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Identifying primer and identifying method for germplasm of hucho taimen and brachymystax lenok and reagent kit

A technology of lecherous salmon and trout, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of identification and damage the development of cold-water fish farming industry. Issues such as resource multiplication and release, germplasm mixing of jelly salmon and lenicho salmon, etc.

Active Publication Date: 2019-08-20
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Salmon and trout are closely related species with similar living environments. Adult fish are easy to distinguish based on their appearance, but juveniles are very similar in appearance and it is difficult to distinguish them from appearance. Therefore, salmon and salmon are very easy to appear The mixed salmon germplasm will damage the development of cold-water fish aquaculture industry and the multiplication and release of salmon and salmon. Therefore, there is an urgent need for a fast and accurate germplasm identification method for salmon and salmon.

Method used

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  • Identifying primer and identifying method for germplasm of hucho taimen and brachymystax lenok and reagent kit
  • Identifying primer and identifying method for germplasm of hucho taimen and brachymystax lenok and reagent kit

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, establishing identification method

[0024] 1. DNA extraction

[0025] Cut some fin rays or 5-10 scales of the sample fish, and add 40 μl of lysate (Protease K 0.5 mg / ml, Tris (pH8.0) 10 mM, KCl 50 mM, Tween 20 0.3% (w / v), NP- 40 0.3% (w / v)), 55°C for 2h, 98°C for 10min to lyse, mix well after lysis, centrifuge at 1000-2000rpm for 1-2min, extract the supernatant, which is the DNA crude extract;

[0026] 2. Design primers

[0027] 53 pairs of SSR primers were designed according to the EST sequence of the Salmonella salmon, 12 pairs of primers failed to amplify the bands in the Salmonella salmon, and 41 pairs of primers that could amplify the bands were screened, and the HtaC1041 primer was found to be specific , the primer sequence is:

[0028] Upstream primer HtaC1041F: 5'-ACTTGGGCATTGGTAAAAGCT-3' (SEQ ID NO.1)

[0029] Downstream primer HtaC1041R: 5'-AGGCATCATGAGAGTGCGTG-3' (SEQ ID NO.2)

[0030] Amplified sequence of salmon HtaC1041:

[0031] ACTTG...

Embodiment 2

[0038] Embodiment 2, sample detection

[0039] Using the identification method of Example 1, collect 121 adult fishes identified as wild salmon and 41 adult fishes identified as wild salmon in different watersheds according to their morphology, and take 5-10 scales each for identification. According to the comparison of the electrophoresis results with the standard spectra of salmon salmon and salmon salmon provided, it was found that a specific band with a size of 166bp appeared in 121 samples identified as salmon salmon according to morphology, indicating that the samples were salmon salmon; according to A specific band of 158bp appeared in the 41 samples that were identified as leucinus morphologically, indicating that the samples were leucinus samples ( figure 2 ), consistent with the morphological identification results.

[0040] The PCR products of 5 samples randomly selected from the jellyfish and slender salmon were sequenced respectively. The SSR sequence of the jer...

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Abstract

The invention discloses an identifying primer and identifying method for germplasm of hucho taimen and brachymystax lenok and a reagent kit. The primer comprises an upstream primer HtaC1041F and a downstream primer HtaC1041R, wherein the sequence of the upstream primer HtaC1041F is as shown in SEQID NO.1, and the sequence of the downstream primer HtaC1041R is as shown in SEQID NO.2. The identifying method disclosed by the invention a new method for identifying the germplasm of the hucho taimen and the brachymystax lenok, established based on a molecular biology technique principle for the first time at home and abroad, a pair of SSR primers are designed through an EST sequence of the hucho taimen, and through a PCR reaction, identification of the hucho taimen and the brachymystax lenok canbe realized. Through the adoption of the identifying primer and the identifying method disclosed by the invention, complete individuals and processed products of the hucho taimen and the brachymystaxlenok can be distinguished, and besides, the identifying primer and the identifying method can be used for identification of roes and offspring which is justly incubated, and have the characteristicsof being accurate, quick, objective, fair, economical and practical.

Description

technical field [0001] The invention relates to germplasm identification primers, a identification method and a kit for salmon and leprosy salmon, and belongs to the technical field of molecular identification. Background technique [0002] Zheluo salmon, whose scientific name is Hucho taimen (Pallas), belongs to the genus Zheluo salmon. Lepidoptera, scientific name Brachymystaxlenok (Pallas), belongs to the genus Lepidoptera. Both belong to the order Salmoniformes and the family Salmonidae, and are rare and valuable cold-water fish in my country. Salmon and trout are endangered species. Both were included in the Red Data Book of Endangered Animals in China in 1998 and included in the Red List of Species in China in 2004. In order to protect and develop these two kinds of rare and valuable fish, researchers successively conquered the artificial propagation and seed cultivation of these two kinds of fish, and successfully carried out large-scale breeding of seedlings and co...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156Y02A40/81
Inventor 佟广香唐国盘匡友谊尹家胜张永泉张庆渔
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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