Microsatellite marker combination and primer pair combination and application of Pseudomonas laevis

Abstract

The present invention discloses a microsatellite marker combination and primer pair composition and application of the Lasberg's gudgeon. The microsatellite marker combination of the Lasberg's gudgeon includes the microsatellite markers PL02, PL08, PL09, PL12, PL17, PL19, PL25, PL27, PL36, PL42, PL44, PL60, PL62, PL63, PL65, and PL68, wherein the nucleotide sequences of the microsatellite markers are shown in SEQ ID NO.1 to SEQ ID NO.16, respectively. The Lasberg's gudgeon satellite marker of the present invention is a microsatellite marker screened from a transcriptome sequence, and has a low probability of null alleles. The microsatellite marker provided by the present invention fills the gap in Lasberg's gudgeon microsatellite markers and can provide a powerful tool for Lasberg's gudgeon population genetics analysis, genetic diversity detection, kinship identification, and molecular marker-assisted breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal microsatellite markers, and particularly relates to a microsatellite marker combination of G. lagerstroemia, a primer pair composition for detecting the microsatellite marker combination of G. lagerstroemia, and application of the primer pair composition in detecting genetic diversity of G. lagerstroemia. Background Art

[0002] Rhynchocypris lagowskii belongs to the Cypriniformes, Cyprinidae, Leuciscinae, genus Phoxinus. Also known as Luo's mullet, long-tailed mullet, and commonly known as "willow root," it is found in the Heilongjiang River system, inland lakes in eastern Inner Mongolia, the Yellow River, Huaihe River, Haihe River, Qiantang River system, and tributaries of the middle and upper reaches of the Yangtze River. A relatively cold-water fish, it typically lives in mountain streams and clear waters. It primarily feeds on aquatic plants, but also consumes aquatic insects, making it an omnivorous fish. Its meat is tender, delicious, free of intramuscular spines, and high in protein and unsaturated fatty acids. It has a promising market prospect as a lactation-promoting food. Due to environmental pollution, spawning ground destruction, and overfishing, the natural resources of this fish have been drastically reduced. Simultaneously, as aquaculture areas continue to expand, the demand for seed has increased dramatically. This shortage of seed has severely hampered the further development of the aquaculture industry. Therefore, large-scale cultivation of the mullet is crucial, both for the protection of natural populations and for the development of new commercial fish species.

[0003] Microsatellite DNA, also known as simple sequence repeats, consists of short tandem repeats of 2-6 bp and is uniformly distributed throughout eukaryotic genomes. Microsatellite markers are co-dominant, stably inherited, follow Mendelian laws of inheritance, are easily accessible, and yield highly reproducible results. They are important molecular markers for monitoring population genetic diversity, analyzing genetic structure, identifying germplasm resources, and genetic breeding. Currently, no microsatellite markers have been reported for the Lassbergian mullet. The development of microsatellite markers for Lassbergian mullet is of great significance for monitoring population genetic diversity and for marker-assisted breeding. Summary of the Invention

[0004] The purpose of the present invention is to fill the blank of microsatellite marker combination of G. lagerstroemia indica and provide a microsatellite marker combination of G. lagerstroemia indica.

[0005] The second object of the present invention is to provide a primer pair composition for detecting the above-mentioned microsatellite marker combination of P. lagerstroemia.

[0006] The third object of the present invention is to provide the use of the above primer pair composition in the detection of genetic diversity of Pseudomonas lagomorpha.

[0007] The technical solution of the present invention is summarized as follows:

[0008] A microsatellite marker combination for P. lagerstroemia indica comprises the following microsatellite markers: PL02, PL08, PL09, PL12, PL17, PL19, PL25, PL27, PL36, PL42, PL44, PL60, PL62, PL63, PL65 and PL68, wherein the nucleotide sequences of the microsatellite markers are shown in SEQ ID NO.1 to SEQ ID NO.16, respectively.

[0009] A primer pair composition for detecting the above-mentioned microsatellite marker combination of Psoralea lagellii, wherein the primer pair composition comprises the following primer pairs:

[0010] Primer pair for detecting the first microsatellite marker PL02: forward primer is SEQ ID NO.17, reverse primer is SEQ ID NO.18;

[0011] Primer pair for detecting the second microsatellite marker PL08: forward primer is SEQ ID NO.19, reverse primer is SEQ ID NO.20;

[0012] Primer pair for detecting the third microsatellite marker PL09: forward primer is SEQ ID NO.21, reverse primer is SEQ ID NO.22;

[0013] Primer pair for detecting the fourth microsatellite marker PL12: forward primer is SEQ ID NO. 23, reverse primer is SEQ ID NO. 24;

[0014] Primer pair for detecting the fifth microsatellite marker PL17: forward primer is SEQ ID NO. 25, reverse primer is SEQ ID NO. 26;

[0015] Primer pair for detecting the sixth microsatellite marker PL19: forward primer is SEQ ID NO.27, reverse primer is SEQ ID NO.28;

[0016] Primer pair for detecting the seventh microsatellite marker PL25: forward primer is SEQ ID NO.29, reverse primer is SEQ ID NO.30;

[0017] Primer pair for detecting the eighth microsatellite marker PL27: forward primer is SEQ ID NO.31, reverse primer is SEQ ID NO.32;

[0018] Primer pair for detecting the ninth microsatellite marker PL36: forward primer is SEQ ID NO.33, reverse primer is SEQ ID NO.34;

[0019] Primer pair for detecting the 10th microsatellite marker PL42: forward primer is SEQ ID NO.35, reverse primer is SEQ ID NO.36;

[0020] Primer pair for detecting the 11th microsatellite marker PL44: forward primer is SEQ ID NO.37, reverse primer is SEQ ID NO.38;

[0021] Primer pair for detecting the 12th microsatellite marker PL60: forward primer is SEQ ID NO.39, reverse primer is SEQ ID NO.40;

[0022] Primer pair for detecting the 13th microsatellite marker PL62: forward primer is SEQ ID NO.41, reverse primer is SEQ ID NO.42;

[0023] Primer pair for detecting the 14th microsatellite marker PL63: forward primer is SEQ ID NO.43, reverse primer is SEQ ID NO.44;

[0024] Primer pair for detecting the 15th microsatellite marker PL65: forward primer is SEQ ID NO.45, reverse primer is SEQ ID NO.46;

[0025] Primer pair for detecting the 16th microsatellite marker PL68: forward primer is SEQ ID NO.47, reverse primer is SEQ ID NO.48;

[0026] Application of the primer pair combination in detecting the genetic diversity of Pseudomonas lagomorpha.

[0027] The above application includes the following steps:

[0028] (1) Extraction of genomic DNA of Pseudomonas laevis;

[0029] (2) PCR amplification of microsatellite markers: The 5' end of the forward primer in the primer pair composition is modified by connecting a fluorescent group, and PCR amplification is performed using the genomic DNA of the scorpion as a template with the corresponding modified forward primer and reverse primer to obtain an amplified product;

[0030] (3) The amplified products were typed on an ABI 3730XL genetic analyzer, using GS-500LIZ as an internal reference, and the individual genotypes were read using Peak Scanner Software v1.0;

[0031] (4) Genetic diversity analysis: Genetic diversity parameters were calculated using PopGene 32 based on the genotype of each individual microsatellite marker.

[0032] Advantages of the present invention:

[0033] 1. The satellite marker of the present invention is a microsatellite marker screened from the transcriptome sequence, and the probability of the occurrence of null alleles is small.

[0034] 2. The microsatellite markers provided by the present invention fill the gap in microsatellite markers for the Lasbergian mullet and can provide a powerful tool for population genetics analysis, genetic diversity detection, kinship identification, and molecular marker-assisted breeding of the Lasbergian mullet. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to the embodiments. It is necessary to point out that the embodiments are part of the embodiments of the present invention, but they should not be understood as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments based on the above contents of the present invention, which fall within the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Acquisition of microsatellite markers of P. lagerstroemia: The present invention uses transcriptome sequencing technology to obtain a large number of microsatellite markers of P. lagerstroemia.

[0038] 2. Extraction of genomic DNA of G. lamarckii: The genomic DNA of G. lamarckii (commercially available) was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit.

[0039] 3. Screening of microsatellite markers:

[0040] From the available microsatellite markers for the scorpionfish, 68 sites with repeat units of 3-5 bases, repeated at least five times, and product lengths of less than 200 bp were selected as candidate microsatellite loci. Primer3 software was used for primer design, and genetic diversity was detected in 105 individuals. Genomic DNA from 5-6 scorpionfish (5 scorpionfish were used in this example) was mixed as a template, and 68 primer pairs were used for temperature gradient PCR amplification to screen for the optimal annealing temperature for each primer pair. PCR amplification products were detected by 2% agarose gel electrophoresis, and 16 primer pairs with single, bright amplified bands (Table 1) were selected to synthesize fluorescent primers for genetic diversity detection. All primers were synthesized at Shanghai Sangon Biotechnology Co., Ltd.

[0041] The microsatellite markers of the present invention, the primers of the microsatellite markers of the Lasbergia serrata and the corresponding primer information are shown in Table 1:

[0042] Table 1 Microsatellite primer sequence information

[0043]

[0044] 4. Microsatellite marker amplification and genetic diversity detection

[0045] Genomic DNA from 105 larvae was used for genetic diversity analysis. The PCR amplification system (15 μL) consisted of 7.5 μL of 2× PCR Mix, 0.2 μL each of forward and reverse primers, 5.6 μL of ultrapure water, and 1.5 μL of DNA template. The PCR reaction procedure consisted of 95°C initial denaturation for 5 min, 33 cycles of 95°C denaturation for 30 s, 60°C annealing for 45 s, and 72°C extension for 1 min, followed by a final extension at 72°C for 10 min. Amplified products were genotyped on an ABI 3730XL genetic analyzer using GS-500LIZ as an internal reference. Individual genotypes were read using peakScanner Software (v1.0). Genetic diversity indicators including allele number (Ao), effective allele number (Ae), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were calculated using PopGene 32 software.

[0046] 5. Results

[0047] The results of genetic diversity detection of 105 fish are shown in Table 2. The average number of alleles at the 16 loci was 4.75, the effective number of alleles was 2.42, the average observed heterozygosity was 0.30, the average expected heterozygosity was 0.51, and the average polymorphic information content was 0.46.

[0048] Table 2 Typing results of microsatellite primers in 105 individuals of P. lamarckii

[0049]

[0050] Note: Ao is the number of alleles, Ae is the effective number of alleles, Ho is the observed heterozygosity, He is the expected heterozygosity, and PIC is the polymorphic content.

[0051] The nucleotide sequences of the 16 microsatellite markers obtained by the present invention: PL02, PL08, PL09, PL12, PL17, PL19, PL25, PL27, PL36, PL42, PL44, PL60, PL62, PL63, PL65, and PL68 are shown in SEQ ID NO.1 to SEQ ID NO.16, respectively.

[0052] The 16 pairs of microsatellite markers provided by the present invention can provide powerful tools for population genetics analysis, genetic diversity detection, kinship identification, and molecular marker-assisted breeding of the mullet.

Claims

1. A microsatellite marker combination of Pseudosphagnum serrata, characterized in that The combination includes microsatellite markers: PL02, PL08, PL09, PL12, PL17, PL19, PL25, PL27, PL36, PL42, PL44, PL60, PL62, PL63, PL65 and PL68, and the nucleotide sequences of the microsatellite markers are shown in SEQ ID NO.1 to SEQ ID NO.

16.

2. A primer pair composition for detecting the microsatellite marker combination of Pseudomonas laevis according to claim 1, characterized in that The primer pair composition includes the following primer pairs: Primer pair for detecting the first microsatellite marker PL02: forward primer is SEQ ID NO.17, reverse primer is SEQ ID NO.18; Primer pair for detecting the second microsatellite marker PL08: forward primer is SEQ ID NO.19, reverse primer is SEQ ID NO.20; Primer pair for detecting the third microsatellite marker PL09: forward primer is SEQ ID NO.21, reverse primer is SEQ ID NO.22; Primer pair for detecting the fourth microsatellite marker PL12: forward primer is SEQ ID NO.23, reverse primer is SEQ ID NO.24; Primer pair for detecting the fifth microsatellite marker PL17: forward primer is SEQ ID NO.25, reverse primer is SEQ ID NO.26; Primer pair for detecting the sixth microsatellite marker PL19: forward primer is SEQ ID NO.27, reverse primer is SEQ ID NO.28; Primer pair for detecting the seventh microsatellite marker PL25: forward primer is SEQ ID NO.29, reverse primer is SEQ ID NO.30; Primer pair for detecting the eighth microsatellite marker PL27: forward primer is SEQ ID NO.31, reverse primer is SEQ ID NO.32; Primer pair for detecting the ninth microsatellite marker PL36: forward primer is SEQ ID NO.33, reverse primer is SEQ ID NO.34; Primer pair for detecting the 10th microsatellite marker PL42: forward primer is SEQ ID NO.35, reverse primer is SEQ ID NO.36; Primer pair for detecting the 11th microsatellite marker PL44: forward primer is SEQ ID NO.37, reverse primer is SEQ ID NO.38; Primer pair for detecting the 12th microsatellite marker PL60: forward primer is SEQ ID NO.39, reverse primer is SEQ ID NO.40; Primer pair for detecting the 13th microsatellite marker PL62: forward primer is SEQ ID NO.41, reverse primer is SEQ ID NO.42; Primer pair for detecting the 14th microsatellite marker PL63: forward primer is SEQ ID NO.43, reverse primer is SEQ ID NO.44; Primer pair for detecting the 15th microsatellite marker PL65: forward primer is SEQ ID NO.45, reverse primer is SEQ ID NO.46; The primer pair for detecting the 16th microsatellite marker PL68: the forward primer is SEQ ID NO.47, and the reverse primer is SEQ ID NO.

48.

3. Use of the primer pair composition of claim 2 in detecting genetic diversity of Pseudomonas lagomorpha.

4. The use according to claim 3, characterized in that The steps include: (1) Extraction of genomic DNA of Pseudomonas laevis; (2) PCR amplification of microsatellite markers: The 5' end of the forward primer in the primer pair composition of claim 2 is modified by connecting a fluorescent group, and PCR amplification is performed using the genomic DNA of the sphenodon as a template with the corresponding modified forward primer and reverse primer to obtain an amplified product; (3) The amplified products were typed on an ABI 3730XL genetic analyzer, using GS-500LIZ as an internal reference, and peakScanner Software v1.0 was used to read the individual genotypes; (4) Genetic diversity analysis: Genetic diversity parameters were calculated using PopGene 32 based on the genotype of each individual microsatellite marker.

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