CN110055335B - Microsatellite molecular marker primer, kit and rapid identification method for identifying female and male tilapia in stellera - Google Patents
Microsatellite molecular marker primer, kit and rapid identification method for identifying female and male tilapia in stellera Download PDFInfo
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Abstract
The invention discloses a microsatellite molecular marker, a kit and a rapid identification method for identifying female and male tilapia mossambica. The invention firstly analyzes the relationship between markers and phenotypes by constructing the Satinus red Holotrichia globosa family and combining the QTLseq linkage mapping technology, determines the sex-determining QTL region, further designs a microsatellite primer, and finally screens out the microsatellite marker-SSR 147 microsatellite molecular marker primer which is closely linked with the sex characters on the LG1 chromosome of the Satinus red Tilapia, wherein the sequences of the microsatellite marker-SSR 147 microsatellite molecular marker primer are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. By applying the microsatellite molecular marker primer SSR147 closely linked with the sex determination region to screen and identify the male and female fishes, the sex of the red tilapia stellera can be identified with high efficiency and high accuracy, so that the accuracy of breeding can be improved. Therefore, the research of the invention can lay a foundation for developing the production of the all-male tilapia mossambica fingerlings.
Description
The technical field is as follows:
the invention belongs to the field of aquatic product genetic breeding, and particularly relates to a microsatellite molecular marker primer, a kit and a rapid identification method for identifying female and male tilapia mossambica in stellera.
Background art:
the Xingzhou red fish has important economic value, is a high-quality variety for interspecific hybridization breeding of tilapia, is successfully introduced to the Guangdong for trial culture at first, and has the growth speed more than 30 percent faster than that of common tilapia. The red fishes in the star continent are all red in body color, but have differentiation, such as peach red, orange red and the like, have variegated spots and few black spots, have omnivory and omnivory, are resistant to low temperature, have fresh and sweet meat quality, have no muscle thorns, and have obvious culture advantages. Due to high environmental adaptability, good meat quality and body color, in recent years, the culture area of the stellera red fish is continuously enlarged, and the yield is greatly increased. At present, the strain becomes a high-quality variety for aquaculture in China.
The growth of male and female stellera chamaejasme has obvious difference. The male fish grows faster than the female fish, and the body weight of the male fish is 30% -50% heavier than that of the female fish. In addition, because the red fishes in stellera have short breeding cycle and early maturity, the breeding process is easy to cause the adverse conditions of excessive breeding, overlarge density, undersize individuals and the like, thereby influencing the improvement of the breeding yield. In order to avoid excessive reproduction, the growth advantages of male fishes can be utilized, hormones are generally adopted in production to carry out sex control on the red fishes in stellera, male fries are produced, and the full-male fishes are bred.
The production of the parthenocarpic hologynic fish mainly comprises the following methods of artificial characterization, hybridization, genetic manipulation, hormone reversion treatment and the like. The production of red fish in total androgens using androgens such as methyltestosterone (17-methyltestosterone) to induce the conversion of genetically female fish into functionally male fish is currently considered to be the most efficient and economically viable method. However, in recent years, research on tilapia shows that the late digestion of methyltestosterone is slow after drug withdrawal, and the higher the concentration of methyltestosterone is, the longer the residual time in the fry is. If complete metabolism before marketing is not ensured, the very small residue of methyltestosterone in the aquatic animal body can be harmful to the human body. Meanwhile, the use of hormone also causes some environmental problems, such as accumulation of methyltestosterone in underwater sediments in the aquatic industry, which can cause negative effects of abnormal production and morphology of yin-yang fish and sterile female fish, endocrine disturbance, hormone level change and the like, and also has great influence on the physical health of aquatic workers actually carrying out hormone treatment. Therefore, due to the potential food safety and environmental hazard, hormones such as methyltestosterone are prohibited from being used in fish sex reversal processes by the ministry of agriculture of many countries including China. In recent years, people pay more and more attention and attention to the quality safety of aquatic products, and the requirements are higher and higher. The quality of imported aquatic products is generally enhanced in countries of the world, particularly developed countries. The quality safety condition of aquatic products in China has become a main factor influencing the competitiveness of the aquatic products in the international trade market. The method is used for producing pollution-free stellera red fish aquatic products, and the first condition for ensuring the sustainable and healthy development of the stellera red fish breeding industry is to solve the problem of quality control of stellera red fish breeding.
The red fishes in the star continent are a high-quality variety for interspecific hybridization of tilapia, and have wide application prospect. However, since the genetic background of the red fishes in stellera is very complicated, the decision mechanism of their sex is still not sufficiently understood by scientists at present. The first premise for carrying out molecular marker-assisted breeding of high-quality Tabanzo red fishes is to perform QTL positioning on sex character genome decision regions and identify molecular markers closely linked with the character QTL.
The invention content is as follows:
the invention aims to provide a microsatellite molecular marker primer for identifying female and male tilapia in stellera.
The invention firstly analyzes the relationship between markers and phenotypes by constructing the Satinus red Holotrichia globosa family and combining the QTLseq linkage mapping technology, determines the sex-determining QTL region, further designs a microsatellite primer, and finally screens out the microsatellite marker-SSR 147 microsatellite molecular marker primer which is closely linked with the sex characters on the LG1 chromosome of the Satinus red Tilapia, wherein the sequences of the microsatellite marker-SSR 147 microsatellite molecular marker primer are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The microsatellite molecular marker primer for identifying the female and male fishes by the red tilapia mossambica is as follows:
f:5'-TACTCTCCCCAACACCCACA-3'; the specific sequence is shown as SEQ ID NO. 1;
r:5'-TGCTGCTTACAGACCTTGTGT-3'; the specific sequence is shown in SEQ ID NO. 2.
The second purpose of the invention is to provide a rapid identification method for identifying female and male tilapia in stellera, which comprises the following steps:
extracting the genome DNA of the tilapia mossambica to be detected, performing PCR amplification by using the microsatellite molecular marker primer for identifying the female and male fishes by the tilapia mossambica as a PCR primer to obtain an amplification product, performing electrophoresis and dyeing on the amplification product, and judging the sex of the tilapia mossambica to be detected according to the electrophoresis result.
The reaction system of the PCR reaction is as follows: 20 mu L of reaction system, which comprises 10 mu L of PCR Master Mix,6.4 mu L of ultrapure water, 0.8 mu L of PCR upstream primer, 0.8 mu L of PCR downstream primer and 2 mu L of template genome DNA, wherein the reaction program of the PCR is as follows: 95 deg.C for 3min;95 ℃ for 30s;61 deg.C, 30s,72 deg.C, 15-30s,34 cycles.
The third purpose of the invention is to provide a detection kit for identifying the female and male tilapia mossambica, which comprises a PCR primer and a PCR reaction reagent, wherein the PCR primer is as follows:
F：5'-TACTCTCCCCAACACCCACA-3'；
R：5'-TGCTGCTTACAGACCTTGTGT-3'。
compared with the prior art, the invention has the following advantages and effects:
by applying the microsatellite molecular marker primer SSR147 closely linked with the sex determination region to screen and identify the male and female fishes, the sex of the red tilapia stellera can be identified with high efficiency and high accuracy, so that the accuracy of breeding can be improved. Therefore, the research of the invention can lay a foundation for developing the production of the all-male tilapia mossambica fries.
Description of the drawings:
FIG. 1 is QTL interval of sex of Satinus red Tilapia located by QTLseq method (Fst =0.12 is genome level threshold);
FIG. 2 is a schematic diagram of genetic male and female fishes of tilapia stellera screened and identified by using a microsatellite marker primer SSR 147; wherein lanes 1-12 are phenotypic females of the population and lanes 13-24 are phenotypic males of the population; it is assumed that K is a male specific band, J is a female specific band, the male genotype is K/J, and the female genotype is J/J.
The specific implementation mode is as follows:
the following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 family construction
In 2016, 1 complete sibling family is successfully constructed by selecting 1 pair of high-quality red tilapia parent fishes from Wulong gang aquaculture development technology Limited in Guangdong province. The experimental fish were cultured and treated experimentally (204 tails total) in Wulong gang aquatic products development Co., ltd, guangzhou city. After the sex glands of the tilapia mossambica grow to maturity for about 4 months, identifying the phenotype sex by manually distinguishing the external genitalia, recording the sex and the weight, shearing the fin samples of the parents and the first filial generation, storing the fin samples in absolute ethyl alcohol, and storing the fin samples at-70 ℃ for later use. Meanwhile, based on sex characters, 30 male and female fishes are selected as breeding groups (contrast), an electronic tag is injected into each fish, and individual identity information is recorded for breeding.
Example 2 genomic DNA Source and preparation
1. Parental and progeny genomic DNA sample extraction
The samples used in the present experiment for the identification of sex-linked molecular markers were: female and male parents of stellera red tilapia, a 30-tailed stellera red tilapia phenotype female individual and a 30-tailed stellera red tilapia phenotype male individual.
DNA extraction method
(1) Parental DNA
DNA is extracted by using the Dongsheng genome DNA rapid extraction kit, and the extracted DNA is diluted by 5 times and then stored at the temperature of minus 20 ℃ for later use.
(2) First-filial generation DNA
a. A1.5 ml 96-well plate was placed on ice, and 300. Mu.l of Set buffer (10mM Tris pH8.0, 50mM EDTA pH8.0, 200mM NaCl,0.5% SDS), 50. Mu.l proteinase K (20 mg/ml) was added to each well;
b. cut 3 x 3mm with sterile scissors 2 Putting a large-size fin sample on filter paper, pressing and sucking residual absolute ethyl alcohol on the sample, and putting the sample into a lysate;
c. simply centrifuging, placing into shaking table, setting temperature at 55 deg.C, rotating speed at 70-100r/min, and cracking for 3hr;
d. taking a silica gel column (96 holes), receiving the filtrate below the silica gel column by using a 96 hole plate, adding 240ul 6M NaI into each hole, adding 80 mu l of lysate, centrifuging for 1min at 2000g, and pouring out the filtrate;
e. adding 240 μ l wash buffer (10mM Tris pH7.5,1mM EDTA pH7.5, 100mM NaCl,50% ethanol) into each well, centrifuging at 2000g for 3min, and standing and drying for 2-5min;
f. adding 120 mul of pure water into each hole, standing for 2min at room temperature, and centrifuging for 2min at 2000g to obtain a DNA sample;
g. electrophoresis was performed on a 1% agarose gel, 120v for 30min, and the gel imaging system photographed. The extracted DNA is diluted by 5 times and stored at the temperature of minus 20 ℃ for later use.
Example 3QTL-seq sequencing and genetic map creation and trait localization
QTL analysis is carried out by adopting a QTL-seq method. Namely, DNA samples of 30 male red tilapia and 30 female red tilapia are selected from 204 red tilapia, each individual extracts 300ng of DNA samples, and the DNA samples are mixed into two large samples, namely a male sample and a female sample, and the genome sequencing is carried out by a company. The genotype data of 30 male and 30 female individuals in the control group were subjected to linkage mapping using PoPoPoposition 2 software to identify the sex trait QTL interval (as shown in FIG. 1).
And after filtering the sequencing result, comparing the sequence data of the posing samples with extreme male and female characters in the population with the genome sequence of the tilapia by utilizing bowtie2 software. The Poposition 2 software can be used to compare the frequencies of alleles in the two populations and calculate genome-wide Fst values by a sliding window method. The parameters when Poposition 2 software is used are set to be min-qual 20, -min-count 5, -max-coverage 500-min-covered-fraction 0.0-window-size 10000-step-size 5000. The window containing less than 20 SNPs was filtered. When at least two highest Fst values of each window exceed the Fst value threshold value, the QTL interval is considered.
Example 4 microsatellite marker development and population analysis
1. Primer design
After obtaining data of sex QTL interval, downloading the sequence of tilapia from the net, searching microsatellite markers in the interval with high Fst value, and designing a primer by using software primer select.
PCR amplification
The obtained nucleic acid sample was prepared into a 20. Mu.L reaction system containing 10. Mu.L of mix, 6.4. Mu.L of ultrapure water, 0.8. Mu.L of the upstream primer, 0.8. Mu.L of the downstream primer, and 2. Mu.L of a sample (template DNA). Setting a PCR reaction program: pre-denaturation at 95 ℃ for 3min, then annealing at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and annealing at 72 ℃ for 30s for 34 cycles, and finally extension at 72 ℃ for 10min. After the reaction is finished, taking 3 mu L of product to perform electrophoresis detection on agarose gel, then taking 3 mu L of product to perform polyacrylamide gel electrophoresis, and finally obtaining the band of the PCR product by using a silver staining method. The microsatellite molecular marker primer is SSR147 (F: 5'-TACTCTCCCCAACACCCACA-3', R: 5'-TGCTGCTTACAGACCTTGTGT-3').
3. Polyacrylamide gel electrophoresis and silver staining detection
1) Glue making
25ml of pure water, 5 XTBE 4ml, 10.7ml of Acr-bis (30%), 26 mul of TEMED and 280 mul of APS (10%) are taken, fully and uniformly stirred by a glass rod, the prepared gel is injected between two assembled glass plates, a comb is gently inserted, and the gel is kept stand for 1 to 2 hours until the gel is solidified.
2) Electrophoresis
After the gel is solidified, taking down the gel together with the glass plate, and placing the gel in an electrophoresis tank; spotting is carried out in sequence, and each well is 3 mu l; the cover is closed, the voltage is adjusted to 200V, after electrophoresis for 10 minutes, the voltage is adjusted to 600V, and electrophoresis is continued for 1.5 hours.
3) Silver staining
After electrophoresis is finished, discharging the buffer solution, taking the glass plates out of the electrophoresis tank, slightly prying the two glass plates by using a thin plate, and putting the gel into a tray filled with pure water to separate the gel from the glass plates; pouring off the pure water and adding 500ml of staining solution (1% 3 ) After dyeing for about 5 minutes, transferring the glue into a tray filled with pure water, rinsing for 5-10 seconds, pouring off the pure water, adding color development liquid (20g NaOH +0.5g Na) 2 CO 3 +4ml of formaldehyde solution (37%) was added to 1L of purified water, pre-cooled in ice before use), and developedAnd (4) after the color is about 10-15min until the strips appear, pouring off the color developing solution, adding pure water to soak for 5min, observing the strips and taking pictures. The silver staining pattern of the microsatellite molecular marker primer SSR147 for identifying the male and female fishes by the red tilapia starzhou is shown in figure 2, and K can be presumed to be a male specific band, J is a female specific band, the male genotype is K/J, and the female genotype is J/J from the figure 2.
4. Population genotype and phenotype data analysis
And (3) carrying out genotype determination on the 204-tailed red tilapia holomorphic family by utilizing a microsatellite molecular marker primer SSR 147. And recording the genotype of each individual by using a table, analyzing genotype data and phenotype data, and identifying the molecular marker which is closely linked with the sex character of the red tilapia stellera. The result shows that the accuracy of identifying the tilapia with male genetic sex in the 204-tailed red tilapia holomorphic family by the microsatellite molecular marker primer SSR147 in the region with the highest Fst value reaches 92.0%, and the reason that the accuracy cannot reach 100% is probably because the sex of the tilapia is influenced by a plurality of factors. In addition to genetic factors, various environmental factors such as temperature, environmental hormones, etc. may also cause sexual reversal during sexual differentiation, resulting in the paradox of genotype and phenotype in some individuals.
Sequence listing
<110> Zhongshan university
GUANGDONG WULONGGANG AQUATIC TECHNOLOGY DEVELOPMENT Co.,Ltd.
<120> microsatellite molecular marker primer, kit and rapid identification method for identifying female and male tilapia in stellera
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgctgcttac agaccttgtg t 21
Claims (2)
1. A microsatellite molecular marker primer for identifying female and male tilapia in stellera is characterized by comprising the following primers:
F：5'-TACTCTCCCCAACACCCACA-3'；
R：5'- TGCTGCTTACAGACCTTGTGT-3'。
2. a detection kit for identifying female and male tilapia in stellera comprises a PCR primer, and is characterized in that the PCR primer is as follows:
F：5'-TACTCTCCCCAACACCCACA-3'；
R：5'- TGCTGCTTACAGACCTTGTGT-3'。
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| CN113621711B (en) * | 2021-07-13 | 2023-09-22 | 武汉中科瑞华生态科技股份有限公司 | Dual PCR microsatellite primer for bighead genetic diversity analysis and application |
| CN114457168B (en) * | 2022-02-16 | 2023-05-23 | 中山大学 | Detection primer of microsatellite related to body color black spot character of red tilapia in wintering period and application of detection primer |
| CN114410804B (en) * | 2022-02-22 | 2023-05-23 | 中山大学 | A pair of detection primers for microsatellite markers related to low temperature resistance of tilapia mossambica |
| CN114752683B (en) * | 2022-04-18 | 2023-08-08 | 广东海洋大学 | Construction method of QTL locus related to sex characteristics of Sillago sihama |
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