CN116064832A - Microsatellite loci of cuttlefish in China and application thereof - Google Patents
Microsatellite loci of cuttlefish in China and application thereof Download PDFInfo
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Abstract
The invention relates to a China gun cuttlefish microsatellite locus and application thereof, wherein 15 microsatellite loci have nucleotide sequences shown in SEQ ID NO:1-15. The sequences of primer pairs designed from these microsatellite loci are SEQ ID NOs: 16-45. According to the invention, 15 microsatellite loci are screened from the genomic DNA of the squid of China, and specific primers are designed at flanking regions at two ends of a microsatellite repetitive sequence, so that the obtained primers can be used for detecting the genetic diversity of the squid population of China, and a reliable molecular marker is provided for researching the genetic structure and the genetic diversity of the squid population of China.
Description
Technical Field
The invention relates to the technical field of molecular biology DNA (deoxyribonucleic acid) markers, in particular to a China gun cuttlefish microsatellite locus and application thereof.
Background
Microsatellite markers (microsatellites), also known as simple repeats (Simple sequence repeat, SSR) or short tandem repeats (Short tandem repeat, STR), are 5-40 tandem repeats composed of 1-6 nucleotide bases as repeat units in the genome, and are widely distributed in eukaryotic genomes. Microsatellites consist of a core sequence and flanking sequences, the core sequence being highly polymorphic due to the different base composition and number of repeat units of the sequence itself. Flanking sequences are typically specific single copy sequences that are conserved. Microsatellite markers are designed based on the high variability of the core sequence and the conservation of the flanking sequences, and relevant analysis is carried out according to the different repetition numbers (polymorphism) of the core sequence of a specific species, so that the polymorphism of different microsatellites in different populations and even among different individuals is revealed. Microsatellite marking technology has been established, is favored by people due to the advantages of high polymorphism, co-dominant inheritance, good repeatability, easy experimental operation and the like, is a powerful tool for genetic breeding, resource protection, phylogenetic and population genetics research, and is widely applied in the fields of human medicine, animals, plants, microorganisms and the like.
China cuttlefish (Uroteuthis chinensis) belongs to Cephalopoda (Cephalopoda), syzygida (Teuthoiida), syzygidae (Loginiidae), and Cylindaceae (Uroteutis) and is a warm water land frame sea species, and is mainly distributed in the regions from south to east China, especially in the middle and south of the south China sea frame and Taiwan strait, the yield of the China is about 60% of the world Syzygidae yield, the Shandong-Taiwan shoal fishing field is most abundant, and the average annual yield is 2.0X10% 4 ～2.5×10 4 t. In order to further understand the information such as the population structure and genetic diversity of the squid of China, microsatellite molecular markers need to be developed aiming at the squid population structure of China.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention aims to provide a China squid microsatellite locus and a corresponding primer, and provides a reliable molecular marker for researching the genetic structure and genetic diversity of China squid population.
To this end, in one aspect of the present invention, the present invention proposes a microsatellite marker for use in the study of squid population genetic information in chinese gun, said microsatellite marker comprising the sequence of SEQ ID NO:1-15.
According to the microsatellite marker for Chinese squid population genetic information research, 15 microsatellite loci are screened from Chinese squid genome DNA, and specific primers are designed at flanking regions at two ends of a microsatellite repetitive sequence, so that the obtained primers can be used for Chinese squid population genetic diversity detection, and reliable molecular markers are provided for Chinese squid population genetic structure and genetic diversity research.
The embodiment of the invention also provides a primer pair designed from the 15 microsatellite loci, wherein the primer pair is prepared from a sequence shown in SEQ ID NO:1, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:16 and SEQ ID NO:17;
from the sequence SEQ ID NO:2, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:18 and SEQ ID NO:19;
from the sequence SEQ ID NO:3, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:20 and SEQ ID NO:21, a step of;
from the sequence SEQ ID NO:4, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:22 and SEQ ID NO:23;
from the sequence SEQ ID NO:5, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:24 and SEQ ID NO:25, a step of selecting a specific type of material;
from the sequence SEQ ID NO:6, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:26 and SEQ ID NO:27;
from the sequence SEQ ID NO:7, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:28 and SEQ ID NO:29;
from the sequence SEQ ID NO:8, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:30 and SEQ ID NO:31;
from the sequence SEQ ID NO:9, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:32 and SEQ ID NO:33;
from the sequence SEQ ID NO:10, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 34 and SEQ ID NO:35;
from the sequence SEQ ID NO:11, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 36 and SEQ ID NO:37, respectively;
from the sequence SEQ ID NO:12, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 38 and SEQ ID NO:39;
from the sequence SEQ ID NO:13, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 40 and SEQ ID NO:41;
from the sequence SEQ ID NO:14, the sequences of which are respectively SEQ ID NOs: 42 and SEQ ID NO: 43.
From the sequence SEQ ID NO:15, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 44 and SEQ ID NO:45.
the primer pair can be used for detecting the genetic diversity of the squid population of the Chinese gun, and provides a reliable molecular marker for researching the genetic structure and the genetic diversity of the squid population of the Chinese gun.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The technical scheme of the invention is described below through specific examples. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; it should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
In order to better understand the above technical solution, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are conventional experiments unless otherwise specified.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.
Example 1 acquisition of microsatellite loci
(1) The genomic DNA of chinese cuttlefish was extracted using GenStar StarSpin Animal DNA Kit standard procedure. The sample of the cuttlefish of China gun is collected from Shandong-Taiwan shoal fishing ground (21 DEG 50 '-23 DEG 30' N,116 DEG 00 '-119 DEG 30' E) and the collection time is 7 months in 2018.
1. Sample pretreatment
1) 5mg of animal tissue material was excised, ground to a powder in liquid nitrogen and transferred to a 1.5mL centrifuge tube.
2) 400. Mu.L of solution A and 10. Mu.L of protease K, if coagulated, were added, and the mixture was broken up by blowing with a gun head to avoid enzyme inactivation.
3) Shaking and mixing for 1min, then placing at 55deg.C for 3h, taking out and mixing for several times during this period to facilitate full cracking.
4) Taking out the sample, and gently shaking and uniformly mixing when the temperature is reduced to room temperature.
2. To the pretreated sample, 300. Mu.L of solution B (PB) and 300. Mu.L of solution C (PC) were added in this order, shaken vigorously, and centrifuged at 12,000rpm for 5min.
The solution will delaminate, the upper layer is the blue extraction layer, the lower layer is the transparent water phase, there may be a partial precipitation layer between the two solutions, and the DNA is in the lower water phase.
3. The gun head penetrates through the upper layer solution and goes deep into the lower layer solution, the lower layer solution is carefully sucked out and is transferred into a centrifugal adsorption column, and the precipitation of the upper layer solution and the middle layer is avoided as much as possible.
4.8,000rpm for 1min, taking down the centrifugal adsorption column, and pouring out the waste liquid in the collecting pipe.
5. The centrifugal adsorption column was put back into the collection tube, 500. Mu.L of universal rinse (WB) was added, and the mixture was centrifuged at 8,000rpm for 1min at room temperature, and the waste liquid in the collection tube was poured out.
6. The step 5 is repeated once.
Centrifugation at 7.12,000rpm at room temperature for 1min to remove residual universal rinse (WB).
8. The column was placed in a fresh clean 1.5mL centrifuge tube, 50-100. Mu.L of universal Eluent (EB) was carefully added and left at room temperature or 55℃for 2min.
9.12,000rpm, and centrifuged at room temperature for 1min. The liquid in the centrifuge tube is genomic DNA, and the sample can be stored at 4 ℃ or-20 ℃ according to the application.
(2) Using IlluminaTruSeq TM Nano DNA Sample Prep Kit method constructs the library. After measuring the original concentration with a Qubit 3.0 fluorometer, 1. Mu.g of DNA was pooled and Covaris M220 sonicated to break the DNA into 300-500bp fragments. The broken DNA can generate a 5' or 3' sticky end and a flat end, all sticky ends are changed into flat ends by using T4 DNA polymerase, a PCR instrument is required to be placed on the end for 20 ℃ for incubation for 30min without a heat cover during the repair of the tail ends, 1.7 x Ampure XP magnetic beads are required to be used for purification and elution after the reaction is finished, one nucleotide ' A ' is added at the 3' end by using Taq DNA polymerase, and the reaction system is also required to be placed on the PCR instrument for incubation for 30min at 20 ℃ and then 1.7 x Ampure XP magnetic beads are required to be used for purification and elution. Joint index (TruSeq) TM Nano DNA Sample Prep Kit) was incubated for 15min at 20℃without a hot lid on the PCR apparatus, and eluted using 0.9XAmpure XP magnetic beads.
(3) Library enrichment and on-machine sequencing, the former PCR amplification 8 cycles (denaturation: 95 ℃ reaction 2min, cycle: 95 ℃ reaction 2min, 55 reaction 30s,72 ℃ reaction 45s, incubation: 72 ℃ 2min.2% agarose gel recovery 350+ -50 bp range band (Certified Low Range UltraAgarose), the latter according to Agilent2100 biological analyzer and real-time fluorescence quantitative PCR detection library fragment distribution meaning molar concentration, according to the required data volume according to molar concentration, using Illunima novaseq 6000 sequencing platform for 2×150bp sequencing, finally obtaining about 10GB of original data.
(4) And (3) obtaining microsatellite loci, performing quality filtration on the obtained original data by using fastp software to obtain clean data, and assembling the obtained clean data by using Spades software to obtain a draft genome. Misa was used to find out the draft genome fragment containing the microsatellite loci for the obtained draft genome.
Example 2 microsatellite primer design
The microsatellite Primer is designed from flanking sequences at two ends of a repeated sequence of a microsatellite locus (Loc 01-Loc 15), the Primer design adopts software Primer Premier 5.0, and the Primer design adopts the following stringency: (1) the primer length is 15-40bp; (2) GC content 30% -70%; (3) annealing temperature is 40-65 ℃; (4) the expected PCR product length is 100-350bp. The primers designed in this example (Table 1) can be used to amplify the sequences SEQ ID NO 1-15 at the microsatellite loci (Loc 01-Loc 15).
Table 1 microsatellite loci sequence information of China cuttlefish
Example 3 Chinese gun cuttlefish diversity detection
(1) Extracting genomic DNA of Chinese cuttlefish: the method comprises the steps of extracting nucleic acid from 20 cuttlefish samples by using an automatic workstation matched with a magnetic bead method animal genome extraction kit, and detecting the concentration and purity of the nucleic acid by using a NanoDROP 8000 ultramicro spectrophotometer.
DNA extraction:
1. taking a proper amount of animal tissue (fresh weight is 30-50 mg), properly cutting, and then placing the animal tissue into a new deep pore plate (marked as (1)) sample hole.
2. To the deep well plate, 400. Mu.L of lysate and 5. Mu.L of proteinase K were added and incubated at 65℃overnight.
3. The next day, the deep-hole plate (1) was placed on station 1 of the nucleic acid extractor, the CTAB-TY program was run, and after cleavage was completed, 280. Mu.L of isopropanol was added to each well.
4. The components are separated into different deep-hole plates, and the magnetic beads are subjected to liquid separation according to 100 mu L/hole, 500 mu L/hole of 75% ethanol and 100 mu L/hole of ultrapure water, and marking is carried out.
5. And (3) placing the deep hole plate (1) on a station 1 of a nucleic acid extraction instrument, placing the deep hole plates filled with magnetic beads, three 75% ethanol and ultrapure water on stations 2-6 respectively, and running a program.
6. After the procedure is completed, the instrument will automatically stop and station 6 will enter the 4 ℃ preservation procedure to temporarily preserve the sample.
DNA detection:
1 run NanoDROP 8000 software, select nucleic acid detect button.
2 washing 8 channels with ultrapure water once, and then using the eluent as a blank.
32 uL of DNA solution was added to each of the 8 wells, and then the start button was clicked to start the detection.
(2) Microsatellite PCR amplification: the genomic DNA of sepia esculenta was amplified using FAM fluorescent-labeled microsatellite primers (obtained in example 2). The amplification reaction system was 30. Mu.L comprising: 3. Mu.L of 10 Xbuffer, 1.5mM MgCl2,0.2mM dNTPs, 0.5. Mu.M each of the primers, 0.5U Taq enzyme (TaKaRa) and 3. Mu.L of template DNA. The PCR amplification procedure was set as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, gradient annealing at 62-52 ℃ for 30s, extension at 72 ℃ for 30s, and 10 cycles of operation; denaturation at 95℃for 30s, annealing at 52℃for 30s, extension at 72℃for 30s, run 25 cycles; extending at 72deg.C for 20min, and preserving at 4deg.C. After the PCR reaction is finished, the amplified product is detected by an ABI PRISM 3730 automatic sequencer.
(3) The results were analyzed using GeneMarker software to obtain the allele, peak map and genotype for each sample. Basic genetic parameters such as allele factors (Na) of each locus of squid are calculated by using GenAlex software. The results are shown in Table 2.
Table 2 basic genetic parameters of microsatellite loci of China cuttlefish
| locus | N | Na | Ne | I | Ho | He | F | Hs | PIC |
| Loc01 | 20 | 4 | 2.58 | 1.09 | 0.714 | 0.612 | -0.166 | 0.806 | 0.541 |
| Loc02 | 20 | 3 | 2.394 | 0.98 | 0.571 | 0.582 | 0.019 | 0.791 | 0.518 |
| Loc03 | 20 | 3 | 1.555 | 0.655 | 0.429 | 0.357 | -0.201 | 0.678 | 0.325 |
| Loc04 | 20 | 4 | 3.919 | 1.376 | 0.857 | 0.745 | -0.151 | 0.872 | 0.697 |
| Loc05 | 20 | 3 | 1.555 | 0.655 | 0.429 | 0.357 | -0.201 | 0.678 | 0.325 |
| Loc06 | 20 | 7 | 4.904 | 1.765 | 0.429 | 0.796 | 0.462 | 0.898 | 0.772 |
| Loc07 | 20 | 3 | 2.335 | 0.956 | 0.714 | 0.572 | -0.249 | 0.786 | 0.502 |
| Loc08 | 20 | 6 | 4.271 | 1.591 | 0.857 | 0.766 | -0.119 | 0.883 | 0.732 |
| Loc09 | 20 | 5 | 4.46 | 1.536 | 0.857 | 0.776 | -0.105 | 0.888 | 0.739 |
| Loc10 | 20 | 4 | 1.58 | 0.753 | 0.429 | 0.367 | -0.168 | 0.684 | 0.348 |
| Loc11 | 20 | 9 | 6.539 | 2.041 | 1 | 0.847 | -0.181 | 0.924 | 0.831 |
| Loc12 | 20 | 6 | 5.434 | 1.749 | 0.714 | 0.816 | 0.125 | 0.908 | 0.791 |
| Loc13 | 20 | 3 | 1.816 | 0.797 | 0.571 | 0.449 | -0.272 | 0.725 | 0.407 |
| Loc14 | 20 | 4 | 2.131 | 0.989 | 0.714 | 0.531 | -0.346 | 0.765 | 0.483 |
| Loc15 | 20 | 6 | 3.768 | 1.535 | 0.857 | 0.735 | -0.167 | 0.867 | 0.701 |
N represents the number of samples, na is the number of alleles, ne is the number of effective alleles, I is the aromatic diversity index, ho is the observed heterozygosity, he is the desired heterozygosity, F is the fixed coefficient, hs is the average heterozygosity in the sub-population, and PIC is the polymorphic information content.
The primers obtained in example 2 were used to amplify 20 squid genomic DNAs, and the results of genetic diversity analysis showed that the number of alleles per microsatellite locus varied from 3 to 9, the effective allele from 1.55 to 6.54, the observed heterozygosity from 0.429 to 1, the expected heterozygosity from 0.357 to 0.847, and the polymorphic information content from 0.325 to 0.831. The microsatellite primers can be used for detecting the genetic diversity of the squid population in China, have high repeatability and are reliable and effective molecular markers.
In conclusion, the microsatellite loci and the primers designed for the microsatellite loci can be used for detecting the genetic diversity of the squid population of China, and provide reliable molecular markers for researching the genetic structure and the genetic diversity of the squid population of China.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (2)
1. The microsatellite marker for studying the genetic information of the squid population of China, which is characterized by comprising a sequence shown in SEQ ID NO:1-15.
2. A primer pair for amplifying the microsatellite loci of claim 1, said primer pair being designed from the microsatellite loci of claim 1;
wherein the sequence is SEQ ID NO:1, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:16 and SEQ ID NO:17;
from the sequence SEQ ID NO:2, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:18 and SEQ ID NO:19;
from the sequence SEQ ID NO:3, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:20 and SEQ ID NO:21, a step of;
from the sequence SEQ ID NO:4, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:22 and SEQ ID NO:23;
from the sequence SEQ ID NO:5, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:24 and SEQ ID NO:25, a step of selecting a specific type of material;
from the sequence SEQ ID NO:6, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:26 and SEQ ID NO:27;
from the sequence SEQ ID NO:7, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:28 and SEQ ID NO:29;
from the sequence SEQ ID NO:8, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:30 and SEQ ID NO:31;
from the sequence SEQ ID NO:9, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NO:32 and SEQ ID NO:33;
from the sequence SEQ ID NO:10, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 34 and SEQ ID NO:35;
from the sequence SEQ ID NO:11, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 36 and SEQ ID NO:37, respectively;
from the sequence SEQ ID NO:12, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 38 and SEQ ID NO:39;
from the sequence SEQ ID NO:13, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 40 and SEQ ID NO:41;
from the sequence SEQ ID NO:14, the sequences of which are respectively SEQ ID NOs: 42 and SEQ ID NO: 43.
From the sequence SEQ ID NO:15, the sequences of the primer pairs designed on the microsatellite loci are SEQ ID NOs: 44 and SEQ ID NO:45.
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