Primer, molecular marker and method for identifying culter alburnus in Yangtze River, culter alburnus in Taihu Lake and culter alburnus in Taihe Lake

Abstract

The invention provides a primer, a molecular marker and a method for identifying culter alburnus Yangtze River, culter alburnus Taihu Lake and culter alburnus Hatahu Lake, specific primers are designed aiming at the molecular marker, specific microsatellite sites are amplified through PCR, the length of amplified fragments is analyzed by capillary electrophoresis, and the specific microsatellite sites are identified. And according to the size of the population specific band, accurate identification of the group or individual of the culter alburnus in Yangtze River, the culter alburnus in Taihu Lake and the culter alburnus in Taihe Lake The molecular marker and the method have the advantages of high accuracy, simplicity, rapidness and accuracy, basic lossless sampling can be realized, a detection result can be accurate to a group and can also be positioned to an individual, the application range is wide, a sampled sample can be normally cultured, the subsequent growth and development of the sample are not influenced, and the method can be realized.

Description

Technical Field

[0001] This invention belongs to the field of aquatic biotechnology and molecular genetic markers, specifically involving a fish population identification technology based on microsatellite molecular markers, which is particularly suitable for the rapid and accurate identification of three important economic geographical populations: Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus. Background Technology

[0002] Culter alburnus ( Alburn knife The Culter alburnus (also known as the mandarin fish) is an important freshwater economic fish in my country, widely distributed in the Pearl River, Yangtze River, and Heilongjiang River. Through long-term geographical isolation and ecological adaptation, different river systems (such as the Yangtze River, Taihu Lake, and Xingkai Lake) have evolved into populations with significant genetic differences. Among them, the Yangtze River Culter alburnus exhibits good genetic diversity and is an important species for aquaculture and stock enhancement in my country; the Taihu Lake Culter alburnus is adapted to artificial aquaculture environments, swims swiftly, is adept at jumping, and has delicate and delicious flesh; the Xingkai Lake Culter alburnus is highly cold-resistant, not good at jumping, has a mild temperament, tolerates low oxygen levels and transportation, is suitable for lure fishing, and has tender and nutritious flesh. Along with the salmon of the Ussuri River and the beach fish of the Suifen River, it is known as one of the "Three Treasures of the Border Region," possessing extremely high economic value and regional characteristics. Three different geographical populations of Culter alburnus exhibit significant differences in spawning methods, sexual maturity cycles, growth rates at different stages, and adapted aquatic environments. However, these three species are remarkably similar in appearance. Traditional identification methods relying on morphological characteristics such as body size and color are highly subjective and prone to error, failing to meet the needs of modern aquaculture, germplasm resource management, and breeding. Especially in seedling distribution, stock enhancement, and genetic conservation, accurately distinguishing Culter alburnus from different water systems is crucial for maintaining population purity and preventing genetic mixing. Therefore, there is an urgent need to develop a rapid, accurate, and stable molecular identification method to achieve efficient identification of Culter alburnus from the Yangtze River, Taihu Lake, and Xingkai Lake.

[0003] Microsatellite markers, also known as simple sequence repeats (SSRs), are DNA sequences composed of tandem repeats of 1-6 base pairs as the core unit, widely and randomly distributed in the genomes of eukaryotic organisms. Microsatellite markers have many outstanding advantages: (1) extremely high polymorphism: due to the very frequent variation in the number of repeats of the core unit, the alleles (manifested as PCR amplification fragment length) at the same microsatellite locus vary greatly among different individuals and populations, resulting in rich information content; (2) codominant inheritance: able to distinguish between homozygotes and heterozygotes, providing complete genetic information; (3) rapid and convenient detection: based on PCR technology, only a small amount of DNA is needed for amplification and detection; (4) good reproducibility: experimental results are stable and reliable; (5) coexistence of conservation and variability: the flanking sequences on both sides of the microsatellite are usually relatively conserved among closely related species or populations, making it easy to design universal primers, while the number of repeat units in the core region is highly variable, suitable for distinguishing closely related groups.

[0004] This invention, based on specific microsatellite markers, develops primers, molecular markers, and methods for identifying the germplasm of *Culter alburnus* from the Yangtze River, Taihu Lake, and Xingkai Lake. This method does not require euthanasia of the fish; only a small amount of fin or tissue samples are needed for accurate identification via PCR amplification and fragment analysis. It offers advantages such as ease of operation, accurate results, and good reproducibility, effectively supporting the conservation and sustainable utilization of *Culter alburnus* germplasm resources.

[0005] The Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus exhibit extremely similar morphology at both the fry and adult stages. Traditional morphological identification methods are highly subjective and prone to significant errors, failing to meet the demands for accurate population identification in areas such as fry tracing, germplasm resource management, breeding, and stock enhancement. Therefore, there is an urgent need to establish an objective, accurate, and rapid molecular identification method. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide primers, molecular markers, and methods for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus. This method distinguishes and identifies Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus from a molecular biological perspective. Furthermore, this method has the advantages of being fast, low-cost, and easy to master.

[0007] As one aspect of the present invention, the present invention provides a DNA molecular marker for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus and Xingkai Lake Culter alburnus, the simple repeating base sequence of the molecular marker being shown in SEQ ID NO.1.

[0008] As one aspect of the present invention, the present invention provides primer pairs for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus and Xingkai Lake Culter alburnus, said primer pairs being SEQ ID NO.2 and SEQ ID NO.3.

[0009] As one aspect of the present invention, the present invention provides a method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus, comprising the following steps: 1) Extract DNA from samples of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus to be identified; 2) The DNA of the sample to be identified was amplified by PCR using the primer pairs SEQ ID NO.2 and SEQ ID NO.3 to obtain PCR products; 3) The PCR products were subjected to capillary electrophoresis on an ABI 3500xl analyzer, and the resulting spectra were analyzed and compared with the standard spectra of the DL600 Marker. If a specific DNA band is amplified at the 330 bp position, it can be identified as Culter alburnus. If a specific DNA band is amplified at the 307 bp position, it can be identified as Culter alburnus from Taihu Lake; If a specific DNA band is amplified at the 290 bp position, it can be identified as Culter alburnus from Lake Xingkai.

[0010] Preferably, the samples of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus are derived from fin rays or blood samples.

[0011] Preferably, the PCR amplification system is 10 μL: 5 μL of 2xTaq PCR mix, 0.5 μL each of forward and reverse primers, 1 μL of the fish DNA sample to be identified, and ddH2O added to 10 μL.

[0012] Preferably, the PCR amplification procedure is as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 45 sec, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃.

[0013] This invention relates to the molecular identification of Culter alburnus germplasm resources, specifically to primers, molecular markers, and methods for identifying Culter alburnus from the Yangtze River, Taihu Lake, and Xingkai Lake, belonging to the field of aquatic biotechnology. This invention can be applied to the accurate identification of these three Culter alburnus geographical populations, germplasm resource protection, and the formulation of germplasm standards in the aquaculture industry.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention relates to the molecular identification of Culter alburnus germplasm resources, specifically to primers, molecular markers, and methods for identifying Culter alburnus from the Yangtze River, Taihu Lake, and Xingkai Lake, belonging to the field of aquatic biotechnology. This invention can be applied to the accurate identification of these three Culter alburnus geographical populations, germplasm resource protection, and the formulation and identification of germplasm standards in the aquaculture industry.

[0015] 1. Based on molecular marker combination techniques such as PCR and SSR typing, a rapid, accurate, economical and reliable method was established to identify three different geographical populations of Culter alburnus from the Yangtze River, Taihu Lake, and Xingkai Lake. The identification method is highly accurate, rapid, precise and practical.

[0016] 2. This method requires only a small number of fin samples, and the sampling operation is simple.

[0017] 3. A small number of fin rays are taken from the tested object, and the sample can survive normally, which can basically achieve non-destructive sampling; the sample can be cultured normally after sampling and will not affect the subsequent growth and development of the sample.

[0018] 4. The test results can be accurate down to the group level or down to the individual level, making it widely applicable.

[0019] 5. This method uses capillary electrophoresis as a detection method, which can achieve large-scale, high-throughput detection and identification, and can significantly reduce detection and identification costs. Attached Figure Description

[0020] Figure 1 The DNA bands are specific for the identification of the Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus in this invention. Detailed Implementation

[0021] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments are now described. Obviously, the accompanying drawings described below are some embodiments of the present invention. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0022] Example 1 This invention, based on microsatellite (SSR) marker technology, designs specific primers to target the genetic differences among Yangtze River, Taihu Lake, and Xingkai Lake Culter alburnus populations, and develops a primer, molecular marker, and method for identifying the germplasm of these three species. This method amplifies specific microsatellite loci via PCR, analyzes the amplified fragment length using capillary electrophoresis, and achieves accurate identification based on the size of the population-specific bands.

[0023] 1. Sample Source The Yangtze River Culter alburnus originates from the natural waters of the Jianli section of the middle reaches of the Yangtze River in Hubei Province, weighing 0.1kg-1.5kg and measuring 21cm-59cm in length. The Taihu Lake Culter alburnus originates from the offspring of parent fish bred in the natural waters of the Balidian Comprehensive Experimental Base in Wuxing District, Huzhou City, Taihu Lake, weighing 0.5kg-0.7kg and measuring 36cm-45cm in length. The Xingkai Lake Culter alburnus originates from the offspring of parent fish bred in the natural waters of the Xingkai Lake Research Institute of Heilongjiang Reclamation Area, weighing 0.8kg-1.4kg and measuring 42cm-55cm in length.

[0024] 2. Specific microsatellite sites This embodiment provides a DNA molecular marker for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus. The base sequence of the molecular marker is SEQ ID NO.1: 5'-AACTCCAGGTTGACATGTAAGCTTGCAT-3'.

[0025] The Yangtze River Culter alburnus lays drifting and slightly sticky eggs, rarely jumps, and its growth rate is between the other two populations, with a sexual maturity cycle of 2-3 years. The Taihu Lake Culter alburnus lays sticky eggs, is prone to jumping, has a rapid growth rate in the seedling stage, and a short sexual maturity cycle of 2 years. The Xingkai Lake Culter alburnus lays drifting eggs, does not jump, has a slow growth rate in the seedling stage, a rapid growth rate after 2 years, and a long sexual maturity cycle of 4-5 years, with larger individuals at sexual maturity. Regarding egg stickiness, jumping behavior, and growth rate, the molecular markers provided in this invention are co-dominantly inherited, with different bands representing the Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus, respectively. This overcomes the difficulty of traditional SSR genotyping, which often requires the joint detection of multiple loci and complex statistical analysis for accurate genotyping.

[0026] Example 2 Based on the aforementioned molecular markers, this embodiment performs identification analysis, specifically designing primer pairs for detecting the molecular markers, with sequences SEQ ID NO.2 and SEQ ID NO.3, respectively: SEQ ID NO.2: 5'-GTTATGACTTAAGTCATTGTATAACC-3'; SEQ ID NO. 3: 5'TTACGTAGACTATCAGTCTGTATGA-3'.

[0027] The method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus in this embodiment includes the following steps: 1) Extract DNA from samples of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus to be identified; Thirty individuals were selected from each of three different populations of Culter alburnus. A 0.5 cm long tail fin tissue or a 0.2 ml tail vein blood sample was taken from each individual.

[0028] DNA was extracted from samples of Culter alburnus using an animal tissue DNA extraction kit. This kit offers advantages over traditional DNA extraction methods, including higher extraction efficiency, shorter processing time, and higher quality DNA.

[0029] 2) Use the above primers to perform PCR amplification on the DNA of the sample to be identified to obtain PCR products; (1) The PCR amplification system is 10 μL: 5 μL of 2xTaq PCR mix, 0.5 μL each of forward and reverse primers, 1 μL of the fish DNA sample to be identified, and ddH2O added to 10 μL.

[0030] (2) PCR amplification program: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 45 sec, for a total of 35 cycles, and finally 72℃ extension for 5 min, and stored at 4℃.

[0031] 3) The PCR products were subjected to capillary electrophoresis on an ABI 3500xl analyzer, and the resulting spectra were analyzed and compared with the standard spectra of the DL600 Marker. If a specific DNA band is amplified at the 330 bp position, it can be identified as Culter alburnus. If a specific DNA band is amplified at the 307 bp position, it can be identified as Culter alburnus from Taihu Lake; If a specific DNA band is amplified at the 290 bp position, it can be identified as Culter alburnus from Lake Xingkai.

[0032] Common microsatellite markers are typically polymorphic, potentially exhibiting 1-20 amplified bands within the same population, and the correspondence between bands and alleles is not strictly one-to-one. The marker used in this study showed only a single band in all three tested populations, with differences in band length among the populations. This difference was closely linked to the three geographical populations, indicating that the marker did not originate from a single related parent and possessed an independent genetic origin across different populations, making it suitable for identifying different varieties. This invention, based on specific microsatellite markers, enables rapid and accurate identification of three geographical populations of Culter alburnus. It is simple to operate, has good reproducibility, and is applicable to seedling identification, germplasm resource management, and related scientific research and production practices.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A DNA molecular marker for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus, characterized in that: The base sequence of this molecular marker is shown in SEQ ID NO.

1.

2. Primer pairs for identifying Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus, characterized in that: The base sequences of the primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

3. A method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus, characterized in that: Includes the following steps, 1) Extract DNA from samples of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus to be identified; 2) The DNA of the sample to be identified was amplified by PCR using the primer pairs SEQ ID NO.2 and SEQ ID NO.3 to obtain PCR products; 3) The PCR products were subjected to capillary electrophoresis on an ABI 3500xl analyzer, and the resulting spectra were analyzed and compared with the standard spectra of the DL600 Marker. If a specific DNA band is amplified at the 330 bp position, it can be identified as Culter alburnus. If a specific DNA band is amplified at the 307 bp position, it can be identified as Culter alburnus from Taihu Lake; If a specific DNA band is amplified at the 290 bp position, it can be identified as Culter alburnus from Lake Xingkai.

4. The method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus as described in claim 3, characterized in that: The samples of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, or Xingkai Lake Culter alburnus were obtained from fin rays or blood samples.

5. The method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus as described in claim 3, characterized in that: The PCR amplification system consisted of 10 μL of the following components: 5 μL of 2xTaq PCR mix, 0.5 μL each of the forward and reverse primers, 1 μL of the fish DNA sample to be identified, and ddH2O added to bring the total volume to 10 μL.

6. The method for identifying the germplasm of Yangtze River Culter alburnus, Taihu Lake Culter alburnus, and Xingkai Lake Culter alburnus as described in claim 3, characterized in that: The PCR amplification procedure was as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 45 sec, for a total of 35 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃.

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