CN114525362B - Primer combination and its application for identification of "Anyuan No. 1" sea cucumber population - Google Patents

Abstract

The invention relates to a primer combination for identifying an 'Anyuan No. 1' stichopus japonicus population and application thereof, belonging to the field of molecular biology, wherein the primer combination is shown in table 1. The invention also provides a method for identifying the stichopus japonicus population of 'Anyuan No. 1' by utilizing the primer combination, extracting DNA of a stichopus japonicus sample by adopting an SDS method, carrying out SNP genotyping of the sample to be detected by utilizing the primer combination, and detecting the special marking site of the stichopus japonicus population of 'Anyuan No. 1'; the judgment standard is as follows: detecting that the individual has all 3 primer marking sites, and judging that the individual is an Anyuan No. 1 stichopus japonicus individual; and the population samples are more than or equal to 30, all 3 primer marking sites of detected individuals are positive detection, the positive detection rate in the population reaches 80% or more, and the Apostichopus japonicus population of Anyuan No. 1 is judged.

Description

Primer combination for identifying the "Anyuan No. 1" sea cucumber population and its application

Technical Field

The invention belongs to the field of molecular biology, and in particular relates to a molecular identification marker primer combination and application for identifying an "Anyuan No. 1" sea cucumber population.

Background Art

Sea cucumbers are traditional high-end health food in my country. They are widely farmed in Liaoning and Shandong, and are the pillar industry of my country's marine aquaculture. The annual output remains at around 200,000 tons. The industry chain is long, the products are diversified, and the annual output value reaches 30-40 billion yuan. As of 2021, after approval by the National Aquatic Species and Breeding Committee, the Ministry of Agriculture and Rural Affairs announced that the sea cucumber "Anyuan No. 1" (GS-01-014-2017) has been approved as a new aquatic variety for national review and promotion. The sea cucumber "Anyuan No. 1" is based on the "Shuiyuan No. 1" with more than 50 spines and a fast growth rate as the parent, with the number of spines, weight and meat yield as the breeding targets, and has been bred for four consecutive generations. Under the same breeding conditions, compared with ordinary sea cucumbers, the "Anyuan No. 1" sea cucumber has brown or dark brown body color, more and longer spines, fast growth rate and high processing yield. It has been widely promoted for breeding in the coastal areas of Shandong, Liaoning and Fujian, with significant economic benefits. There are sea cucumber seedlings pretending to be "Anyuan No. 1" on the market, disrupting the market. The development of molecular markers that can be used to identify "Anyuan No. 1" sea cucumbers can not only effectively resolve disputes, but also has important significance for regulating the sea cucumber market and effectively protecting sea cucumber germplasm resources.

As the third generation of molecular markers, single nucleotide polymorphism (SNPs) markers are co-dominant markers with high polymorphism and can be quickly detected by sequencing technology. This study used resequencing data to mine the unique SNP sites of "Anyuan No. 1", combined with KASP typing technology to design primer combinations that can be used to identify "Anyuan No. 1" sea cucumbers, providing technical support for parentage identification and germplasm resource protection of the new sea cucumber variety "Anyuan No. 1".

Summary of the invention

The technical problem to be solved by the present invention is to provide a total of SNP marker primers that can be used to identify the "Anyuan No. 1" sea cucumber population based on resequencing technology and KASP typing technology. The application of this primer can successfully identify the "Anyuan No. 1" sea cucumber population and individuals.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

A primer combination for identifying the "Anyuan No. 1" sea cucumber population, the primer combination is shown in Table 1:

Table 1 SNP marker primer combinations used to identify “Anyuan No. 1” sea cucumber

The present invention provides a method for identifying a population of a new sea cucumber variety "Anyuan No. 1" using the above three groups of primers, and the method is specifically as follows:

The DNA of the sea cucumber samples was extracted by SDS method, and the SNP genotyping of the samples was performed with KASP combination markers to detect the unique marker sites of the "Anyuan No. 1" sea cucumber population; the judgment standard was: the detected individual had all three of the above primer marker sites, and was determined to be an "Anyuan No. 1" sea cucumber individual. The population sample was greater than or equal to 30 individuals, and the detected individual had all three of the above primer marker sites as positive detection, and the positive detection rate in the population reached 80% or more, and the "Anyuan No. 1" sea cucumber population was determined.

The beneficial effects of the present invention compared with the prior art are as follows:

The present invention utilizes resequencing data to mine SNP sites unique to the sea cucumber "Anyuan No. 1" population, and combines KASP typing technology to design primer combinations that can be used to identify the sea cucumber "Anyuan No. 1" population, providing technical support for paternity identification and germplasm resource protection of the sea cucumber "Anyuan No. 1".

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a genotyping diagram of the sea cucumber population to be tested using the primers of the present invention (partial results are shown).

DETAILED DESCRIPTION

The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

Example 1

1. Acquisition of specific SNP markers of “Anyuan No. 1” sea cucumber

1.1 Sample selection

A total of 256 sea cucumbers were collected from seven representative farmed and wild populations, including the new sea cucumber species "Anyuan No. 1" and from Yantai, Shandong, Muping, Shandong, Changdao County, Shandong, Weihai, Shandong, Dalian, Liaoning, and Zhuanghe, Liaoning.

1.2 DNA extraction

The total genomic DNA was extracted by SDS method. The kit used was TIANGEN amp Marine Animals DNA Kit (TIANGEN, Beijing, China), and the muscle tissue of sea cucumbers was extracted. The purity and integrity of nucleic acid were preliminarily tested by 1% agarose gel electrophoresis. The main band of qualified DNA samples was clear, with no degradation or slight degradation. The mass concentration of the qualified sea cucumber DNA samples was adjusted to about 100 ng/ul for storage.

1.3 Genome resequencing

The qualified samples were randomly fragmented into 350bp fragments using BioruptorPico ultrasonic disruptor, and then the library was prepared using the genomic DNA library construction kit. The library was constructed according to the following steps:

(1) The genomic DNA is fragmented using a BionuptorPico ultrasonic disruptor;

(2) DNA fragment end filling: repair the genome ends into blunt ends and add a phosphate group to the 5' end;

(3) Adding 'A' to the 3' end of the DNA fragment;

(4) DNA end connection adapter: Adapter is added to both ends of the DNA fragment through TA connection;

(5) PCR amplification: The ligation products to which the adapters have been added are amplified by PCR to form a library that can be sequenced.

The constructed library was quality checked by three methods: Qubit library concentration quantification, agarose electrophoresis detection of library size characteristics, and qPCR to accurately quantify the effective concentration of the library. The concentration of the library that passed the quality inspection was normalized and high-throughput sequencing was performed on the Illumina HiSeq2500 sequencing platform.

1.4SNP acquisition and quality control

After obtaining clean reads, BWA software was used to align clean reads with the reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/002/754/855/GCA_002754855.1_ASM275485v1/). The software GATK (v3.8) was used to perform population SNP detection on the samples, and the obtained results were filtered with the filtering criteria of "--minQ 200--minDP 3--min-meanDP 3--max-missing 0.8--maf0.05".

1.5 Association typing

Genome association analysis was performed using Plink software.

The "Anyuan No. 1" sea cucumber and sea cucumbers of other populations are divided into two subgroups.

The Logistic model was used for association analysis to obtain the significant P value corresponding to each SNP. The obtained P was corrected using Bonferroni, and finally the significantly associated SNP sites of "Anyuan No. 1" sea cucumber were obtained.

1.6 KASP primer design

According to the above-mentioned significant SNP sites of "Anyuan No. 1", 42 pairs of PCR 3' end specific amplification primers were designed. The design software was Primer 5. Each sequence required 3 primers, namely two specific upstream primers and one universal downstream primer. The primer design principles are: (1) The primer itself has no hairpin structure, and primer dimers will not be formed between the upstream and downstream primers; (2) The GC content of the primer is between 40 and 60%, and the Tm value is between 55 and 65 °C.

2KASP tag verification

2.1 Validation of population DNA extraction and marker detection

"Anyuan No. 1" sea cucumber and 15 sea cucumbers from other groups were randomly selected, and DNA was extracted by SDS method. Labeling detection was performed on Douglas ArrayTapeRPlatform, and fluorescent quantitative PCR reaction was performed in SOELLEX high-throughput PCR water bath environment. TB GreenTM Premix Ex TaqTMⅡ reagent was used, and 20μl mixed system was prepared to perform specific amplification by the two-step reaction procedure of "denaturation-annealing extension".

2.2 KASP marker typing

The signals of the two fluorescent groups, FAM and VIC, were read using the ARAYA fluorescence reader, and the results were imported into the database.

In Douglas Scientific Dashboard, sample SNP typing was performed according to the principles of clear typing, NTC (no sample negative control), and no specific amplification. The typing results were analyzed using SNP viewer genotype reading software. The typing was successful when the KASP marker read a heterozygous genotype in the sample. In the typing diagram, X:X represents homozygous type, including five types: A:A, T:T, C:C, G:G, and -:- (missing), X:Y represents heterozygous type, including three types: A:G, C:T, and A:- (partial missing), ? represents no signal or a weak signal, and Uncallable represents a signal but no clear typing.

Three markers were screened out from the 42 KASP markers. The screening condition was that the detection rate in the "Anyuan No. 1" population reached 100% and that they did not appear in other populations. The sequences of the three KASP markers are shown in Table 1.

2.3 Identification standards and application of “Anyuan No. 1” sea cucumber

The judgment criteria are: the DNA of the sea cucumber samples is extracted by SDS method, the SNP genotyping of the samples to be tested is performed with KASP combination markers, and the unique marker sites of the "Anyuan No. 1" sea cucumber population are detected; the judgment criteria are: the detected individual has all three of the above primer marker sites, and is determined to be an "Anyuan No. 1" sea cucumber individual. If the population sample is greater than or equal to 30, the detected individual has all three of the above primer marker sites as a positive detection, and the positive detection rate in the population reaches 80% or more, it is determined to be an "Anyuan No. 1" sea cucumber population.

Explanation of setting the judgment standard: Based on the previous survey, it is very easy for unintentional mixing of individuals and groups to occur in sea cucumber ponds and bottom seeding farming processes. Setting an 80% positive detection rate as the "Anyuan No. 1" group discrimination indicator is more objective.

application:

1. Thirty sea cucumbers of known germplasm (5 "Anyuan No. 1" sea cucumbers and 25 common sea cucumbers from different regions) were identified. The results showed that all five "Anyuan No. 1" sea cucumbers had three primer markers; 0 of the 25 common sea cucumbers from different regions had all three markers detected (4 had two markers and 8 had one marker), and were determined to be non-"Anyuan No. 1" sea cucumbers. The detection accuracy rate was 100%.

2. 4 groups were tested (1 group was a pond culture group of purchased "Anyuan No. 1" sea cucumber seedlings, 32 individuals; the other 4 groups were randomly selected pond sampling groups in Dalian and Shandong, 30 individuals in each pond, a total of 120 individuals). The SNPviewer test results showed (partial results, see Figure 1): There were 32 individuals in the "Anyuan No. 1" seedling culture group, 29 individuals with 3 markers, and the positive detection rate of the "Anyuan No. 1" group was 90.63%, which was determined to be the "Anyuan No. 1" sea cucumber group. Among the other 4 groups, 0 individuals had all 3 markers detected (17 individuals with 2 markers and 32 individuals with 1 marker), which was determined to be a non-"Anyuan No. 1" sea cucumber group.

Sequence Listing

<110> Dalian Ocean University

<120> Primer combination for identification of the “Anyuan No. 1” sea cucumber population and its application

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> DNA/RNA

<213> Artificial Sequence

<400> 1

ttttcttcgt acttcgtaga ctgtga 26

<210> 2

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 2

tcttcgtact tcgtagactg tgg 23

<210> 3

<211> 29

<212> DNA/RNA

<213> Artificial Sequence

<400> 3

gtgcaatgca ataaacaact ttccctcat 29

<210> 4

<211> 25

<212> DNA/RNA

<213> Artificial Sequence

<400> 4

ggagccatcc attgatacta tgaag 25

<210> 5

<211> 25

<212> DNA/RNA

<213> Artificial Sequence

<400> 5

ggagccatcc attgatacta tgaaa 25

<210> 6

<211> 29

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

cagagactac actgggtata cagtaataa 29

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

ccagggatga ctatcgctga g 21

<210> 8

<211> 22

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

cccagggatg actatcgctg aa 22

<210> 9

<211> 29

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

ttacacagtc agtccttcaa acaacacat 29

Claims (2)

1. A primer combination for identifying a "anderson No. 1" stichopus japonicus population, wherein the primer combination is as shown in the following table

2. A method for identifying a population of "anderson No. 1" stichopus japonicus using the primer combination of claim 1, wherein the method is specifically as follows:

extracting DNA of the stichopus japonicus sample by adopting an SDS method, carrying out SNP genotyping of the sample to be detected by using the primer combination, and detecting the unique marker locus of the stichopus japonicus population of Anyuan No. 1; the judgment standard is as follows: detecting that the individual has all 3 primer marking sites, and judging that the individual is an Anyuan No. 1 stichopus japonicus individual; the number of the population samples is more than or equal to 30, the detected individuals are judged to be positive individuals when the number of the detected individuals is G/G, AX-741061 at the AX-294749 site and T/T at the AX-762538 site, the positive detection rate in the population reaches 80% or more, and the Apostichopus japonicus population of Anyuan No. 1 is judged.

Images