TWI690263B - Marker and method for molecular assisted breeding of grouper - Google Patents

Abstract

The present invention provides a marker for selecting larva and/or breeding stock and/or accelerating the breeding in grouper. The marker is a simple sequence repeat polymorphism marker of genes associated with high growth. The invention also provides a primer pair for selecting larva and/or breeding stock and/or accelerating the breeding in grouper, and a method and kit for selecting larva and/or breeding stock and/or accelerating the breeding in grouper.

Description

Signs and methods of grouper molecular assisted breeding

The present invention relates to an aquaculture technology. In detail, it relates to a technology for screening grouper seed and/or seed fish and/or accelerated breeding.

According to the prediction of the World Organization (FAO), the world's aquatic products will be in short supply in a few years. Due to overfishing and sea pollution, traditional fishing has been declining year after year, and the catch of the fishing industry is estimated to have reached the limit It must be aquaculture to make up for the insufficient supply of fishery products to meet the global market demand for aquatic products.

The aquaculture industry has appeared very early in the development of human civilization, but it has only begun to receive attention and rapid development in the past three decades. my country's aquaculture industry is famous internationally for its ability to grow nearly 120 species of shellfish, and it ranks among the three largest aquaculture kingdoms in the world along with Japan and Norway.

The grouper (Epinephelus spp.) is classified as the grouper (Epinephelus) of the family Perciformes (Serranidae) in the order of the bony fish (Perciformes). Grouper is a warm-water fish that eats meat. It is distributed in tropical and subtropical waters. This fish has been regarded as a table delicacy since ancient times. Because of its living habits, it has not been possible to increase the catch with science and technology. Due to its high market value, it is the most important economically farmed fish in Taiwan and Southeast Asian countries. In the past, grouper fry had to be obtained through fishing and then farmed into big fish for sale. After years of continuous attempts and research improvements, Taiwan first bred Epinephelus malabaricus grouper by injection of hormones in 1982. Since then, it has opened the era of complete grouper farming, in feeding management and seed production. , Currently in a leading position in the world. It generally takes at least 8 to 12 months for grouper farming to reach the listed body size, but the current final growth rate is not high, only 0.3% of the stocked fish eggs, which is largely due to the advantages and disadvantages of the grouper fry purchased by the farmers. In general, the farming site is fixed at regular intervals, that is to say, the grouping of groupers of different sizes must be divided into nets to avoid the occurrence of a large number of eating behaviors. Therefore, it can be shown that the fish grown in the same period have a significant gap in growth rate .

On the other hand, an important indicator of egg quality is the incubation rate, because the incubation rate of eggs depends on the ability of egg development, and the ability of egg development is defined as having a fertilizable and fully developed embryo to form a normal, especially the embryogenesis genes Before performance. At the same time, according to previous studies, it is pointed out that the number of eggs during oogenesis, the accumulation of various growth factors, and the process of embryonic development are the main keys to determine egg quality. For lower vertebrates, especially fish, eggs accumulate a large amount of nutrients in the yolk for embryonic development to hatch until the first feeding. Therefore, many studies have shown that egg mass and vitellogenesis may be importantly related to the isolation of egg lipoprotein nutrients, vitamins, or hormonal factors from maternal sources during egg production. Although there have been many studies on the role of finite factors in the egg hatching process, there are currently very few molecular indicators that can be used as credible egg qualities. At the same time, there is no reasonable biochemical basis for how to determine the quality of the egg. Therefore, the uncertainty of the quality of the egg quality has caused serious economic losses to the farming fish industry. Since many gene expressions and environmental factors can affect gene expression and early embryonic development during egg production, compared with previous studies focusing on the composition of yolk, there is still a lack of the role of fish maternal information RNA (mRNA) accumulation in eggs With understanding, although they speculate and determine ovum is of considerable importance (Brooks et al., 1997, Rev Fish Biol Fish 7 387-416).

Looking around the structure of the grouper aquaculture industry, the control of mortality and the acquisition of high-quality grouper seed are still the bottlenecks that the aquaculture industry has not broken through, so the development can screen out grouper seed and/or seed fish and/or accelerate breeding to enhance the grouper seed industry Competitiveness is no longer urgent.

The present invention successfully uses the screened signs, methods and test kits to screen out grouper seed and/or seed fish and/or accelerated breeding. This gene signature can be further used to enhance the production of grouper quality seed and develop the cutting-edge grouper seed industry For the future development of high-quality high-quality grouper seed strains, it has great help, with great technical applicability and commercial development value.

Therefore, the present invention provides a marker for screening grouper fry and/or breeding fish and/or accelerated breeding, which is selected from a multi-type satellite (Simple Sequence Repeat) marker of myostatin (MSTN), ER- Microsatellite polymorphism marker of ER-C protein 2-like Nerve growth factor-like gene and FK506-binding protein-like protein and protein Wnt-2b-A (FK506-binding protein-like protein & protein Wnt-2b-A) a group of microsatellite polymorphism markers of genes.

The present invention also provides a pair of primers for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which is selected from primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), primer 3 ( SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) and primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6).

The invention further provides a method for screening grouper seed and/or seed fish and/or accelerated breeding, which comprises detecting the aforementioned signs of grouper seed and/or seed fish.

The present invention further provides a kit for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which comprises reagents that can detect the markers according to the foregoing.

The embodiments or examples described in this disclosure are now described in specific languages, and it should be understood that they are not intended to limit the scope of this disclosure. Any changes or modifications to the described embodiments, as well as any further application of the principles described herein, should be considered as generally conceivable by those with ordinary knowledge in the technical field to which the invention belongs.

Ranges are generally expressed herein as "about" one specific value and/or to "about" another specific value. When such a range is expressed, one aspect includes a specific value and/or a range to another specific value. Similarly, when the value is expressed as an approximate value by using the word "about", it should be understood that the specific value may form another aspect. In addition, it should be understood that each end point of each range is significant, and one end point is related to the other end point and independent of each other.

The terminology used herein is for describing particular illustrative embodiments only, and is not intended to limit the concepts of the present invention. Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" as used herein are also meant to include the plural forms. It should also be understood that when used in this specification, the terms "including" and "including" refer to the presence of the described features, wholes, steps, operations, elements or components, but do not exclude the presence or addition of one or more other Feature, whole, step, operation, element or assembly.

The invention provides a marker for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which is selected from microsatellite polymorphism markers of muscle multiplication genes, microsatellites of ER-C protein 2 like nerve growth factor-like genes A group consisting of polymorphic markers and microsatellite polymorphic markers of FK506 binding protein-like protein and protein Wnt-2b-A gene.

The term "microsatellite" used in the present invention is also called simple sequence repeats (SSRs) or short tandem repeats (STRs), which is a type of polymorphism. Refers to a form in which two or more nucleotides are arranged repeatedly, and different repeating sequences are adjacent to each other, about 2 to 10 base pairs in length, commonly found in non-coding introns. Due to the different repetition units and the number of repetitions, the distribution is very different and constitutes polymorphism. Different individuals have different repetitions at a homologous microsatellite site, which can be used as a marker for molecular breeding.

The microsatellite according to the present invention may be located upstream or downstream of its associated structural gene segment, for example, it may be located within about 500 base pairs upstream or downstream of its associated structural gene segment, preferably at approximately 350 bases Inwardly, the better line is within about 250 base pairs.

The microsatellite polymorphism according to the present invention includes, but is not limited to, microsatellite sequence, length or repeat number polymorphism. Preferably, it refers to the microsatellite repeat number polymorphism.

The microsatellite polymorphism of the muscle multiplication gene according to the present invention refers to the polymorphism of the microsatellite associated with the muscle multiplication gene among different individuals in the grouper genome. Preferably, the microsatellite polymorphism of the muscle multiplication gene is composed of primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), using the grouper genome as a template, and the polymerase chain reaction synthesized Product microsatellite polymorphism.

The microsatellite polymorphism of the ER-C protein 2 like nerve growth factor-like gene according to the present invention refers to the microsatellite that is associated with the ER-C protein 2 like nerve growth factor-like gene in the grouper genome is displayed among different individuals Out of many types. Preferably, the microsatellite polymorphism of the ER-C protein 2 like nerve growth factor-like gene consists of primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), using the grouper genome as a template , Microsatellite polymorphism of the polymerase chain reaction product synthesized.

The microsatellite polymorphism of the FK506 binding protein-like protein and protein Wnt-2b-A gene according to the present invention refers to the microsatellite associated with the FK506 binding protein-like protein and protein Wnt-2b-A gene in the grouper genome It shows polymorphism among different individuals. Preferably, the microsatellite polymorphism of the FK506 binding protein-like protein and protein Wnt-2b-A gene is composed of primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6), using the grouper genome As a template, the microsatellite polymorphism of the polymerase chain reaction product synthesized.

The present invention also provides a pair of primers for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which is selected from primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), primer 3 ( SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) and primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6).

The invention further provides a method for screening grouper seed and/or seed fish and/or accelerated breeding, which comprises detecting the aforementioned signs of grouper seed and/or seed fish.

According to the present invention, it uses microsatellite marker molecular detection method to detect the difference of the marker.

Based on the decoding of the grouper genome sequence, each of the microsatellite fragments and related positions according to the present invention are well known to those with ordinary knowledge in the technical field to which the present invention belongs, so those with ordinary knowledge in the technical field to which the present invention belongs can design appropriate A detection method to detect the polymorphism of the microsatellites, for example, a polymerase chain reaction method is used to detect the polymorphism of the microsatellite, preferably, the length of the microsatellite fragment is detected, and more preferably, the polymerase is detected The length of the chain reaction product. Methods for detecting the length of polymerase chain reaction products include but are not limited to gel electrophoresis.

In a specific embodiment of the present invention, it detects the marker in the fin of the grouper fry or the breeding fish, and the detection method includes but is not limited to obtaining the gene body in the fin or muscle cell, and detecting the marker according to the present invention .

The present invention further provides a kit for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which comprises reagents that can detect the markers according to the foregoing.

Preferably, the set includes primer pairs for screening grouper seedlings and/or breeding fish and/or accelerated breeding, wherein the primer pairs are selected from primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), a group consisting of primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) and primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6).

Preferably, the kit according to the present invention contains reagents for polymerase chain reaction.

On the other hand, the kit according to the present invention contains reagents for the detection method of microsatellite marker molecules.

In the embodiments of the present invention, microsatellite polymorphism markers for muscle multiplication genes, microsatellite polymorphism markers for ER-C protein 2 like nerve growth factor-like genes, and FK506 binding protein-like protein and protein Wnt-2b- The microsatellite polymorphism marker of the A gene detects the difference between the high-growth and low-growth fish, and can be used as a genetic marker for screening grouper seed and/or fish and/or accelerated breeding. The characteristics of accuracy, and the detection method is the simultaneous detection of multiple signs, so it has the advantages of speed, convenience and low cost, which is beneficial to actually enter the aquaculture industry to assist fishermen in the detection operation.

The following non-limiting examples help those with ordinary knowledge in the technical field to which the invention belongs to implement the invention. These examples should not be considered as excessively limiting the invention. Those with ordinary knowledge in the technical field to which the present invention belongs can modify and change the embodiments discussed herein without departing from the spirit or scope of the present invention, and still belong to the scope of the present invention. Example 1 Materials and Methods grouper seedlings sample collection

Collect the grouper specimens from the southern area, and dissect or decompose the grouper specimens from each farm to carry out the subsequent relevant mark detection. Detection of multiple markers using polymerase chain reaction technology (1) Extraction of tissue DNA

Take about 30 mg of grouper tissue into a microcentrifuge tube, add 1 mL of dissolution buffer and treat it with proteolytic enzyme K at 60°C for one hour, add a high salt solution to help precipitate the remaining protein, and centrifuge at 13,000 rpm to remove the precipitate Protein, take the supernatant and add an equal volume of isopropanol to mix and then inject into the DNA microtube column of the genome, centrifuge at 8000 rpm to precipitate DNA, pour out the effluent and centrifuge at 13000 rpm for two minutes to remove the remaining isopropyl Alcohol, and finally dissolve the DNA in 200 μL of pure water and collect it by centrifugation. After measuring the concentration and purity with a spectrophotometer, store it in a refrigerator at -20°C until use. (2) Detection of polymerase chain reaction

(3)毛細管電泳分析 The DNA samples drawn in the previous step are amplified using the specific primers shown in Table 1 below. Add 1 µL each of 10 µM forward and reverse primers, 5 µL of 2.5 mM dNTP mixture, 5 µL of 10X PCR buffer, 0.5 µL of taq polymerase, and finally add secondary water to a total volume of 50 µL per tube. Polymerase chain reaction. Temperature cycle setting Stage 1: Pretreatment at 94°C, 2 minutes, Stage 2: Denaturation at 94°C, 1 minute, primer annealing at 37°C, 30 seconds, polymerization at 72°C, 50 seconds, second stage three The steps are repeated 5 cycles, the third stage: denaturation 94°C, 1 minute, primer annealing 60°C, 30 seconds, polymerization reaction 72°C, 30 seconds, the third stage three steps repeat 8 cycles, the fourth stage: Polymerize at 72°C for 10 minutes. Table 1:

(3) Capillary electrophoresis analysis

For the results of multiple marker amplification, Fragment Analyzer ^TM Automated CE System was used for capillary electrophoresis analysis technology. result

After extracting muscle tissue DNA from samples of 20 groups of high and low growth, three sets of microsatellite polymorphic marker primers were used for polymerase chain reaction detection. Using the Fragment AnalyzerTM Automated CE System for capillary electrophoresis analysis technology, it can be known that high-growth stock fish can obtain 228bp, 260bp and 263bp muscle multiplication gene microsatellite polymorphism marker DNA fragments after amplification of primers 1 and 2 (Figure 1A); after amplification of primers 3 and 4, microsatellite polymorphic marker fragments of ER-C protein 2 -like nerve growth factor-like genes of 138 bp and 140 bp in size can be obtained (Figure 2A); after amplification of primers 5 and 6 A microsatellite polymorphic marker fragment of 163 bp-sized FK506 binding protein-like protein and protein Wnt-2b-A gene was obtained (Figure 3A).

Low-growth breeding fish can obtain a microsatellite polymorphic marker fragment of the muscle multiplying gene of 228 bp after amplification by primers 1 and 2 (Figure 1B); after amplification of primers 3 and 4 can obtain sizes of 138 bp, 140 bp, 142 bp and 144 bp The microsatellite polymorphic marker fragment of the ER-C protein 2-like nerve growth factor-like gene (Figure 2B); after amplification of primers 5 and 6, FK506-binding protein-like protein and protein Wnt-2b of 128bp and 163bp are available -A microsatellite polymorphic marker fragment of the A gene (Figure 3B).

Therefore, the microsatellite polymorphism marker of muscle multiplication gene, microsatellite polymorphism marker of ER-C protein 2 like nerve growth factor-like gene, microsatellite polymorphism of FK506 binding protein-like protein and protein Wnt-2b-A gene It is marked in the high-growth and low-growth fish species, there are indeed different types, and the grouper seedlings and/or fish species and/or accelerated breeding can be screened accordingly.

The above-mentioned embodiments are only to illustrate the principle and efficacy of the present invention, but not to limit the present invention. Modifications and changes made by those skilled in the art to the above embodiments still do not violate the spirit of the present invention. The scope of the rights of the present invention shall be as listed in the patent application scope described later.

FIG. 1A shows the results of capillary electrophoresis analysis of the microsatellite polymorphism marker polymerase chain reaction product of the muscle multiplication gene of high-growing stock fish. FIG. 1B shows the capillary electrophoresis analysis result of the microsatellite polymorphism marker polymerase chain reaction product of the muscle multiplication gene of low-growing stock fish.

FIG. 2A shows the capillary electrophoresis analysis result of the microsatellite polymorphism marker polymerase chain reaction product of the ER-C protein 2-like nerve growth factor-like gene of high-growing stock fish. FIG. 2B shows the capillary electrophoresis analysis result of the microsatellite polymorphism marker polymerase chain reaction product of the ER-C protein 2-like nerve growth factor-like gene of low-growing stock fish.

Figure 3A shows the results of capillary electrophoresis analysis of the microsatellite polymorphism marker polymerase chain reaction product of FK506 binding protein-like protein and protein Wnt-2b-A gene of high-growing stock fish. Figure 3B shows the results of capillary electrophoresis analysis of microsatellite polymorphism marker polymerase chain reaction products of FK506 binding protein-like protein and protein Wnt-2b-A gene of low-growing stock fish.

Claims (8)

A marker for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which comprises a muscle multiplication gene synthesized by a polymerase chain reaction between primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2) (myostatin, MSTN) microsatellite polymorphism marker, primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4) ER-C protein 2 like nerve synthesized by polymerase chain reaction Microsatellite polymorphism marker of ER-C protein 2-like Nerve growth factor-like gene and primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6) by polymerase chain reaction Microsatellite polymorphism markers of the synthesized FK506-binding protein-like protein and protein Wnt-2b-A (FK506-binding protein-like protein & protein Wnt-2b-A) gene; of which, high-growth stock fish expand at primers 1 and 2 After amplification, fragments of 228bp, 260bp and 263bp can be obtained; after amplification of primers 3 and 4, fragments of 138bp and 140bp can be obtained; after amplification of primers 5 and 6, fragments of 163bp can be obtained. A pair of primers for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which comprises primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), primer 3 (SEQ ID NO: 3) And primer 4 (SEQ ID NO: 4) and primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 6). A method for screening grouper fingerlings and/or fingerlings and/or accelerated breeding, which includes detecting the grouper fingerlings and/or fingerlings according to item 1 of the request. According to the method of claim 3, it uses microsatellite marker molecular detection method to detect the difference of the mark. According to the method of claim 3, it is to detect the mark in the fins or muscles of grouper fry or fish. A kit for screening grouper seedlings and/or breeding fish and/or accelerated breeding, which comprises a reagent capable of detecting the mark according to item 1 of the claim and primers for screening grouper seedlings and/or breeding fish and/or accelerated breeding Yes, where the primer pair includes primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2), primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), and primer 5 ( SEQ ID NO: 5) and Primer 6 (SEQ ID NO: 6). According to the set of claim 6, it contains reagents for polymerase chain reaction. According to the set of claim 6, it contains reagents for microsatellite marker molecular detection method.

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