Primer group of microsatellite marker HA88 related to growth of belly-expanding hippocampus and application of primer group

Abstract

The invention relates to a primer group of a microsatellite marker HA88 related to growth of belly-expanding hippocampus and application of the primer group, and belongs to the field of molecular biology, and the sequence of the primer group is shown as SEQ ID NO.1-2. The invention also provides an application of the primer group in breeding of growth traits of hippocampus swelling, DNA of a hippocampus swelling individual is subjected to PCR amplification by using the primer group, an individual capable of amplifying a specific fragment with the size of 245bp is screened as a basic parent, and a heterozygous individual or a homozygous individual with the fragment is produced for breeding of a rapid growth variety, so that the breeding of the growth traits of the hippocampus swelling individual is realized, and the breeding of the growth traits of the hippocampus swelling individual is realized. Compared with a conventional breeding method, the method can shorten the breeding period, accelerate the breeding process and improve the breeding efficiency.

Description

Technical Field

[0001] This application belongs to the field of fish DNA marker technology and application, and specifically involves a primer set of a microsatellite marker HA88 related to the growth of Hippocampus inflatus and its application. Background Art

[0002] The puffy seahorse is a special marine aquaculture fish introduced to my country in recent years. It has high nutritional and medicinal value. In the actual production and breeding process, the same batch of seahorses showed different specifications, that is, within the same breeding cycle, the growth rate of the same batch of seahorses was different. The average weight of the fast-growing seahorses at one month old was 0.260g heavier than the slow-growing seahorses, and the average body length was 2.533cm. The breeding growth rate of seahorses has a significant impact on improving the enthusiasm of farmers and promoting the development of the puffy seahorse breeding industry. Growth is an important economic and technical indicator. Breeding a fast-growing puffy seahorse strain can effectively shorten the breeding cycle, reduce breeding costs, and increase breeding income.

[0003] Molecular marker-assisted breeding is the direct selection and breeding of individuals with alleles or genotypes that have trait advantages with the help of molecular markers that are closely related to the trait. Compared with traditional breeding methods, molecular marker-assisted selection has a large amount of information, is not easily affected by the environment, has a high selection intensity, and has high selection efficiency and accuracy. Among the common molecular marker technologies, microsatellite molecular markers (simple sequence repeats, SSR) are widely and randomly distributed in the genome, have the advantages of co-dominant inheritance, high polymorphism, good stability and simple operation, and have been widely used in aquatic animal breeding research.

[0004] Using molecular markers to assist in the breeding of fast-growing seahorses is an important way to reduce costs and improve economic benefits. Currently, the screening of seahorse SSR markers is mainly focused on variety identification, and there are few studies on growth. There are no reports on molecular markers for seahorse growth traits at home and abroad. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a primer set of a microsatellite marker HA88 related to the growth of hippocampus distended and its application.

[0006] The present invention is achieved through the following technical solutions:

[0007] A primer set for a microsatellite marker HA88 associated with the growth of hippocampus distended, wherein the sequence of the forward primer in the primer set is GACGCCATTAAGCAGTGACA (SEQ ID NO. 1), and the sequence of the reverse primer is CCATGACACAATTTCAAGCG (SEQ ID NO. 2).

[0008] The present invention also provides the application of the primer set in the selection of growth traits of Hippocampus inflatus. The application method is to use the primer set to perform PCR amplification on the DNA of Hippocampus inflatus individuals, screen individuals that can amplify a specific fragment with a size of 245bp as basic parents, produce heterozygous individuals or homozygous individuals with the fragment, and the offspring have a faster growth rate.

[0009] Furthermore, the PCR reaction system was 50 μL: 1 μL DNA solution, 1 μL upstream primer, 1 μL downstream primer, 25 μL 2×HS Taq PreMix (TORO IVD), and 22 μL sterile double distilled water.

[0010] Furthermore, the PCR amplification reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 10 min.

[0011] The beneficial effects of the present invention compared with the prior art are as follows: the present invention obtains molecular markers related to the growth of Hippocampus inflatus for the first time, and the primer set is used in the breeding of growth traits of Hippocampus inflatus for the breeding of fast-growing varieties. Compared with conventional breeding methods, this method can shorten the breeding period, speed up the breeding process, and improve the breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Banding of microsatellite markers in group F and group S (mixed pool population) electropherogram and peak diagram;

[0013] Figure 2 Electropherograms of the band distribution of microsatellite markers in groups F and S (all individuals);

[0014] Figure 3 Electrophoresis of amplified bands of HA88 in a mixed pool of DNA from 46 full-sib families;

[0015] Figure 4 Electrophoresis of amplified bands of HA88 in 30 individuals of family A08. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention is further explained below by examples in combination with the accompanying drawings, but the protection scope of the present invention is not limited in any form by the examples. Unless otherwise specified, the experimental methods used in the examples are conventional methods and techniques well known to those skilled in the art, and the materials and reagents are all purchased commercially.

[0017] Example 1

[0018] 1. Acquisition of materials related to the growth traits of Hippocampus bulging

[0019] The experimental seahorses came from Weihai Yinze Biotechnology Co., Ltd. About 10,000 seahorse fry (body length 1.6±0.2cm, weight 0.008±0.002g) produced on the same day were placed in the same breeding pond for 30 days. During the experiment, they were fed normally, and the water temperature was maintained at 18±0.5℃, salinity 30ppt, and dissolved oxygen>6.0mg / L. After the experiment, they stopped feeding for 24h, and 1,000 seahorses were randomly picked from the breeding pond, and their growth indicators were measured. The weight of each seahorse was recorded, and 60 seahorses with a weight greater than 0.2g and 60 seahorses with a weight less than 0.1g were taken.

[0020] After the measurement, the hippocampus samples were placed in anhydrous ethanol and frozen in a -80°C refrigerator for later use. 60 tails with fast growth rate were selected as the fast growth rate group (F group), and 60 tails with slow growth rate were selected as the slow growth rate group (S group). The T test was used to verify whether there was a significant difference between the two groups.

[0021] 2. Microsatellite primers

[0022] According to the genome data of Hippocampus inflatus (https: / / ngdc.cncb.ac.cn / gwh / Ass embly / 18745 / show), the microsatellite identification tool MISA was used to search for microsatellite loci, and 95 microsatellite loci were selected. Primers were designed according to their flanking conserved sequences. The primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0023] 3. Extraction and detection of genomic DNA

[0024] The Tiangen marine animal DNA extraction kit was used to extract DNA from 60 tails of Hippocampus inflatus F and S groups. The quality and integrity of the extracted DNA were determined by 1% agarose gel electrophoresis, and the concentration was determined by UV spectrophotometer. The DNA was diluted to 50 ng / μL with double distilled water and stored at -80℃ for later use.

[0025] 4. Establishment of BSA gene pool

[0026] The 30 samples with the fastest growth rate in group F were selected, and an equal amount of 5 μL of DNA solution was taken from each sample to form a fast-growing gene pool (F pool). Similarly, the 30 samples with the slowest growth rate in group S were selected, and an equal amount of 5 μL of DNA solution was taken from each sample to form a slow-growing gene pool (S pool).

[0027] 5. Microsatellite marker screening

[0028] 95 pairs of microsatellite primers were used to perform PCR amplification on two DNA pools. PCR reaction system: 1 μL DNA solution, 1 μL upstream primer, 1 μL downstream primer, 25 μL 2×HS Taq PreMix (TOROIVD), and 22 μL sterilized double distilled water. PCR amplification reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, reaction at the actual annealing temperature of each pair of primers for 30 s, 72°C extension for 30 s, a total of 35 cycles, and 72°C extension for 10 min.

[0029] The PCR products were analyzed by Fragment Analyzer TM The electrophoresis was performed by fully automatic capillary electrophoresis system, with a sample volume of 2 μL, constant voltage of 6 kV, and electrophoresis for 1 h. After the electrophoresis, the results were analyzed using ProSize data analysis software. By analyzing the PCR amplification of DNA from the two gene pools, a microsatellite locus that could amplify differential allele fragments in the two pools was preliminarily screened, such as Figure 1 shown.

[0030] 6. Preliminary verification of microsatellite markers

[0031] Using the technical means in step 4 (PCR amplification and capillary electrophoresis), the differential bands of a microsatellite marker in group F and group S were analyzed, and the frequency of the differential bands was tested by Pearson test, and finally a microsatellite marker HA88 with significant difference was obtained. Forward primer: GACGCCATTAAGCAGTGACA (SEQ ID NO.1), reverse primer: CCATGACACAATTTCAAGCG (SEQ ID NO.2).

[0032] like Figure 2 As shown in Table 1, the frequency of the 245 bp fragment of the marker in the F group was 35, and the frequency in the S group was 24. Pearson correlation analysis showed that the fragment was significantly correlated with the growth of hippocampus distended. The specific statistical data and correlation analysis results are shown in Table 1.

[0033] Table 1 Statistics of the occurrence of HA88 allele fragments in individual amplified band patterns

[0034]

[0035]

[0036] 7. Family individual re-verification

[0037] The primers of microsatellite locus HA88 were used to amplify the pooled DNA of 46 families by PCR, and capillary gel electrophoresis was performed. The results showed that 37 families had a 245 bp band of HA88 locus ( Figure 3 ), select one family with significant growth difference from these 37 families, select 15 fastest growing samples (F group) and 15 slowest growing samples (S group) from this family, perform SSR differential band analysis on all individuals in the family, and count the amplification of 245bp differential alleles in the F group and S group in the family ( Figure 4 ), the results are shown in Table 2. After Pearson test, there was a significant positive correlation between the differential alleles of family A8 and the growth traits (P<0.05), which further verified that the microsatellite locus HA88 was significantly correlated with the growth traits of Hippocampus distensus.

[0038] Table 2 Statistics of the occurrence of HA88 allele fragments in the amplified band patterns of individuals in family A8

[0039]

[0040] Example 2: Application of microsatellite markers for growth traits in the selection of high growth rate strains of hippocampus bulging

[0041] Before strengthening the broodstock, part of the dorsal fin of the candidate parent was cut, fixed in 95% anhydrous ethanol and stored at low temperature for genomic DNA extraction. The extraction and detection of DNA, PCR and capillary electrophoresis processes, and analysis of electrophoresis results are as described in Example 1. Individuals with a genotype of 245bp bands were selected as basic parents to construct a core breeding group, and the offspring produced were collected for cultivation. 1,000 seahorses were randomly picked up regularly every month for weight measurement, and the measurement was repeated 5 times. The weight of the offspring of the ordinary breeding group of the same period was measured in the same way. The results showed that under the same breeding and management conditions, the offspring of the core breeding group of parents with 245bp bands always had a faster growth rate than the offspring of the ordinary group at different developmental stages. The results are shown in Table 3;

[0042] Table 3 Mean body weight of offspring in the core breeding group and the common breeding group at different developmental stages

[0043]

Claims

1. A primer set for a microsatellite marker HA88 associated with the growth of hippocampus bulging, characterized in that: The sequence of the primer set is shown in SEQ ID NO.1-2.

2. The use of the primer set according to claim 1 in the breeding of growth traits of hippocampus bulging, characterized in that: The application method is to use the primer set described in claim 1 to perform PCR amplification on the DNA of Hippocampus inflatus individuals, screen individuals that can amplify a specific fragment with a size of 245 bp as basic parents, and produce heterozygous individuals or homozygous individuals with the fragment.

3. The use according to claim 2, characterized in that: The PCR reaction system is 50 μL: 1 μL DNA solution, 1 μL upstream primer, 1 μL downstream primer, 25 μL 2×HS Taq PreMix, and 22 μL sterilized double distilled water.

4. The use according to claim 2, characterized in that: The PCR amplification reaction conditions were as follows: pre-denaturation at 94° C. for 5 min; denaturation at 94° C. for 30 s, annealing at 55° C. for 30 s, and extension at 72° C. for 30 s, for a total of 35 cycles; and extension at 72° C. for 10 min.

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