CN109943642A - A method of for No. 1 Molecular Identification of exopalaemon carinicauda section Su Hong - Google Patents
A method of for No. 1 Molecular Identification of exopalaemon carinicauda section Su Hong Download PDFInfo
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- CN109943642A CN109943642A CN201910150554.2A CN201910150554A CN109943642A CN 109943642 A CN109943642 A CN 109943642A CN 201910150554 A CN201910150554 A CN 201910150554A CN 109943642 A CN109943642 A CN 109943642A
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Abstract
The invention belongs to aquatic livestock Conservation and application fields, and in particular to a method of it is used for exopalaemon carinicauda " section Su Hong 1 " Molecular Identification.Utilize high throughput sequencing technologies and association analysis method, 2 specific single nucleotide polymorphism (SNP) for developing exopalaemon carinicauda " section Su Hong 1 " new varieties mark Ec53862-167-A/T and Ec53862-181-A/T, the two labels are A/T mutation.Genotype of the two labels in " section Su Hong 1 " individual is AA, and in common exopalaemon carinicauda is TT or AT genotype.According to the two SNP markers of exploitation, the method that exopalaemon carinicauda " section Su Hong 1 " new varieties Molecular Identification is carried out by SNP classifying method is established.The present invention provides technological means for Molecular Identification and the kind protection of exopalaemon carinicauda " section Su Hong 1 ", also provides important technology support for the cultivation and popularization and application of exopalaemon carinicauda new varieties.
Description
Technical field
The invention belongs to aquatic livestock Conservation and application fields, and in particular to one kind is used for exopalaemon carinicauda " section Su Hong 1
Number " method for identifying molecules of new varieties.
Background technique
Exopalaemon carinicauda also known as small white shrimp, suede shrimp, shrimp of welcoming spring are that warm tropical seas bottom is dwelt shrimps.Exopalaemon carinicauda has breeding
The advantages that ability is strong, the speed of growth is fast and environmental suitability is wide had been developed as Deposits in Eastern Coastal China area weight in recent years
The shrimps in culture kind wanted.It is growing for the demand of high-quality seed along with the rise of exopalaemon carinicauda aquaculture, however mesh
The preceding main cultivation germplasm of exopalaemon carinicauda is both from wild fishing, and germplasm is degenerated serious after the cultivation of many years, constrains
The sound development of exopalaemon carinicauda aquaculture, exopalaemon carinicauda aquaculture industry are badly in need of the excellent excellent variety of character.
Artificially breeding is the effective way cultivated improved seeds, and by continuous multi-generation systematic breeding, can significantly improve mesh
Character is marked, the excellent new varieties of character are cultivated.Exopalaemon carinicauda " section Su Hong 1 " is the exopalaemon carinicauda new varieties of artificially breeding,
It is authorized by the Ministry of Agriculture within the kind 2018, cultivar registration number is GS-01-004-2017.Exopalaemon carinicauda " section Su Hong 1 " new product
Kind the characteristics of be epidermis and muscle is red, and be rich in carotenoid and astaxanthin, with high nutrition, high added value it is excellent
Point, therefore the kind has broad popularization and application prospect.During popularization and application, for protection kind power and vast cultivation
Family, consumer interests, need to set up accurate cultivar identification method, to ensure that variety popularization orderly carries out.Phenotype mirror
Fixed and Molecular Identification is the common identification of means of plant and animal species, and compared to phenotypic evaluation, method for identifying molecules has not by feeding
The advantage for growing the influence of the factors such as environment, Individual Size, growth and development, is answered in more and more cultivar identifications at present
With.
Single nucleotide polymorphism (SNP) label is DNA sequence polymorphism caused by the variation of single nucleotide acid, is divided extensively
It is distributed in animal-plant gene group, since SNP marker is suitble to high-pass typing, SNP marker answering in kind Molecular Identification
With more and more extensive.Exopalaemon carinicauda " section Su Hong 1 " have passed through the hereditary and selection in continuous 4 generation, form the unique heredity of kind
Structure feature, therefore the SNP mark of the varietY specificity by high-flux sequence means combination related analysis technology, can be screened
Note, thus the method for establishing exopalaemon carinicauda " section Su Hong 1 " Molecular Identification.
The present invention is to obtain the SNP mark that can be used for exopalaemon carinicauda " section Su Hong 1 " Molecular Identification by screening
Note, so that a kind of method for establishing " section Su Hong 1 " new varieties Molecular Identification, has protection kind intellectual property important
Meaning.
Summary of the invention
The object of the present invention is to provide a kind of methods for being used for exopalaemon carinicauda " section Su Hong 1 " new varieties Molecular Identification, use
In the property right protection of new varieties.
To achieve the above object, the invention adopts a technical scheme as:
A method of it being used for exopalaemon carinicauda " section Su Hong 1 " Molecular Identification, passes through primer Ec53862F:
CGACGACACATCAAAAATCGACA and Ec53862R:GTCTGTTTCACACTCAAGTCGAC expands exopalaemon carinicauda tissue gene
Group DNA sequence dna is simultaneously sequenced, and nucleotide sequence shown in SEQIDEc53862 is obtained, according to sequencing result pair
The SNP marker Ec53862-167-A/T and Ec53862-181-A/T of the position SEQIDEc53862 sequence 167bp and 181bp are carried out
Parting, if the genotyping result of two labels is AA homozygosis, which is " section Su Hong 1 " new varieties, miscellaneous if AT
It closes or TT is homozygous, be then common exopalaemon carinicauda.
SEQID Ec53862 has DNA (DNA) sequence of SEQ ID NO.1 in sequence table；
Sequence table
The information of SEQ ID No.1
(a) sequence signature
Length: 601 nucleotide
Type: nucleotide
Chain: single-stranded
(b) molecule type: DNA
Sequence description:
The nucleotide sequence of SEQID Ec53862 is as follows:
ATTAAGTGAAAAGGTTGCTTTTATATACCCATAGACTTGACGACGACACATCAAAAATCGACAACAAAC
AAGAGGTCACCTCAGCCGTTTGTGTTATTAAAACAACTTTCACCTAAATTTGGTGGAGTTATCATGCCCAATAGCAT
ACGATACCAGCCAGTAAGTANTTTTAGAAGCATTNAGTACATATTTAATTGAAAATCCACTAGTAATAACGTAGAAT
TTGCAATTGTTATGGGAAATGGCAGTAGACTCTGCCTATCTGTCAGAGTAGATGACGTAAGTCAAAATCAACTGGAC
GACGATAACCTTGATATCAACATTGTGTCTGTCACTTTGAGGAACTGGATGTTCAGGAAACACTTTTGATGTGGTGA
CTTTGAAAAAAAAAATCCAGAAAATCCCTGTTTAGACAAGTCCTCCTTAGTCTGTCGTTGACTACGTTACCTTCAAA
GAATAGGCTTAAACTACCTTTCTTGTCTACCGTAGGTTTTAAGGAGAAATTCTTCCTGACCATATTACAGGTCGACT
TGAGTGTGAAACAGACTTTATTGGCTCCTCTAATCTAATTAAGGTTTGTAGTTCGGTAAGCA
N=[T/A].
The present invention utilizes high throughput sequencing technologies and association analysis method, develops exopalaemon carinicauda " section Su Hong 1 " new product
The specific single nucleotide polymorphism (SNP) of 2 of kind mark Ec53862-167-A/T and Ec53862-181-A/T, the two marks
Note is A/T mutation.Genotype of the two labels in " section Su Hong 1 " individual is AA, and in common exopalaemon carinicauda
It is TT or AT genotype.According to the two SNP markers of exploitation, establishes and exopalaemon carinicauda " section is carried out by SNP classifying method
The method of Su Hong 1 " new varieties Molecular Identification.The present invention is the Molecular Identification and kind protection of exopalaemon carinicauda " section Su Hong 1 "
Technological means is provided, also provides important technology support for the cultivation and popularization and application of exopalaemon carinicauda new varieties.
Advantage for present invention:
1. the method for identifying molecules that this method provides is by exopalaemon carinicauda " section Su Hong 1 " developmental stage, Individual Size and
The influence of the external factors such as breeding environment can be accurately realized the identification of new varieties.
2. the method for identifying molecules established only needs 1 amplification and sequencing, parting, Ke Yizhun can be carried out to 2 SNP
Really identify exopalaemon carinicauda " section Su Hong 1 ", this method is easy to operate, low in cost.
Specific embodiment
Embodiment 1: the foundation of exopalaemon carinicauda " section Su Hong 1 " method for identifying molecules
(1) exopalaemon carinicauda DNA is extracted
It is individual to randomly choose 30 tails " section Su Hong 1 ", the wild exopalaemon carinicauda of 10 tail Shandong Binzhou Prefectures, 10 tail Nantong
The common exopalaemon carinicauda of the cultivation in area, the common exopalaemon carinicauda of 10 tail Lianyungang Areas, above-mentioned material collectively constitute experimental material.
Above-mentioned experimental material extracts the DNA of musculature using Tiangeng Plant Genome extracts kit, and passes through
Nanodrop and agarose gel electrophoresis detection DNA concentration and quality.
(2) PCR amplification and sequencing and typing
Utilize the target area sequence of each individual of following primer pair amplifies:
Ec53862F:CGACGACACATCAAAAATCGACA
Ec53862R:GTCTGTTTCACACTCAAGTCGAC
Pcr amplification product carries out generation sequencing using ABI3730xl sequenator, and sequencing primer is Ec53862F primer, root
Parting is carried out to two SNP sites of Ec53862-167-A/T and Ec53862-181-A/T according to sequencing peak figure, if the position is only
There is 1 A base peak, then genotyping result is AA type, if an only T base peak, genotyping result is TT type, if the position was both
There is A base peak to have T base peak again, then genotyping result is AT type.
(3) interpretation of result
According to above-mentioned genotyping result, the wild exopalaemon carinicauda of " section Su Hong 1 " individual, Shandong Binzhou Prefecture, 10 tails are obtained
The common exopalaemon carinicauda of the cultivation of Nantong Area, judgement that the common exopalaemon carinicaudas of 10 tail Lianyungang Areas determines as a result, result such as
Shown in table 1, two SNP sites of Ec53862-167-A/T and Ec53862-181-A/T of exopalaemon carinicauda " section Su Hong 1 " are
AA is homozygous, and the kind in other sources is TT type, is 100% by Su Hong 1 accuracy rate of the Marker Identification section.
Table 1: two SNP site genotyping results of different population
| Source | Ec53862-167-A/T | Ec53862-181-A/T |
| Section Su Hong 1 | AA | AA |
| Binzhou group | TT | TT |
| Lianyun Harbour group | TT | TT |
| Nantong group | TT | TT |
Embodiment 2: one kind for exopalaemon carinicauda " section Su Hong 1 " Molecular Identification method application (1) material collect with
DNA is extracted
Each 32 tail of exopalaemon carinicauda is bought in different stands from the fishery market of Qingdao South Mountain, and 96 tails make for detecting in total
The DNA of musculature is extracted with Tiangeng genome DNA extracting reagent kit, and is detected by Nanodrop and agarose gel electrophoresis
DNA concentration and quality.Each DNA sample is diluted to 50ng/ μ l later.
(2) PCR amplification and sequencing and typing
PCR amplification is carried out to DNA sample using Ec53862F and Ec53862R, amplified production is sequenced using ABI3730xl
Instrument carries out generation sequencing, and sequencing primer is Ec53862F primer, according to sequencing peak figure to Ec53862-167-A/T and Ec53862-
Two SNP sites of 181-A/T carry out parting, if the position only has 1 A base peak, genotyping result is AA type, if only one
A T base peak, then genotyping result is TT type, if there is T base peak at the position existing A base peak again, genotyping result is AT type.
(3) genotyping result is analyzed
According to above-mentioned genotyping result, the genotype distribution of two SNP sites is analyzed in 96 individuals, as the result is shown all 96
Individual is TT homozygosis in the site Ec53862-167-A/T, is also that TT type is homozygous in the site Ec53862-181-A/T, says
Bright above-mentioned material is common exopalaemon carinicauda material.
The above are the descriptions to the embodiment of the present invention to keep this field special by the foregoing description of the disclosed embodiments
Industry technical staff can be realized or using the present invention.Various modifications to these embodiments carry out those skilled in the art
Saying will be apparent, and the general principles defined herein can be the case where not departing from the spirit or scope of the present invention
Under, it realizes in other embodiments.Therefore, the present invention will not be limited to the embodiments shown herein, but to accord with
Close the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. a kind of method for exopalaemon carinicauda section No. 1 Molecular Identification of Su Hong, it is characterised in that: by using primer
Ec53862F:CGACGACACATCAAAAATCGACA and Ec53862R:GTCTGTTTCACACTCAAGTCGAC amplification spine end is white
Shrimp genomic dna sequence is simultaneously sequenced, and nucleotide sequence shown in SEQIDEc53862 is obtained, according to sequencing result pair
The SNP marker Ec53862-167-A/T and Ec53862-181-A/T of the position SEQIDEc53862 sequence 167bp and 181bp are carried out
Parting, if the genotyping result of two labels is AA homozygous, which is " section Su Hong 1 " new varieties, if AT heterozygosis
Or TT is homozygous, then is the common exopalaemon carinicauda of non-" section Su Hong 1 ".
2. according to the method described in claim 1, it is characterized by: for identifying the 2 of exopalaemon carinicauda " section Su Hong 1 " new varieties
A SNP marker Ec53862-167-A/T and Ec53862-181-A/T, the label is in exopalaemon carinicauda " section Su Hong 1 " new varieties
It is AA homozygosis, it is homozygous for AT heterozygosis or TT in common exopalaemon carinicauda group.
3. method according to claim 1 or 2, it is characterised in that:
SEQID Ec53862 has DNA (DNA) sequence of SEQ ID NO.1 in sequence table；
The nucleotide sequence of SEQID Ec53862 is as follows:
ATTAAGTGAAAAGGTTGCTTTTATATACCCATAGACTTGACGACGACACATCAAAAATCGACAACAAACAAG
AGGTCACCTCAGCCGTTTGTGTTATTAAAACAACTTTCACCTAAATTTGGTGGAGTTATCATGCCCAATAGCATAC
GATACCAGCCAGTAAGTA[T/A]TTTTAGAAGCATT[T/A]AGTACATATTTAATTGAAAATCCACTAGTAATAAC
GTAGAATTTGCAATTGTTATGGGAAATGGCAGTAGACTCTGCCTATCTGTCAGAGTAGATGACGTAAGTCAAAATC
AACTGGACGACGATAACCTTGATATCAACATTGTGTCTGTCACTTTGAGGAACTGGATGTTCAGGAAACACTTTTG
ATGTGGTGACTTTGAAAAAAAAAATCCAGAAAATCCCTGTTTAGACAAGTCCTCCTTAGTCTGTCGTTGACTACGT
TACCTTCAAAGAATAGGCTTAAACTACCTTTCTTGTCTACCGTAGGTTTTAAGGAGAAATTCTTCCTGACCATATT
ACAGGTCGACTTGAGTGTGAAACAGACTTTATTGGCTCCTCTAATCTAATTAAGGTTTGTAGTTCGGTAAGCA。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755315A (en) * | 2016-11-21 | 2017-05-31 | 中国农业科学院烟草研究所 | Tobacco glandular hairs secretion characteristic related gene Nt te Molecular mappings and preparation method and application |
| CN108570509A (en) * | 2018-07-19 | 2018-09-25 | 中国水产科学研究院黄海水产研究所 | A kind of detection method of exopalaemon carinicauda EC16 SNP markers |
| CN108913784A (en) * | 2018-07-19 | 2018-11-30 | 中国水产科学研究院黄海水产研究所 | A kind of detection method of exopalaemon carinicauda EC12 SNP marker |
| CN109234403A (en) * | 2018-10-09 | 2019-01-18 | 石河子大学 | A kind of method and primer special for identifying sheep lumbar vertebrae data/coherency shape |
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- 2019-02-28 CN CN201910150554.2A patent/CN109943642B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755315A (en) * | 2016-11-21 | 2017-05-31 | 中国农业科学院烟草研究所 | Tobacco glandular hairs secretion characteristic related gene Nt te Molecular mappings and preparation method and application |
| CN108570509A (en) * | 2018-07-19 | 2018-09-25 | 中国水产科学研究院黄海水产研究所 | A kind of detection method of exopalaemon carinicauda EC16 SNP markers |
| CN108913784A (en) * | 2018-07-19 | 2018-11-30 | 中国水产科学研究院黄海水产研究所 | A kind of detection method of exopalaemon carinicauda EC12 SNP marker |
| CN109234403A (en) * | 2018-10-09 | 2019-01-18 | 石河子大学 | A kind of method and primer special for identifying sheep lumbar vertebrae data/coherency shape |
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