CN113005204A - Vibrio-resistant related EST-STR marker of litopenaeus vannamei, specific primer and detection method thereof - Google Patents
Vibrio-resistant related EST-STR marker of litopenaeus vannamei, specific primer and detection method thereof Download PDFInfo
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Abstract
The invention discloses a Vibrio-resistant related EST-STR marker of Litopenaeus vannamei, a specific primer and a detection method thereof. The invention utilizes the constructed transcriptome library to develop and obtain 6 EST-STR marks of the litopenaeus vannamei with high polymorphism, the serial numbers of the EST-STR marks are Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv-Vp023, Lv-Vp029 and Lv-Vp041 respectively, and specific primers of the EST-STR marks are designed respectively. The EST-SSR marker and the primer of the litopenaeus vannamei can be used for genetic diversity research of the litopenaeus vannamei and auxiliary breeding of excellent varieties.
Description
Technical Field
The invention belongs to the technical field of biomolecule labeling, and particularly relates to a Vibrio-resistant related EST-STR label of litopenaeus vannamei, a specific primer and a detection method thereof.
Background
Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus pacificus (White pacific shrimp). Wild populations are distributed in central and south america, from mexico to the pacific coastal waters where chile's natural water temperatures are above 20 ℃ throughout the year, typical tropical prawns. The coastal waters from south to north and the seawater and fresh water of a plurality of inland areas of China are both cultured. At present, the litopenaeus vannamei is the first cultured shrimp in the world, and accounts for about 70 percent of the worldwide cultured prawn yield and 80 to 90 percent of the national prawn culture yield. At present, the breeding yield of litopenaeus vannamei per year in China exceeds 160 million tons. In recent years, the White Spot Syndrome Virus (WSSV) disease, the early death syndrome (EMS) bacterial disease and the like frequently occur, the cultured prawns die in large quantities due to abnormal fluctuation of the culture water environment, and the quality requirements of the market on the cultured prawns are higher and higher. Market changes, disease epidemics and breeding environment changes lead to increasingly urgent needs for new disease-resistant breeding.
The traditional breeding method has long period and low efficiency. With the rapid transformation of modern biotechnology and genetic technology in the field of aquatic products, molecular marker technology based on trait-related functional genes becomes one of the key technologies for genetic breeding of aquatic products. Molecular marker assisted breeding technology based on molecular marker technology such as Simple repeat sequences (SSRs) or Short Tandem Repeat Sequences (STRs) has become a key technology of current aquatic product breeding. Currently, few SSR markers based on antibacterial ESTs (expressed Sequence tag) have been developed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides an EST-STR marker of the litopenaeus vannamei, an amplification primer, a detection method and application thereof, and provides a molecular marker with high polymorphism for genetic diversity analysis and molecular marker-assisted breeding of the litopenaeus vannamei.
In order to realize the aim, the invention utilizes the transcriptome library data constructed by the litopenaeus vannamei seedlings constructed by the team under the infection of Vibrio harveyi, and the constructed sequencing library is sequenced on the Illumina HiSeq 4000 platform (Illumina, San Diego, USA) of the Huada Gene GmbH (Huada gene, Shenzhen). The method comprises the steps of developing a high polymorphic EST-STR marker of the litopenaeus vannamei from an EST-SSR sequence of the litopenaeus vannamei obtained from a database, providing a polymorphic primer, and providing a high polymorphic molecular marker for genetic diversity research of the litopenaeus vannamei and auxiliary breeding of excellent varieties.
According to the invention, 50 pairs of primers are selected from the Vibrio harveyi infected Litopenaeus vannamei EST-SSR database to perform PCR amplification on the genomic DNA of the Litopenaeus vannamei according to the results of function prediction and core region repeated difference, wherein 6 pairs of primers can stably amplify a target band. The STR sample is detected by adopting a 3730xl device, SSR data are analyzed by using Genemapper software, and finally 6 vannamei prawn EST-STR markers with high polymorphism are determined.
The first purpose of the invention is to provide an EST-STR mark for the litopenaeus vannamei, wherein the EST-STR mark is numbered as Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv-Vp023, Lv-Vp029 and Lv-Vp 041;
the nucleotide sequence of the Lv-Vp001 is shown as SEQ ID NO. 1;
the nucleotide sequence of the Lv-Vp011 is shown in SEQ ID NO. 2;
the nucleotide sequence of the Lv-Vp012 is shown in SEQ ID NO. 3;
the nucleotide sequence of the Lv-Vp023 is shown in SEQ ID NO. 4;
the nucleotide sequence of the Lv-Vp029 is shown as SEQ ID NO. 5;
the nucleotide sequence of the Lv-Vp041 is shown as SEQ ID NO. 6.
The second purpose of the invention is to provide an amplification primer marked by the EST-STR of the litopenaeus vannamei, wherein the amplification primer comprises:
for the Lv-Vp001 site:
Lv-Vp001-F：5’-ACTGGAGGATCCTGAGAGATAGC-3’；
Lv-Vp001-R：5’-GCTTCGTTCTCTCACTCTTCTCTT-3’；
for the Lv-Vp011 site:
Lv-Vp011-F：5’-AAGGATTTCTCTTACACGAACCC-3’；
Lv-Vp011-R：5’-ACCAAAAAGAAAAACAGAATGGC-3’；
for the Lv-Vp012 site:
Lv-Vp012-F：5’-AATCTACAGGACTCGAATGCACA-3’；
Lv-Vp012-R：5’-CTTTTGTCCTCCACTTTACATGC-3’；
for Lv-Vp23 site:
Lv-Vp23-F：5’-CGAGAGAAAGAGGGAGAAAAAGA-3’；
Lv-Vp23-R：5’-TCATGGAGATGATGACTGGATAA-3’；
for Lv-Vp29 site:
Lv-Vp29-F：5’-CACCATATTCTCCAAGAAGCAGT-3’；
Lv-Vp29-R：5’-TTACTCATCCTGCTTCAACACCT-3’；
for Lv-Vp41 site:
Lv-Vp41-F：5’-CTTTTCGTTGATGAGTCTTTCCA-3’；
Lv-Vp41-R：5’-AGGGAGAAAGAGTGGAGTGAGAT-3’。
preferably, the forward primer of the amplification primer is labeled at the 5' end with a fluorophore.
Preferably, the fluorophore is FAM or HEX.
The third purpose of the invention is to provide a method for detecting the EST-STR mark of the litopenaeus vannamei, which comprises the following steps:
(1) extracting genomic DNA of the litopenaeus vannamei;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and respectively using the primer pair Lv-Vp001-F/R aiming at the Lv-Vp001 site, the primer pair Lv-Vp011-F/R aiming at the Lv-Vp011 site, the primer pair Lv-Vp012-F/R aiming at the Lv-Vp012 site, the primer pair Lv-Vp023-F/R aiming at the Lv-Vp023 site, the primer pair Lv-Vp029-F/R aiming at the Lv-Vp029 site and the primer pair Lv-Vp041-F/R aiming at the Lv-Vp041 site;
(3) and (4) typing the PCR amplification product by using a sequencer.
Preferably, the PCR amplification reaction system is 25 μ L, and comprises: containing Mg 2+10 XPCR buffer 2.5. mu.L, 10mM dNTPs 1. mu. L, Taq enzyme 2U, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu. L, DNA template 10ng, the remainder being made up to 25. mu.L by sterile double distilled water.
Preferably, the PCR amplification comprises the following reaction procedures: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 10 cycles; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 35 cycles total, and re-extension at 72 ℃ for 5-8 minutes.
The fourth purpose of the invention is to provide the application of the Litopenaeus vannamei EST-STR marker or the amplification primer in the group genetic diversity research, gene positioning, genetic linkage map construction, germplasm identification, variety classification or molecular marker-assisted breeding of the Litopenaeus vannamei.
Preferably, the application is the application in the application of the Litopenaeus vannamei EST-STR marker or amplification primer related to the antibacterial and/or insect-resistant property.
Preferably, the antibacterial and/or insect-resistant agent is vibrio harveyi and/or prawn enterobacter hepatica.
The invention utilizes the EST sequence of the litopenaeus vannamei obtained from the NCBI database to develop and obtain 6 EST-STR marks of the litopenaeus vannamei with high polymorphism, and the serial numbers are Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv-Vp023, Lv-Vp029 and Lv-Vp041 respectively. The expressed sequence tag EST-STR marker of the litopenaeus vannamei can be used for genetic relationship analysis and molecular marker assisted breeding of the litopenaeus vannamei and lays a foundation for genetic diversity research of the litopenaeus vannamei and assisted breeding of good varieties.
The molecular marker related to the antibacterial property of the litopenaeus vannamei, which is explored by the invention, has great application value for the screening of the offspring seeds with strong antibacterial capability and the screening of excellent antibacterial germplasm of the litopenaeus vannamei.
Drawings
FIG. 1 is an STR typing diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by Lv-Vp001 site primers; wherein S1-S20 represent 20 samples.
FIG. 2 is an STR typing diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by Lv-Vp011 site primer; wherein S1-S20 represent 20 samples.
FIG. 3 is an STR typing diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by Lv-Vp012 locus primers; wherein S1-S20 represent 20 samples.
FIG. 4 is an STR typing chart of 20 Litopenaeus vannamei genomic DNAs amplified by Lv-Vp023 site primers; wherein S1-S20 represent 20 samples.
FIG. 5 is an STR typing diagram of 20 Litopenaeus vannamei genomic DNAs amplified by Lv-Vp029 site primers; wherein S1-S20 represent 20 samples.
FIG. 6 is an STR typing diagram of 20 genomic DNAs of Litopenaeus vannamei amplified by Lv-Vp041 locus primers; wherein S1-S20 represent 20 samples.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The experimental procedures in the following examples were carried out in a conventional manner or according to the kit instructions unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Primer synthesis and sequencing were performed by Shanghai bioengineering, Inc.
Example 1:
1. screening of EST-STR sequence of Litopenaeus vannamei
According to the invention, 50 pairs of EST-STR primers (shown in Table 1) are selected from an EST-SSR database of Vibrio harveyi infected litopenaeus vannamei and are subjected to polymorphism screening according to the function prediction related to diseases and the result of repeated difference of core regions.
TABLE 1 Litopenaeus vannamei EST-STR labeled primer characteristics Table
2. Screening and result analysis of EST-STR labeled primers of litopenaeus vannamei
2.1 extraction of genomic DNA of Litopenaeus vannamei
The DNA samples are respectively 5 Vibrio sensitive individuals and 5 insensitive individuals; the method comprises the following steps of respectively resisting 5 prawn enterobacter hepatica individuals and 5 contrasts, totaling 20 samples, taking muscle tissues of 20 litopenaeus vannamei tails from 20 farms such as Guangdong Shenzhen, Zhuhai, Zhanjiang, Xuweng and Maocai respectively according to the sample conditions shown in table 2, extracting genomic DNA of litopenaeus vannamei by adopting a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), and strictly performing the operation steps according to the instruction. Genomic DNA quantification was performed using NanoDropTM2000 spectrophotometer, and the quality is detected by agarose electrophoresis.
TABLE 2 sample conditions
2.2 preliminary screening of primers
Randomly extracting 5 parts of the extracted genomic DNA of the litopenaeus vannamei as a template, respectively adopting 50 pairs of primers in the table 1 to perform PCR gradient amplification on the genomic DNA, and performing 25 mu L reverse amplificationA reaction system comprises: containing Mg 2+10 XPCR buffer 2.5. mu.L, 10mM dNTPs 1. mu. L, Taq enzyme 2U, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu. L, DNA template 10ng, the remainder being made up to 25. mu.L by sterile double distilled water. Reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 50-60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 10 cycles; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 35 cycles total, and re-extension at 72 ℃ for 5-8 minutes.
The PCR amplification product is detected by 2% agarose electrophoresis, and 6 pairs of primers can stably amplify a band at a specific annealing temperature. Sequencing the amplification products respectively, wherein the results show that the 6 pairs of primers can amplify target bands, and the sites are respectively: Lv-Vp001 (shown as SEQ ID NO. 1), Lv-Vp011 (shown as SEQ ID NO. 2), Lv-Vp012 (shown as SEQ ID NO. 3), Lv-Vp023 (shown as SEQ ID NO. 4), Lv-Vp029 (shown as SEQ ID NO. 5) and Lv-Vp041 (shown as SEQ ID NO. 6). The screened primers are shown in Table 3.
EST-SST primers screened in Table 3
2.3 screening and result analysis of polymorphic primers
And respectively carrying out fluorescence labeling on the 5' ends of the upstream primers of the 6 pairs of primers preliminarily screened by using FAM and HEX, and carrying out PCR amplification by using the extracted 20 genomic DNAs of the litopenaeus vannamei as templates. The reaction system is the same as step 2.2, and the reaction procedures are the same as those described in step 2.2 except that the annealing temperature is 60 ℃. Typing the PCR amplification products by adopting a 3730XL sequencer, judging specific numerical values of allele fragments by using GeneMapper3.2 software, and determining 6 functional genes EST-STR markers (shown in figures 1-6) of the polymorphic litopenaeus vannamei, wherein the sites are respectively as follows: Lv-Vp001 (shown as SEQ ID NO. 1), Lv-Vp011 (shown as SEQ ID NO. 2), Lv-Vp012 (shown as SEQ ID NO. 3), Lv-Vp023 (shown as SEQ ID NO. 4), Lv-Vp029 (shown as SEQ ID NO. 5) and Lv-Vp041 (shown as SEQ ID NO. 6).
Expected heterozygosity is calculated by using Cervus 3.0 software, and the heterozygosity and Polymorphism Information Content (PIC) are observed. The results show that: the allele factors of the 6 polymorphic microsatellite markers are respectively 5, 2, 14, 6, 9 and 4; the observed heterozygosity is 0.45, 0.00, 0.35, 0.70, 0.60 and 0.20 respectively; desired heterozygosity of 0.7654, 0.1846, 0.9013, 0.8231, 0.8500, and 0.6679, respectively; PIC values are 0.7040, 0.1638, 0.8687, 0.7738, 0.8071 and 0.5834 (Table 4), except Lv-Vp011, the rest is more than 0.5, which shows that the 6 EST-STR markers have high polymorphism and can be used for genetic relationship analysis and molecular marker assisted breeding of litopenaeus vannamei.
TABLE 4 functional gene EST-STR mark characteristic table of Litopenaeus vannamei
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> Vibrio resistance related EST-STR mark of litopenaeus vannamei, specific primer and detection method thereof
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cgagagaaag agggagaaaa agagagaaag agaaagagag agagagagag aggacgcaag 60
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Claims (10)
1. The EST-STR mark of the litopenaeus vannamei is characterized in that the EST-STR mark is numbered as Lv-Vp001, Lv-Vp011, Lv-Vp012, Lv-Vp023, Lv-Vp029 and Lv-Vp 041;
the nucleotide sequence of the Lv-Vp001 is shown as SEQ ID NO. 1;
the nucleotide sequence of the Lv-Vp011 is shown in SEQ ID NO. 2;
the nucleotide sequence of the Lv-Vp012 is shown in SEQ ID NO. 3;
the nucleotide sequence of the Lv-Vp023 is shown in SEQ ID NO. 4;
the nucleotide sequence of the Lv-Vp029 is shown as SEQ ID NO. 5;
the nucleotide sequence of the Lv-Vp041 is shown as SEQ ID NO. 6.
2. The amplification primer for the EST-STR marker of the litopenaeus vannamei of claim 1, which comprises:
for the Lv-Vp001 site:
Lv-Vp001-F：5’-ACTGGAGGATCCTGAGAGATAGC-3’；
Lv-Vp001-R：5’-GCTTCGTTCTCTCACTCTTCTCTT-3’；
for the Lv-Vp011 site:
Lv-Vp011-F：5’-AAGGATTTCTCTTACACGAACCC-3’；
Lv-Vp011-R：5’-ACCAAAAAGAAAAACAGAATGGC-3’；
for the Lv-Vp012 site:
Lv-Vp012-F：5’-AATCTACAGGACTCGAATGCACA-3’；
Lv-Vp012-R：5’-CTTTTGTCCTCCACTTTACATGC-3’；
for Lv-Vp23 site:
Lv-Vp23-F：5’-CGAGAGAAAGAGGGAGAAAAAGA-3’；
Lv-Vp23-R：5’-TCATGGAGATGATGACTGGATAA-3’；
for Lv-Vp29 site:
Lv-Vp29-F：5’-CACCATATTCTCCAAGAAGCAGT-3’；
Lv-Vp29-R：5’-TTACTCATCCTGCTTCAACACCT-3’；
for Lv-Vp41 site:
Lv-Vp41-F：5’-CTTTTCGTTGATGAGTCTTTCCA-3’；
Lv-Vp41-R：5’-AGGGAGAAAGAGTGGAGTGAGAT-3’。
3. the amplification primer of claim 2, wherein the forward primer of the amplification primer is labeled at its 5' end with a fluorescent group.
4. The amplification primer of claim 3, wherein the fluorescent group is FAM or HEX.
5. A detection method of an EST-STR mark of a litopenaeus vannamei is characterized by comprising the following steps:
(1) extracting genomic DNA of the litopenaeus vannamei;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and respectively using the primer pair Lv-Vp001-F/R aiming at the Lv-Vp001 site, the primer pair Lv-Vp011-F/R aiming at the Lv-Vp011 site, the primer pair Lv-Vp012-F/R aiming at the Lv-Vp012 site, the primer pair Lv-Vp023-F/R aiming at the Lv-Vp023 site, the primer pair Lv-Vp029-F/R aiming at the Lv-Vp029 site and the primer pair Lv-Vp041-F/R aiming at the Lv-Vp041 site;
(3) and (4) typing the PCR amplification product by using a sequencer.
6. The detection method according to claim 5, wherein the PCR amplification reaction system comprises 25 μ L:containing Mg2+10 XPCR buffer 2.5. mu.L, 10mM dNTPs 1. mu. L, Taq enzyme 2U, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu. L, DNA template 10ng, the remainder being made up to 25. mu.L by sterile double distilled water.
7. The detection method according to claim 5, wherein the PCR amplification is performed by the following reaction procedures: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds for 10 cycles; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 35 cycles total, and re-extension at 72 ℃ for 5-8 minutes.
8. The application of the Litopenaeus vannamei EST-STR marker of claim 1 or the amplification primer of claim 2 in the research of the genetic diversity of Litopenaeus vannamei populations, gene localization, genetic linkage map construction, germplasm identification, variety classification or molecular marker-assisted breeding.
9. The use of claim 8, wherein the use is as an EST-STR marker or amplification primer for the Litopenaeus vannamei related to the antibacterial and/or insect-resistant traits.
10. The use according to claim 9, wherein the antibacterial and/or insect-resistant is/are against vibrio harveyi and/or against enterobacter prawn hepaticus.
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| PCT/CN2021/094239 WO2022068215A1 (en) | 2021-04-15 | 2021-05-18 | Litopenaeus vannamei vibrio-resistance related est-str marker, and specific primers thereof and detection method therefor |
| JP2023548987A JP2023546627A (en) | 2021-04-15 | 2021-05-18 | Vibrio resistance-related EST-STR markers in vannamei shrimp, their specific primers, and detection methods |
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| CN107287296B (en) * | 2017-06-20 | 2021-06-01 | 中国科学院南海海洋研究所 | Functional gene EST-SSR (expressed sequence tag-simple sequence repeat) marker of litopenaeus vannamei as well as specific primer and detection method thereof |
| CN108034729B (en) * | 2017-12-01 | 2021-03-05 | 中国科学院南海海洋研究所 | EST-STR marker Lv-F36a related to growth traits of litopenaeus vannamei, and amplification primer and application thereof |
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