CN109943642B

CN109943642B - Method for identifying Suhong No.1 molecule of Clariaceae Exopalaemon carinicauda

Info

Publication number: CN109943642B
Application number: CN201910150554.2A
Authority: CN
Inventors: 李富花; 于洋; 金月; 张成松; 相建海; 张晓军
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2022-05-06
Anticipated expiration: 2039-02-28
Also published as: CN109943642A

Abstract

The invention belongs to the field of germplasm protection and application of aquatic animals, and particularly relates to a method for identifying a molecule of 'Suhong No. 1' of a family of palaemon carinicauda. By utilizing a high-throughput sequencing technology and a correlation analysis method, 2 specific Single Nucleotide Polymorphism (SNP) markers Ec53862-167-A/T and Ec53862-181-A/T of a new variety of the Cladonia chinensis 'Kesu red No. 1' are developed, and the two markers are A/T mutations. Both markers were of AA genotype in individuals "Cosu Red No. 1" and of TT or AT genotype in common Exopalaemon carinicauda. According to the two developed SNP markers, a method for carrying out molecular identification on a new variety of the exopalaemon carinicauda 'Kesu red No. 1' by an SNP typing method is established. The invention provides a technical means for molecular identification and variety protection of the Exopalaemon carinicauda 'Kesu red No. 1', and also provides an important technical support for cultivation, popularization and application of new Exopalaemon carinicauda varieties.

Description

Method for identifying Suhong No.1 molecule of Clariaceae Exopalaemon carinicauda

Technical Field

The invention belongs to the field of germplasm protection and application of aquatic animals, and particularly relates to a molecular identification method for a new variety of Exopalaemon carinicauda 'Kesu red No. 1'.

Background

The exopalaemon carinicauda is also called small white shrimp, fine shrimp, winter spring shrimp, and is benthic shrimp in warm zone sea area. The exopalaemon carinicauda has the advantages of strong reproductive capacity, high growth speed, wide environmental adaptability and the like, and has been developed into important cultured shrimp varieties in coastal areas of the eastern part of China in recent years. Along with the rise of the palaemon carinicauda breeding industry, the demand of high-quality offspring seeds is increased day by day, however, at present, the main breeding germplasm of the palaemon carinicauda is obtained by wild fishing, and the germplasm is seriously degraded after years of breeding, so that the healthy development of the palaemon carinicauda breeding industry is restricted, and the palaemon carinicauda breeding industry urgently needs excellent varieties with excellent characters.

The artificial breeding is an effective way for cultivating excellent varieties, and the target characters can be obviously improved through continuous multi-generation system breeding, so that new varieties with excellent characters can be cultivated. The palaemon carinicauda 'Kesu red No. 1' is a new palaemon carinicauda variety which is artificially bred, passes approval of Ministry of agriculture in 2018, and has the registration number of GS-01-004-2017. The new variety of the exopalaemon carinicauda 'the family Suhong No. 1' is characterized in that the epidermis and the muscle are red, and the new variety is rich in carotenoid and astaxanthin and has the advantages of high nutrition and high added value, so the new variety has wide popularization and application prospect. In the process of popularization and application, an accurate variety identification method needs to be established for protecting the variety right and the interests of a large number of farmers and consumers, so that the ordered popularization of the variety is guaranteed. Phenotypic identification and molecular identification are common identification means for animal and plant varieties, and compared with phenotypic identification, the molecular identification method has the advantage of being not influenced by factors such as breeding environment, individual size, growth and development state and the like, and is applied to more and more variety identification at present.

Single Nucleotide Polymorphism (SNP) markers are DNA sequence polymorphisms caused by single nucleotide variation and are widely distributed in animal and plant genomes, and because the SNP markers are suitable for high-throughput typing, the application of the SNP markers in variety molecular identification is more and more extensive. The exopalaemon carinicauda 'Kesu red No. 1' is subjected to continuous 4-generation genetic breeding, and forms the unique genetic structural characteristics of the variety, so that the specific SNP marker of the variety can be screened by combining a high-throughput sequencing means with an association analysis technology, and the method for molecular identification of the exopalaemon carinicauda 'Kesu red No. 1' is established.

The invention obtains an SNP marker which can be used for identifying the molecule of the Kesu red No.1 of the exopalaemon carinicauda by screening, thereby establishing a method for identifying the molecule of a new variety of the Kesu red No.1 and having important significance for protecting the intellectual property of the variety.

Disclosure of Invention

The invention aims to provide a method for identifying a new variety of palaemon carinicauda 'Kesu red No. 1', which is used for protecting property rights of the new variety.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for identifying a molecule of the 'Suhong No. 1' of the family palaemon carinicauda is implemented by a primer Ec 53862F: CGACGACACATCAAAAATCGACA and Ec 53862R: GTCTGTTTCACACTCAAGTCGAC amplifying the DNA sequence of the palaemon carinicauda tissue genome and sequencing to obtain the nucleotide sequence shown by SEQIDIC 53862, typing SNP markers Ec53862-167-A/T and Ec53862-181-A/T AT positions of 167bp and 181bp of the SEQIDIC 53862 sequence according to the sequencing result, if the typing results of the two markers are both AA homozygous, the palaemon carinicauda is a new variety 'Kesu red No. 1', and if the two markers are AT heterozygous or TT homozygous, the palaemon carinicauda is a common palaemon carinicauda.

SEQ ID Ec53862 has the deoxyribonucleic acid (DNA) sequence of SEQ ID No.1 of the sequence Listing;

sequence listing

Information of SEQ ID No.1

(a) Sequence characterization

Length: 601 nucleotide

Type (2): nucleotide, its preparation and use

Chain type: single strand

(b) Molecular type: DNA

Description of the sequence:

the nucleotide sequence of seq id Ec53862 is as follows:

ATTAAGTGAAAAGGTTGCTTTTATATACCCATAGACTTGACGACGACACATCAAAAATCGACAACAAACAAGAGGTCACCTCAGCCGTTTGTGTTATTAAAACAACTTTCACCTAAATTTGGTGGAGTTATCATGCCCAATAGCATACGATACCAGCCAGTAAGTANTTTTAGAAGCATTNAGTACATATTTAATTGAAAATCCACTAGTAATAACGTAGAATTTGCAATTGTTATGGGAAATGGCAGTAGACTCTGCCTATCTGTCAGAGTAGATGACGTAAGTCAAAATCAACTGGACGACGATAACCTTGATATCAACATTGTGTCTGTCACTTTGAGGAACTGGATGTTCAGGAAACACTTTTGATGTGGTGACTTTGAAAAAAAAAATCCAGAAAATCCCTGTTTAGACAAGTCCTCCTTAGTCTGTCGTTGACTACGTTACCTTCAAAGAATAGGCTTAAACTACCTTTCTTGTCTACCGTAGGTTTTAAGGAGAAATTCTTCCTGACCATATTACAGGTCGACTTGAGTGTGAAACAGACTTTATTGGCTCCTCTAATCTAATTAAGGTTTGTAGTTCGGTAAGCA

N＝[T/A]。

the invention utilizes a high-throughput sequencing technology and a correlation analysis method to develop 2 specific Single Nucleotide Polymorphism (SNP) markers Ec53862-167-A/T and Ec53862-181-A/T of a new variety of the Spanish carinicauda 'Kesu red No. 1', wherein the two markers are A/T mutations. Both markers were of AA genotype in individuals "Cosu Red No. 1" and of TT or AT genotype in common Exopalaemon carinicauda. According to the two developed SNP markers, a method for carrying out molecular identification on a new variety of the exopalaemon carinicauda 'Kesu red No. 1' by an SNP typing method is established. The invention provides a technical means for molecular identification and variety protection of the Exopalaemon carinicauda 'Kesu red No. 1', and also provides an important technical support for cultivation, popularization and application of new Exopalaemon carinicauda varieties.

The invention has the advantages that:

1. the molecular identification method provided by the method is not influenced by external factors such as the development period, the individual size, the culture environment and the like of the Spanish carinica 'Kesu red No. 1', and can accurately realize the identification of new varieties.

2. The established molecular identification method can be used for typing 2 SNP only by 1 amplification and sequencing, can accurately identify the 'Kesu red No. 1' of the exopalaemon carinicauda, and has the advantages of simple operation and low cost.

Detailed Description

Example 1: establishment of molecular identification method of exopalaemon carinicauda' Kesu red No.1

(1) DNA extraction of exopalaemon carinicauda

Randomly selecting 30 individuals of 'Kesu hong No. 1', 10 wild palaemon carinicauda in Shandong Binzhou, 10 cultured common palaemon carinicauda in south-east China, and 10 common palaemon carinicauda in Caesalpinia, wherein the materials jointly form experimental materials.

The experimental materials were prepared by extracting the DNA of muscle tissue using a Tiangen plant genome extraction kit and detecting the DNA concentration and quality by Nanodrop and agarose gel electrophoresis.

(2) PCR amplification and sequencing typing

The target region sequence of each individual was amplified using the following primer pairs:

Ec53862F：CGACGACACATCAAAAATCGACA

Ec53862R：GTCTGTTTCACACTCAAGTCGAC

and carrying out first-generation sequencing on the PCR amplification product by using an ABI3730xl sequencer, wherein the sequencing primer is an Ec53862F primer, typing is carried out on two SNP loci of Ec53862-167-A/T and Ec53862-181-A/T according to a sequencing peak map, if the position has only 1A base peak, the typing result is an AA type, if the position has only one T base peak, the typing result is a TT type, and if the position has both the A base peak and the T base peak, the typing result is an AT type.

(3) Analysis of results

According to the typing results, the judgment results of the judgment of individuals of 'Kesu hong No. 1', wild palaemon carinicauda in Shandong Binzhou areas, cultured common palaemon carinicauda in 10-tailed Nantong areas and common palaemon carinicauda in 10-tailed Hongyong areas are obtained, the results are shown in Table 1, the two SNP loci Ec53862-167-A/T and Ec53862-181-A/T of the palaemon carinicauda 'Kesu hong No. 1' are AA homozygote, the varieties of other sources are TT type, and the accuracy of the identification of the Kesu hong No.1 through the marker is 100%.

Table 1: results of two SNP site typing of different populations

Source	Ec53862-167-A/T	Ec53862-181-A/T
			Kesu hong No.1	AA	AA
Bin nationality group	TT	TT
			Lianyuankang group	TT	TT
Nantong group	TT	TT

Example 2: application of method for identifying molecule of 'Suhong No. 1' of family palaemon carinicauda in (1) material collection and DNA extraction

32 tails of exopalaemon carinicauda are purchased from different stalls on the Qingdao south mountain aquatic product market, 96 tails are used for detection in total, the Tiangen genome DNA extraction kit is used for extracting DNA of muscle tissues, and the concentration and the quality of the DNA are detected through Nanodrop and agarose gel electrophoresis. Each DNA sample was then diluted to 50 ng/. mu.l.

(2) PCR amplification and sequencing typing

PCR amplification is carried out on a DNA sample by utilizing Ec53862F and Ec53862R, an ABI3730xl sequencer is used for carrying out first-generation sequencing on an amplification product, a sequencing primer is an Ec53862F primer, two SNP loci of Ec53862-167-A/T and Ec53862-181-A/T are typed according to a sequencing peak map, if the position has only 1A base peak, the typing result is an AA type, if the position has only one T base peak, the typing result is a TT type, and if the position has both A base peak and T base peak, the typing result is an AT type.

(3) Analysis of typing results

According to the typing result, genotype distribution of two SNP loci in 96 individuals is analyzed, and the result shows that all 96 individuals are TT homozygous at Ec53862-167-A/T loci and are TT homozygous at Ec53862-181-A/T loci, which indicates that the materials are common palaemon carinicauda materials.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying Suhong No.1 molecules in the family of Cerriopsis carinatus is characterized in that: by using primer Ec 53862F: CGACGACACATCAAAAATCGACA and Ec 53862R: GTCTGTTTCACACTCAAGTCGAC amplifying the genomic DNA sequence of the palaemon carinicauda and sequencing to obtain the target region sequence of each individual, typing SNP markers Ec53862-167-A/T and Ec53862-181-A/T at positions 167bp and 181bp of the sequence SEQIDE Ec53862 according to the sequencing result, if the typing results of the two markers are AA homozygous, the palaemon carinicauda is a new variety 'Kesu red No. 1', and if the typing results of the two markers are TT homozygous, the palaemon carinicauda is a common palaemon carinicauda which is not 'Kesu red No. 1';

the nucleotide sequence of seq id Ec53862 is as follows:

ATTAAGTGAAAAGGTTGCTTTTATATACCCATAGACTTGACGACGACACATCAAAAATCGACAACAAACAAGAGGTCACCTCAGCCGTTTGTGTTATTAAAACAACTTTCACCTAAATTTGGTGGAGTTATCATGCCCAATAGCATACGATACCAGCCAGTAAGTA[T/A]TTTTAGAAGCATT[T/A]AGTACATATTTAATTGAAAATCCACTAGTAATAACGTAGAATTTGCAATTGTTATGGGAAATGGCAGTAGACTCTGCCTATCTGTCAGAGTAGATGACGTAAGTCAAAATCAACTGGACGACGATAACCTTGATATCAACATTGTGTCTGTCACTTTGAGGAACTGGATGTTCAGGAAACACTTTTGATGTGGTGACTTTGAAAAAAAAAATCCAGAAAATCCCTGTTTAGACAAGTCCTCCTTAGTCTGTCGTTGACTACGTTACCTTCAAAGAATAGGCTTAAACTACCTTTCTTGTCTACCGTAGGTTTTAAGGAGAAATTCTTCCTGACCATATTACAGGTCGACTTGAGTGTGAAACAGACTTTATTGGCTCCTCTAATCTAATTAAGGTTTGTAGTTCGGTAAGCA 。