CN115976229A - Vinca bream sex specific molecular marker and application - Google Patents
Vinca bream sex specific molecular marker and application Download PDFInfo
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- CN115976229A CN115976229A CN202211631691.6A CN202211631691A CN115976229A CN 115976229 A CN115976229 A CN 115976229A CN 202211631691 A CN202211631691 A CN 202211631691A CN 115976229 A CN115976229 A CN 115976229A
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Abstract
The invention discloses a specific molecular marker of vinca bream sex and application thereof, belonging to the field of fish genetics. The invention utilizes a 2b-RAD high-throughput sequencing method to obtain a male peripeh masanthus pekinensis specific short sequence, and obtains a male sex identification molecular marker of the peripeh masanthus pekinensis through comparison with a whole genome sequence of the peripeh masanthus pekinensis, wherein the sequence of the marker is shown as SEQ ID NO. 2. The invention can be used as a powerful tool for genetic identification of parabramis pekinensis genders, and has the advantages of accuracy, high efficiency, simplicity, convenience and the like.
Description
Technical Field
The invention belongs to the field of fish genetics, and particularly relates to a vinca bream sex specific molecular marker and application thereof.
Background
The research on sex determination mechanism of fish is considered as one of the hot problems of evolutionary biology, has important theoretical and practical significance, and is concerned by biologists. Teleosts are ancient vertebrates with a complex variety of sex determination systems (Devlin RH, nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental infections. Aquaculture.2002;208 (3-4): 191-364). Therefore, research on fish sex determination will help to understand the evolution of vertebrate sex determination. There are three main approaches to the current gender determination system: cytogenetic methods, breeding experiments and the discovery of sex-specific molecular markers (Gamble T, zarkower D. Identification of a seat-specific molecular markers using restriction site-associated DNAsequencing. Mol Ecol resource 2014;14 (5): 902-913). Wherein cytogenetic methods are limited to most species lacking a heteromorphic sex chromosome; while the subjects of breeding experiments have mainly focused on species with successful breeding techniques. Therefore, the identification of sex-specific molecular markers is considered to be a powerful method to study the genetic basis for sex determination in species.
The bream of pernicious pekinensis belongs to the family Cyprinidae (Cyprinidae) of bream, also called long-body bream, grass bream and the like, and is commonly found in rivers and lakes in China. The parabramis pekinensis generally lives in the middle and lower layers of a water body, is herbivorous fish, and is suitable for culture in ponds, reservoirs, lakes and other water areas due to rapid growth, strong adaptability, wide bait sources and high population yield. The fresh-water fish is a good product in fresh-water fish because of delicious taste, tender and smooth meat quality and high protein content, and is deeply favored by consumers.
There are currently significant phenotypic differences between males and females in many farmed fish, such as body size and growth rate. For example, nile tilapia grows faster in males than in females, while sea bass and rainbow trout, and the like, grow faster and larger in females than in males. Most fish can not be sexed at the sexual maturity stage or until adult, which greatly hinders sex identification of early fish. The growth speed of female bream of Changchun is 10-15% faster than that of male, and the sex control breeding research of the bream of Changchun has not been reported. Therefore, the development of a rapid, economic, accurate and reliable molecular biology method for sex determination is of great significance for understanding the reproductive biology of artificially cultured species and the genetic factors determining sex differences and guiding artificial breeding. The development of the sex-specific molecular marker of the bream with catharanthus roseus is of great significance to breeding research and practice such as sex-controlled breeding of the bream with catharanthus roseus or molecular marker-assisted breeding.
Simplified genome sequencing is a technology developed in recent years that combines restriction enzymes with high throughput sequencing. The method utilizes different restriction enzymes to perform enzyme digestion on specific cuts of genome DNA, collects enzyme digestion fragments, then uses the enzyme digestion fragments as a template to construct a high-throughput sequencing library and perform sequencing, and finally obtains sequences of a large number of enzyme digestion fragments. The method greatly reduces the complexity of genome, has lower cost, and is a universal and efficient typing method (Davey, J.W.et. Genome-wide genetic marker discovery and genotyping next-generation sequencing. Nat Rev Genet.2011.12,7 499-510) at present.
Disclosure of Invention
The invention aims to provide a specific molecular marker of the bream of peruvian periwinkle and application thereof aiming at the situation that the prior art has no convenient method for identifying the sex of the bream of peruvian periwinkle. The method can accurately identify the genetic sex of the bream under the periwinkle, and lays a technical foundation for identifying the sex of the bream under the periwinkle in batches.
The purpose of the invention is realized by the following technical scheme:
the development method of the vinca bream sex specific molecular marker comprises the following steps:
(1) Identification of male peribramis pekinensis specific short sequence
5, taking 5-female 5-male pernema pekinensis with known sex as a sample, extracting total DNA by using tail-cutting fins, constructing a sequencing library according to a 2b-RAD method, and performing sequencing on a computer after the sequencing library is qualified. After filtering the sequencing data, performing sex specific sequence identification by using Stacks v1.31 to obtain a sequence which is completely present in 5-tailed male pernema rosea but is not present in 5-tailed female pernema rosea, namely a male pernema rosea special short sequence, and the nucleic acid sequence of the sequence is shown as SEQ ID NO. 1.
(2) Male vinca bream genome sequencing
The short sequence identified in step (1) is only 32bp, and common PCR primer design cannot be carried out, so that a longer genome sequence is required. Thus, one tail of the 5-tailed male perrama vinata was subjected to high-throughput whole genome sequencing and then de novo assembly using the Soap Denovo v1.2.0 software to obtain the whole genome sequence of the male perrama vinata.
(3) Performing BLAST search on the specific short sequence of the male pernema pekinensis obtained in the step (1) in the whole genome sequence of the male pernema pekinensis obtained in the step (2) to obtain a 450bp long sequence, wherein the nucleic acid sequence of the sequence is shown as SEQ ID NO. 2.
A sex specific molecular marker of bream belonging to Catharanthus roseus is a sequence containing a sequence shown by SEQ ID NO.1 in genome of bream belonging to Male Catharanthus roseus.
In some embodiments, the nucleotide sequence of the vinca bream sex-specific molecular marker is shown in SEQ ID No. 2.
A primer capable of amplifying the full length or part of the vinca bream sex-specific molecular marker.
In some embodiments, the primers have the following sequences: f: CCTTCAATGCTCATCCAT (SEQ ID NO. 3), R: TTGTTGCTTTCCGTGAGT (SEQ ID NO. 4).
The specific molecular marker or primer of the bream with catharanthus roseus sex is applied to identification of the bream with catharanthus roseus sex.
A method for identifying sex of parabramis pekinensis comprises the following steps: extracting genome DNA of the bream to be detected, performing PCR amplification by using the primers, wherein if a specific fragment can be amplified, the sex of the bream to be detected is male, and if the specific fragment cannot be amplified, the sex of the bream to be detected is female.
In the method, the tail fin of the bream to be detected is cut to extract genome DNA.
In the method, when the sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO.4, the length of the amplified specific fragment is 450bp.
The sex specific molecular marker of the bream cultured in the periwinkle is applied to breeding of the periwinkle.
The invention has the advantages and beneficial effects that: the molecular marker and the primer can be used as a powerful tool for genetic identification of the parabramis pekinensis and have the advantages of accuracy, high efficiency, simplicity and convenience and the like.
Drawings
FIG. 1 shows the accuracy of PapSex-11-1 primer using peripekana pekinensis of known sex.
Fig. 2 shows that 8 male individuals and 7 female individuals with sex ratio close to 1:1 are shown in the figure for sex identification of progeny of the parabramis pekinensis population with unknown sex by using the primer paplex-11-1, which indicates that the parabramis pekinensis population is in a random mating state, the female and male proportions are close, and the sex determination mechanism may be male and female concordant.
Detailed Description
The invention uses low-cost simplified genome sequencing to search a specific short sequence of bream under male periwinkle, and takes the specific sequence as an index to carry out sequence extension in the whole genome of bream under male periwinkle so as to obtain a long fragment sequence which can be used for designing a common PCR primer. The method specifically comprises the following steps:
(1) Identification of male peribramis pekinensis specific short sequence
5, taking 5-female 5-male pernema pekinensis with known sex as a sample, extracting total DNA by using tail-cutting fins, constructing a sequencing library according to a 2b-RAD method, and performing sequencing on a computer after the sequencing library is qualified. After filtering the sequencing data, performing sex specific sequence identification by using Stacks v1.31 to obtain a sequence which is completely present in 5-tailed male pernema rosea but is not present in 5-tailed female pernema rosea, namely a male pernema rosea special short sequence, and the nucleic acid sequence of the sequence is shown as SEQ ID NO. 1.
(2) Male vinca bream genome sequencing
The short sequence identified in step (1) is only 32bp, and common PCR primer design cannot be carried out, so that a longer genome sequence is required. Thus, one tail of the 5-tailed male perrama vinata was subjected to high-throughput whole genome sequencing and then de novo assembly using the Soap Denovo v1.2.0 software to obtain the whole genome sequence of the male perrama vinata.
(3) Performing BLAST search on the specific short sequence of the male pernema pekinensis obtained in the step (1) in the whole genome sequence of the male pernema pekinensis obtained in the step (2) to obtain a 450bp long sequence, wherein the nucleic acid sequence of the sequence is shown as SEQ ID NO. 2. PCR primer design is carried out on the basis of the long sequence to obtain a pair of primers capable of accurately distinguishing the sex of the parabramis pekinensis with 100 percent, the name of the primer is PapSex-11-1, and the sequence of the primer is shown in a table 1.
TABLE 1 information table of male specific molecular marker primers of bream of Changchun
The invention is further illustrated by the following examples.
Example 1 identification of specific short sequence of bream of male periwinkle. This example mainly comprises the following steps, i.e., digestion, ligation, amplification and recovery.
(1) Enzyme digestion:
2b-RAD library construction is carried out on 5-male periantha pekinensis with known sex, and the specific process is as follows: (the amount used per individual is as follows)
gDNA 5.0μL
BSAXI(1U/μL)0.5μL
CutSmart Buffer 0.5μL
After mixing, the mixture was incubated at 37 ℃ for 1 hour.
(2) Connecting:
first, a connection joint is prepared. For linker 1, 10. Mu.M P1 (. Beta.) and an equal volume of 10. Mu.M anti-P primer were mixed and stored at-20 ℃ until use. For linker 2, 10. Mu.M P2 (5. Beta.) and an equal volume of 10. Mu.M anti-P primer were mixed and stored at-20 ℃ until use.
mu.L of the mixture was added to the reaction product of the first step and incubated at 4 ℃ for 8 hours for further use.
(3) Amplification:
the following mixtures were prepared for each individual:
placing the mixture into a PCR instrument, and setting program parameters of the PCR instrument as follows: pre-denaturation at 70 ℃ for 30 seconds, (denaturation at 95 ℃ for 30 seconds, annealing at 65 ℃ for 2 minutes, and extension at 72 ℃ for 30 seconds) for 16 cycles; the PCR product was stored at 4 ℃.
(4) And (3) recovering:
the PCR products were electrophoresed on a 2% agarose gel, the voltage was set at 220V, and the electrophoresis time was 20 minutes. The gel after electrophoresis was stained with a 1% Ethidium Bromide (EB) solution, each gel was scanned with a JS-a380 (shanghai culture) gel imager and stored by photographing, and finally the target band (about 170 bp) was cut off and DNA was recovered using a D2501-02 kit from e.z.n.a company, and the recovered product was directly sequenced using illuminhiseq 2500.
After filtering the sequencing-off FASTQ-formatted sequences, site reconstruction and sex-specific site identification were performed using Stacks v1.31 software. Finally, a short sequence specific to male perianthus pekinensis was obtained, which sequence is present in 5-tailed male perianthus pekinensis but absent in 5-tailed female perianthus pekinensis. The nucleic acid sequence is shown in SEQ ID NO. 1.
Example 2 Whole genome sequencing and extension of specific short sequence of bream of Male Catharanthus roseus
(1) One of 5 male perrama vinosa was selected for whole genome sequencing followed by whole genome assembly using SOAPDENOVO v 1.20.
(2) The specific short sequence of the male pernema rosea identified in the example 1 is subjected to BLAST search in the whole genome to obtain a long sequence with the length of 450bp, and the nucleic acid sequence of the long sequence is shown as SEQ ID NO. 2.
Example 3 primer design and Performance verification based on specific Long sequence of bream of Male Catharanthus roseus
A) 36 tails (female 17 tails and male 19 tails) are selected from cultured vinca bream populations with known sexes for PCR verification, and the experimental conditions are as follows:
the total PCR amplification reaction was 12.5. Mu.L:
PCR primer information:
PapSex-11-1F：CCTTCAATGCTCATCCAT，
PapSex-11-1R：TTGTTGCTTTCCGTGAGT。
the PCR reaction conditions were as follows:
the amplification is carried out for 35 cycles, and the effect is optimal.
The PCR products were electrophoresed on a 2% agarose gel, the voltage was set at 220V, and the electrophoresis time was 20 minutes. The gel after electrophoresis was stained with a 1% Ethidium Bromide (EB) solution, and each gel was scanned and stored by photographing with a JS-A380 (Shanghai Peizhen) gel imager.
The results showed that amplification of the PapSex-11-1 primer verified 36 tails, of which 17 tails female and 19 tails male. The sex molecule identification result completely corresponds to the sex gland dissection result one by one. A partial individual electropherogram is shown in FIG. 1.
B) Verification is carried out by using cultured perianthus pekinensis populations with unknown sexes under the verification conditions shown in step A of the embodiment, and 126 offspring are verified in total, wherein the proportion of female 64 tail, male 62 tail and sex is close to 1:1. A partial individual electropherogram is shown in FIG. 2.
Claims (10)
1. A pecan bream sex specific molecular marker is characterized in that: the molecular marker is a segment of sequence containing a sequence shown in SEQ ID NO.1 in the genome of bream of male periwinkle.
2. The vinca bream sex-specific molecular marker of claim 1, wherein: the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 2.
3. A development method of a vinca bream sex specific molecular marker is characterized in that: the method comprises the following steps:
(1) Identification of male peribram peribracteatum specific short sequence
Taking 5-female 5-male perbractea pekinensis with known sex as a sample, extracting total DNA (deoxyribonucleic acid) by using tail-cutting fins, constructing a sequencing library according to a 2b-RAD (radio-assisted radiation) method, and performing sequencing on a computer after the sequencing library is qualified; filtering sequencing data, and performing sex specific sequence identification by using Stacks v1.31 to obtain a sequence which is completely appeared in 5-tailed male pernema rosea and is not appeared in 5-tailed female pernema rosea, wherein the sequence is a male pernema rosea specific short sequence, and the nucleic acid sequence of the sequence is shown as SEQ ID NO. 1;
(2) Male vinca bream genome sequencing
The short sequence identified in the step (1) is only 32bp, can not be used for common PCR primer design, and needs a longer genome sequence; carrying out high-throughput whole genome sequencing on one tail of the 5-tailed male pernema pekinensis, and carrying out de novo assembly by using Soap Denovo v1.2.0 software to obtain a whole genome sequence of the male pernema pekinensis;
(3) Performing BLAST search on the specific short sequence of the male pernema pekinensis obtained in the step (1) in the whole genome sequence of the male pernema pekinensis obtained in the step (2) to obtain a 450bp long sequence, wherein the nucleic acid sequence of the sequence is shown as SEQ ID NO. 2.
4. A primer, characterized by: the primers can amplify the full length or part of the vinca bream sex-specific molecular marker in claim 1 or 2.
5. The primer according to claim 4, wherein: the sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4.
6. Use of the vinca bream sex-specific molecular marker of claim 1 or 2 for identification of vinca bream sex.
7. Use of the primers of claim 4 or 5 for identifying the sex of parabramis pekinensis.
8. A method for identifying sex of parabramis pekinensis is characterized by comprising the following steps: the method comprises the following steps: extracting genome DNA of the bream to be detected, performing PCR amplification by using the primer in claim 4 or 5, wherein if a specific fragment can be amplified, the sex of the bream to be detected is male, and if the specific fragment cannot be amplified, the sex of the bream to be detected is female.
9. The method for identifying the sex of vinca bream according to claim 8, wherein: when the sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO.4, the length of the amplified specific fragment is 450bp.
10. Use of the vinca bream sex-specific molecular marker of claim 1 or 2 in breeding of vinca bream.
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| CN116836989A (en) * | 2023-06-30 | 2023-10-03 | 中国长江三峡集团有限公司中华鲟研究所 | Male molecular marker of bream and application thereof |
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| CN116836989A (en) * | 2023-06-30 | 2023-10-03 | 中国长江三峡集团有限公司中华鲟研究所 | Male molecular marker of bream and application thereof |
| CN116836989B (en) * | 2023-06-30 | 2024-01-05 | 中国长江三峡集团有限公司中华鲟研究所 | Male molecular marker of bream and application thereof |
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