Method for determining salmon migration time based on genetic distance and principal coordinate analysis

Abstract

The application provides a method for determining salmon run time based on genetic distance and principal coordinate analysis, and belongs to the technical field of molecular biology. On the basis of obtaining salmon microsatellite data, the application makes accurate determination of salmon run time at the molecular level by using a genetic differentiation coefficient in combination with a technical means of principal coordinate component analysis. Based on the application, technical support is provided for accurate determination of the run time of anadromous fish, and the application has important significance for the study of anadromous fish.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and more specifically, it relates to a method for determining the migration time of salmon based on genetic distance and principal coordinate analysis. Background Technology

[0002] Chum salmon are cold-water, anadromous fish that spawn in rivers and are widely distributed in the Bering Sea, North Pacific Ocean, Sea of ​​Okhotsk, Sea of ​​Japan, and their coastal rivers. In my country, they are mainly distributed in the Tumen River and Heilongjiang River basins in Jilin Province. The salmon's migration route is long and arduous, and the timing of their migration varies in different river basins.

[0003] Currently, there is no accurate method to determine the migration time of salmon; only traditional methods using markers can provide a rough estimate. Continuous observation of salmon migration times in the Tumen River and Heilongjiang River basins of my country using the traditional fin-cutting method revealed differences in migration times between the two basins. Different scholars hold differing opinions, suggesting that the migration time varies from 3 to 5 years, but the exact migration time remains uncertain. The accuracy of traditional marking methods is extremely low because markers easily fall off during growth, cut-off tail fins regrow, and the migration process is arduous; only 1 out of 2000 salmon reaches their spawning grounds. According to surveys, in Jilin Province, since the implementation of salmon stock enhancement and release programs over more than ten years, fewer than 5 marked salmon have been caught, posing a significant challenge to determining the migration time.

[0004] Therefore, there is an urgent need to develop a method that can accurately determine the migration of salmon. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the migration time of salmon based on genetic distance and principal coordinate analysis, so as to make up for the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] A method for determining the migration time of salmon based on genetic distance and principal coordinate analysis includes the following steps:

[0008] (1) Sample processing: Take salmon scales or fin tissue and extract DNA;

[0009] (2) Perform microsatellite sequencing and determine genotypes based on microsatellite primer amplification;

[0010] (3) Data analysis based on the determined genotypes: calculate the pairwise genetic differentiation index. The smaller the genetic differentiation index, the closer the genetic distance. Perform principal coordinate analysis. The principal coordinate results determine the degree of population overlap. The greater the overlap, the greater the probability that they are from the same population. Use a combination of genetic distance and principal coordinate analysis for double verification.

[0011] (4) Determine the migration time: Combine the results of genetic differentiation index and principal coordinate analysis. The genetic differentiation coefficient is the smallest in the genetic differentiation index results and the population overlap is large in the principal coordinate analysis structure to determine the migration time of salmon.

[0012] Furthermore, in step (2), during microsatellite sequencing, after synthesizing microsatellite primers, sequencing is performed using a fully automated sequencer. Allele sizes are obtained and data is processed using Genemapper 4.0 software to obtain allele fragment sizes.

[0013] Furthermore, in step (3), the pairwise genetic differentiation index is calculated using Arlequin software. F ST The smaller the genetic differentiation index, the closer the genetic distance. Principal coordinate analysis was performed using GenALEx 6.502. The principal coordinate results determined the degree of population overlap. The greater the overlap, the greater the likelihood that they belong to the same population. The method of combining genetic distance and principal coordinate analysis was used for double verification.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0015] Based on microsatellite data of salmonid chum salmon, this invention accurately determines the migration time of salmonid chum salmon at the molecular level using a combination of genetic differentiation coefficient and principal coordinate component analysis. This invention provides technical support for the accurate determination of the migration time of migratory fishes and is of great significance for the study of migratory fishes. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of allele fragment sizes; where (a) is the paternal homozygote, (b) is the maternal homozygote, and (c) is the heterozygote of both the paternal and maternal parents.

[0017] Figure 2 This is the principal coordinate analysis diagram in Example 1.

[0018] Figure 3 This is a schematic diagram of fish fins in a comparative scale. Detailed Implementation

[0019] The technical solution of the present invention will be further described and illustrated below with reference to the embodiments.

[0020] Example 1

[0021] The experimental materials used in this embodiment were: randomly collected salmon from the lower reaches of the Tumen River estuary, scales or tail fin tissue were taken and preserved in 95% anhydrous ethanol.

[0022] A method for determining the migration time of salmon based on genetic distance and principal coordinate analysis includes the following steps:

[0023] 1. Genomic DNA extraction: Genomic DNA was extracted using a marine animal tissue genomic DNA extraction kit. The extraction quality was assessed by 0.8% agarose gel electrophoresis and micro spectrophotometer, and the DNA was stored at -20°C for later use.

[0024] 2. Microsatellite sequencing: After synthesizing microsatellite primers, sequencing was performed using a fully automated sequencer. Allele sizes were obtained and data was processed using Genemapper 4.0 software to determine allele fragment sizes (e.g., Figure 1 (As shown).

[0025] 3. Data Processing: The pairwise genetic differentiation index was calculated using Arlequin software. F ST The smaller the genetic differentiation index, the closer the genetic distance. GenALEx 6.502 is used for principal coordinate analysis. The principal coordinate graph is used to determine the degree of population overlap. The greater the overlap, the closer the kinship between the same population or different populations. The method of combining genetic distance and principal coordinate analysis is used for double verification.

[0026] 4. Determination time: Combining the genetic differentiation index and principal coordinate analysis results.

[0027] The verification experiment was conducted using the method provided in Example 1, and the results are as follows:

[0028] As shown in Table 1, the genetic differentiation index results show that the genetic differentiation coefficients of the WSA and WSD populations are the smallest (WSA is a 14-year population, and WSD is a 17-year population), the genetic differentiation coefficients of the WSB and WSE populations are the smallest (WSB is a 15-year population, and WSE is an 18-year population), and the genetic differentiation coefficients among the WSC, WSF, RSA, and RSB populations are small (WSC is a 16-year population, WSF is a 19-year population, RSA is a 16-year population, and RSB is a 19-year population). The principal coordinate analysis structure shows that the WSA and WSD populations have a large degree of overlap, the WSB and WSE populations have a large degree of overlap, and the WSC, WSF, RSA, and RSB populations completely overlap. The results are consistent, so it can be determined that the migration time of salmon is 3 years.

[0029] Table 1. Population pairwise genetic differentiation coefficient

[0030]

[0031] In the table above, the populations are: WSA-2014, WSB-2015, WSC-2016, WSD-2017 (wild), WSE-2018 (wild), WSF-2019 (wild), RSA-2016 (released), and RSB-2019 (released).

[0032] Analysis of the results indicates that the migration time of salmon in the Tumen River basin is 3 years. The genetic differentiation index results show that WSA and WSD have the lowest genetic differentiation coefficients (WSA is a 14-year population, WSD is a 17-year population), WSB and WSE have the lowest genetic differentiation coefficients (WSB is a 15-year population, WSE is an 18-year population), and the genetic differentiation coefficients among WSC, WSF, RSA, and RSB populations are small (WSC is a 16-year population, WSF is a 19-year population, RSA is a 16-year population, and RSB is a 19-year population). The principal coordinate analysis results (see...) Figure 2 As can be seen from the results, the WSA and WSD populations overlap significantly, the WSB and WSE populations overlap significantly, and the WSC, WSF, RSA, and RSB populations overlap together. Since the results are consistent, it can be determined that the migration time of salmon is 3 years.

[0033] Comparative example:

[0034] This comparison uses traditional fin clipping marking to determine migration time. The clipped fins will regrow (e.g., ...). Figure 3 As shown in the figure, this traditional method is not suitable for determining the migration time of salmon, because salmon grow in the sea for more than 3 years and are very likely to regrow the cut-off fins, so it cannot be used as a criterion.

[0035] As can be seen from the above embodiments and comparative examples, the present invention can provide an effective and accurate method for determining the migration time of migratory fish.

[0036] Based on the above embodiments, the present invention continues to describe in detail the technical features involved therein and the functions and roles of these technical features in the present invention, so as to help those skilled in the art to fully understand the technical solution of the present invention and reproduce it.

[0037] Finally, although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for determining the migration time of salmon based on genetic distance and principal coordinate analysis, characterized in that, The method includes the following steps: (1) Sample processing: Take salmon scales or fin tissue and extract DNA; (2) Perform microsatellite sequencing and determine genotypes based on microsatellite primer amplification; (3) Data analysis based on the determined genotypes: Calculate the pairwise genetic differentiation index. The smaller the genetic differentiation index, the closer the genetic distance. Perform principal coordinate analysis. The principal coordinate results determine the degree of population overlap. The greater the overlap, the closer the kinship between the same population or different populations. Use a combination of genetic distance and principal coordinate analysis for double verification. Calculate the pairwise genetic differentiation index using Arlequin software. F ST The smaller the genetic differentiation index, the closer the genetic distance. Principal coordinate analysis was performed using GenALEx 6.

502. The principal coordinate results determined the degree of population overlap. The greater the overlap, the closer the kinship between the same population or different populations. The method of combining genetic distance and principal coordinate analysis was used for double verification. (4) Determine the migration time: Combine the results of genetic differentiation index and principal coordinate analysis. The genetic differentiation coefficient is the smallest in the genetic differentiation index results and the population overlap is large in the principal coordinate analysis structure to determine the migration time of salmon.

2. The method as described in claim 1, characterized in that, In step (2), during microsatellite sequencing, microsatellite primers are synthesized and sequenced using a fully automated sequencer. Allele sizes are obtained and data is processed using Genemapper 4.0 software to obtain allele fragment sizes.

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