A method for monitoring Siniperca chuatsi germplasm using microsatellite genetic markers

Abstract

The present invention discloses a method for monitoring the germplasm of chuatsi (Siniperca chuatsi) using microsatellite genetic markers, including extraction and detection of genomic DNA, search and analysis of microsatellite sequences, screening and optimization of polymorphic microsatellite primers, detection of polymorphic microsatellites, and monitoring of different geographical chuatsi populations and their genetic traits; the present invention screens a large number of ESTs generated by chuatsi transcriptome sequencing for microsatellite sites and develops and verifies polymorphic microsatellite markers, and develops 15 EST-SSR polymorphic microsatellite markers, which can monitor different geographical populations of chuatsi and their genetic traits. The method can make up for the limitations of the current chuatsi germplasm detection, improved variety breeding and germplasm improvement research, such as the relatively vague genetic background and the small number of molecular markers, and provide a basis for the chuatsi germplasm detection, improved variety breeding and germplasm improvement, thereby promoting the progress of chuatsi molecular marker germplasm detection, assisted genetic breeding and genomics research.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for monitoring mandarin fish germplasm using microsatellite genetic markers. Background Art

[0002] Mandarin fish Siniperca chuatsi ), also known as Guiyu, osmanthus fish, fat mandarin fish, seasonal flower fish, claw fish, and flower crucian carp. In biological classification, it belongs to the order Perciformes, subfamily Siniperinae, and genus Siniperca. It is a precious fish with high economic value, enjoying the reputation of "freshwater grouper". It has excellent traits such as fast growth, tender meat, delicious taste, and high nutritional content. It is a famous and special aquatic product deeply loved by consumers and breeders, and it is also a nourishing treasure suitable for all ages. However, in recent years, due to overfishing of rivers, deterioration of the ecological environment of water bodies, and inbreeding in aquaculture production, the current situation of natural germplasm resources decline, breeding germplasm traits degradation, and continuous outbreaks of fish diseases has affected the continuation of the germplasm of Siniperca chuatsi and the normal development of its aquaculture industry. It is urgently needed to use modern molecular biological methods to carry out research on germplasm detection, resource protection, and breeding of fine varieties of Siniperca chuatsi.

[0003] At present, the germplasm detection of mandarin fish mainly adopts appearance trait indicators such as body shape, body color, and pattern, but these indicators will be interfered by various factors such as age, gender, and living environment, and it is impossible to correctly detect germplasm of different ages, genders, and living environments. In recent years, the development of genetic markers has become an important means of germplasm detection and molecular marker-assisted breeding, but the development of microsatellite molecular markers for mandarin fish is relatively small. Researchers analyzed the types and frequencies of microsatellite sequences in some EST data of mandarin fish transcriptomes, proving the feasibility of discovering SSR markers from mandarin fish transcriptome ESTs. However, the development of mandarin fish EST-SSR markers in germplasm detection has not been reported at home and abroad. Based on the screening of microsatellite loci and the development and verification of polymorphic microsatellite markers by a large number of ESTs generated by transcriptome sequencing of mandarin fish, the present invention has developed a new method for germplasm detection of mandarin fish using EST-SSR, which can make up for the limitations of the current mandarin fish germplasm detection and improved variety breeding research, such as the vague genetic background and the small number of molecular markers, and provide a basis for mandarin fish germplasm detection and improved variety breeding, thereby promoting the progress of molecular marker germplasm detection, assisted genetic breeding and genomics research of mandarin fish. Summary of the invention

[0004] In view of the above problems existing in the prior art, the object of the present invention is to provide a method for monitoring the germplasm of mandarin fish using microsatellite genetic markers to solve the many problems caused by the shortcomings of the prior art.

[0005] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A method for monitoring mandarin fish germplasm using microsatellite genetic markers comprises the following steps:

[0007] (1) Extraction and detection of genomic DNA

[0008] 0.2 g of tail fins of several mandarin fish were taken and genomic DNA was extracted by phenol-chloroform method. The integrity of DNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of DNA were determined by nucleic acid protein analyzer. Finally, the DNA was diluted to about 100 ng / μL with sterile water and stored at -20℃ for future use.

[0009] (2) Search and analysis of microsatellite sequences

[0010] Mandarin fish of different growth ages were selected for transcriptome sequencing. Based on the transcriptome library sequences measured, SSR sites in all non-redundant gene databases were retrieved. PCR primers, gene annotations and GO secondary classifications were designed based on the SSRs flanking sequences. Primers designed for SSR sites on genes in the three GO secondary classification entries of growth, proliferation and metabolism were selected.

[0011] (3) Screening and optimization of polymorphic microsatellite primers

[0012] From all primers of the three secondary classification items in step (2), 120 pairs of primers were randomly selected for pre-amplification and polymorphic primer screening.

[0013] (4) Monitoring of different geographical populations of Siniperca chuatsi and their genetic traits

[0014] 15 pairs of primers with good stability were screened from step (3). The same number of wild geographical populations of mandarin fish from the Yangtze River, Huaihe River, Pearl River and Heilongjiang River were selected for pre-amplification and polymorphic microsatellite primer screening. Individual gene scanning, population genetic structure analysis and diversity detection were performed respectively. The amplification system, procedure and annealing temperature were referred to the pre-amplification process. Polyacrylamide gel electrophoresis and silver nitrate staining were followed and photographs were taken. The genotype data of each mandarin fish were obtained using the software Gel-Pro Analyzer 4.5. The allele frequency of each microsatellite marker locus was calculated using the software PopGen32 ( P ), number of alleles ( Na ), effective number of alleles ( Ne ), observed heterozygosity ( Ho ), expected heterozygosity ( He ), Hadwinberg equilibrium P HW , calculate the polymorphism information content PIC,

[0015] The calculation formula of the polymorphism information content PIC is:

[0016] Where Pi and Pj are the frequencies of the i-th and j-th alleles in the population, respectively, j = i + 1, and n is the number of alleles at a certain locus. The genetic distance and genetic similarity between different strains, as well as related genetic parameters, were calculated, and the molecular phylogenetic tree of different strains was constructed using UPGMA.

[0017] Preferably, the screening conditions in step (3) are that the minimum length of SSRs is 15 bp; the minimum number of repetitions of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides are 8, 5, 4, 3 and 3 times respectively, and the product size is between 150 bp and 500 bp.

[0018] Preferably, the mandarin fish sample in step (1) is taken from 30 mandarin fish in the same geographical population.

[0019] Preferably, in step (2), the mandarin fish samples are taken from at least 30 individuals from different populations, and the 15 pairs of microsatellite loci and primer sequences in step (4) are:

[0020] .

[0021] The advantages of the present invention are that the selected primers have high amplification efficiency, clear bands, rich polymorphisms, and good repeatability. The present invention screens microsatellite sites and develops and verifies polymorphic microsatellite markers for a large number of ESTs generated by transcriptome sequencing of mandarin fish, and develops 15 EST-SSR polymorphic microsatellite markers, which can monitor different geographical populations of mandarin fish and their genetic traits. It can make up for the limitations of the current mandarin fish germplasm detection, improved breeding, and germplasm improvement research, such as the vague genetic background and the small number of molecular markers, and provide a basis for the germplasm detection, improved breeding, and germplasm improvement of mandarin fish, thereby promoting the progress of germplasm detection, assisted genetic breeding, and genomics research of mandarin fish molecular markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] like Figure 1 This is a diagram showing the results of amplifying the C6713 site in Siniperca chuatsi in an embodiment of the present invention;

[0023] like Figure 2 This is a diagram showing the result of amplifying the C12534 site in Siniperca chuatsi in an embodiment of the present invention;

[0024] like Figure 3 This is a diagram showing the result of amplifying the C18691 site in Siniperca chuatsi in an embodiment of the present invention;

[0025] like Figure 4 This is a UPGMA cluster analysis diagram of four populations of mandarin fish in the embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments. The following embodiments may enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0027] A method for monitoring mandarin fish germplasm using microsatellite genetic markers comprises the following steps:

[0028] (1) Extraction and detection of genomic DNA

[0029] 0.2 g of tail fins of 10 mandarin fish in the same population were taken and genomic DNA was extracted by phenol-chloroform method. The integrity of DNA was tested by 1% agarose gel electrophoresis, and the concentration and purity of DNA were determined by nucleic acid protein analyzer. Finally, the DNA was diluted to about 100 ng / μL with sterile water and stored at -20℃ for future use.

[0030] (2) Search and analysis of microsatellite sequences

[0031] Three mandarin fish of different growth ages from the same population were selected for transcriptome sequencing. Based on the measured transcriptome library sequences, SSR sites in all non-redundant gene databases were retrieved. PCR primers, gene annotations and GO secondary classifications were designed based on the SSRs flanking sequences, and primers designed for SSR sites on genes in the three GO secondary classification entries of growth, proliferation, and metabolism were selected.

[0032] (3) Screening and optimization of polymorphic microsatellite primers

[0033] From all primers of the three secondary classification items in step (2), 120 pairs of primers were randomly selected for pre-amplification and polymorphic primer screening. The screening conditions were that the minimum length of SSRs was 15 bp; the minimum number of repeats of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides was 8, 5, 4, 3, and 3, respectively, and the product size was between 150 bp and 500 bp.

[0034] (4) Monitoring of Mandarin fish populations in different geographic locations and their genetic traits

[0035] From step (3), 15 pairs of primers with good stability were selected. Thirty mandarin fish from four cultured mandarin fish populations, namely, the Pearl River mandarin fish population, the Heilongjiang mandarin fish population, the Yangtze River mandarin fish population, and the Huaihe River mandarin fish population, totaling 120 mandarin fish, were respectively selected for individual gene scanning, population genetic structure analysis, and diversity detection. The amplification system, procedure, and annealing temperature were referred to the pre-amplification process. Polyacrylamide gel electrophoresis and silver nitrate staining were followed and photographs were taken. The genotype data of each mandarin fish were obtained using the software Gel-Pro Analyzer 4.5. The allele frequencies of each microsatellite marker locus were calculated using the software PopGen32 ( P ), number of alleles ( Na ), effective number of alleles ( Ne ), observed heterozygosity ( Ho ), expected heterozygosity ( He ), Hadwinberg equilibrium P HW , calculate the polymorphism information content PIC.

[0036] The calculation formula of the polymorphism information content PIC is:

[0037] Where Pi and Pj are the frequencies of the i-th and j-th alleles in the population, respectively, j = i + 1, and n is the number of alleles at a certain locus. The genetic distance and genetic similarity between different strains, as well as related genetic parameters, were calculated, and the molecular phylogenetic tree of different strains was constructed using UPGMA.

[0038] After PCR reaction and polyacrylamide gel electrophoresis, the 15 pairs of microsatellite primers had ideal amplification effects in the four Siniperca chuatsi populations, with clear and stable bands and a certain degree of polymorphism, such as Figure 1 , Figure 2 and Figure 3 They are the polymorphism amplification results of sites C6713, C12534, and C18691 in some populations, respectively. Lanes 1-30 represent an individual of Siniperca chuatsi.

[0039]

[0040] The average effective allele number, average allele number, average observed heterozygosity, average expected heterozygosity and average polymorphic information content of four wild geographical populations of Siniperca chuatsi in the Yangtze River system, Huaihe River system, Pearl River system and Heilongjiang River system are shown in Table 1. Among them, 15 pairs of primers were used to perform gene scanning on 120 individuals in the four populations, and a total of 354 alleles were obtained. The number of alleles obtained by each microsatellite primer ranged from 2 to 7, with an average number of alleles of 2.95 and an average effective number of alleles of 2.46. Among them, C12534 had the largest number of alleles. In addition, the average observed heterozygosity was 0.40, the average expected heterozygosity was 0.55, and the average polymorphic information content was 0.48, reaching a moderate polymorphism level (0.5> P >0.25); in each of the four populations, the observed heterozygosity was lower than the expected heterozygosity, indicating that these populations were heterozygous deficient.

[0041]

[0042] As shown in Table 2, based on the allele frequency of each locus, the effective number of alleles, observed heterozygosity, expected heterozygosity and polymorphic information content reflecting the genetic diversity of the population were calculated. Among them, the average effective number of alleles, average observed heterozygosity, average expected heterozygosity and average polymorphic information content of the Huaihe River mandarin fish population were the highest, which were 2.74, 0.45, 0.60, and 0.53, respectively, indicating that the Huaihe River mandarin fish population had the highest genetic diversity, followed by the Heilongjiang River mandarin fish population and the Yangtze River mandarin fish population, while the Pearl River mandarin fish population had the lowest genetic diversity.

[0043]

[0044] As shown in Table 3, according to the genetic similarity coefficient and genetic distance results among the four populations, the genetic distance between the Yangtze River Siniperca chuatsi population and the Heilongjiang Siniperca chuatsi population was the largest (0.157), and the genetic similarity coefficient was the smallest (0.8547), indicating that the two were more distantly related; the Yangtze River Siniperca chuatsi population and the Huaihe River Siniperca chuatsi population had the smallest genetic distance (0.0841) and the largest genetic similarity coefficient (0.9194), indicating that the two were more closely related. The UPGMA method was used to construct the phylogenetic tree of offspring and parents (such as Figure 4 As shown in the figure, we can see that the Yangtze River mandarin fish population and the Huaihe River mandarin fish population first gather into one group, then aggregate with the Pearl River mandarin fish population, and finally aggregate with the Heilongjiang River mandarin fish population.

[0045] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A method for monitoring Siniperca chuatsi germplasm using microsatellite genetic markers, characterized in that: The steps include: (1) Extraction and detection of genomic DNA Take 0.2g of the tail fin of several mandarin fish, extract genomic DNA by phenol-chloroform method; detect the integrity of DNA by 1% agarose gel electrophoresis, and measure the concentration and purity of DNA by nucleic acid protein analyzer, and finally dilute to 100ng / μL with sterile water and store at -20℃ for future use; (2) Search and analysis of microsatellite sequences Mandarin fish of different growth ages were selected for transcriptome sequencing. Based on the transcriptome library sequences measured, SSR loci in all non-redundant gene databases were retrieved. PCR primers, gene annotations and GO secondary classifications were designed based on the SSRs flanking sequences. Primers designed for SSR loci on alleles in the three GO secondary classification entries of growth, proliferation and metabolism were selected. (3) Screening and optimization of polymorphic microsatellite primers From all primers of the three secondary classification items in step (2), 120 pairs of primers were randomly selected for pre-amplification and polymorphic primer screening; (4) Monitoring of different geographical populations of Siniperca chuatsi and their genetic traits Fifteen pairs of primers with good stability were screened from step (3). The same number of wild geographical populations of mandarin fish from the Yangtze River, Huaihe River, Pearl River, and Heilongjiang River were selected for individual gene scanning, population genetic structure analysis, and diversity detection. The amplification system, procedure, and annealing temperature were based on the pre-amplification process. Polyacrylamide gel electrophoresis and silver nitrate staining were followed and photographs were taken. The genotype data of each mandarin fish were obtained using the software Gel-Pro Analyzer 4.

5. The allele frequencies of each microsatellite marker locus were calculated using the software PopGen32 ( P ), number of alleles ( Na ), effective number of alleles ( Ne ), observed heterozygosity ( Ho ), expected heterozygosity ( He ), Hadwinberg equilibrium P HW , calculate the polymorphism information content PIC, The calculation formula of the polymorphism information content PIC is: Where Pi and Pj are the frequencies of the i-th and j-th alleles in the population, respectively, j = i + 1, and n is the number of alleles at a certain locus; the genetic distance and genetic similarity between different strains, as well as related genetic parameters, were calculated, and the molecular phylogenetic tree of different strains was constructed using UPGMA; In step (2), the mandarin fish samples are taken from at least 30 individuals from different populations, and the 15 pairs of microsatellite loci and primer sequences in step (4) are: 。 2. The method for monitoring Siniperca chuatsi germplasm using microsatellite genetic markers according to claim 1, characterized in that: The screening conditions in step (3) are that the minimum length of SSRs is 15 bp; the minimum number of repetitions of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides are 8, 5, 4, 3 and 3 times respectively, and the product size is between 150 bp and 500 bp.

3. The method for monitoring Siniperca chuatsi germplasm using microsatellite genetic markers according to claim 1, characterized in that: In the step (1), the mandarin fish samples are taken from 30 mandarin fish in the same geographical population.

Images