CN107841561A - The SNP marker and screening technique that long oyster shell form and aspect are closed - Google Patents
The SNP marker and screening technique that long oyster shell form and aspect are closed Download PDFInfo
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- CN107841561A CN107841561A CN201610826171.9A CN201610826171A CN107841561A CN 107841561 A CN107841561 A CN 107841561A CN 201610826171 A CN201610826171 A CN 201610826171A CN 107841561 A CN107841561 A CN 107841561A
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Abstract
The SNP genetic markers and screening technique closed the invention discloses long oyster shell form and aspect, the present invention use the long oyster shell colo(u)r group body of high-resolution melting curve analysis technology and 133 SNP sites excavated from EST public databases to seed selection（Golden, white, purple and black）Genotyping is carried out, analyzes to obtain 4 marks closed with long oyster life shell form and aspect by specific position.The present invention educates for long oyster shell color sorting provides molecule foundation, while will also have the function that to long oyster shell color Protection of Diversity important.
Description
Technical field
The invention belongs to field of molecular biotechnology, and in particular to a kind of SNP marker closed with long oyster shell form and aspect and its
Screening technique.
Background technology
Long oyster（Crassostrea gigas）Also known as Pacific oyster, has that environmental suitability is strong, the speed of growth
Hurry up, meat is fine and smooth and it is nutritious the features such as.In China's seawater aquaculture industry, long oyster is important economic shellfish, simultaneously
And worldwide it is distributed the cultivation species most wide, yield is maximum.The basic task of oyster genetic breeding be research and
On the premise of grasping oyster genetic variations rule, the basis of breeding objective as needed and original kind, using suitable
Breeding methods and method, select the high yield and high quality kind for meeting production development requirement, and then promote the development of oyster industry.Mesh
Before, except growth and disease resistance trait, long oyster shell color affect the hobby of consumer in breeding, so as to affect long oyster
Value, also turns into important breeding character.In addition, in long-term long oyster genetic improvement work, find under nature
Long oyster shell that thing is covered often is attached to there is shell color polymorphism, has filtered out white shell color, black shell color, golden shell color at present
With purple four kinds of shell color types of shell color.Existing result of study confirms that long oyster shell color is inhereditary feature.
SNP (Single Nucleotide Polymorphism) marks, referred to as SNP marker, is base
In the mutation of mononucleotide, have the characteristics that number is more, distribution is wide, compared to tandem sequence repeats microsatellite（SSR）Mark, SNP marker
Genetic stability and accuracy are of a relatively high.SNP marker is widely applied in aquatic livestock at present, including affiliation point
Analysis, hybrid vigour judgement, germplasm identification, genetic diversity sex investigation, map construction, QTL positioning and molecular labeling auxiliary
Seed selection etc..The shortcomings of time-consuming with effect difference, compared with traditional breeding way, molecular breeding be present in traditional breeding method
Inhereditary feature can not only be improved in a short time, save breeding time and cost, additionally it is possible on a molecular scale
Constantly explore and select more improved seeds.With the fast development of Protocols in Molecular Biology, SNP as with the direct phase of character
The mark of pass turns into the important tool of molecular mark.Therefore, the SNP marker closed with long oyster shell form and aspect is excavated, can
To educate offer molecular labeling for current long oyster shell color sorting, accelerate breeding process, breeding cost is reduced, for oyster culture industry
Sustainable health development is significant.
The content of the invention
It is an object of the invention to provide 4 SNP markers closed with long oyster shell form and aspect, established for long oyster shell color molecule seed selection
Fixed basis.
To reach above-mentioned purpose, the concrete technical scheme that the present invention takes is as follows：
The SNP marker that long oyster shell form and aspect are closed, it is characterised in that：
First SNP marker is named as CgSNP82, and it is located at the of the est sequence that accession number on NCBI websites is FQ667678
At 39 bases, mutation type is：A/C；
Second SNP marker is named as CgSNP273, and it is located at the of the est sequence that accession number on NCBI websites is HS243031
At 401 bases, mutation type is：A/G；
3rd SNP marker is named as CgSNP646, and it is located at the of the est sequence that accession number on NCBI websites is HS166834
At 222 bases, mutation type is：A/G；
4th SNP marker is named as CgSNP1131, and it is located at the est sequence that accession number on NCBI websites is HS225071
At 477th base, mutation type is：A/G.
The screening technique of four kinds of described SNP markers, comprises the following steps：
The acquisition of experimental population：Breeding parent, choosing are used as using golden, white, purple and the long oyster culture individual of black respectively
Select breeding and cultivate four kinds of different Ke Sechang oysters colonies（Gold, white, purple and black）；
There is polymorphism and the good SNP marker of parting effect using what is developed from est sequence, with high-resolution melting curve
（HRM）Carry out Genotyping；
FDIST2 methods are used respectively, and hierarchical island methods and Bayesian likelihood methods are entered
Row specific position is analyzed, and it is the consensus sites that three kinds of methods detect to have 4 sites.
The amplimer of 4 described SNP markers is respectively：
CgSNP82-F:ATGGCTGGACTCACCAACCGT,
CgSNP82-R: CACATTTCCGCTTCCTTGCTG；
CgSNP273-F:CAGCCTGAGATAGACAATGGAAG,
CgSNP273-R: TACATAGGAGGAGCGTGTGGTG；
CgSNP646-F:GAGAAAACAGTAGAAATTGACGCC,
CgSNP646-R: GAAATGGAGTTCTGATCTCAAGTTC；
CgSNP1131-F:ATGTGCTTTTTACCCGAACTGC,
CgSNP1131-R: ACCTGTTTTGGTTGCTCGTCTT。
Advantages of the present invention：
Excavation with shell form and aspect pass SNP marker is the pith of long oyster shell color breeding research, and molecule auxiliary is educated at present
The focus of kind research, but the SNP marker closed with shell form and aspect developed is very limited, and it is auxiliary to constrain long oyster shell color molecular labeling
Help the progress of breeding.After the present invention carries out parting with HRM, provided and long oyster shell by specific position analysis
4 SNP markers that form and aspect are closed, have saved time and cost, have improved operating efficiency.Also educate offer for long oyster shell color sorting simultaneously
Molecule foundation, have the function that to long oyster shell color Protection of Diversity important.
Embodiment
The present invention is further elaborated below by specific embodiment.
Embodiment 1
（1）The selection of experiment material：White shell color, black shell color, gold are chosen in the long oyster that in June, 2013 cultivates from Rushan Gold Mine, Shandong
Shell color and the excellent individual of 4 kinds of shell color shapes of purple shell color establish 4 kinds of shell colo(u)r group bodies, smart ovum are collected using post-mortem method as close shellfish,
Carry out artificial insemination.Each colony randomly chooses 38 individuals, and 152 individuals are as experiment material altogether.
（2）The DNA of long oyster is extracted using phenol chloroform method.
（3）Using developed from EST storehouses there is polymorphism and good 133 SNP markers of parting effect are to experimental population
Carry out Genotyping, the reaction system of HRM analyses
HRM analysis reaction condition be：95 DEG C of pre-degeneration 5mim；95 DEG C of denaturation 10s, (63 DEG C -60 of touchdown programs 10s
DEG C, each .5 DEG C of circulation decline 0), single fluorescence, 40 circulations are collected in 72 DEG C of extension 10s, 10s ends；95 DEG C reaction 1min, 40
DEG C reaction 1min, while collects fluorescence signal, 1 circulation；70-90 DEG C of reading fluorescence curve, 25 times/DEG C.
（4）FDIST2 methods, hierarchical island methods and Bayesian likelihood are used respectively
Method carries out specific position analysis to genotyping result, obtains 4 specific positions.
One of ordinary skill in the art will appreciate that, within the scope of the present invention, be carried out for above-described embodiment
Modification, addition and replacement are all possible, and it is all without departing from protection scope of the present invention.
<110>Chinese Marine University
<120>The SNP marker that long oyster shell form and aspect are closed
<130> 1
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 747
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP82
<400> 1
gaccaaccgt ccgtggacca agaacggacg tggaacatcg acgacatcgt tctagttatc 60
tggttcccta ccagcaagga agcggaaatg tggcttcgtc atgaaaaaca tttccgtaat 120
gcagcttttc cggaattcta tggatctgac gtcgtgtctc taccaattaa ctggaacccg 180
acaaacgatc gacattaccc aacattcctt gtaacggaat tcgaaggaat cgttgaccca 240
gatcagttca gggagaagtt cgccgtcaac atgagtgacg tgttacaacg tcacgaggcc 300
gaacgcttcg tcatccagac agtcggaatc aagcatctgc gtgggaactg gttcaaaccc 360
aagagcgtgg ttacgtgcac cagattccca agcagtgaag cggccctgcg gttttttcat 420
gacccggagt atgaacgttt gaggcggtca gtgagggaca tctccaagaa ccgcaccacc 480
gtcatgttca ccctgaccga gcgccccgtg tgagccccac ccccggcaat caatcgctgc 540
caaagcaacc tccaaacaat ctgtgataaa aactgaatac tagctacacg ttgtagaacc 600
agtggatcag tattactcag agatagacag cccttatcct aagattaaag tcctctttct 660
aatattcatt ccgtcttctg cttctaagtt ttgaatttat ttaaaataga attatgtccg 720
tccactgcct gaatactgtt tagttat 747
<210> 2
<211> 631
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP273
<400> 2
attgtttacg ttctgcagga agaataatgt cggtgacgga gacagatccc ccttttcggg 60
cgatggtgag agcaagtggt ttgggtgaag tatggactca cagcacggag agtagtaagt 120
ttaatcagtt tggatggagg tgcacaaata aagagaattc cttctctaac gacactctga 180
ttggaaactg gaatgaggaa aggttcgata acaatatatc gagacaggct caaagaatac 240
cgggccagca cgagcactat tttcagtcaa catacggcgt tggttacaat aaatcaccac 300
catatgaagt tcctaaggaa ttaattcatt taaaagagag gcattctcat gcctttccgg 360
gacatcagcc tgagatagac aatggaaggc taaagtcagt atataacagc tgggagacca 420
ccacacgctc ctcctatgta gaccccaaga tccgccaaca gccacttcag aacccaacca 480
gctagtttat ccatgggggt cacccgggtc aggagttatg gatgaatttt atgctaatga 540
atattgaagt ttattgatat ccaatatctg tctctgagtt ttcaatatca attttacatg 600
taatatagat ttttttttaa ttcaaatata g 631
<210> 3
<211> 682
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP646
<400> 3
tgttgatcaa aataaaattc acgcagaggg tcttttcaga caaaagacca tcgaaatcga 60
cggagaactc tcagctaaga aaggcagtct ttctatcaaa acacctttcg atggttttaa 120
gaaccagtat attaagttta ctgcagaggg acaaaagcta attgcagaag gaacattaat 180
ggagaaaaca gtagaaattg acgccctttt taacaaagac ggaaaaatag aaggagaact 240
tgagatcaga actccatttc aaggttttaa gaaacaagca attagatttt cgcatcatat 300
gacaaataaa ggttgccaaa cacacgctga tgttctcttt aaccaagaaa agtctgaatt 360
cgatctcagt atcattaatg aattgaataa ggaattcaaa gtctcgttga aaacaccgat 420
cacaggctat aaagaacagg ttttttgcgt ggaaacagta ccatcgccaa acggcttaaa 480
agtacactca gaagtccatt tcgataaaaa gaagttgagg ctgattttaa ttatgaacgt 540
gaaccgcagt tgttggccaa ttttgattgt aaaaaccccc ttttactggt tttgaatcga 600
cttcaatatc tttccaaaaa gatgggtctt tgagaaacat gcagctatct agcagtcttg 660
agtatggctc ttctcaaacc gg 682
<210> 4
<211> 634
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP1131
<400> 4
aaaaatactc aaatttatat ttttaaaaaa atatgtcgaa gaaatattca acactagtta 60
aagtctgaac attaagaaaa gagtcagtga ctaatatttc taaaaggttt attgttcgaa 120
tccattatcc tgcacgtaaa gttcaattca tcttgaccat tataatattt ccactcgacg 180
ttatccatgt aacatttatg gtttcgttga gtctgtgctt cccaaacgtg ctgaattcca 240
ataacgaaga agttttacac aaagttcaga tcgaggctct aggactgacg attttctttc 300
gtctttggtg tcatccccat cagaaccatt aggcgaagca gacccattct caccctgtcc 360
ttgactcttt gaggccagac ctcggctcag taaaccaatg acgatgtcct tattttcagg 420
cgtgatatct atcccatatt tcttcaaaac ctgttttggt tgctcgtctt cgagattatg 480
agaacgtttg cagttcgggt aaaaagcaca tcttcccgca ataaacgaac agacatgaag 540
atgtggacaa tctttaggtt ttttacaacc ttgggatata attatagaaa ctgcatattg 600
gaaggaatag tgactccctt tctatgattc aggt 634
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP82-F
<400> 5
atggctggac tcaccaaccg t 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP82-R
<400> 6
cacatttccg cttccttgct g 21
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP273-F
<400> 7
cagcctgaga tagacaatgg aag 23
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP237-R
<400> 8
tacataggag gagcgtgtgg tg 22
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP646-F
<400> 9
gagaaaacag tagaaattga cgcc 24
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP646-R
<400> 10
gaaatggagt tctgatctca agttc 25
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP1131-F
<400> 11
atgtgctttt tacccgaact gc 22
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> CgSNP1131-R
<400> 12
acctgttttg gttgctcgtc tt 22
Claims (3)
1. the SNP marker closed with long oyster shell form and aspect, it is characterised in that：
First SNP marker is named as CgSNP82, and it is located at the of the est sequence that accession number on NCBI websites is FQ667678
At 39 bases, mutation type is：A/C；
Second SNP marker is named as CgSNP273, and it is located at the of the est sequence that accession number on NCBI websites is HS243031
At 401 bases, mutation type is：A/G；
3rd SNP marker is named as CgSNP646, and it is located at the of the est sequence that accession number on NCBI websites is HS166834
At 222 bases, mutation type is：A/G；
4th SNP marker is named as CgSNP1131, and it is located at the est sequence that accession number on NCBI websites is HS225071
At 477th base, mutation type is：A/G.
2. the screening technique of the SNP marker described in claim 1, it is characterised in that comprise the following steps：
The acquisition of experimental population：Respectively using golden, white, purple and the long oyster culture colony of black as breeding parent, lead to
Cross four kinds of different Ke Sechang oysters colonies that selection and use is cultivated（Gold, white, purple and black）；
There is polymorphism and the good SNP marker of parting effect using what is developed from est sequence, Genotyping is carried out with HRM；
FDIST2 methods are used respectively, and hierarchical island methods and Bayesian likelihood methods are entered
Row specific position is analyzed, wherein 4 sites are the site that every kind of method can detect.
3. SNP marker according to claim 1, it is characterised in that the primer of amplification 4 SNP markers is respectively：
CgSNP82-F:ATGGCTGGACTCACCAACCGT,
CgSNP82-R: CACATTTCCGCTTCCTTGCTG；
CgSNP273-F:CAGCCTGAGATAGACAATGGAAG,
CgSNP273-R: TACATAGGAGGAGCGTGTGGTG；
CgSNP646-F:GAGAAAACAGTAGAAATTGACGCC,
CgSNP646-R: GAAATGGAGTTCTGATCTCAAGTTC；
CgSNP1131-F:ATGTGCTTTTTACCCGAACTGC,
CgSNP1131-R: ACCTGTTTTGGTTGCTCGTCTT。
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| CN201610826171.9A CN107841561B (en) | 2016-09-18 | 2016-09-18 | SNP (Single nucleotide polymorphism) marker related to oyster shell color and screening method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742531A (en) * | 2012-07-31 | 2012-10-24 | 中国海洋大学 | Construction method of superior strains of rapid-growing crassostrea gigas |
| CN104152444A (en) * | 2014-07-24 | 2014-11-19 | 中国科学院海洋研究所 | SNP (single-nucleotide polymorphism) marker related to Crassostrea gigas glycogen content character and application thereof |
-
2016
- 2016-09-18 CN CN201610826171.9A patent/CN107841561B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742531A (en) * | 2012-07-31 | 2012-10-24 | 中国海洋大学 | Construction method of superior strains of rapid-growing crassostrea gigas |
| CN104152444A (en) * | 2014-07-24 | 2014-11-19 | 中国科学院海洋研究所 | SNP (single-nucleotide polymorphism) marker related to Crassostrea gigas glycogen content character and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 王家丰: "长牡蛎基因区SNP标记规模开发及其在遗传育种研究中的应用", 《中国科学院大学博士学位论文》 * |
| 王庆志: "长牡蛎品种选育与生长性状的遗传参数估计", 《中国博士学位论文全文数据库（农业科技辑）》 * |
| 王庆志等: "长牡蛎成体生长性状的遗传参数估计", 《中国水产科学》 * |
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