Primers for Molecular Marking of Chromosome X of Siniperca chuatsi and Its Application

Abstract

The invention discloses a molecular marker primer for X chromosome of Siniperca chuatsi, which is characterized in that: the primer is a primer pair 1 or a primer pair 2, the primer pair 1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair 2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the Contig-1 downstream primer is shown in SEQ ID NO: 2, the nucleotide sequence of the Contig-2 upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the Contig-2 downstream primer is shown in SEQ ID NO: 4. Also disclosed is a method for identifying YY super male fish of Siniperca chuatsi and the application of the primer or method in all-male breeding of Siniperca chuatsi.

Description

Technical Field

[0001] The invention belongs to the technical field of fish sex chromosome composition identification, and particularly relates to a molecular marker primer for a Siniperca chuatsi X chromosome and an application thereof. Background Art

[0002] In recent years, with the rapid development of molecular biology technology, sex chromosome-specific markers have been developed for many fish species, such as Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss), yellow catfish (Pelteobagrus fulvidraco), semi-smooth tongue sole (Cynoglossus semilaevis) and redfin pufferfish (Takifugurubripes), etc. However, these markers are mainly obtained through molecular marker development technologies such as microsatellite (SSR), restriction site associated DNA (RAD) and amplified fragment length polymorphism (AFLP). These molecular marker development methods are time-consuming, labor-intensive and have a low success rate. The emergence of high-throughput sequencing technology provides a new and efficient means for molecular marker development. At present, sex chromosome-specific molecular markers for Channa argus and large yellow croaker (Larimichthyscrocea) have been successfully developed through high-throughput sequencing methods.

[0003] Sinipercachuatsi belongs to the order Perciformes, family Sinipercidae, genus Siniperca, commonly known as Mandarin fish, Osmanthus fish, and its English name means Chinese perch, Mandarin fish. Sinipercachuatsi is a rare freshwater fish unique to my country. Its meat is delicious, nutritious, and has no intramuscular spines. It is deeply loved by consumers at home and abroad. From February to May every year, there is a great demand for mandarin fish in China, and the selling price is high, but the stock of mandarin fish in the pond is small. In recent years, in order to obtain higher profits, farmers have chosen to sell mandarin fish after the wintering period. The growth of Sinipercachuatsi is sexually dimorphic. During the 2-9 growth months before the wintering period, the growth rate of female fish is faster than that of male fish. During the 9-14 growth months after the wintering period, female fish reach sexual maturity and slow down their growth rate. At this time, the growth rate of male fish is faster than that of female fish. Therefore, breeding all-male mandarin fish has extremely high economic value.

[0004] In the previous karyotype analysis of Siniperca chuatsi, no obvious sex chromosomes were found. However, through the sequencing of male and female genomes and the development of male-specific molecular markers of Siniperca chuatsi, we confirmed that there are Y chromosome-specific DNA fragments in the male fish genome, so the sex determination of Siniperca chuatsi is determined to be XX / XY type, that is, male heterogamety. The first step in the monosexual breeding of all-male Siniperca chuatsi is to obtain YY super male fish. The main way to obtain super male fish is to mate the pseudo female fish induced by estrogen with normal male fish, so as to obtain YY super male fish offspring. In theory, super male fish account for 25% of the offspring. Secondly, the obtained super male fish is mated with normal female fish to obtain male fish offspring with all XY genotypes. The traditional method of screening XY male fish and YY super male fish is to determine the sex chromosome composition of the father through test cross experiments. It takes a lot of experimental costs to verify the sex of the test cross offspring through male-specific markers of Siniperca chuatsi, and the test cross parents need to occupy more breeding resources to breed alone, so the test cross method is time-consuming, labor-intensive and resource-consuming. So far, there has been no report on the successful identification of X chromosome-specific markers in Siniperca chuatsi at home and abroad. Therefore, the development of X chromosome molecular markers for rapid screening of YY super male fish has great production application value for all-male breeding of Siniperca chuatsi. Summary of the invention

[0005] The first object of the present invention is to provide a molecular marker primer for X chromosome of Siniperca chuatsi, which can identify YY super male Siniperca chuatsi and XY male Siniperca chuatsi at the molecular level.

[0006] The second purpose of the present invention is to provide a method for identifying YY super male fish of Siniperca chuatsi, which is time-saving and has accurate detection results.

[0007] The third object of the present invention is to provide the application of the above primers or methods in all-male breeding of Siniperca chuatsi.

[0008] To achieve the above first purpose, the present invention adopts the following technical solutions:

[0009] The invention discloses a molecular marker primer for chromosome X of Siniperca chuatsi. The primer is a primer pair 1 or a primer pair 2. The primer pair 1 comprises a Contig-1 upstream primer and a Contig-1 downstream primer. The primer pair 2 comprises a Contig-2 upstream primer and a Contig-2 downstream primer. The nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the Contig-1 downstream primer is shown in SEQ ID NO: 2, the nucleotide sequence of the Contig-2 upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the Contig-2 downstream primer is shown in SEQ ID NO: 4.

[0010] Specifically, the nucleotide sequences of each primer from 5' to 3' are as follows:

[0011] Contig-1 upstream primer: 5′-ATTCCCATCAACCTGT-3′;

[0012] Contig-1 downstream primer: 5′-CACAATCTGTGCCTAC-3′;

[0013] Contig-2 upstream primer: 5′-TTGCGTTTAGTGTTGATTGT-3′;

[0014] Contig-2 downstream primer: 5′-AGACCTGCACGTCCTAA-3′.

[0015] To achieve the above second purpose, the present invention adopts the following technical solutions:

[0016] A method for identifying super male fish of Siniperca chuatsi YY, comprising the following steps:

[0017] (1) Preparing DNA samples of Siniperca chuatsi;

[0018] (2) using the DNA sample in step (1) as a template, and using the primer pair 1 or the primer pair 2 in claim 1 to perform PCR amplification, and performing electrophoresis detection after the amplification reaction is completed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

[0019] In the above method for identifying the super male fish of Siniperca chuatsi YY:

[0020] Preferably, in step (2), the DNA sample in step (1) is used as a template, and primer pair 1 is used to perform PCR amplification. After the amplification reaction is completed, electrophoresis detection is performed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band of about 935 bp, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band of about 935 bp, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

[0021] Preferably, in step (2), the DNA sample in step (1) is used as a template, and primer pair 2 is used to perform PCR amplification. After the amplification reaction is completed, electrophoresis detection is performed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band of about 850 bp, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band of about 850 bp, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

[0022] Preferably, in step (1), the DNA sample is prepared by column centrifugation to extract DNA from Siniperca chuatsi.

[0023] Preferably, during PCR amplification in step (2), a 20 μL PCR reaction system is used, wherein the 20 μL PCR reaction system comprises 50 ng DNA, 0.8 μL each of the Contig-1 upstream primer and the Contig-1 downstream primer of primer pair 1 or 0.8 μL each of the Contig-2 upstream primer and the Contig-2 downstream primer of primer pair 2, 10 μL of 2×Taq MasterMix, and the rest is ddH 2 OFill in.

[0024] Preferably, during PCR amplification in step (2), the PCR reaction procedure adopted is: first, pre-denaturation at 94°C for 2 min; then denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; then final extension at 72°C for 5 min.

[0025] Preferably, during the electrophoresis detection in step (2), 1% by mass agarose gel electrophoresis is used to detect the length of the amplified fragment.

[0026] To achieve the third objective, the present invention adopts the following technical solutions:

[0027] Application of the primers or methods in all-male breeding of Siniperca chuatsi.

[0028] The present invention has the following beneficial effects:

[0029] (1) Based on the high-throughput sequencing data of the genomes of YY super males and XX females, the present invention uses analysis methods such as genome alignment and assembly to quickly and accurately screen out X chromosome molecular markers of Siniperca chuatsi, and designs primers for identifying X chromosome molecular markers of Siniperca chuatsi for the first time. The primers can quickly and accurately identify YY super males of Siniperca chuatsi;

[0030] (2) The method of the present invention establishes a method for identifying the YY super male fish of Siniperca chuatsi using the molecular marker primers of the X chromosome of Siniperca chuatsi. By using two pairs of specific primers, the YY super male fish of Siniperca chuatsi can be identified by completing one PCR reaction. Compared with the traditional method of determining the sex chromosome composition of the father through test cross experiments, the method is time-saving, efficient, and resource-saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of the breeding of YY super male fish in Example 1;

[0032] Figure 2 The electrophoresis diagram of the YY fish offspring screened for sequencing in Example 1, wherein af is the offspring of the YY test cross, and g is the offspring of the XY test cross;

[0033] Figure 3 A schematic diagram of obtaining the X chromosome-specific candidate fragment in Example 1;

[0034] Figure 4 The electrophoresis diagram of the PCR product in Example 1 in which the primers Contig-1 and Contig-2 detected amplified fragments of 935 bp and 850 bp in XX female and XY male samples, respectively, and no corresponding amplified fragments were detected in YY super male fish;

[0035] Figure 5 The electrophoresis diagram of the PCR products of the other four pairs of primers in Example 1 for amplifying non-X chromosome specific fragments in XX females, XY males and YY super males;

[0036] Figure 6 The electrophoresis diagram of the PCR products of primer Contig-1 in Example 2 in 8 XX females, 8 XY males and 6 YY super males;

[0037] Figure 7 This is the electrophoresis diagram of the PCR products of primer Contig-2 in Example 2 in 8 XX females, 8 XY males and 6 YY super males. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical solution of the present invention. The reagents or materials used in the embodiments, unless otherwise specified, are all from commercial channels.

[0039] Example 1

[0040] This embodiment provides X chromosome molecular marker primers for Siniperca chuatsi, the primers include primer pair 1 or primer pair 2, the primer pair 1 includes a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair 2 includes a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the Contig-1 downstream primer is shown in SEQ ID NO: 2, the nucleotide sequence of the Contig-2 upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the Contig-2 downstream primer is shown in SEQ ID NO: 4.

[0041] Specifically, the nucleotide sequences of each primer from 5' to 3' are as follows:

[0042] Contig-1 upstream primer: 5′-ATTCCCATCAACCTGT-3′;

[0043] Contig-1 downstream primer: 5′-CACAATCTGTGCCTAC-3′;

[0044] Contig-2 upstream primer: 5′-TTGCGTTTAGTGTTGATTGT-3′;

[0045] Contig-2 downstream primer: 5′-AGACCTGCACGTCCTAA-3′.

[0046] The above-mentioned X chromosome molecular marker primer of Siniperca chuatsi in the present invention is obtained by the following method, which comprises the steps of:

[0047] (I) Sequencing library construction and resequencing

[0048] 1. Preparation of DNA samples of mandarin fish:

[0049] During the breeding period, the tail fins of three super male mandarin fish were cut, and the super male fish were selected by test cross method ( Figure 1 and Figure 2 The super male fish were numbered SM1-SM3 in sequence, and DNA samples were prepared by conventional column centrifugation method (Universal Column Genome Extraction Kit, Beijing Kangwei Century Biotechnology Co., Ltd.) according to the instructions. The quality and concentration were tested by 1% agarose gel electrophoresis and micro-spectrophotometer (NanoDrop2000) to meet the requirements of high-throughput sequencing.

[0050] 2. Sequencing library construction and high-throughput sequencing:

[0051] DNA samples of super male fish SM1-SM3 were taken to construct 350-500bp fragment sequencing libraries respectively, and paired-end (Pair-End) PE150 sequencing was performed using the Illumina HiSeq X Ten platform to obtain super male fish genome sequencing clean data: 33,810,178,500bp.

[0052] The library construction steps were specifically referred to the Novogene NGS DNA Library Prep Kit instructions, and sequencing was completed by Beijing Novogene Technology Co., Ltd.

[0053] (II) Enrichment of X chromosome-specific DNA fragments

[0054] 1. Obtaining X chromosome-specific candidate DNA fragments:

[0055] The female fish reference genome (PRJNA612372, Females: SRR11300855, SRR11300854, SRR11300853) obtained in the laboratory was aligned with the super male fish SM1-SM3 sequencing reads using bwa v0.7.17-r1188 (http: / / bio-bwa.sourceforge.net / ) software, using a double-end alignment strategy, the base error rate was set to 0.04, and the rest of the parameters were default. Contigs in the female fish reference genome that could be aligned with the super male fish sequencing reads were filtered out, and contigs in the male fish reference genome that could not be aligned with the super male fish sequencing reads were selected. These regions are possible X chromosome-specific DNA fragments ( Figure 3 ).

[0056] 2. Sequencing depth analysis of X chromosome specific fragments:

[0057] Samtools (version 1.9) was used to perform sequencing depth analysis and screening on the candidate X-specific fragments obtained in the previous step. The reference genomes of male (XY) and female (XX) mandarin fish previously obtained in the laboratory (PRJNA612372, Males: SRR11300858, SRR11300857, SRR11300856; Females: SRR11300855, SRR11300854, SRR11300853) were selected for reference analysis. In the comparative analysis of the X chromosome and Y chromosome sequencing depth, in order to avoid the error in sequence alignment leading to abnormal calculation of depth, the depth calculation selected sequences homologous to XY chromosomes and with a similarity of less than 95% for statistics. The screening principle is that the sequencing depth of the fragment is 0.3-0.7 times the total depth of the male genome and 0.8-1.2 times the total depth of the female genome. The sequences that meet this sequencing principle are candidate X chromosome-specific sequences. A total of 49 X chromosome-specific contig sequences that met the above screening criteria were obtained.

[0058] (III) Screening of X chromosome molecular markers

[0059] 1. Primer design and synthesis:

[0060] Based on the above 49 X chromosome-specific Contig sequences, 6 Contig sequences were randomly selected, and PCR primers were designed using Primer5 software (the software usage refers to Primer PREMIER Version5.0 for Windows and PowerMacintosh). The primers were numbered according to Contig-xxF / R (xx represents the Contig number), such as the primer number of Contig-1 is Contig-1F / R (F represents the upstream primer, R represents the downstream primer, the same below). The primers were synthesized by Invitrogen (Shanghai) Trading Co., Ltd.

[0061] 2. Preparation of DNA samples of mandarin fish:

[0062] During the breeding period, the tail fins of four female, four male and four super male mandarin fish were clipped and numbered as female fish F1-F4, male fish M1-M4 and super male fish SM1-SM4, respectively. DNA samples were prepared by conventional column centrifugation method (Universal Column Genome Extraction Kit, Beijing Kangwei Century Biotechnology Co., Ltd.) according to the instructions, and the quality and concentration were detected by 1% agarose gel electrophoresis and a micro spectrophotometer (NanoDrop2000).

[0063] 3. Screening and confirmation of X chromosome molecular markers:

[0064] The above six primer pairs were selected to perform PCR detection on four female fish, four male fish and four super male fish. The total PCR amplification reaction system was 20 μL, including 10 μL of Kangwei Century 2×Taq MasterMix (Beijing Kangwei Century Biotechnology Co., Ltd.), 0.8 μL of upstream and downstream primers (10 μmol / L), 50 ng of template DNA, and the rest of the reaction was carried out in ddH 2 OFill in.

[0065] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles; and final extension at 72°C for 5 min.

[0066] Agarose gel electrophoresis results showed that two pairs of primers only obtained X chromosome-specific amplification bands in male and female (XX, XY) individuals, and no X chromosome-specific amplification fragments were detected in super male (YY) individuals ( Figure 4 ), two pairs of primers are Contig-1F / R and Contig-2F / R, which means that the corresponding contigs are Contig-1 and Contig-2; the other four pairs of primers have the same amplification products in females, males and super males ( Figure 5 ), demonstrating that these fragments are not X-chromosome-specific fragments.

[0067] Through PCR amplification with 6 pairs of primers in 4 female, 4 male and 4 super male individuals, it was confirmed that 2 contigs: Contig-1 and Contig-2 were X chromosome specific fragments and X chromosome molecular markers of mandarin fish.

[0068] Example 2

[0069] This embodiment provides a method for identifying super male fish of Siniperca chuatsi YY using molecular marker primers of Siniperca chuatsi X chromosome, comprising the following steps:

[0070] (1) Preparation of DNA samples of Siniperca chuatsi:

[0071] Sexually mature mandarin fish were obtained from Sanshui Baijin Aquatic Seed Co., Ltd., Foshan City, Guangdong Province. Eight male and eight female mandarin fish were obtained after dissection, and six super male fish were obtained after cross-cross verification. The tail fins were cut off respectively, and DNA samples were prepared by conventional column centrifugation method (Universal Column Genome Extraction Kit, Beijing Kangwei Century Biotechnology Co., Ltd.) according to the instructions.

[0072] (2) PCR amplification verification:

[0073] The primers Contig-1F / R and Contig-2F / R (obtained in Example 1) were used to perform PCR verification on the above-extracted mandarin fish DNA samples.

[0074] The total PCR amplification reaction system was 20 μL, including 10 μL of Kangweishiji 2× Taq MasterMix (Beijing Kangweishiji Biotechnology Co., Ltd.), 0.8 μL of upstream and downstream primers (10 μmol / L), 50 ng of template DNA, and the rest was washed with ddH 2 O added.

[0075] The PCR reaction procedure was as follows: first, pre-denaturation at 94°C for 2 min; then denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and then final extension at 72°C for 5 min.

[0076] After the PCR reaction, the product was subjected to 1% agarose gel electrophoresis. The results showed that primers Contig-1F / R and Contig-2F / R could amplify bands in 16 normal male and female fish DNA samples. At this time, the molecular size of the X chromosome-specific band amplified by primers Contig-1F / R was about 935bp; the molecular size of the X chromosome-specific band amplified by Contig-2F / R was about 850bp. No X chromosome-specific band could be amplified in the DNA samples of 6 super male fish ( Figure 6 :Contig-1F / R amplification results and Figure 7 :Contig-2F / R amplification result diagram). This indicates that molecular markers Contig-1F / R and Contig-2F / R can be used to identify the sex chromosome composition of male mandarin fish.

[0077] Example 3

[0078] This embodiment provides an application of the above-mentioned Siniperca chuatsi X chromosome molecular marker primers for all-male breeding of Siniperca chuatsi, comprising the following steps:

[0079] (1) When the fry of Siniperca chuatsi was cultured to 10 days old, 500 mg / kg of dihydroxyestrone (E 2 The eel feed was fed to the bait fish, and the hormone feed was fed to the mandarin fish. The feeding was continued for two months. Natural light was used during the breeding period. The water temperature was 26-28℃ and the fish were fed with full food.

[0080] (2) Using the Y chromosome molecular marker primers previously obtained by this laboratory, XY pseudo-female fish were screened out (see application number 201911100842.3), and then mated with XY normal male fish. The offspring fry were cultured to 30 days of age, and the offspring were screened using the method in Example 2 to obtain super male fish with a YY genotype. The specific process is referred to Example 2.

[0081] (3) The YY super male fish identified by the marker is raised until sexual maturity, and then mated with normal XX female fish during the breeding season. The offspring obtained are all male fish.

[0082] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and modification based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be as described in the claims.

Claims

1. A molecular marker primer for chromosome X of Siniperca chuatsi, Its characteristics are: The primers are primer pair 1 or primer pair 2, the primer pair 1 includes a Contig-1 upstream primer and a Contig-1 downstream primer, the primer pair 2 includes a Contig-2 upstream primer and a Contig-2 downstream primer, wherein the nucleotide sequence of the Contig-1 upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the Contig-1 downstream primer is shown in SEQ ID NO: 2, the nucleotide sequence of the Contig-2 upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the Contig-2 downstream primer is shown in SEQ ID NO:

4.

2. A method for identifying super male fish of Siniperca chuatsi YY, Its characteristics are The following steps are involved: (1) Preparing DNA samples of Siniperca chuatsi; (2) using the DNA sample in step (1) as a template, and using the primer pair 1 or the primer pair 2 in claim 1 to perform PCR amplification, and performing electrophoresis detection after the amplification reaction is completed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

3. The method for identifying the super male fish of Siniperca chuatsi YY according to claim 2, Its characteristics are: In step (2), the DNA sample in step (1) is used as a template, and primer pair 1 is used to perform PCR amplification. After the amplification reaction is completed, electrophoresis detection is performed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band of about 935 bp, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band of about 935 bp, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

4. The method for identifying super male fish of Siniperca chuatsi YY according to claim 2, Its characteristics are: In step (2), the DNA sample in step (1) is used as a template, and primer pair 2 is used to perform PCR amplification. After the amplification reaction is completed, electrophoresis detection is performed. If the electrophoresis result shows that the amplified product has an X chromosome-specific band of about 850 bp, the Siniperca chuatsi DNA sample detected at this time is an XX female Siniperca chuatsi or an XY male Siniperca chuatsi. If the electrophoresis result shows that the amplified product does not have an X chromosome-specific band of about 850 bp, the Siniperca chuatsi DNA sample detected at this time is a YY super male Siniperca chuatsi.

5. Use of the primers according to claim 1 in all-male breeding of Siniperca chuatsi.

6. Application of the method according to claim 2 in all-male breeding of Siniperca chuatsi.

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