CN112813172B - Primer and method for identifying zander population - Google Patents
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Abstract
A primer for identifying a zander population and an identification method thereof relate to a primer for identifying a fish population and an identification method thereof. The primers for identifying the zander population consist of two groups of primer pairs of HLJSL041 and HLJSL 073. The identification method for identifying the zander population comprises the following steps: firstly, extracting genome DNA; secondly, performing SSR-PCR reaction on the sample DNA by adopting two primer pairs of HLJSL041 and HLJSL 073; and thirdly, performing gel electrophoresis, reading genotype data, and distinguishing and identifying the zander population in the Heilongjiang river water system. The specific microsatellite marker is developed from the genome sequence of the zander, so that the zander population of a black Longjiang river system can be identified and distinguished, particularly, the zander population can be distinguished from main zander populations of black Longjiang black river sections, Duck greens river, Goering Qisi river basin and the like, and a reference is provided for breeding and grouping the zander.
Description
Technical Field
The invention relates to a primer for identifying fish populations and an identification method thereof.
Background
The zander Lucioperca belongs to Perciformes, Percifae, and zander. The fish is fat and thick in body and meat, tender in meat quality, delicious in taste, rich in nutrition, high in protein and amino acid content and called as freshwater fish king. The zander is originally distributed in rivers and lakes of European salty sea, black sea, interior sea and baltic sea water systems. Due to natural diffusion and artificial transplantation, the water system is distributed in most regions in Europe, China and China, the water system of the frontier Qisi in Xinjiang and the water system of the Heilongjiang in China at present, and the water system becomes an important fresh water culture object in Europe and China in China. The zander is also introduced into and cultured as an important famous and special-quality fish species in a plurality of provinces and cities in China.
The Heilongjiang river water system is the water system with the largest basin area in the east Asia region. Through Mongolia, Russia and Heilongjiang province and inner Mongolia autonomous region of China, the annual distribution of precipitation in a drainage basin is extremely uneven, and the icing period is as long as 4-6 months. The biggest branch of Heilongjiang is Songhua river, and other major branches include Huma river, Jingqi river (Jieya river), Wusulijiang, etc.
At present, artificial propagation of zander is successfully realized in China, and used parent fishes are mainly fished from rivers and lakes in the Qizis basin or the Heilongjiang basin. It was reported that zander entered the black longjiang river basin via xingkian lake. Currently, zander populations are formed in Wusullijiang, Heilongjiang and Duck-Lvjiang. The problems of unclear seed source and germplasm mixing of the zander are difficult to solve by using morphological characteristics, the development of artificial propagation, large-scale seed production and fine seed breeding of the zander is not facilitated, and the identification of zander populations with different sources has important significance for the development of seed production and seed breeding.
Disclosure of Invention
Aiming at the problems of unclear seed source and mixed germplasm of a zander population, the invention aims to provide a primer for identifying the zander population and an identification method thereof.
The primers for identifying the zander population consist of two groups of primer pairs of HLJSL041 and HLJSL 073; the upstream primer of the primer pair HLJSL041 is 5'-TGCCTAACACCTGAACACCA-3', and the downstream primer is 5'-AAGGCAAACTGTGGGGTAGG-3'; the upstream primer of the primer pair HLJSL073 is 5'-AGAAATCGCCTCTGTGTTGT-3', and the downstream primer is 5'-TTGCTGCTGTAACACTGTCA-3'.
The identification method for identifying the zander population comprises the following steps:
firstly, extracting genome DNA;
secondly, performing SSR-PCR reaction on the sample DNA by adopting two primer pairs of HLJSL041 and HLJSL 073;
and thirdly, performing gel electrophoresis, reading genotype data, and distinguishing and identifying the zander population in the Heilongjiang river water system.
Furthermore, a non-destructive method is adopted to collect and extract the genomic DNA of the sample, about 0.1g of reproducible fin ray tissue of the sample individual is cut, and the sample is placed into 75 percent alcohol for preservation after being numbered.
Furthermore, the digestion time is strictly controlled to be 30min in the process of extracting DNA.
The core repeat sequence amplified by the primer of the invention is (AGAT)n。
The specific microsatellite marker is developed from the genome sequence of the zander, so that the zander population of a black Longjiang river system can be identified and distinguished, particularly, the zander population can be distinguished from main zander populations of black Longjiang black river sections, Duck greens river, Goering Qisi river basin and the like, and a reference is provided for breeding and grouping the zander.
The invention relates to a new method for distinguishing zander populations established for the first time based on a molecular biology technology, wherein the zander populations of Wusul river are specifically marked as homozygous individuals, while the black Longjiang black river segment populations and the Duck Lvjiang populations are mainly heterozygous individuals and have population specific alleles. The method is used for identifying the main zander population in the Heilongjiang water system, the population identification proportion is more than 90%, and the exclusion proportion is more than 96%. The method has the advantages of high accuracy, good technical repeatability and high cost performance, can detect a large number of samples simultaneously, and reduces the complexity of the quality resources and the group judgment of the zander.
Drawings
FIG. 1 is an amplification map of the HLJSL041 primer pair in example 1;
FIG. 2 is an amplification map of the primer pair HLJSL073 in example 1;
FIG. 3 is a graph of the genotype amplified based on primer pair HLJSL041 and HLJSL073 in example 1, with Strcuture2.3.4 output.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the identification method for identifying the zander population in the embodiment comprises the following steps:
firstly, extracting genome DNA;
secondly, performing SSR-PCR reaction on the sample DNA by adopting two primer pairs of HLJSL041 and HLJSL 073;
and thirdly, performing gel electrophoresis, reading genotype data, and distinguishing and identifying the zander population in the Heilongjiang river water system.
After the SSR-PCR reaction of the present embodiment was completed, capillary gel electrophoresis was performed using a 3730XL genetic Analyzer (ABI), and image collection and data analysis were performed using GeneMappre V4.1 software to obtain genotype data.
After reading the genotype data of the population, the embodiment can assist in further identifying and distinguishing the population by means of structure2.3.4 software.
The upstream primer of the primer pair HLJSL041 is 5'-TGCCTAACACCTGAACACCA-3', and the downstream primer is 5'-AAGGCAAACTGTGGGGTAGG-3'; the upstream primer of the primer pair HLJSL073 is 5'-AGAAATCGCCTCTGTGTTGT-3', and the downstream primer is 5'-TTGCTGCTGTAACACTGTCA-3'. The annealing temperature of the HLJSL041 upstream primer is 59.16 ℃, and the GC content is 50%; the annealing temperature of the HLJSL041 downstream primer is 59.89 ℃, and the GC content is 55 percent; the annealing temperature of the HLJSL073 upstream primer is 57.45 ℃, and the GC content is 45%; the annealing temperature of the HLJSL073 downstream primer is 57.68 ℃, and the GC content is 45%; the SSR-PCR reaction annealing temperatures of the two groups of primers are both 58 ℃.
The genotype data in this embodiment are:
the amplification map of the individual HLJSL041 of the zandrin perch is 128/128bp homozygote, and the amplification map of the individual HLJ073 is 103/103bp homozygote;
140bp strips appear in the HLJSL041 amplification map of the zander individual at the river section of the black Longjiang black river, and 107bp strips appear in the HLJ073 amplification map;
144bp or 132bp bands appear in the HLJSL041 amplification map of the individual zanthoxylum schlegelii, and 137bp, 132bp or 120bp bands appear in the HLJ073 amplification map.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: the 5' end of the forward primer of the primer pair is labeled with blue fluorescence. Other steps and parameters are the same as those in the first embodiment.
Example 1
The identification method for identifying the zander population comprises the following steps:
firstly, extracting genome DNA;
secondly, performing SSR-PCR reaction on the sample DNA by adopting two primer pairs of HLJSL041 and HLJSL 073;
thirdly, gel electrophoresis is carried out, genotype data are read, and the zander population of the Heilongjiang river water system is distinguished and identified;
firstly, collecting and extracting sample genome DNA by adopting a non-destructive method, shearing about 0.1g of reproducible fin ray tissue of a sample individual, numbering and then storing in 75% alcohol;
the digestion time is strictly controlled to be 30min in the process of extracting DNA;
the genotype data were: the amplification map of the individual HLJSL041 of the zandrin perch is 128/128bp homozygote, and the amplification map of the individual HLJ073 is 103/103bp homozygote; 140bp strips appear in the HLJSL041 amplification map of the zander individual at the river section of the black Longjiang black river, and 107bp strips appear in the HLJ073 amplification map; 144bp or 132bp bands appear in the HLJSL041 amplification map of the individual zang perch in the duck green river, and 137bp, 132bp or 120bp bands appear in the HLJ073 amplification map;
labeling the 5' end of the forward primer of the primer pair with blue (FAM) fluorescence;
the annealing temperature of SSR-PCR reaction in the second step is 58 ℃;
the upstream primer of the primer pair HLJSL041 is 5'-TGCCTAACACCTGAACACCA-3', and the downstream primer is 5'-AAGGCAAACTGTGGGGTAGG-3'; the upstream primer of the primer pair HLJSL073 is 5'-AGAAATCGCCTCTGTGTTGT-3', and the downstream primer is 5'-TTGCTGCTGTAACACTGTCA-3'.
Parent zander fishes are collected from a black longjiang water system, and 153 tails of samples are collected, wherein the 153 tails of the parent zander fishes comprise 32 tails of source xingkai lake (XK) of Wusul river, 47 tails of black longjiang black river (HH) and 74 tails of Dulvjiang Dandongjiang section (YJ). Samples of genomic DNA were taken and about 0.1g of each sample was added to 200. mu.LThe lysate (0.5% sodium lauryl sarcosinate, 200. mu.g/mL proteinase K, 10mmol/L EDTA (pH8.0)) is incubated at 55 ℃ for 30min, extracted once with a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), precipitated by adding 2 times of precooled absolute ethanol for 24h, centrifuged at 12500r/min for 15min, washed once with 70% ethanol, dried at room temperature and dissolved by adding 100. mu.L 0.1 × TE. The purity and concentration were measured by UV spectrometer and diluted to 50 ng/. mu.L. The 5' ends of the forward primers of the two primer pairs are marked with blue (FAM) fluorescence, and a 15 mu L PCR amplification reaction system (comprising 2 XPCR MIX 7.5 mu L, upstream and downstream primers (10 mu mol/L) each 0.3 mu L, DNA template 1 mu L, ddH is established2O5.9 μ L); the PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 20s, renaturation at 58 ℃ for 20s, extension at 72 ℃ for 40s, 30 cycles; finally, extension is carried out for 5min at 72 ℃. After the reaction, 1 μ L of PCR product was added with 9 μ L of sample HiDi (ABI), denatured at 95 ℃ for 3min, immediately subjected to ice-water bath, subjected to capillary gel electrophoresis with 3730XL gene sequencer (ABI), and subjected to image collection and data analysis with GeneMappre V4.1 software.
Reading individual genotype data, wherein 128/128bp homozygous individuals in the HLJSL041 comprise 38 individuals, wherein 32 tails of the individuals are from Wusul river, 4 tails of the individuals are from the river segment of the black river, and 2 tails of the individuals are from Dulvjiang; the 103/103bp homozygous individual 32 tails in the HLJSL073 are from the Wusul river population, and the homozygous individuals of the target segment amplified by combining the two specific markers are from the Wusul river zander population, the identification proportion is 100%, and the exclusion proportion is 100%. The HLJSL041 amplification map contains 40 individual 140bp, 38 individual 40 individual sources from the black river segment population, and 2 individual sources from the Duck green river segment population; the HLJ073 amplification map has 107bp individual 22 tails which are all from the black river segment population, and the combination of the two markers can identify 91.49% of individuals in the black river segment population, with the exclusion ratio of 98.11%. The HLJSL041 is used for amplifying 61 individual tails of 144bp or 132bp in a map, wherein 60 tails are from the Duck-green river, and 1 tail is from the black river section; the HLJ073 amplification map comprises individual 62 tails of 137bp, 132bp or 120bp, wherein 60 tails are from the Yangtze river of Duck-Lujiang, 2 tails are from the Yangtze river segment of black river, and the combination of the two markers can identify 97.30% of individuals in the Yangtze river population, wherein the exclusion ratio is 96.20% (as shown in figure 1 and figure 2). Further analysis was performed using Structure software, and at k 3, 3 zander populations could be distinguished from each other using two primer sets, HLJSL041 and HLJSL073, with a small number of confounding individuals in the black river and duck green river populations (as shown in fig. 3).
Example 2
A total of 117 zander samples collected from the pacifying river section (XK)37 tails of the ukrainian river, the black dragon river (HH)40 tails of the black dragon river and the dong river section (YJ)40 tails of the cananga river were identified in the same manner as in example 1.
The experimental results are as follows:
128/128bp homozygous individuals 41 in the HLJSL041, wherein 37 tails are from the Yangtze river segment of Wusuli river, and 4 tails are from the river segment of Black Longjiang river; 103/103bp homozygous individuals 37 in the HLJSL073 are obtained from the Yangtze river section of Wusul river, the homozygous individuals 37 of the target segment are amplified by combining two groups of primer pairs and are all obtained from the zandrin river zander population, and the identification proportion of the zandrin river zander in the Heilongjiang water system is 100 percent, and the exclusion proportion is 100 percent.
The HLJSL041 amplification map contains 33 individual tails of 140bp, 31 individual tails are from a black dragon river segment group, 2 individual tails are from a duck green river segment group, and 17 individual tails of 107bp in the HLJ073 amplification map are all from the black river segment group, and the two groups of primer pairs are combined, so that 90.00 percent of the individuals in the black dragon river segment group can be identified by the method; the exclusion ratio was 97.40%.
The HLJSL041 is used for amplifying individual 36 tails of 144bp or 132bp in a map, wherein 35 tails are from Duck-green river, and 1 tail is from the black river section; HLJ073 amplification map has individual 38 tails of 137bp, 132bp or 120bp, wherein 36 tails are from Duck-green river, and 2 tails are from black river segments; the combination of the two groups of primer pairs can identify 100.00 percent of individuals in the Heilongjiang water-washed Duanjiang group, and the exclusion proportion is 97.40 percent.
Further using Structure software to analyze, when k is 3, using the two groups of primer pairs of the invention can distinguish 3 zander groups, and the black river group and the Duck-green river group have a small amount of mixed individuals.
Claims (5)
1. The primer for identifying the zander population is characterized in that the primer consists of two groups of primer pairs of HLJSL041 and HLJSL 073; the upstream primer of the primer pair HLJSL041 is 5'-TGCCTAACACCTGAACACCA-3', and the downstream primer is 5'-AAGGCAAACTGTGGGGTAGG-3'; the upstream primer of the primer pair HLJSL073 is 5'-AGAAATCGCCTCTGTGTTGT-3', and the downstream primer is 5'-TTGCTGCTGTAACACTGTCA-3'.
2. The identification method for identifying the zander population is characterized by comprising the following steps of:
firstly, extracting genome DNA;
secondly, performing SSR-PCR reaction on the sample DNA by using two groups of primer pairs of HLJSL041 and HLJSL073 of claim 1;
and thirdly, performing gel electrophoresis, reading genotype data, and distinguishing and identifying the zander population in the Heilongjiang river water system.
3. The method for identifying a zander population according to claim 2, wherein said genotype data is:
the amplification map of the individual HLJSL041 of the zandrin perch is 128/128bp homozygote, and the amplification map of the individual HLJ073 is 103/103bp homozygote;
140bp strips appear in the HLJSL041 amplification map of the zander individual at the river section of the black Longjiang black river, and 107bp strips appear in the HLJ073 amplification map;
144bp or 132bp bands appear in the HLJSL041 amplification map of the individual zanthoxylum schlegelii, and 137bp, 132bp or 120bp bands appear in the HLJ073 amplification map.
4. The method for identifying a zander population according to claim 2, wherein the 5' -end of the forward primer of the primer pair is labeled with blue fluorescence.
5. The identification method for identifying a zander population according to claim 2, wherein the SSR-PCR reaction annealing temperatures of the two primer pairs of the HLJSL041 and the HLJSL073 in the step two are both 58 ℃.
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