CN114058715B - Microsatellite molecular marker for polymorphism of goby and primer pair and application thereof - Google Patents

Microsatellite molecular marker for polymorphism of goby and primer pair and application thereof Download PDF

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CN114058715B
CN114058715B CN202111470481.9A CN202111470481A CN114058715B CN 114058715 B CN114058715 B CN 114058715B CN 202111470481 A CN202111470481 A CN 202111470481A CN 114058715 B CN114058715 B CN 114058715B
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CN114058715A (en
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秦蛟
程飞
谢松光
张磊
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Abstract

The invention belongs to the technical field of DNA molecular markers of invasive species population genetics, and provides a microsatellite molecular marker of a goby polymorphism of eel and a primer pair and application thereof. The invention builds a simplified genome library of the goby, performs high-throughput sequencing, screens microsatellite loci in the genome, and performs polymorphism experiment verification of a large sample population on 14 loci. 14 polymorphic microsatellite markers, including 10 high-polymorphic markers and 4 medium-polymorphic markers, were obtained from the first screening. The invention provides molecular technical support for research of the genetics of the invasion population of the goby, and is beneficial to retrospective research of the invasion population source and invasion path.

Description

Microsatellite molecular marker for polymorphism of goby and primer pair and application thereof
Technical Field
The invention relates to the technical field of DNA molecular markers of invasive species population genetics, in particular to a microsatellite molecular marker of a goby polymorphism of eel and goby, and a primer pair and application thereof.
Background
Anguillar Japonica Taenioides cirratus, also known as Leptoradix Erythrinae, is belonging to genus Taenioides of family Gobiidae, class Perciformes, order Perciformes. The fish is taken as carnivorous fish, has great fertility and small primary mature age, and can quickly establish self-bred invasive population in a new environment; a plurality of large lakes invaded into inland in China are reported to cause a sharp reduction in the amount of small fish and shrimp resources in the lakes (Liang Y, fang T, li J, yang K, zhao X.X, cui K, and Lu W.X, age, growth and reproductive traits of invasive goby Taenioides cirratus in the Chaohu Lake, china. JAppl Ichthyol,2020,36 (2): 219-226). In the 80 s of the 20 th century, the fish is seen in the Gaoyou lake, and after 2000, the resource amount is obviously increased, so that the fish becomes a common species in lakes; subsequently, in 2005 and 2011 in the down-and in 2011 in the top-of-the-south lakes, respectively, and in 2014 in the top-of-the-south lakes (Qin J, cheng F, zhang L, schmidtB.V, liuJ, xie S.G, invasions oftwo estuarine gobiid species interactively induced from water diversion and saltwater introsion.manag Biol investment, 2019,10 (1): 139-150); appears in the nest lake in 2012. The eel, goby and lake invade population is large in scale, the risk of increasing, bursting and even further spreading is increased, and the damage to the structural and functional integrity of the lake ecosystem is large. Therefore, researches on source places, invasion paths and the like of invasion populations are urgently needed to be carried out, and further prevention and control countermeasures of invasion fishes are scientifically and reasonably formulated.
Aiming at the problems of fuzzy population source and invasion path of invasive species, the problems can be accurately traced back by using a population genetics method of genetic relativity in and among the populations reflected by molecular markers, and the problems can be applied and verified in a plurality of invasive species at home and abroad. Microsatellites (microsatellites), also known as simple sequence repeats (Simple sequence repeat, SSR) or short tandem sequence repeats (STRs), consist of a core sequence and flanking sequences at both ends thereof, are widely present in eukaryotic genomes, have the characteristics of co-dominant inheritance, wide polymorphism, wide distribution and easy detection, and are the most widely applied molecular markers in current invasive species population source and invasive path research. In order to rapidly and effectively identify the source places and invasion paths of different lake invasion populations of the goby, the development of microsatellite primers is particularly important. At present, microsatellite marker molecules of gobies palustris have not been developed and applied.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a polymorphic microsatellite molecular marker of the goby and a primer pair and application thereof by constructing a goby simplified genome library, carrying out high-throughput sequencing and screening microsatellite loci in a genome.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an eel goby polymorphism microsatellite molecular marker which comprises 14 eel goby polymorphism microsatellite molecular marker loci, wherein the molecular marker loci are TC144, TC145, TC176, TC208, TC220, TC119, TC149, TC155, TC203, TC13, TC35, TC33, TC37 and TC48.
The invention also provides a primer pair of the microsatellite molecular marker of the polymorphism of the goby,
the forward and reverse primers of TC144 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the forward and reverse primers of TC145 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
the positive and negative primers of TC176 are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the forward and reverse primers of TC208 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the forward and reverse primers of TC220 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
the forward and reverse primers of TC119 are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;
the positive and negative primers of TC149 are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;
the forward and reverse primers of TC155 are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;
the forward and reverse primers of TC203 are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;
the forward and reverse primers of TC13 are respectively shown as SEQ ID NO.19 and SEQ ID NO. 20;
the forward and reverse primers of TC35 are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22;
the forward and reverse primers of TC33 are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;
the forward and reverse primers of TC37 are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26;
the forward and reverse primers of TC48 are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28.
Preferably, the 5' end of the positive primer of the polymorphic microsatellite molecular marker locus of the goby is provided with a fluorescent marker, wherein the fluorescent markers of TC144, TC208, TC149, TC13 and TC37 are FAM, the fluorescent markers of TC145, TC220, TC155, TC35 and TC48 are HEX, and the fluorescent markers of TC176, TC119, TC203 and TC33 are TAMRA.
The invention also provides application of the Anguillar Japonica polymorphic microsatellite molecular marker or the Anguillar Japonica polymorphic microsatellite molecular marker primer pair in research of Anguillar Japonica genetics.
Preferably, the genetic study includes backtracking of the source of the invasive population and the path of the invasion.
Compared with the prior art, the invention has the following beneficial effects:
the invention builds a simplified genome library of the goby, performs high-throughput sequencing, screens microsatellite loci in the genome, and performs polymorphism experiment verification of a large sample population on 14 loci. 14 polymorphic microsatellite markers, including 10 high-polymorphic markers and 4 medium-polymorphic markers, were obtained from the first screening. The invention provides molecular technical support for research of the genetics of the invasion population of the goby, and is beneficial to retrospective research of the invasion population source and invasion path.
Drawings
FIG. 1 is a graph showing the genotyping peaks (homozygotes) of TC144 according to the present invention;
FIG. 2 shows the TC144 genotyping peaks (multiplex amplification) according to the invention.
Detailed Description
The invention provides an eel goby polymorphism microsatellite molecular marker which comprises 14 eel goby polymorphism microsatellite molecular marker loci, wherein the molecular marker loci are TC144, TC145, TC176, TC208, TC220, TC119, TC149, TC155, TC203, TC13, TC35, TC33, TC37 and TC48.
The invention also provides a primer pair of the microsatellite molecular marker of the polymorphism of the goby,
the forward and reverse primers of TC144 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the forward and reverse primers of TC145 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
the positive and negative primers of TC176 are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the forward and reverse primers of TC208 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the forward and reverse primers of TC220 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
the forward and reverse primers of TC119 are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;
the positive and negative primers of TC149 are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;
the forward and reverse primers of TC155 are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;
the forward and reverse primers of TC203 are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;
the forward and reverse primers of TC13 are respectively shown as SEQ ID NO.19 and SEQ ID NO. 20;
the forward and reverse primers of TC35 are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22;
the forward and reverse primers of TC33 are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;
the forward and reverse primers of TC37 are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26;
the forward and reverse primers of TC48 are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28.
In the invention, the 5' end of the positive primer of the polymorphic microsatellite molecular marker locus of the goby is preferably provided with a fluorescent marker, wherein the fluorescent markers of TC144, TC208, TC149, TC13 and TC37 are preferably FAM, the fluorescent markers of TC145, TC220, TC155, TC35 and TC48 are preferably HEX, and the fluorescent markers of TC176, TC119, TC203 and TC33 are preferably TAMRA.
The invention also provides application of the Anguillar Japonica polymorphic microsatellite molecular marker or the Anguillar Japonica polymorphic microsatellite molecular marker primer pair in research of Anguillar Japonica genetics.
In the present invention, the genetic study preferably includes backtracking of the source of the invasive population and the path of the invasion.
The technical scheme provided by the invention is described in detail below in connection with experimental examples, but they are not to be construed as limiting the scope of the invention.
Experimental example 1
(1) Extraction of Anguillar Japonica genomic DNA:
55 adult fish of Anguillar Japonica and goby are collected, 1g of muscle tissue is taken and stored with 95% alcohol. Extracting total genomic DNA of Anguillar Japonica with animal DNA extraction kit (Wuhan division of Beijing qing family biotechnology Co., ltd.) according to the instruction of the kit, and storing at-20deg.C.
(2) Simplified construction of genomic libraries and high throughput sequencing:
genomic DNA samples of 1-tailed eel goby were selected, whole genome random sequencing was performed using Mi Seq sequencer Illumina USA (sequencing service was provided by the institute of aquatic biology public technical service center of China academy of sciences), the sequencing amount was set to 1Gbp, and then the measured fragments were spliced and aligned, and microsatellite loci were searched from the spliced fragments using MISA (MIcroSAtellite) software. Search criteria: repeating 2 bases 6 times or more; the 3 bases, 4 bases, 5 bases and 6 bases are repeated 5 times and more.
(3) Microsatellite primer synthesis, detection and screening:
based on the searched microsatellite sequences, primer Premier 5.0 was used to design PCR amplification primers for microsatellite loci. Major parameters of primer design: GC content is between 40-60%, primer length is between 18-25bp, annealing temperature is between 45-60 ℃, and expected length of PCR product is between 130-350 bp. 2 samples were taken from each of 2 origin populations (Yangtze river mouth) and 4 lake invasion populations (Gaoyou lake, camara lake, nanfu lake and Chaohu lake) for PCR amplification, and the PCR reaction system was 30. Mu.l: 26 μl 1.1X1.times.T3 Super PCR Mix,1 μl 10 μM Primer F,1 μl 10 μM Primer R and 2 μl DNA template. The PCR amplification procedure was: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 52-60℃for 10s (the temperatures for specific primers are shown in Table 1), extension at 72℃for 10s,35 cycles; extending at 72deg.C for 10min, and preserving at 4deg.C. And (3) performing agarose gel electrophoresis (2 μl sample+6 μl bromophenol blue) on the amplified PCR product, obtaining an identification gel diagram at 300V voltage for 12min, determining the template concentration through the gel diagram, adding water to dilute to the concentration required by capillary electrophoresis, and then performing capillary electrophoresis monitoring on the amplified PCR product by a 3730 sequencer. The analysis of the accurate site of the data is carried out by using software Gene mapper4.1, and the accurate size of the site is determined according to the core base repetition number of the corresponding relation of the primer by the analysis data. Judging whether the detection primer has site polymorphism according to the analyzed site information, and finally successfully designing 14 pairs of microsatellite marker primers.
TABLE 1 14 microsatellite marker primer Table
(4) Large sample verification of microsatellite markers:
the microsatellite loci are verified by using 55 samples of the original producing field population of the goby Yangtze river of the goby, and the genotyping result is analyzed by using Cervus v.3.0.7 software. The specific results are shown in Table 2.
TABLE 2 polymorphism parameter Table for 14 microsatellite loci
As can be seen from Table 2, the distribution range of alleles is 2-13, with an average value of 5.5; the variation range of the observed heterozygosity is 0.327 to 1.000, and the average value is 0.610; the expected heterozygosity varies from 0.345 to 0.866 with an average value of 0.605; the variation range of the site Polymorphism Information Content (PIC) is 0.317-0.841, the average value is 0.550, wherein 4 microsatellite sites with the value of 0.25 < PIC less than or equal to 0.5 (moderate polymorphism) and 10 microsatellite sites with the value of PIC more than 0.5 (high polymorphism) are provided; of the 14 polymorphic sites, 2 were significantly off Ha Diwen berg equilibrium (p < 0.001).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (4)

1. A molecular marker composition of a palaeda salsa polymorphism microsatellite, which is characterized by comprising 14 molecular markers of the palaeda salsa polymorphism microsatellite, wherein the molecular markers are TC144, TC145, TC176, TC208, TC220, TC119, TC149, TC155, TC203, TC13, TC35, TC33, TC37 and TC48;
the 14 polymorphic microsatellite molecular markers of the goby are amplified by the following forward and reverse primers:
the repeated element of TC144 is (ATA) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the repeated element of TC145 is (TA) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
the repeated element of TC176 is (TAGA) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the repeated element of TC208 is (GTA) 10, and the forward and reverse primers are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the repetitive element of TC220 is (TAGAA) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
the repeated element of TC119 is (TA) 7, and the forward and reverse primers are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;
the repeated element of TC149 is (CTTT) 7, and the forward and reverse primers are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;
the repeated element of TC155 is (AC) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;
the repeated element of TC203 is (AC) 13, and the forward and reverse primers are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;
the repetitive element of TC13 is (ATAATG) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.19 and SEQ ID NO. 20;
the repeated element of TC35 is (ATA) 20, and the forward and reverse primers are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22;
the repetitive element of TC33 is (TAG) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;
the repeated element of TC37 is (TA) 6, and the forward and reverse primers are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26;
the repeated element of TC48 is (TTTA) 6, and the forward and reverse primers are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28.
2. The primer pair composition of the microsatellite molecular marker of the goby polymorphism of claim 1, which is characterized by comprising a forward and reverse primer of TC144, a forward and reverse primer of TC145, a forward and reverse primer of TC176, a forward and reverse primer of TC208, a forward and reverse primer of TC220, a forward and reverse primer of TC119, a forward and reverse primer of TC149, a forward and reverse primer of TC155, a forward and reverse primer of TC203, a forward and reverse primer of TC13, a forward and reverse primer of TC35, a forward and reverse primer of TC33, a forward and reverse primer of TC37, and a forward and reverse primer of TC48;
the repeated element of TC144 is (ATA) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the repeated element of TC145 is (TA) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
the repeated element of TC176 is (TAGA) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
the repeated element of TC208 is (GTA) 10, and the forward and reverse primers are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;
the repetitive element of TC220 is (TAGAA) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;
the repeated element of TC119 is (TA) 7, and the forward and reverse primers are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;
the repeated element of TC149 is (CTTT) 7, and the forward and reverse primers are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;
the repeated element of TC155 is (AC) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;
the repeated element of TC203 is (AC) 13, and the forward and reverse primers are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;
the repetitive element of TC13 is (ATAATG) 9, and the forward and reverse primers are respectively shown as SEQ ID NO.19 and SEQ ID NO. 20;
the repeated element of TC35 is (ATA) 20, and the forward and reverse primers are respectively shown as SEQ ID NO.21 and SEQ ID NO. 22;
the repetitive element of TC33 is (TAG) 11, and the forward and reverse primers are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;
the repeated element of TC37 is (TA) 6, and the forward and reverse primers are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26;
the repeated element of TC48 is (TTTA) 6, and the forward and reverse primers are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28.
3. The primer pair composition of claim 2, wherein the positive primer 5 'is fluorescently labeled, wherein the positive primers 5' of TC144, TC208, TC149, TC13 and TC37 are FAM, the positive primers 5 'of TC145, TC220, TC155, TC35 and TC48 are HEX, and the positive primers 5' of TC176, TC119, TC203 and TC33 are TAMRA.
4. Use of the polymorphic microsatellite molecular marker composition of goby of claim 1 or the primer pair composition of any one of claims 2 to 3 in research of goby genetics of goby.
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