CN107354234B - Method for screening parent oysters with high glycogen content and related primer pair thereof - Google Patents

Abstract

The invention relates to a method for screening crassostrea gigas parent scallops with high glycogen content and a primer pair of related SNP markers. The SNP locus has A and G allelic forms in genome DNA; the nucleotide sequence of 500bp of the upstream and the downstream is shown as SEQ ID No. 1. The implementation result shows that the SNP locus can be accurately detected by using the method, and individuals with high glycogen content can be screened. Meanwhile, compared with an AA genotype individual, the glycogen content of the GG genotype individual at the site is remarkably increased by 4.5%. Subsequently, by the method, parent oysters of GG genotypes are screened to guide oyster breeding. The invention provides SNP marker development and potential application of individuals with high glycogen content of crassostrea gigas, and has the advantages that genotype identification can be carried out on parent scallops before offspring seed breeding, and the content of glycogen of offspring is improved. The reliability of the SNP marker obtained by the research result is higher, the population adaptation range is wider, and the effect is more stable.

Description

Method for screening parent shellfish with high glycogen content in long oyster and related primer pairs

technical field

The invention belongs to the field of genetic engineering and genetic breeding, and relates to a method for screening parent oysters with high glycogen content and a primer pair for related SNP markers.

Background technique

Oysters are an important marine aquatic resource and one of the most important marine aquaculture shellfish in the world. my country's oyster farming scale and output have ranked first in the world for many years, but for a long time, my country's oysters can only be produced and sold by themselves, and it is difficult to enter the high-end market. The main reason is that in the process of breeding and breeding, only the high yield and high quantity of oysters are paid attention to, and the content of its nutritional quality is ignored. The high content of nutrients such as glycogen is one of the most important characteristics of oysters, which not only affects the plumpness and yield of oysters, but also affects the taste of oyster consumption, and is an important part of the quality of oyster products. Therefore, improving the content of nutritional qualities such as glycogen and genetic improvement is an important way to solve the current situation of high yield and low efficiency in my country's oyster industry.

The research on aquatic animal breeding started late, and traditional group breeding methods are mainly used to construct family lines. The main drawback is that the breeding cycle is long and the effect is slow. In recent years, with the development of breeding technology, molecular marker-assisted selection and whole genome selection have been gradually applied in aquatic animal breeding, which has greatly improved the genetic and breeding level of shellfish. Compared with traditional breeding, the selection of traits has been significantly improved. Efficiency and Precision. At the same time, the use of molecular marker-assisted selection can reduce blindness, shorten breeding years, and improve breeding efficiency. The key to molecular breeding is to obtain reliable molecular markers. Currently, the commonly used methods are QTL mapping of traits and genome-wide association analysis (GWAS). Although QTL mapping methods can rapidly locate chromosome segments associated with traits, they have the characteristics of high accuracy in trait analysis. However, based on the linkage analysis of limited meiosis, the regions of the mapped large segments of the genome are linked together, making it difficult to precisely locate. The genome-wide association analysis GWAS method can perform overall association analysis of genetic variation genes in the whole genome, and locate the associated loci in a small interval, which significantly improves the accuracy and precision of the positioning, especially for complex traits. genetic analysis.

Although GWAS have played an important role in agrobiological studies of crops and livestock, only a few reports have been reported in aquatic animals. In the existing studies on shellfish, the development of molecular markers mainly focuses on the known functional genes. The number of markers is relatively small, and the major loci may not necessarily be obtained, so it is impossible to have a comprehensive understanding of the genetic regulation mechanism of phenotype. . So far, there have been no reports on obtaining major loci based on genome-wide association analysis. However, with the completion of the whole genome sequencing of the long oyster and the acquisition of a high-density genetic linkage map, our research team took the lead in conducting GWAS analysis of key quality traits in oysters, and obtained key loci and genes that affect quality traits, including the control of glycogen. The major polymorphism sites and key genes of the content. On the basis of whole-genome association analysis, the present invention develops a large number of SNP markers, analyzes the genetic basis for determining the glycogen content of oysters, and selects the SNP markers that are extremely significantly related to the glycogen content for screening individuals with high glycogen content. Compared with previously developed single SNP loci, the results of this study are more reliable and applicable to a wider range of populations.

Invention content:

The purpose of the present invention is to provide a SNP marker related to the glycogen content of the oyster, and to provide a reference for the molecular marker-assisted selection of the oyster.

The specific methods for obtaining SNPs are as follows: (1) Material collection and homogenization and domestication: 486 wild oysters were collected and homogenized. (2) Determination of phenotypic data: The glycogen content of individual oysters was detected by anthrone colorimetry. (3) Genotyping: On the Illumina next-generation sequencing platform, 486 oyster parents were re-sequenced, SNPs loci were screened and individuals were typed to obtain valid SNPs loci for association analysis. (4) Association analysis: Genome-wide association analysis was performed using mixed linear model, and 7 SNP loci (P-value<10 ^-6 ) significantly associated with the trait were obtained, which were located in the range of 36,512-37,583bp of the long oyster genome scaffold1597. By LD block analysis, 7 SNP sites were tightly linked (LD>0.7). The Manhattan plot of the genome-wide association analysis is shown in Figure 1. Then, a SNP site located at base 36,675 of scaffold1597 (P-value of 2.49×10 ^-7 ) was selected as the SNP site for subsequent development. There are two base forms of A and G at this site. The same identification method was applied to other SNP sites located in the 36,512-37,583 bp range of scaffold 1597.

The present invention is achieved through the following technical solutions:

A SNP marker related to the glycogen content of oyster: The marker is located at base 36,675 of the oyster genome scaffold1597, and this base exists in the form of A and G. The sequence of 500 bp upstream and downstream of the site is shown in SEQ ID No. 1. The main detection steps are as follows:

1. DNA was extracted with phenol-chloroform, and the specific steps were as follows:

1) Take a 1.5 ml centrifuge tube, add 700 μl of DNA extraction buffer (100 mM Tris-HCl, 5 mM EDTA), take 3-10 mg of oyster gill tissue, and cut into pieces with scissors. The utensils used must be flame sterilized and rinsed with ddH2O between _two individuals to prevent cross-contamination between individuals. Add 35 μl SDS and mix. Add 2 μl proteinase K and mix.

2) Incubate the centrifuge tubes in a metal bath at 65°C, add 2 μl of proteinase K to each tube after 1.5 hours, gently shake the centrifuge tubes during the period and continue to incubate for more than half an hour after the tissue is completely digested, with a total incubation time of at least 3 hours.

3) Cool the incubated samples to room temperature, add an equal volume of PCI, mix well, and centrifuge at 13,000 rpm for 10 min. Repeat the above steps once. PCI is a mixed solution of phenol:chloroform:isoamyl alcohol (25:24:1).

4) The supernatant of step 3) was extracted once with chloroform:isoamyl alcohol (24:1), and centrifuged at 13000 rpm for 5 min.

5) Transfer the supernatant of step 4) into an equal volume of -20°C isopropanol, invert back and forth to mix well, and place at -20°C for 20 minutes.

6) Centrifuge the mixed solution of step 5) at 4°C and 13000rpm for 5min. Pour out all liquid carefully, taking care not to allow the white sediment particles at the bottom to move or pour out.

7) Wash twice with 75% ethanol at -20°C, pour out the liquid, and place a centrifuge tube in the opening to dry the ethanol. When the white precipitate becomes colorless and transparent, add 50-100 μl (depending on the amount of DNA) ddH2O to dissolve the DNA. Store at -20°C for later use.

2. Use specific primers for peripheral amplification.

Take the above-mentioned long oyster genomic DNA as a template, and use primers to prepare a reaction system: 1uL of genomic DNA, 5uL of general PCR mix, 0.2uL of primers F and R, 3.6uL of sterilized double-distilled water; the reaction system can be amplified year-on-year;

The primer is: upstream primer F: 5'-TCCGAACTTGGAATCCTCTC-3'

Downstream primer R: 5'-GCAAATGTTAAGGTGGCTCA-3'

The reaction procedure for PCR amplification is as follows:

3. SnaPshot template preparation

Add 5U SAP and 2U ExoI to 15μL PCR product, shake and mix, incubate at 37°C for 1 hr, and then incubate at 75°C for 15 minutes to inactivate SAP and ExoI enzymes.

4. SnaPshot PCR amplification and purification.

The PCR product was used as the template for SNaPshot PCR. After purification, 2 μL of each was mixed. The specific steps of SNaPshotPCR are:

The specific primer sequences are:

5’-TTTTTTTTTTTTTTTTGAGAATTGTAAACCACACACACGG-3’

Product purification: Add 1 U SAP to 10 μL of the above SNaPshot PCR product, shake and mix, incubate at 37°C for 1 hr, at 75°C for 15 min to inactivate the enzyme, and store at 4°C for 24hr or -20°C for long-term storage.

5. Capillary electrophoresis.

1) Electrophoresis sample preparation: First, dilute the SNaPshot product 20 times:

Denaturation at 95°C for 5 min, followed by rapid ice cooling for 4 min.

2) Capillary electrophoresis. The prepared samples were subjected to capillary electrophoresis using a 3730XL DNA Analyzer and the signal was collected. Environmental conditions: laboratory temperature: 18-25°C; capillary length: 50cm; furnace temperature: 60°C; operating voltage: 15kV. Results The experimental results were analyzed using GeneMapper V4.0. Identify different genotypes.

Potential applications of SNP markers related to glycogen content in oysters: So far, except for this patent, there has been no report on the development of SNP markers based on genome-wide association analysis in oysters. Compared with the previously developed SNP markers, the results of this study have higher reliability, a wider range of adaptation groups, and a more stable effect. Before seed breeding, oyster genomic DNA was extracted by non-lethal sampling, and the genotype of the parent shellfish was determined by using the SNP marker and its identification method as described above.

Description of drawings

Figure 1 is a Manhattan plot of the genome-wide association analysis of glycogen content in long oysters.

Detailed ways:

The present invention is further illustrated below in conjunction with the examples, but the examples do not limit the present invention in any form.

Example 1:

a) Collection of samples: 288 individuals from the wild population that hatched seedlings in Jiaonan were collected, dissected, and the adductor muscle and remaining tissues were taken, which were snap-frozen with liquid nitrogen and stored at -80°C for later use.

b) DNA extraction: extract the genomic DNA of 288 samples and determine the concentration using ultraviolet absorptiometry, and use sterilized water to dilute the genomic DNA to 10-20ng/uL according to the determined concentration;

c) SNP locus genotype detection using SnaPshot: (1) Peripheral amplification using specific primers. Take the above-mentioned long oyster genomic DNA as a template, and use primers F and R to prepare a reaction system: 1uL of genomic DNA, 5uL of general PCR mix, 0.2uL of primers F and R, 3.6uL of sterilized double-distilled water; the reaction system can be amplified year-on-year; The primer sequences are:

Upstream primer F: 5'-TCCGAACTTGGAATCCTCTC-3'

Downstream primer R: 5'-GCAAATGTTAAGGTGGCTCA-3'

The reaction procedure for PCR amplification is as follows:

(2) SnaPshot template preparation. Add 5U SAP and 2U ExoI to 15μL PCR product, shake and mix, incubate at 37°C for 1 hr and then at 75°C for 15 min to inactivate SAP and ExoI enzymes.

(3) SnaPshot PCR amplification and purification. The PCR product was used as the template for SNaPshot PCR. After purification, 2 μL of each was mixed. The specific steps of SNaPshot PCR are:

The specific primer sequences are:

5’-TTTTTTTTTTTTTTGAGAATTGTAAACCACACACACGG-3’

Product purification: Add 1 U SAP to 10 μL of the above SNaPshot PCR product, shake and mix, incubate at 37°C for 1 hr, at 75°C for 15 minutes to inactivate the enzyme, and store at 4°C for 24hr or -20°C for long-term storage.

(4) Capillary electrophoresis. Electrophoresis sample preparation: First dilute the SNaPshot product 20-fold:

Denaturation at 95°C for 5 min, followed by rapid ice cooling for 4 min. The prepared samples were subjected to capillary electrophoresis using the 3730XL DNA Analyzer and the signal was collected. Environmental conditions: laboratory temperature: 18-25°C; capillary length: 50cm; furnace temperature: 60°C; operating voltage: 15kV. Results The experimental results were analyzed using GeneMapper V4.0. Identify different genotypes.

d) Result analysis: After detection, there are 86 individual genotypes of G/G, 141 individual genotypes of A/G, and 61 individual genotypes of A/A.

e) Detection of glycogen content: The data shows that the order of obtaining glycogen content of different genotypes is GG>AG>AA. Compared with the AA type, the glycogen content of the GG genotype was increased by 4.5%. Therefore, the typing of this locus was significantly correlated with glycogen content. By screening GG-type individuals in breeding, the glycogen content of offspring can be significantly improved.

Table 1: Glycogen content and genotyping analysis of 288 individuals.

Sequence listing (1) Information sequence characteristic length of SEQ ID NO.1: 1001bp

Type: Nucleic Acid

Chain type: single chain

Topology: Linear

Molecular Type: DNA

Source: Long Oyster

Sequence description:

>scaffold1597

CAATGGTTTTTTTTTTACTACTACCTCAGAATTAAAAAATTGTACATCTGATACAAAATGGC GGGGCAGGGTGCGGTCTAGCGGTGGAGTTTAATATCCTTGATGTGGATAAAAAGCAATTAGT GGAACTGTCAGAGAAGAGAAAAATGGCGATGTTTCTTCGATTCGCAAATCATATGCCTGACA GGTTCTCAAATTTTACAATTTACGATTTATTGGCCTTGATTTATAACAGCAGATCATTTTTG GTCAATGTCCCGAGTGTCTTGTTTCTGTACTGAGCAGCCACGCGTGCACAATAGTAATTTCC GAACTTGGAATCCTCTCATATCGGCGAAGACGGGCCGAAAATAACTGATACCGCGAATAGTC AAAAAATGTCTGTTAACAGTGAAGCGTTAACAGTGAAATTTTGCACCATCTTTTTCAAAACG TCCCATCTTCAGCAACATTTGACTGTTTAGTGTTGTTTAAAAGAGAACGATGTCCAATAGGT TTTTACCGTGTGTGGTTTACAATTCTCCCTATAATTCTCCTTCCCACAGGGCCGTAGATTAC GGGTCGGCGGAACATAAGCGGGCCTCAACCATGCCGTCACAGGGTTCACTGTTCACCAATTT TGAGGAAGTTCAGGTAAGATCTCACCTTTGGCAATCTAATATACTAGTAGCAGTATCCTAGT GAGCCACCTTAACATTTGCTTTTCGTTTTTGAAGATTTTGAAACAGACCAAAAAAAATATCA TGTTCATTTCATCATTTTGAGAATTTTGAATTAACTAGATAAATAAGTGTAAATACAAAATT TACAATTTTTAAAACAAAGTATACTTGTATTGTGTGAAAATACATAGTTATTTCTAGCCCTG AAAAACTAAAAATATACACTTGTAGTATGTATTATAGATCGTGTGTCGTGGGAATATTTTGG GAAAACTGTGGTTTCAACATTATTTATAGGCATCAATTTAGAAATTAAAGCACAG TTACAAA CTATGGTTA.

The SNP site of the present invention has two allele forms of A and G in the genomic DNA; the 500bp nucleotide sequence of its upstream and downstream is shown in SEQ ID No.1. The implementation results show that the method can accurately detect the SNP site and screen individuals with high glycogen content. At the same time, the glycogen content of individuals with GG genotype at this locus was significantly increased by 4.5% compared with individuals with AA genotype. This method can be used to screen parent shellfish of GG genotype to guide oyster breeding. The invention provides the development and potential application of a SNP marker for screening individuals with high glycogen content in oyster oysters. The results of this study have higher reliability of SNP markers, a wider range of adaptation groups, and a more stable effect.

sequence listing

<110> Institute of Oceanography, Chinese Academy of Sciences

<120> A method for screening oysters with high glycogen content in long oysters and primer pairs marked by related SNPs

<141> 2017-09-20

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caatggtttt ttttttacta ctacctcaga attaaaaaat tgtacatctg atacaaaatg 60

gcggggcagg gtgcggtcta gcggtggagt ttaatatcct tgatgtggat aaaaagcaat 120

tagtggaact gtcagagaag agaaaaatgg cgatgtttct tcgattcgca aatcatatgc 180

ctgacaggtt ctcaaatttt acaatttacg atttattggc cttgatttat aacagcagat 240

catttttggt caatgtcccg agtgtcttgt ttctgtactg agcagccacg cgtgcacaat 300

agtaatttcc gaacttggaa tcctctcata tcggcgaaga cgggccgaaa ataactgata 360

ccgcgaatag tcaaaaaatg tctgttaaca gtgaagcgtt aacagtgaaa ttttgcacca 420

tctttttcaa aacgtcccat cttcagcaac atttgactgt ttagtgttgt ttaaaagaga 480

acgatgtcca ataggttttt accgtgtgtg gtttacaatt ctccctataa ttctccttcc 540

cacagggccg tagattacgg gtcggcggaa cataagcggg cctcaaccat gccgtcacag 600

ggttcactgt tcaccaattt tgaggaagtt caggtaagat ctcacctttg gcaatctaat 660

atactagtag cagtatccta gtgagccacc ttaacatttg cttttcgttt ttgaagattt 720

tgaaacagac caaaaaaaat atcatgttca tttcatcatt ttgagaattt tgaattaact 780

agataaataa gtgtaaatac aaaatttaca atttttaaaa caaagtatac ttgtattgtg 840

tgaaaataca tagttatttc tagccctgaa aaactaaaaa tatacacttg tagtatgtat 900

tatagatcgt gtgtcgtggg aatattttgg gaaaactgtg gtttcaacat tatttatagg 960

catcaattta gaaattaaag cacagttaca aactatggtt a 1001

Claims (1)

1. A method for screening parent oysters with high glycogen content by using primers for detecting SNP marker loci is characterized by comprising the following steps:

the SNP marker locus is located at the 501bp position of a sequence SEQ ID No.1, the locus has the forms of A and G bases, genotype identification of the SNP marker locus is carried out on the parent oyster before seedling breeding, glycogen content of different genotypes is GG > AG > AA, and the glycogen content of a progeny population is improved by screening the parent oyster with the genotype of the locus GG;

the method comprises the following steps:

(1) extracting genomic DNA of parent oysters, namely extracting the genomic DNA of the parent oysters, and diluting the genomic DNA to 10-20 ng/mu L by using sterilized water or TE buffer solution;

(2) taking the genomic DNA of the parent oyster of the crassostrea gigas as a template, and preparing a reaction system by using primers:

the specific reaction system is as follows: 1 muL of genome DNA, 5 muL of universal PCR mix, 0.2 muL of each of an upstream primer F and a downstream primer R, and 3.6 muL of sterilized double-distilled water;

the upstream primer F is as follows: 5'-TCCGAACTTGGAATCCTCTC-3' the flow of the air in the air conditioner,

the downstream primer R is as follows: 5'-GCAAATGTTAAGGTGGCTCA-3', respectively;

(3) the reaction procedure for PCR amplification was:

(4) preparation of SnaPshot template: adding 5U of SAP and 2U of ExoI into 15 muL of PCR product, shaking, mixing uniformly, preserving heat at 37 ℃ for 1 hour, and then preserving heat at 75 ℃ for 15min to inactivate SAP and ExoI enzyme;

(5) performing amplification and purification by using SnaPshot PCR: taking the PCR product as a template of the SnaPshot PCR, wherein the specific primer sequence of the SnaPshot PCR is as follows:

5’- TTTTTTTTTTTTTTTGAGAATTGTAAACCACACACGG-3’

and (3) purifying a product: adding 1U of SAP into 10 muL of the SnaPshot PCR product, shaking and uniformly mixing, preserving heat at 37 ℃ for 1 hour, preserving heat at 75 ℃ for 15min to inactivate enzyme, and preserving at 4 ℃ for 24 hours or preserving at-20 ℃ for a long time;

(6) capillary electrophoresis

1) Preparing an electrophoresis sample: the SnaPshot product was first diluted 20-fold:

the reagent range is as follows: the 10 muL total volume comprises Hi-Di Formamid 9.25 muL, GS-120LIZ 0.25 muL and SnaPshot product 0.5 muL;

denaturation at 95 deg.C for 5min, and rapidly cooling with ice for 4 min;

2) capillary electrophoresis: capillary electrophoresis was performed on the prepared samples using 3730 XLDDNA Analyzer and signals were collected, environmental conditions: laboratory temperature: 18-25 ℃; capillary length: 50 cm; temperature of the heating furnace: 60 ℃; operating voltage: 15kV, results the results of the experiment were analyzed using GeneMapper V4.0 to determine the different genotypes.

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