Primer group, detection method, kit and application for identifying coilia ectenes, coilia brachygnathus and hybrids of coilia ectenes and coilia brachygnathus

Abstract

The invention provides a primer group for identifying coilia ectenes, coilia brachygnathus and hybrids thereof, a detection method, a kit and application, and belongs to the technical field of molecular marker identification. In order to overcome the defects that morphological differences of coilia ectenes, coilia brachygnathus and hybrid adults are not obvious and the like, a primer pair A or a primer pair B is designed for gene amplification, and the coilia ectenes, the coilia brachygnathus and the hybrid adults are identified by comparing sequence differences after amplification. The sequences of amplification products of the coilia ectenes, the coilia brachygnathus and the hybrids have obvious differences, the types of the amplification products can be classified through molecular markers, and the reaction result is directly visualized in the form of a gel electrophoretogram without a sequencing means. The method is simple to operate, intuitive in result, low in requirement on experimental equipment and low in cost, and all work can be completed within several hours without statistics. According to the invention, the hybrid can be identified through a sequencing peak map, and compared with an SNP site identification type, the difference is more obvious, the statistical frequency is not needed, and the accuracy is high.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker identification, and in particular to a primer set, a detection method, a kit and an application thereof for identifying anchovy, short-jawed anchovy and their hybrids. Background Art

[0002] Coilia nasus is a fish of the family Anchovy in the order Herringiformes. It is rich in high-quality protein, minerals (such as calcium, phosphorus, and iron) and vitamins (such as vitamins A, D, and E), as well as unsaturated fatty acids, especially ω-3 fatty acids. These nutrients have many benefits for the human body, such as maintaining the health of muscles, bones, and the immune system, promoting vision maintenance, calcium absorption, and anti-oxidation. Its meat has a unique and delicate flavor and is known as one of the three delicacies of the Yangtze River. With the large-scale expansion of ports, docks, and water conservancy projects along the Yangtze River estuary, environmental pollution, and overfishing, its spawning grounds, fattening grounds, and migration routes have been destroyed. Due to the reduction in the amount of wild scimitar fish resources and the growing market demand for scimitar fish, the artificial breeding and farming technology of scimitar fish has developed rapidly in the past decade, with huge economic benefits. As the sister species of the razor-sharp coilia, the short-jawed coilia (Coilia brachygnathus) has little morphological difference from the razor-sharp coilia. Fishermen and ecological surveyors often use the length of the maxilla as a simple mark to distinguish the two. In addition, in the artificial breeding of razor-sharp coilia, short-jawed coilia are often mistakenly used as parents, resulting in the offspring group showing the trait of different lengths of maxillae, which seriously affects the germplasm resources and market of razor-sharp coilia and reduces consumers' trust in farmed razor-sharp coilia. Therefore, it is urgent to carry out genetic breeding and screening of razor-sharp coilia to improve the germplasm resources of razor-sharp coilia and increase the breeding yield, so as to promote the high-quality development of the razor-sharp coilia breeding industry.

[0003] Traditional morphology uses the ratio of maxillary length to head length as the classification standard for razor-sharp and short-jawed coilfish: maxillary length greater than the posterior edge of the operculum (maxillary length greater than head length) is considered razor-sharp coilfish; maxillary length less than the posterior edge of the operculum (maxillary length less than head length) is considered short-jawed coilfish. However, these identification features are easily affected by environmental factors (such as sampling of the incomplete maxillary bones of some populations), measurement errors, and ratios approaching 1, making it difficult to effectively identify.

[0004] With the continuous breakthroughs in comparative genomic technology, molecular marker technology with SNP site screening as the core has continued to develop, with obvious differences and higher accuracy compared to morphological classification; many patents are based on specific SNP sites to determine the molecular markers in specific areas of the razor-sharp and short-jawed anchovy for identification, but relatively speaking, sequencing a large number of individuals takes a long time, is expensive, and requires more experimental instruments; in addition to SNP, InDel markers mostly need to be identified by polyacrylamide gel electrophoresis to distinguish small fragments, which is difficult to prepare, time-consuming, has a small sample capacity, and the results are not obvious; in addition, there is no report on the identification method of the hybrids of the razor-sharp and short-jawed anchovy, so it is necessary to develop a new identification method based on ordinary agarose gel electrophoresis. Summary of the invention

[0005] The purpose of the present invention is to provide a primer set, a detection method, a kit and an application for identifying razor-sharp anchovy, short-jawed anchovy and their hybrids, which can distinguish razor-sharp anchovy, short-jawed anchovy and their hybrids based on ordinary agarose gel electrophoresis, and the method is simple, the result is intuitive and the cost is low.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a primer set for identifying razorfish, short-jawed anchovy and their hybrids. The primer set for identifying razorfish, short-jawed anchovy and their hybrids comprises a primer pair A shown in sequences SEQ ID NO.1 to SEQ ID NO.2 or a primer pair B shown in sequences SEQ ID NO.3 to SEQ ID NO.4.

[0008] The present invention also provides a method for identifying Coilia edulis, Coilia serrata and their hybrids, comprising the following steps:

[0009] (1) extracting sample genomic DNA and amplifying the genomic DNA using primer pair A or primer pair B;

[0010] (2) Gel electrophoresis was used as the detection method. The amplified product was loaded and the nucleic acid dye was used for color development. Obvious differences in the number and size of the bands were observed, and the sample was judged to be an eriphthalmoid fish, a short-jawed fish, or a hybrid.

[0011] Further, when primer pair A is used for amplification in step (2), the band size difference is:

[0012] The length of the band amplified by primer pair A for Coilia otarius was 666bp, and the length of the band amplified by Coilia shortnata was 629bp. 37bp was missing in the sequencing peak graph; the base sequences before and after 269bp in the sequencing peak graph showed single peaks and overlapping peaks, which were hybrids.

[0013] Further, when primer pair B is used for amplification in step (2), the band size difference is:

[0014] The length of the band amplified by primer pair B for Coilia otarius is 213 bp, and the length of the band amplified for Coilia brevis is 176 bp. Two bands appear when amplifying the hybrid. The number and size of the bands can be used to determine the

[0015] The present invention also provides a kit for identifying erithacus scaly, erithacus brachyurae and their hybrids, characterized in that the kit comprises a primer pair A or a primer pair B.

[0016] The invention also provides a primer set for identifying razorfish, short-jawed anchovy and their hybrids, and a kit for identifying razorfish, short-jawed anchovy and their hybrids, and uses the kit in identifying razorfish, short-jawed anchovy and their hybrids.

[0017] The beneficial effects of the present invention compared with the prior art are:

[0018] (1) The present invention aims at the defects and shortcomings of the lack of obvious morphological differences among the adults of razor-sharp coil, short-jawed coil and hybrids, designs primer pair A or primer pair B for gene amplification, and identifies razor-sharp coil, short-jawed coil and hybrids by comparing the sequence differences after amplification. The sequences of the amplified products of razor-sharp coil, short-jawed coil and hybrids are obviously different, and their types can be classified by molecular markers. The results can be directly reflected in the form of gel electrophoresis without sequencing. The method is simple to operate, the results are intuitive, all work can be completed in a few hours, no statistics are required, and the experimental equipment requirements are low and the cost is low.

[0019] (2) The present invention can also identify hybrids through sequencing peak graphs. By comparison, it is found that when primer pair A is used for amplification, the base sequences before and after 269bp in the sequencing peak graph present single peaks and overlapping peaks, which are hybrids; when primer pair B is used for amplification, two bands appear, which are hybrids. Compared with SNP site discrimination type, the present invention has more significant differences, does not require statistical frequency, and has a high accuracy rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 It is a schematic diagram of the cutlassfish, short jawed anchovy and hybrid in Example 1 of the present invention;

[0022] Figure 2 The electrophoresis diagram of the genomic DNA (partial) of some cutlassfish, short-jawed fish and hybrids extracted in Example 1 of the present invention, including 10 cutlassfish, 10 short-jawed fish and 4 hybrids;

[0023] Figure 3 The corresponding band diagram after agarose gel electrophoresis of the amplified products after PCR with primer pairs A and B using genomic DNA templates of Coilia otarius, Coilia shortnata and hybrids;

[0024] Figure 4 The sequence alignment diagram of the amplified products of the genomic DNA templates of the cutlass fish, short-jawed fish and hybrid after being tested using primer pair A in Example 1 of the present invention, from top to bottom are the sequencing peak diagrams of the cutlass fish, short-jawed fish and hybrid individuals, wherein the 37 bp deletion annotated in the short-jawed fish peak diagram is the base difference of the identification method, and the hybrid peak diagram is annotated with its unique overlapping peak;

[0025] Figure 5 The band differences in the agarose gel electrophoresis of the amplified products after PCR with primer pair B using genomic DNA templates of Coilia otarius, Coilia brachygnathus and hybrid in Example 2 of the present invention are shown. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Table 1 Specific primers for identifying Coilia nasus, Coilia brachygnatha and their hybrids

[0032]

[0033]

[0034] Example 1

[0035] In Example 1 of the present invention, genes of Coilia lanceolata, Coilia brachyurae and their hybrids were amplified, and primer pair A shown in SEQ ID NO.1 to SEQ ID NO.4 in Table 1 was used to amplify genes of Coilia lanceolata, Coilia brachyurae and their hybrids, and sequencing was performed, and the specific steps were as follows:

[0036] 1. Extraction of genomic DNA

[0037] According to the jaw length / head length, the cutlass fish, short jaw fish, and hybrids (preserved in 95% alcohol) were initially selected. Figure 1 ( Figure 1 There is no obvious morphological difference between the long-jaw hybrid and the cutthroat fish, and between the short-jaw hybrid and the short-jaw fish). The genomic DNA was extracted using a kit (MolPure Cell / Tissue DNA Kit Cell / Tissue DNA Extraction Kit). The kit operation steps are as follows:

[0038] (1) Take 20-30 mg of muscle sample, place it in a centrifuge tube and place it in liquid nitrogen to grind the tissue into powder. Transfer it to a 1.5 mL centrifuge tube, insert DNA adsorption column T1 into a 2 mL collection tube, add 200 μL of buffer AC to DNA adsorption column T1, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0039] (2) Add 400 μL of lysis buffer LB and 10 μL of Proteinase K, use a pipette to break up the tissue clots in the centrifuge tube, vortex to mix, and instantly collect the liquid on the tube wall. Place in a 55°C warm bath for 1 to 2 hours. Vortex and mix the sample 2 to 3 times per hour, each time for 15 seconds, until the sample is clear and transparent.

[0040] (3) Remove the centrifuge tube and cool it to room temperature, then gently shake it to mix. Add 300 μL of deproteinization solution PL and 300 μL of binding solution BD in sequence, shake vigorously, centrifuge at 12,000 rpm for 5 min, and carefully aspirate the lower layer after the solution is separated.

[0041] Add the sample pretreatment solution to the DNA adsorption column T1, centrifuge at 12,000rpm for 1min, and discard the waste liquid. Put the DNA adsorption column T1 back into the collection tube, add 500μL of rinsing solution, centrifuge at 12,000rpm for 30s, and discard the waste liquid. Repeat the rinsing step and put the DNA adsorption column T1 back into the collection tube, centrifuge the empty column at 12,000rpm at room temperature for 2min to remove the residual rinsing solution.

[0042] Place DNA adsorption column T1 in a new 1.5mL centrifuge tube, add 50μL of 65℃ preheated eluent to the center of the adsorption column, place at room temperature for 2min, and centrifuge at 12,000rpm for 1min. Collect the filtrate, apply the DNA filtrate to the column again, place at room temperature for 2min, and elute to obtain the DNA solution. Use NaNOdrop to determine the DNA concentration and use 1% agarose gel to detect the integrity of the genomic DNA ( Figure 2 (Results of partial genomic DNA electrophoresis of Coilia otarius, Coilia shortnata and their hybrids).

[0043] 2. Primer synthesis and PCR amplification

[0044] (1) Based on the difference analysis of the whole genome resequencing results of C. erigeron and C. brachygnathus, primer pairs A and B were designed for identifying C. erigeron and C. brachygnathus. The upstream and downstream primer sequences, annealing temperatures and amplified fragment lengths are shown in Table 1.

[0045] (2) Primer pairs A and B were sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis. After obtaining the primers, they were diluted to a working concentration. The primer abbreviations were set as FA, RA and FB, RB. PCR amplification was performed on Coilia scaly, Coilia brachyurae and hybrids using primer pairs A and B respectively. The results were as follows: Figure 3 shown.

[0046] Figure 3 The results showed that primer pairs A and B could successfully amplify Coilia otarius, Coilia brachygnathus and their hybrids.

[0047] (3) Primer pair A was used as the upstream and downstream primers of Example 1. The PCR amplification system was as follows: 10 μL of 2×Hieff Canace Gold PCR Master Mix, 1 μL of extracted genomic DNA (diluted to 50 ng / μL in advance), 1 μL of each of the upstream and downstream primers of primer pair A, and the mixture was filled up to 20 μL with ddH2O.

[0048] PCR program: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min, 34 cycles, 72℃ final extension for 10min, and storage at 4℃ after completion.

[0049] The amplified products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed using SNAPGENE software. By comparing the base sequence with the sequencing peak graph, the cutlassfish, short-jawed fish and hybrids could be identified. The results are as follows: Figure 4 shown.

[0050] Figure 4 The 37 bp deletion annotated in the peak image of short-jawed Coilia intermedius is the base difference of the identification method, and the hybrid peak image is annotated with its unique overlapping peaks.

[0051] Example 2

[0052] In Example 2 of the present invention, the primer pair B shown in the sequences SEQ ID NO.3 to SEQ ID NO.4 in Table 1 was used to amplify the genes of Coilia otarius, Coilia brevis and their hybrids, and the specific steps were as follows:

[0053] (1) The genomic DNA was extracted using the method in Example 1. Primer pair B in Table 1 was used as the upstream and downstream primers used in Example 2. After obtaining the primers, the primers were diluted to a working concentration of 1 nmol / μL.

[0054] The PCR amplification reaction system was set, the reaction system volume was set to 20 μL, and the primer abbreviations were set to FB and RB.

[0055] Add 10 μL of 2×HieffPCRMasterMix, 1 μL of genomic DNA extracted in Example 1 (the concentration was diluted to 50 ng / μL in advance), and 1 μL of each of the upstream and downstream primers of primer pair B, and fill up to 20 μL system with ddH2O.

[0056] PCR program: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 36 cycles, 72℃ final extension for 10min, and storage at 4℃ after completion.

[0057] (2) Agarose gel preparation

[0058] According to the PCR product, a suitable size of gel plate and comb were selected to prepare 1.2% agarose gel electrophoresis, nucleic acid dye was loaded and stained, and the electrophoresis parameters were voltage 200V, current 400mA, and running time 25min. After the end, the gel image was observed with a gel imager. The band differences between the cutlass fish, short jaw fish and hybrids can be clearly distinguished according to the gel image. The results are as follows Figure 5 shown.

[0059] Depend on Figure 5 It can be seen that primer pair B can successfully identify ectodontia, short-jawed anchovy and their hybrids. The length of the band amplified by primer pair B for ectodontia is 213 bp, and the length of the band amplified by short-jawed anchovy is 176 bp. Two bands appear when amplifying the hybrid.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for identifying Coilia lanceolata, Coilia brachyurae and their hybrids, characterized in that: The primer set for identifying Coilia otarius, Coilia brachyurae and their hybrids comprises a primer pair A shown in the sequences of SEQ ID NO.1 to SEQ ID NO.2 or a primer pair B shown in the sequences of SEQ ID NO.3 to SEQ ID NO.

4.

2. A method for identifying Coilia lanceolata, Coilia brachyurae and their hybrids, characterized in that: The steps include: (1) extracting sample genomic DNA and amplifying the genomic DNA using primer pair A or primer pair B; (2) Gel electrophoresis was used as the detection method. The amplified product was loaded and the nucleic acid dye was used for color development. Obvious differences in the number and size of the bands were observed, and the sample was judged to be an eriphthalmoid fish, a short-jawed fish, or a hybrid.

3. The method for identifying Coilia lanceolata, Coilia brachyurae and their hybrids according to claim 2, characterized in that: When primer pair A is used for amplification in step (2), the band size difference is: The length of the band amplified by primer pair A for Coilia otarius was 666bp, and the length of the band amplified by Coilia shortnata was 629bp. 37bp was missing in the sequencing peak graph; the base sequences before and after 269bp in the sequencing peak graph showed single peaks and overlapping peaks, which were hybrids.

4. The method for identifying Coilia lanceolata, Coilia brachyurae and their hybrids according to claim 2, characterized in that: When primer pair B is used for amplification in step (2), the band size difference is: The length of the band amplified by primer pair B for Coilia otarius was 213 bp, and the length of the band amplified for Coilia brachygnathus was 176 bp. Two bands appeared when amplifying the hybrid.

5. A kit for identifying Coilia lanceolata, Coilia brachyurae and their hybrids, characterized in that: The kit comprises the primer pair A or the primer pair B according to claim 1.

6. Use of the primer set for identifying erithecus scaly, anchovy and their hybrids as claimed in claim 1 or the kit for identifying erithecus scaly, anchovy and their hybrids as claimed in claim 5 in identifying erithecus scaly, anchovy and their hybrids.

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