer and application.
Background technique:
Pteria martensii is China and dominant species of seawater pearl culture in the world.Successfully carry out from nineteen sixty-five in China
Since pteria martensii artificial breeding, seawater pearl industry development is rapid, it has also become Guangdong, Guangxi and Hainan part littoral
One of area's sea-farming pillar industry.Nearly more than 10 years, the quality and yield of China's sea water pearls were decreased obviously, and international competitiveness subtracts
Weak, economic benefit of aquaculture glides.To find out its cause, in addition to the deterioration of the breeding environment and backward breeding technology, there are one major reasons
It is: does not focus on the breeding and reservation of kind of shellfish for many years, lacks excellent variety；Seedling quality is irregular, and pinctada character is moved back
Change, such as growth is slow, individual smalltype, reduced capability of growing cultured pearls.For the healthy and stable development of seawater pearl breeding industry in our country, it cultivates
It is suitble to the pteria martensii excellent variety of China sea area cultivation extremely urgent.
Breeding technique has hybridization, selection and use, a variety of methods such as biotechnology breeding and molecular breeding.Molecular breeding is mesh
The preceding hot spot studied both at home and abroad is the Main way of breeding technique development, it has the characteristics that efficient, quick.Molecular breeding from
The exploitation of molecular labeling is not opened, and single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is by gene
The horizontal single base-pair variation of group causes DNA sequence polymorphism (Sachidanandam et al., 2001), it is after RFLP
With the third generation molecular labeling to grow up after SSR molecular marker.The condition for meeting SNP should be one of allele
The frequency of appearance in group should be not less than 1% (Kang et al., 2002).This variation includes turning for single base
(exchange of homotype base), transversion (special-shaped base exchange), insertion and missing are changed, but general signified SNP does not include two kinds next
Situation.The SNP found at present, the overwhelming majority all only have 2 kinds of nucleotide, and three kinds or more of nucleotide co-exists in a population
It there's almost no, therefore SNP is two polymorphic alleles.SNP marker has widely distributed, inheritance stability and dimorphism etc. special
Point, thus achieve in the pathogenic mechanism of human diseases and treatment, important crops and domestic animal genetic breeding it is huge at
Effect.Especially in recent years, a large amount of SNP sites are obtained using high-throughput Roche454 sequencing technologies and be associated with character
The report of analysis and discovery new gene, shows that SNP marker has become science of heredity and the main tool of MAS.And in aquaculture
In animal, MAS breeding technique is carried out using SNP marker and is also in early stage, such as population genetic diversity analysis, kind mirror
In terms of the genetic breedings fields such as fixed, trait associations analysis, genetic linkage maps building and QTL positioning.Molecular labeling is
Realize that genotype selection provides possibility, because the genotype of molecular labeling can identify.If target gene and some
Molecular labeling close linkage, then being detected by the genotype to molecular labeling, it will be able to obtain the gene of target gene
Type.Therefore, it can be selected by genotype of the molecular labeling to objective trait, here it is molecular marker assisted selections
(marker-assisted selection,MAS).Compared with conventional breeding methods, MAS has apparent superiority, especially
Those are difficult to measure, genetic force is lower and could show to obtain character in the later period, and MAS is not easy to be protected from environmental, and does not have
The limitation of gender and age, therefore, MAS can be selected in early days, shorten the generation inteval, and then improved the intensity of selection and educated
Kind efficiency (Lande and Thompson, 1990).And it is marked currently, excavating with the associated SNP of pteria martensii growth traits
Note also seldom report, the object of the present invention is to provide a kind of new with the associated SNP marker of pteria martensii growth traits, provides
A kind of method of the identification of living body SNP has genotype selection and use technology in favor of carrying out the practice of genotype selection and use
Practical operability.
Summary of the invention:
The first purpose of the invention is to provide a kind of detections with the associated SNP marker of pteria martensii growth traits to draw
Object.
Detection primer with the associated SNP marker of pteria martensii growth traits of the invention, which is characterized in that including with
Lower primer:
ASSN1184FI:5 '-CGATTTGCTAGAAAGCCATTA-3 '；
ASSN1184RI:5 '-AATTTGTTACAACTTACCTATAATGCAA-3 '；
ASSN1184FO:5 '-GGTAAAACATTAACACGGATTGA-3 '；
ASSN1184RO:5 '-ATTGTAATGACTTACCTAAAACGGAT-3 '.
A second object of the present invention is to provide a kind of and associated SNP marker of pteria martensii growth traits, features
It is, the sequence of the SNP marker is as shown in SEQ ID NO.1, overall length 1100bp, shown in the SEQ ID NO.1
Sequence the 457th bit base from 5 ' ends is A or T, indicates the site with w in SEQ ID NO.1 sequence.
Shown in specific as follows:
GTGCATGTCCACTTGATGCGACCACTAAAACAGATGCAATAATTGCGAAAGATCGTGCCAAACTAAAA
TATTTCGATCGCAAAGTGGTCGTGCACCAAATGAGGAGGGGGCTTTAAAAATGTGATGGCATTTCAGAGAAATATA
ACTTGGGTTGATTTTGGTTAGATATGAGAAGGTAAAACATTAACACGGATTGATGGTATTTGGGACAGCTGAAAGT
AGATATGAAAAATAAATACATGTACTGGGTTCGTAGTGAATAAAGTGGAGGGAGGGATATTCAGTACCCTTGTAGT
TTGACGTCTTGCTGTTTGACTGTGATGATGTTTTCATATTGTAGGAGATCGTGCTGGTAGTATTCTTTGGCCTGGA
GTACGTAGTACGCTTATGGGCAGCAGGCTGTCGTAGTAAATACATTGGTGTTAGAGGACGTCTACGATTTGCTAGA
AAGCCAATW (W is A or T) TCCATTATAGGTAAGTTGTAACAAATTTAAGGTGTTGACCGCCGCCGCCCCCTCCT CT
GTCATATCTCTTATGTCCGTCTCCCGATTCGTCTGTGTATTACAAAATGTTATAATCCCCACTAAGAGGCTCTAAA
TTTCTCCGACCGTTAGAGAGCAAATATCCGTTTTAGGTAAGTCATTACAATTTTTTAGCTTAGCTTCCCTTCCCTA
CCTAGTCAATGCCTTGTCCGTCTCTCATCTCGTCTGTGTATTATAGAAAACTGCTTTCTCCACACTGAGGCTCCCT
ATTTTCACCAACAGAGAGTTAGAACAAATATGAGATAGCCCCCTTCCCTTCCTGTCCCATCTCTTGTCCGTCTTTC
AGTTTGTCTGTGTATTACAGAAAACTGCAACCTCCACTTGCATATGTAGAGGCTCCCAATTTTCACACACAGAGAC
ACATGATTCGATATAAGAATCATTAACTCTTTATGTCTTTTACCTTTGCAAATTGTCGTTATTTACGTCTGCAGGT
CGACCAGAGCGTCCCGTTACTAGTAGATTAACTTTTTTTAGCAGTGTAAATAATCATGCGTGGAAAAAAGCGTCCT
TACCATCTAAATGTCATCGTTAAAGCATCGAATCAATACGGTACGGGAACCGT。
Third object of the present invention is to provide with the associated SNP marker of pteria martensii growth traits in pteria martensii
Application in molecular mark.
Fourth object of the present invention is to provide a kind of method of individual of the detection pteria martensii with growth vigor,
It is characterized in that, by being examined to the above-mentioned and associated SNP marker of pteria martensii growth traits in pteria martensii to be measured
It surveys, determines whether the pteria martensii to be measured is the individual with growth vigor, determine if to carry out as standby parent
Breeding.
It is preferred that specific steps are as follows:
A, pteria martensii sample gene group DNA to be measured is extracted；
B, using above-mentioned detection primer ASSN1184FI, ASSN1184RI, ASSN1184FO, ASSN1184RO to geneva
Nacre sample gene group DNA carries out PCR amplification；
C, genotyping is carried out to pteria martensii sample according to pcr amplification product, when only occurring 310bp size 1
Band is TT genotype, which is determined as the individual with growth vigor, carries out breeding as standby parent.
It is preferred that the PCR amplification, PCR reaction system are as follows: 25 μ L；PCR reaction system is as follows: including geneva pearl oyster
Shellfish sample gene group DNA 40ng, 1 × PCR Mixture, each 0.8 μM of detection primer ASSN1184FI, ASSN1184RI,
Each 0.2 μM of ASSN1184FO, ASSN1184RO, 0.1U Taq DNA enzymatic, surplus is water；Reaction condition are as follows: 94 DEG C, 4min；94
DEG C 30s, 57 DEG C of 30s, 72 DEG C of 45s, 35 circulations；72 DEG C of extension 10min.
It is preferred that the extraction pteria martensii sample gene group DNA to be measured is to extract pteria martensii sample to be measured
The genomic DNA of gill tissue.
Fifth object of the present invention is to provide one kind for detecting and the associated SNP marker of pteria martensii growth traits
Kit, which is characterized in that including the above-mentioned detection primer with the associated SNP marker of pteria martensii growth traits.
Using the detection primer of SNP marker associated with pteria martensii growth traits of the invention, can amplify
With the associated SNP genotype locus of pteria martensii growth traits, growth can be intuitively filtered out further according to PCR amplification electropherogram
Advantage individual is used as standby parent and carries out breeding, realizes molecular mark.
Detailed description of the invention:
Fig. 1 is the amplification electrophoretogram with the associated SNP marker of pteria martensii growth traits, wherein swimming lane 1-12 and swimming lane
14-25 is pteria martensii sample, and 13 be DL2000Maker, and band is followed successively by 2000bp, 1000bp, 750bp from top to bottom,
500bp, 250bp, 100bp.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1:
120 pteria martensii individuals from the same group are taken at random, after cleaning up, measure the growth of each shellfish
Character (shell is high, shell is long, shell is wide and weight), opens shell with opener, one piece of gill tissue is placed in 95% ethyl alcohol with scissors clip
In, it is saved in -20 DEG C, corresponds record, the shellfish after sampling is continued to cultivate.Use HiPure Universal DNA Kit
(guangzhou meiji biology company) extracts the genomic DNA of the gill tissue from 120 pteria martensiis, relevant operation ginseng
See that kit specification carries out.After DNA is extracted, the agarose gel electrophoresis in 1.0% detects its integrality, uv-spectrophotometric
The concentration and purity of meter measurement DNA, saves in -20 DEG C.
The detection primer of SNP marker associated with pteria martensii growth traits are as follows:
ASSN1184FI:5 '-CGATTTGCTAGAAAGCCATTA-3 '；
ASSN1184RI:5 '-AATTTGTTACAACTTACCTATAATGCAA-3 '；
ASSN1184FO:5 '-GGTAAAACATTAACACGGATTGA-3 '；
ASSN1184RO:5 '-ATTGTAATGACTTACCTAAAACGGAT-3 '.
With above-mentioned primer ASSN1184FI, ASSN1184RI, ASSN1184FO, ASSN1184RO to pteria martensii sample
Genomic DNA carries out PCR amplification in PCR instrument.PCR reaction system is as follows: 25 μ L of amplification system, includes pteria martensii sample
2.5 μ L of genomic DNA 0.5 μ L (40ng), 10 × PCR Mixture, 10 μM of detection primers ASSN1184FI, ASSN1184RI
Each 2 μ L, each 0.5 μ L, 0.1U Taq DNA enzymatic (Takara company) of 10 μM of detection primers ASSN1184FO, ASSN1184RO are double
It steams water and complements to 25 μ L.Reaction condition are as follows: 94 DEG C, 4min；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 45s, 35 circulations；72 DEG C of extensions
10min。
PCR product 3% agarose (guangzhou kanglong science and technology biology company) gel electrophoresis observes electricity with gel imaging system
Swimming map.Typing electrophoresis chart as shown in Figure 1, occur have 2 band of 310bp and 206bp size be AT genotype (swimming lane 3,4,
6,8,9,10,11,12,15,17,18,23), only occur 1 band of 310bp size be TT genotype (swimming lane 1,2,5,7,21,
22,24) 1 band of 206bp size, only occur is AA genotype (swimming lane 14,16,19,20,25).In this 120 individuals,
TT genotype individuals 22, shell is high, shell is long, shell is wide and weight is respectively 62.39cm, 62.93cm, 23.88cm, 38.98g, AT
Genotype individuals 59, shell is high, shell is long, shell is wide and weight is respectively 61.29cm, 62.78cm, 23.56cm, 37.22g, AA base
Because of type individual 37, shell is high, shell is long, shell is wide and weight is respectively 58.80cm, 59.80cm, 23.23cm, 33.8g, has TT base
Because the growth traits value of the individual of type is maximum, the growth traits value of AT genotype individuals is taken second place, the character value of AA genotype individuals
It is minimum.The correlation analysis with growth traits, gene is marked using the general linear model (GLM) in SPSS19.0 software
Difference between type corresponding trait mean value carries out Multiple range test analysis with Duancan, and the shell of TT genotype individuals is high, shell is long, shell
Wide and weight is noticeably greater than AT genotype individuals and AA genotype individuals (P < 0.05), that is, thinks to have between them significant
Statistical difference.Beneficial gene type is TT, can pick out the individual of TT genotype as standby parent, to cultivate superior progeny.
Claims (7)
1. a kind of and associated SNP marker of pteria martensii growth traits detection primer group, which is characterized in that draw including following
Object:
ASSN1184FI:5 '-CGATTTGCTAGAAAGCCATTA-3 '；
ASSN1184RI:5 '-AATTTGTTACAACTTACCTATAATGCAA-3 '；
ASSN1184FO:5 '-GGTAAAACATTAACACGGATTGA-3 '；
ASSN1184RO:5 '-ATTGTAATGACTTACCTAAAACGGAT-3 '.
2. with the application of the associated SNP marker of pteria martensii growth traits in molecular marker-assisted breeding of pinctada martensii；
The sequence of the SNP marker is as shown in SEQ ID NO.1, and sequence shown in the SEQ ID NO.1 is the 457th from 5 ' ends
Base is A or T.
3. a kind of method that detection pteria martensii has the individual of growth vigor, which is characterized in that by geneva pearl to be measured
The as claimed in claim 2 and associated SNP marker of pteria martensii growth traits in female shellfish is detected, and is determined described to be measured
Whether pteria martensii is the individual with growth vigor, determines if to carry out breeding as standby parent.
4. according to the method described in claim 3, characterized by comprising the following steps:
A, pteria martensii sample gene group DNA to be measured is extracted；
B, using detection primer ASSN1184FI, ASSN1184RI, ASSN1184FO, ASSN1184RO couple in claim 1
Pteria martensii sample gene group DNA carries out PCR amplification；
C, genotyping is carried out to pteria martensii sample according to pcr amplification product, when only there is 1 band of 310bp size
For TT genotype, which is determined as the individual with growth vigor, carries out breeding as standby parent.
5. according to the method described in claim 4, it is characterized in that, the PCR amplification, PCR reaction system are as follows: 25 μ L；
PCR reaction system is as follows: including pteria martensii sample gene group DNA 40ng, 1 × PCR Mixture, detection primer
Each 0.8 μM of ASSN1184FI, ASSN1184RI, each 0.2 μM of ASSN1184FO, ASSN1184RO, 0.1U Taq DNA enzymatic, surplus
For water；Reaction condition are as follows: 94 DEG C, 4min；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 45s, 35 circulations；72 DEG C of extension 10min.
6. according to the method described in claim 4, it is characterized in that, extraction pteria martensii sample gene group to be measured
DNA is the genomic DNA for extracting the gill tissue of pteria martensii sample to be measured.
7. a kind of for detecting and the kit of the associated SNP marker of pteria martensii growth traits, which is characterized in that including power
Benefit require 1 described in detection primer group with the associated SNP marker of pteria martensii growth traits.
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| CN108715896B (en) * | 2018-04-28 | 2020-10-27 | 中国科学院南海海洋研究所 | Method for screening pinctada martensii living parent with excellent growth characteristics |
| CN110218800A (en) * | 2019-07-10 | 2019-09-10 | 广东海洋大学 | One kind SNP marker relevant to pteria martensii growth traits and its application |
| CN110218801A (en) * | 2019-07-10 | 2019-09-10 | 广东海洋大学 | One kind EGFR gene SNP marker relevant to pteria martensii growth traits and its application |
| CN111607652B (en) * | 2020-05-29 | 2022-02-11 | 中国科学院南海海洋研究所 | InDel marker associated with growth traits of pinctada martensii, detection primer group and development method |
| CN111705142B (en) * | 2020-07-09 | 2023-04-11 | 广东海洋大学 | MIP gene SNP molecular marker related to growth traits of pinctada martensii and application thereof |
| CN111676298A (en) * | 2020-07-09 | 2020-09-18 | 广东海洋大学 | Pinctada martensii growth trait related KSPI gene SNP molecular marker and application thereof |
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