Search

CN-122012732-A - Primers, molecular markers and method for identifying culter alburnus of Yangtze river, culter alburnus of Taihu lake and culter alburnus of Xingshai lake

CN122012732ACN 122012732 ACN122012732 ACN 122012732ACN-122012732-A

Abstract

The invention provides a primer, a molecular marker and a method for identifying culter alburnus of the Yangtze river, culter alburnus of the Tai lake and culter alburnus of the Xingshai lake, wherein a specific primer is designed for the molecular marker, a specific microsatellite site is amplified through PCR, the amplified fragment length is analyzed through capillary electrophoresis, and the accurate identification of the population or individuals of the culter alburnus of the Yangtze river, the culter alburnus of the Tai lake and the culter alburnus of the Xingshai lake is realized according to the size of a population specific strip. The molecular marker and the method have the advantages of high accuracy, simplicity, rapidness and accuracy, capability of realizing basic nondestructive sampling, accurate detection result to the group, positioning to the individual, wide application range, normal culture of the sampled sample, no influence on the subsequent growth and development of the sample, and realization.

Inventors

  • LIU WEI
  • Pei Xueying
  • Dai Yafan
  • SUN HAOBO
  • ZHANG LEIMING
  • MA XINGYU
  • ZHOU GUOQIN
  • SHI XIAOLAN

Assignees

  • 南京市水产科学研究所(南京市水产技术推广站、南京市水生动物疫病预防控制中心)

Dates

Publication Date
20260512
Application Date
20260224

Claims (6)

  1. 1. The DNA molecular marker for identifying the culter alburnus of the Yangtze river, the culter alburnus of the Taihu lake and the culter alburnus germplasm of the Xingshai lake is characterized in that the base sequence of the molecular marker is shown as SEQ ID NO. 1.
  2. 2. The primer pair for identifying the culter alburnus of the Yangtze river, the culter alburnus of the Taihu lake and the culter alburnus of the Xingshai lake is characterized in that the base sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
  3. 3. A method for identifying the germplasm of the culter of the Erythroculter Yangtze river, the Erythroculter taipaiensis and the Erythroculter Xingshi lake is characterized by comprising the following steps, 1) Extracting DNA of a sample of the culter ilishaeformis or the culter ilishaeformis of the Pacific lake to be identified; 2) Carrying out PCR amplification on DNA of a sample to be identified by using the primer pair SEQ ID NO.2 and SEQ ID NO.3 to obtain a PCR product; 3) The PCR products were subjected to capillary electrophoresis on an ABI 3500xl analyzer and subjected to profile analysis by comparison with the DL600Marker standard profile: if 1 specific DNA band is amplified at the position 330 bp, identifying as the culter ilishaeformis; if 1 specific DNA band is amplified at 307 bp, identifying as the Erythroculter ilishaeformis; If 1 specific DNA band was amplified at position 290 bp, it was identified as Erythroculter ilishaeformis.
  4. 4. The method of identifying the germplasm of culter alburnus, culter alburnus and culter alburnus in Xingshai lakes of claim 3, wherein the sample of culter alburnus, culter alburnus in Taihu lake or culter alburnus in Xingshai lake is derived from a fin or blood sample.
  5. 5. The method for identifying the culter of the Yangtze river, the culter of the Tai lake and the culter of the Xingshi lake according to claim 3, wherein the PCR amplification system is 10 mu L, 2xTaq PCR mix 5 mu L, forward and reverse primers are 0.5 mu L respectively, the DNA sample of the fish to be identified is 1 mu L, and ddH 2 O is added to 10 mu L.
  6. 6. The method for identifying the germplasm of the culter of the Yangtze river, the culter of the Tai lake and the culter of the Xingshi lake according to claim 3, wherein the PCR amplification is carried out by a process of pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s and extension at 72 ℃ for 45sec for 35 cycles, and finally extension at 72 ℃ for 5min and preservation at 4 ℃.

Description

Primers, molecular markers and method for identifying culter alburnus of Yangtze river, culter alburnus of Taihu lake and culter alburnus of Xingshai lake Technical Field The invention belongs to the field of aquatic biotechnology and molecular genetic markers, and particularly relates to a fish population identification technology based on microsatellite molecular markers, which is particularly suitable for rapid and accurate identification of three important economic geographic populations, namely, culter alburnus of the Yangtze river, culter alburnus of the Taihu lake and culter alburnus of the Xingshai lake. Background Culter ilishaeformis (Culter alburnus) is an important freshwater economic fish in China and is widely distributed in water areas such as Zhujiang, changjiang and Heilongjiang. In the process of long-term geographic isolation and ecological adaptation, the culter ilishaeformis of different water systems (such as Yangtze river, tai lake and Xingshai lake) have evolved to form populations with significant genetic differences. The Erythroculter ilishaeformis in the Yangtze river system has good genetic diversity, is an important breeding and proliferation releasing object in China, is suitable for artificial breeding environments, has rapid swimming, good jump, fine and delicious meat quality, has strong cold resistance, bad jump, mild property, low oxygen resistance and transportation resistance, is suitable for road sub-fishing, has fresh and tender meat and rich nutrition, is suitable for the fish of the beach of the salmon and the seism of the Usuli river, and is called as 'side plug three delicacies', and has extremely high economic value and regional characteristics. The spawning mode, sexual maturation period, growth speed in different periods and adaptive water area environment of three types of ilishaeformis of different geographical populations are quite different, however, the three types of ilishaeformis are quite similar in appearance and morphology, and the traditional identification method relying on morphological characteristics such as body type, body color and the like is strong in subjectivity and large in error, so that the requirements of modern aquaculture, germplasm resource management and fine breed breeding are difficult to meet. Particularly in the processes of seed circulation, proliferation and release and genetic protection, the method for accurately distinguishing the culter ilishaeformis of different water systems has important significance for keeping the population pure and avoiding germplasm mixing. Therefore, there is an urgent need to develop a rapid, accurate and stable molecular identification method to realize efficient identification of the culter alburnus of the Yangtze river, the culter alburnus of the Taihu lake and the culter alburnus of the Xingshai lake. Microsatellite (Microsatellite) markers, also known as simple sequence repeats (Simple Sequence Repeat, SSR), are DNA sequences which are made up of tandem repeats of 1-6 base pairs as core units, widely and randomly distributed in eukaryotic genomes. The microsatellite marker has the advantages of extremely high polymorphism, extremely high difference of alleles (expressed as PCR amplified fragment length) of different individuals and different populations on the same microsatellite locus due to extremely frequent variation of repetition times of core units, abundant information content, co-dominant inheritance, capability of distinguishing homozygotes and heterozygotes and providing complete genetic information, rapid and simple detection, capability of carrying out amplification detection by only trace DNA (deoxyribonucleic acid) based on PCR technology, good repeatability, stable and reliable experimental result, and compatibility of conservation and variability, wherein flanking sequences on two sides of the microsatellite are generally conserved among species or populations with similar relativity, so that a universal primer is convenient to design, and the number of the repeated units of the core region has high variability and is suitable for distinguishing near-edge groups. The invention discloses a primer, a molecular marker and a method for identifying the germplasm of the culter alburnus of the Yangtze river, the culter alburnus of the Taihu lake and the culter alburnus of the Xingshi lake based on specific microsatellite markers. According to the method, the fish body is not required to be killed, only a small amount of fin strips or tissue samples are required to be cut, accurate identification can be realized through PCR amplification and fragment analysis, the method has the advantages of simplicity and convenience in operation, accurate results and good repeatability, and the protection and sustainable utilization of the culter ilishaeformis germplasm resources can be effectively supported. The forms of the culter alburnus of the Yangtze river, the culter alburnus of the Taihu la