A sex-specific molecular marker for Changchun bream and its application

Abstract

The invention discloses a sex-specific molecular marker and application of Changchun bream, belonging to the field of fish genetics. The invention uses the 2b-RAD high-throughput sequencing method to obtain a male Changchun bream-specific short sequence, and obtains a male-specific sex identification molecular marker of Changchun bream by comparing it with the whole genome sequence of male Changchun bream, and its sequence is shown in SEQ ID NO.2. The invention can be used as a powerful tool for genetic identification of sex of Changchun bream, and has the advantages of accuracy, efficiency, simplicity, etc.

Description

Technical Field

[0001] The invention belongs to the field of fish genetics, and in particular relates to a sex-specific molecular marker of Changchun bream and its application. Background Art

[0002] The study of the sex determination mechanism of fish is considered to be one of the hot issues in evolutionary biology. It has important theoretical and practical significance and has attracted much attention from biologists. Bony fish are ancient vertebrates with complex and diverse sex determination systems (Devlin RH, Nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture. 2002; 208(3–4): 191–364). Therefore, the study of sex determination in fish will help to understand the evolution of sex determination in vertebrates. There are currently three main methods for studying the sex determination system: cytogenetic methods, breeding experiments, and the discovery of sex-specific molecular markers (Gamble T, Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. Mol Ecol Resour. 2014; 14(5): 902–913). Among them, the cytogenetic method is limited to most species that lack heteromorphic sex chromosomes; while the objects of breeding experiments are mainly concentrated on species with successful breeding techniques. Therefore, identification of sex-specific molecular markers is considered a powerful approach to study the genetic basis of sex determination in species.

[0003] Changchun bream (Parabramis pekinensis) belongs to the genus Parabramis of the family Cyprinidae. It is also known as long-bodied bream and grass bream. It is commonly found in rivers and lakes across China. Changchun bream generally lives in the middle and lower layers of water bodies and is a herbivorous fish. Due to its rapid growth, strong adaptability, wide source of bait, and high population yield, it is suitable for breeding in ponds, reservoirs, lakes and other waters. Because of its delicious taste, tender meat and high protein content, it is deeply loved by consumers and is a top-quality freshwater fish.

[0004] Currently, there are obvious phenotypic differences between males and females of many farmed fish, such as body size and growth rate. For example, male Nile tilapia grow faster than females, while female sea bass and rainbow trout grow faster and larger than males. Most fish can only be sexed at sexual maturity or until adulthood, which greatly hinders the sex identification of early fish. Female Changchun bream grows 10-15% faster than males, and no research on sex-controlled breeding of Changchun bream has been reported. Therefore, the development of rapid, economical, accurate and reliable molecular biological methods for sex identification is of great significance for understanding the reproductive biology of artificially cultured species and the genetic factors that determine sex differences, as well as guiding artificial breeding. The development of sex-specific molecular markers for Changchun bream is of great significance for breeding research and practice such as sex-controlled breeding or molecular marker-assisted breeding of Changchun bream.

[0005] Simplified genome sequencing is a technology that combines restriction endonucleases and high-throughput sequencing that has been developed in recent years. It uses different restriction endonucleases to perform enzyme digestion at specific incisions of genomic DNA, enriches the digested fragments and uses them as templates to construct a high-throughput sequencing library and sequence them, and finally obtains the sequences of a large number of digested fragments. This method greatly reduces the complexity of the genome and is relatively low in cost. It has become a general and efficient typing method (Davey, J Wetal. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet. 2011.12, 7 499-510). Summary of the invention

[0006] The purpose of the present invention is to provide a sex-specific molecular marker and application of Changchun bream in view of the fact that there is no convenient method for identifying the sex of Changchun bream in the prior art. The present invention can accurately identify the genetic sex of Changchun bream and lay a technical foundation for batch identification of the sex of Changchun bream.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The method for developing sex-specific molecular markers of bream of Changchun comprises the following steps:

[0009] (1) Identification of male bream-specific short sequences

[0010] Five female and five male Changchun breams of known sex were used as samples, and the tail fin was cut to extract total DNA. The sequencing library was constructed according to the 2b-RAD method, and the sequencing library was sequenced after passing the inspection. After filtering the sequencing data, Stacks v1.31 was used to identify sex-specific sequences, and a sequence was obtained that appeared in all five male Changchun breams, but not in the five female Changchun breams. This sequence was a male Changchun bream-specific short sequence, and its nucleic acid sequence was shown in SEQ ID NO.1.

[0011] (2) Genome sequencing of male Changchun bream

[0012] The short sequence identified in step (1) is only 32 bp, which cannot be used for common PCR primer design, so a longer genome sequence is required. Therefore, one of the five male Changchun breams was subjected to high-throughput whole genome sequencing, and then SoapDenovo v1.2.0 software was used for de novo assembly to obtain the whole genome sequence of the male Changchun bream.

[0013] (3) The male Changchun bream-specific short sequence obtained in (1) was subjected to BLAST search in the male Changchun bream whole genome sequence obtained in (2) to obtain a 450 bp long sequence, the nucleic acid sequence of which is shown in SEQ ID NO.2.

[0014] A sex-specific molecular marker for Changchun bream is a sequence in the genome of male Changchun bream containing the sequence shown in SEQ ID NO.1.

[0015] In some embodiments, the nucleotide sequence of the sex-specific molecular marker of Changchun bream is shown as SEQ ID NO.2.

[0016] A primer capable of amplifying the full length or part of the Changchun bream sex-specific molecular marker.

[0017] In some embodiments, the sequences of the primers are as follows: F: CCTTCAATGCTCATCCAT (SEQ ID NO. 3), R: TTGTTGCTTTCCGTGAGT (SEQ ID NO. 4).

[0018] Application of the above-mentioned Changchun bream sex-specific molecular markers or primers in identifying the sex of Changchun bream.

[0019] A method for identifying the sex of Changchun bream comprises the following steps: extracting genomic DNA of the Changchun bream to be tested, performing PCR amplification using the above primers, if a specific fragment can be amplified, the sex of the Changchun bream to be tested is male, and if no specific fragment can be amplified, the sex of the Changchun bream to be tested is female.

[0020] In the method described, the tail fin of the Changchun bream to be tested is cut to extract genomic DNA.

[0021] In the method, when the sequences of the primers are as shown in SEQ ID NOs. 3 and 4, the length of the amplified specific fragment is 450 bp.

[0022] Application of the above-mentioned Changchun bream sex-specific molecular markers in Changchun bream breeding.

[0023] Advantages and beneficial effects of the present invention: The molecular markers and primers of the present invention can be used as powerful tools for genetic identification of sex of Changchun bream, and have the advantages of accuracy, high efficiency, simplicity and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The accuracy of primer PapSex-11-1 was verified using bream of known sex.

[0025] Figure 2 In order to use primer PapSex-11-1 to identify the sex of the offspring of a Changchun bream population of unknown sex, there are 8 male individuals and 7 female individuals as shown in the figure, and the sex ratio is close to 1:1, indicating that the Changchun bream population is in a random mating state, with a similar sex ratio of females and males, and the sex determination mechanism may be female isogametic and male anisogametic. DETAILED DESCRIPTION

[0026] The present invention uses low-cost simplified genome sequencing to find male Changchun bream-specific short sequences, and uses the specific sequences as indexes to extend the sequences in the male Changchun bream genome to obtain long fragment sequences that can be used to design common PCR primers. Specifically, the process includes the following steps:

[0027] (1) Identification of male bream-specific short sequences

[0028] Five female and five male Changchun breams of known sex were used as samples, and the tail fin was cut to extract total DNA. The sequencing library was constructed according to the 2b-RAD method, and the sequencing library was sequenced after passing the inspection. After filtering the sequencing data, Stacks v1.31 was used to identify sex-specific sequences, and a sequence was obtained that appeared in all five male Changchun breams, but not in the five female Changchun breams. This sequence was a male Changchun bream-specific short sequence, and its nucleic acid sequence was shown in SEQ ID NO.1.

[0029] (2) Genome sequencing of male Changchun bream

[0030] The short sequence identified in step (1) is only 32 bp, which cannot be used for common PCR primer design, so a longer genome sequence is required. Therefore, one of the five male Changchun breams was subjected to high-throughput whole genome sequencing, and then SoapDenovo v1.2.0 software was used for de novo assembly to obtain the whole genome sequence of the male Changchun bream.

[0031] (3) The male Changchun bream specific short sequence obtained in (1) was subjected to BLAST search in the male Changchun bream whole genome sequence obtained in (2) to obtain a 450 bp long sequence, the nucleic acid sequence of which is shown in SEQ ID NO. 2. Based on this long sequence, PCR primers were designed to obtain a pair of primers that can accurately distinguish the sex of Changchun bream with 100%, the primer name is PapSex-11-1, and the primer sequence is shown in Table 1.

[0032] Table 1 Information of male-specific molecular marker primers of Changchun bream

[0033]

[0034] The present invention is further described below in conjunction with specific embodiments.

[0035] [Example 1] Identification of male bream specific short sequences This example mainly includes the following steps, namely, enzyme digestion, ligation, amplification and recovery.

[0036] (1) Enzyme digestion:

[0037] The 2b-RAD database was constructed for 5 female and 5 male Changchun breams of known sex. The specific process is as follows: (The following is the amount used for each individual)

[0038] gDNA 5.0 μL

[0039] BSAXI (1U / μL) 0.5μL

[0040] CutSmart Buffer 0.5μL

[0041] After mixing, incubate at 37°C for 1 hour.

[0042] (2) Connection:

[0043] First, prepare the ligation adapter. For adapter 1, mix 10 μM P1 (3β) and an equal volume of 10 μM anti-P primer and store at -20°C for later use. For adapter 2, mix 10 μM P2 (5β) and an equal volume of 10 μM anti-P primer and store at -20°C for later use.

[0044]

[0045] The above 20 μL mixture was added to the first step reaction product, incubated at 4°C for 8 hours and then used.

[0046] (3) Amplification:

[0047] Prepare the following mixture for each individual:

[0048]

[0049]

[0050] The above mixture was placed in a PCR instrument, and the program parameters of the PCR instrument were set as follows: 70°C pre-denaturation for 30 seconds, (95°C denaturation for 30 seconds, 65°C annealing for 2 minutes, 72°C extension for 30 seconds) for 16 cycles; the PCR product was stored at 4°C.

[0051] (4) Recycling:

[0052] The PCR product was electrophoresed on a 2% agarose gel, the voltage was set to 220V, and the electrophoresis time was 20 minutes. The gel after electrophoresis was stained with 1% ethidium bromide (EB) solution, and each gel was scanned and photographed with a JS-A380 (Shanghai Peiqing) gel imager. Finally, the target band (about 170bp) was cut out and DNA was recovered using the EZNA D2501-02 kit. The recovered product can be directly sequenced using ILLUMINA HiSeq2500.

[0053] After filtering the FASTQ format sequence generated by the sequencing machine, Stacks v1.31 software was used to reconstruct the site and identify the sex-specific site. Finally, a short sequence specific to male Changchun bream was obtained. This sequence was present in all 5 male Changchun breams, but not in all 5 female Changchun breams. Its nucleic acid sequence is shown in SEQ ID NO.1.

[0054] [Example 2] Sequencing of the whole genome of male Changchun bream and extension of male Changchun bream-specific short sequences

[0055] (1) One male bream was selected from five for whole genome sequencing, and then the whole genome was assembled using SOAPDENOVO v1.20.

[0056] (2) The male Changchun bream-specific short sequence identified in Example 1 was subjected to BLAST search in the whole genome to obtain a 450 bp long sequence, whose nucleic acid sequence is shown in SEQ ID NO.2.

[0057] [Example 3] Primer design based on male Changchun bream specific long sequence and its performance verification

[0058] A) 36 breams (17 females and 19 males) of known sex were selected from a cultured population of breams for PCR verification. The experimental conditions were as follows:

[0059] The total PCR amplification reaction system is 12.5 μL:

[0060]

[0061] PCR Primer Information:

[0062] PapSex-11-1F:CCTTCAATGCTCATCCAT,

[0063] PapSex-11-1R:TTGTTGCTTTCCGTGAGT.

[0064] The PCR reaction conditions are as follows:

[0065]

[0066]

[0067] The best effect was achieved after 35 cycles of amplification.

[0068] The PCR products were electrophoresed on 2% agarose gel at 220 V for 20 minutes. The gel after electrophoresis was stained with 1% ethidium bromide (EB) solution, and each gel was scanned and photographed using a JS-A380 (Shanghai Peiqing) gel imager.

[0069] The results showed that 36 pigs were amplified and verified by PapSex-11-1 primers, including 17 females and 19 males. The results of molecular sex identification corresponded completely to the results of gonadal anatomy of male and female. Figure 1 .

[0070] B) A cultured population of unknown sex of Changchun bream was used for verification. The verification conditions were as shown in step A of this embodiment. A total of 126 offspring were verified, including 64 females and 62 males, with a sex ratio close to 1:1. The electrophoresis diagram of some individuals is shown in Figure 2 .

Claims

1. A sex-specific molecular marker for bream, characterized in that: The nucleotide sequence of the molecular marker is shown as SEQ ID NO.

2.

2. A primer, characterized in that: The primers are capable of amplifying the full length of the sex-specific molecular marker of Changchun bream described in claim 1.

3. The primer according to claim 2, characterized in that: The sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. Use of the Changchun bream sex-specific molecular marker described in claim 1 in identifying the sex of Changchun bream.

5. Use of the primers described in claim 2 or 3 in identifying the sex of bream.

6. A method for identifying the sex of bream, characterized in that: The method comprises the following steps: extracting genomic DNA of the Changchun bream to be tested, performing PCR amplification using the primers described in claim 2 or 3, if a specific fragment can be amplified, the gender of the Changchun bream to be tested is male, and if no specific fragment can be amplified, the gender of the Changchun bream to be tested is female.

7. The method for identifying the sex of bream according to claim 6, characterized in that: When the sequences of the primers are as shown in SEQ ID NO.3 and SEQ ID NO.4, the length of the amplified specific fragment is 450 bp.

8. Use of the Changchun bream sex-specific molecular marker described in claim 1 in Changchun bream breeding.

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