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

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

A kind of method for molecular identification of white shrimp family Suhong No. 1

technical field

The invention belongs to the field of aquatic animal germplasm protection and application, and in particular relates to a molecular identification method for a new variety of white shrimp "Kesuhong No. 1".

Background technique

Ridgetail white shrimp, also known as small white shrimp, velvet shrimp, spring prawn, is a benthic shrimp in the warm tropical sea area. Ridgetail white shrimp has the advantages of strong reproductive ability, fast growth rate and wide environmental adaptability. In recent years, it has developed into an important cultured shrimp species in the eastern coastal areas of my country. With the rise of white shrimp aquaculture, the demand for high-quality seed is increasing. However, the main cultured germplasm of white shrimp are from wild fishing. The healthy development of the aquaculture industry is in urgent need of excellent varieties with excellent traits.

Artificial breeding is an effective way to cultivate excellent varieties. Through continuous multi-generation systematic breeding, target traits can be significantly improved and new varieties with excellent traits can be cultivated. Ridgetail white shrimp "Kesuhong No. 1" is a new artificially bred variety of ridgetail white shrimp, which was approved by the Ministry of Agriculture in 2018, and the variety registration number is GS-01-004-2017. The new variety of white shrimp "Kesuhong No. 1" is characterized by red skin and muscle, rich in carotenoids and astaxanthin, and has the advantages of high nutrition and high added value, so this variety has a wide range of promotion. application prospects. In the process of promotion and application, in order to protect the rights of varieties and the interests of farmers and consumers, it is necessary to establish an accurate method for identification of varieties, so as to ensure the orderly promotion of varieties. Phenotypic identification and molecular identification are commonly used identification methods for animal and plant varieties. Compared with phenotypic identification, molecular identification methods have the advantage of not being affected by factors such as breeding environment, individual size, and growth and development status. application in species identification.

Single nucleotide polymorphism (SNP) markers are DNA sequence polymorphisms caused by the variation of a single nucleotide and are widely distributed in the genomes of animals and plants. Since SNP markers are suitable for high-throughput typing, SNP markers are widely used in cultivars. Molecular identification has become more and more widely used. The white shrimp "Kesuhong 1" has undergone 4 consecutive generations of genetic selection, and it has formed a unique genetic structure of the species. Therefore, high-throughput sequencing methods combined with association analysis technology can be used to screen the species-specific SNP markers , so as to establish a molecular identification method for the white shrimp "Kesuhong No. 1".

The present invention obtains a SNP marker that can be used for molecular identification of "Kesuhong No. 1" of white shrimp through screening, thereby establishing a method for molecular identification of a new variety of "Kesuhong No. 1", which is useful for protecting species knowledge. Property rights are important.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for molecular identification of a new variety of white shrimp "Kesuhong No. 1" for the protection of property rights of the new variety.

To achieve the above object, the present invention adopts the technical scheme as follows:

A method for molecular identification of white shrimp "Kesuhong No. 1", by using primers Ec53862F: CGACGACACATCAAAAAATCGACA and Ec53862R: GTCTGTTTTCACACTCAAGTCGAC to amplify and sequence the DNA sequence of white shrimp tissue to obtain the nucleotide sequence shown in SEQIDEc53862 , according to the sequencing results, the SNP markers Ec53862-167-A/T and Ec53862-181-A/T at the 167bp and 181bp positions of the SEQIDEc53862 sequence were typed. If the typing results of the two markers are both AA homozygous, the ridge tail The white shrimp is a new variety of "Kesuhong No. 1". If it is AT heterozygous or TT homozygous, it is a common ridge-tailed white shrimp.

SEQID Ec53862 has the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

sequence listing

Information on SEQ ID No.1

(a) Sequence features

Length: 601 nucleotides

Type: Nucleotide

Chain type: single chain

(b) Molecular type: DNA

Sequence description:

The nucleotide sequence of SEQID Ec53862 is as follows:

ATTAAGTGAAAAGGTTGCTTTTATATACCCATAGACTTGACGACGACACATCAAAAATCGACAACAAACAAGAGGTCACCTCAGCCGTTTGTGTTATTAAAACAACTTTCACCTAAATTTGGTGGAGTTATCATGCCCAATAGCATACGATACCAGCCAGTAAGTANTTTTAGAAGCATTNAGTACATATTTAATTGAAAATCCACTAGTAATAACGTAGAATTTGCAATTGTTATGGGAAATGGCAGTAGACTCTGCCTATCTGTCAGAGTAGATGACGTAAGTCAAAATCAACTGGACGACGATAACCTTGATATCAACATTGTGTCTGTCACTTTGAGGAACTGGATGTTCAGGAAACACTTTTGATGTGGTGACTTTGAAAAAAAAAATCCAGAAAATCCCTGTTTAGACAAGTCCTCCTTAGTCTGTCGTTGACTACGTTACCTTCAAAGAATAGGCTTAAACTACCTTTCTTGTCTACCGTAGGTTTTAAGGAGAAATTCTTCCTGACCATATTACAGGTCGACTTGAGTGTGAAACAGACTTTATTGGCTCCTCTAATCTAATTAAGGTTTGTAGTTCGGTAAGCA

N=[T/A].

The present invention utilizes high-throughput sequencing technology and correlation analysis method to develop two specific single nucleotide polymorphism (SNP) markers Ec53862-167-A/T of the new species of white shrimp "Kesuhong 1" and Ec53862-181-A/T, both markers are A/T mutations. The genotypes of these two markers in the individual "Kesuhong 1" are both AA, while those of the common white shrimp are both TT or AT genotypes. According to the two SNP markers developed, a method for molecular identification of the new species of white shrimp "Kesuhong 1" by SNP typing was established. The invention provides technical means for molecular identification and variety protection of the white shrimp "Kesuhong No. 1", and also provides important technical support for the cultivation, popularization and application of new varieties of white shrimp.

The advantages of the present invention:

1. The molecular identification method provided by this method is not affected by external factors such as the developmental period of the white shrimp "Kesuhong No. 1", the size of the individual and the breeding environment, and can accurately realize the identification of new species.

2. The established molecular identification method only needs one amplification and sequencing to type two SNPs, and can accurately identify the white shrimp "Kesuhong No. 1". The method is simple to operate and low cost.

Detailed ways

Example 1: Establishment of the molecular identification method of white shrimp "Kesuhong No. 1"

(1) DNA extraction of white shrimp

Randomly selected 30 individuals of "Kesuhong No. 1", 10 wild white shrimp from Binzhou area, Shandong, 10 cultured common white shrimp from Nantong area, and 10 common white shrimp from Lianyungang area. The above materials together form the experiment. Material.

The above experimental materials were used to extract DNA from muscle tissue using the Tiangen plant genome extraction kit, and the DNA concentration and quality were detected by Nanodrop and agarose gel electrophoresis.

(2) PCR amplification and sequencing typing

Amplify the target region sequence of each individual using the following primer pairs:

Ec53862F:CGACGACACATCAAAAATCGACA

Ec53862R: GTCTGTTTCACACTCAAGTCGAC

PCR amplification products were sequenced by ABI3730xl sequencer, and the sequencing primer was Ec53862F. According to the sequencing peak map, the two SNP loci Ec53862-167-A/T and Ec53862-181-A/T were typed. If there is only one A base peak, the typing result is AA type. If there is only one T base peak, the typing result is TT type. If the position has both A base peak and T base peak, The typing result is AT type.

(3) Analysis of results

According to the above typing results, the determination results of the individual "Kesuhong No. 1", wild white shrimp from Binzhou area, Shandong, 10 cultured common white shrimp from Nantong area, and 10 common white shrimp from Lianyungang area were obtained. The results are shown in Table 1. The two SNP loci Ec53862-167-A/T and Ec53862-181-A/T of the white shrimp "Kesuhong 1" are both homozygous for AA, while the varieties from other sources are homozygous for AA. All were TT type, and the accuracy of identifying Kesuhong 1 by this marker was 100%.

Table 1: Typing results of two SNP loci in different populations

source Ec53862-167-A/T Ec53862-181-A/T Kesuhong No. 1 AA AA Binzhou group TT TT Lianyungang Group TT TT Nantong Group TT TT

Example 2: Application of a method for molecular identification of white shrimp "Kesuhong No. 1" (1) Material collection and DNA extraction

Purchased 32 white shrimp from different stalls in Qingdao Nanshan Aquatic Products Market, and a total of 96 were used for detection. The DNA of muscle tissue was extracted using Tiangen genomic DNA extraction kit, and the DNA concentration was detected by Nanodrop and agarose gel electrophoresis. and quality. Each DNA sample was then diluted to 50ng/μl.

(2) PCR amplification and sequencing typing

The DNA samples were amplified by PCR using Ec53862F and Ec53862R, and the amplified products were sequenced by ABI3730xl sequencer. The sequencing primer was the Ec53862F primer. If there is only one A base peak at the position, the typing result is AA type; if there is only one T base peak, the typing result is TT type. If the position has both The A base peak has a T base peak, and the typing result is AT type.

(3) Analysis of typing results

According to the above typing results, the genotype distribution of the two SNP loci in 96 individuals was analyzed, and the results showed that all 96 individuals were homozygous for TT at the Ec53862-167-A/T locus, The T sites were also homozygous for the TT type, indicating that the above materials were all common white shrimp materials.

The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in 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)

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 。