Sex identification marks, identification methods and applications of black carp

Abstract

The present invention relates to the field of molecular ecology, and in particular to a sex identification marker, an identification method and an application of a black carp. The black carp sex identification marker is selected from at least one of Mar26, Mar28, Mar37 and Mar38; wherein Mar26 is composed of an upstream primer shown in SEQ ID NO: 51 and a downstream primer shown in SEQ ID NO: 52; Mar28 is composed of an upstream primer shown in SEQ ID NO: 55 and a downstream primer shown in SEQ ID NO: 56; Mar37 is composed of an upstream primer shown in SEQ ID NO: 73 and a downstream primer shown in SEQ ID NO: 74; Mar38 is composed of an upstream primer shown in SEQ ID NO: 75 and a downstream primer shown in SEQ ID NO: 76. Furthermore, the sex identification marker is Mar28, and the provided sex identification marker can accurately and efficiently identify the sex of the black carp without being affected by the developmental period. The method for genetic sex identification of black carp established by the present invention can be helpful for application in the monosexual breeding of black carp.

Description

Technical Field

[0001] The invention relates to the field of molecular ecology, and in particular to a sex identification mark, an identification method and an application of a carp. Background Art

[0002] The black carp is an important freshwater farmed fish that exhibits sexual dimorphism in terms of body shape and color. Male black carps not only grow faster than females, but also have a blue-green body color during the breeding season, which makes them highly ornamental. Females are bland and unpopular among consumers. Therefore, achieving all-male farming of black carps can effectively increase species production and economic benefits. The sexual dimorphism of these farmed fish makes sex-controlled breeding increasingly important in aquaculture.

[0003] In terms of the development of sex identification markers, Qiu Qing et al. conducted RAPD amplification analysis on the testes and ovaries of the horsemouth fish and screened out 11 pairs of primers that can amplify the difference between male and female bands. However, there is no obvious external morphological difference between male and female fish in the early stage of gonadal development, so it is necessary to develop a fast and efficient sex identification marker.

[0004] In recent decades, methods such as RAPD, SSR, and AFLP have been successfully used to develop sex identification markers for a variety of fish. However, these molecular markers have defects such as low efficiency and poor repeatability. The technology of screening sex identification markers based on high-throughput sequencing can not only reduce sequencing costs and shorten sequencing cycles, but also has great advantages in the universality and repeatability of markers. Whole genome resequencing is a method of resequencing the whole genome of a species based on the genome at the chromosome level. By comparing with the reference genome, it can detect SNP, Indel and other variation information in the whole genome, thereby obtaining molecular genetic information of individuals or groups. Compared with other sequencing technologies, whole genome resequencing has the advantages of low sequencing cost and simple bioinformatics analysis, and has become an important means of developing sex identification markers. BSA is developed based on whole genome resequencing technology. It can quickly identify specific genes and variation regions in the genome and screen out markers associated with phenotypes. It is a simple and efficient trait positioning technology.

[0005] This application aims to perform BSA sequencing on male and female black carp and establish a rapid, accurate, PCR-based genetic sex identification method for black carp, laying a foundation for research related to sex determination and differentiation, sex-controlled breeding and aquaculture management of black carp. Summary of the invention

[0006] Based on the deficiencies of the prior art, the present invention aims to provide a sex identification marker, identification method and application of the black carp, the sex identification marker provided can accurately and efficiently identify the sex of the black carp without being affected by the developmental period. The method for genetic sex identification of black carp established by the present invention can be helpful for the application in the monosexual breeding of black carp.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide a sex identification marker for black carp, wherein the sex identification marker is selected from at least one of Mar26, Mar28, Mar37 and Mar38;

[0009] Wherein, Mar26 is composed of the upstream primer shown in SEQ ID NO: 51 and the downstream primer shown in SEQ ID NO: 52;

[0010] Mar28 consists of the upstream primer shown in SEQ ID NO: 55 and the downstream primer shown in SEQ ID NO: 56;

[0011] Mar37 consists of the upstream primer shown in SEQ ID NO: 73 and the downstream primer shown in SEQ ID NO: 74;

[0012] Mar38 consists of an upstream primer shown in SEQ ID NO: 75 and a downstream primer shown in SEQ ID NO: 76.

[0013] Furthermore, the sex identification marker is Mar28.

[0014] Another object of the present invention is to provide a method for identifying sex of a croaker, the method comprising:

[0015] Collect samples to be tested;

[0016] Extracting DNA from the sample to be tested to obtain DNA of the sample to be tested;

[0017] Using the above sex identification markers to perform PCR amplification on the DNA of the sample to be tested;

[0018] PCR amplification products were detected by agarose gel electrophoresis;

[0019] The sex of the mackerel was determined using electrophoresis test results.

[0020] Furthermore, the sample to be tested is muscle or tail fin tissue of black carp.

[0021] Furthermore, the method for extracting DNA from the sample to be tested is to add the cut muscle or tail fin tissue of the black carp to the lysis solution and proteinase K for thorough homogenization, then add RNase A, incubate, transfer to a fully automatic nucleic acid extraction and purification instrument to run the BMGB program, and after the operation is completed, the genomic DNA of the black carp can be obtained.

[0022] Furthermore, the method also includes using a NanoDrop One spectrophotometer to detect the concentration and purity of the genomic DNA, and using agarose gel electrophoresis to detect the integrity of the genomic DNA.

[0023] Furthermore, the PCR amplification system used in the PCR amplification includes:

[0024] 2×Rapid Taq Master Mix12.5 μL;

[0025] 1 μL each of 10 μmol / L upstream and downstream primers;

[0026] DNA 1 μL;

[0027] ddH2O9.5 μL.

[0028] Furthermore, the PCR amplification program used in the PCR amplification is:

[0029] Pre-denaturation at 94 °C for 5 min;

[0030] 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min, 30 cycles;

[0031] 72 ℃ for 10 min.

[0032] Furthermore, the concentration of the agarose gel is 1%.

[0033] Another object of the present invention is to provide an application of the above-mentioned sex identification marker or the above-mentioned identification method in the parthenogenetic breeding of black carp.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention screened 59,637 sex-related SNP sites of black carp (male heterozygous, female homozygous) by BSA, and screened 45 sex-specific regions by combining the CQ method, developed black carp sex identification markers based on sex-specific sequences, and established a method for identifying the genetic sex of black carp based on PCR. The PCR-based detection method is an accurate, rapid and economical genetic sex identification tool. For sexually mature black carp, males and females can be distinguished by body surface color during the breeding season. However, it is difficult to distinguish male and female individuals of black carp in the early developmental stage and non-breeding season by body surface morphology. The sex identification marker can accurately and efficiently identify the sex of black carp without being affected by the developmental stage. The method for genetic sex identification of black carp established by the present invention will be helpful for the development of its molecular breeding work, especially in the application of black carp unisexual breeding.

[0036] 2. The sex-specific region fragments screened by the CQ method in this application are relatively long, and the detection process is convenient and low-cost. After completing the primer design, the genetic sex of the detected species can be determined by PCR amplification and gel electrophoresis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The electrophoresis diagram of the genomic DNA of the black carp used for the construction of the resequencing library in the embodiment (Marker: 15000bp);

[0038] Figure 2 The electrophoresis diagram of PCR amplification of 4 pairs of primers in 6 tails of black carp;

[0039] Figure 3 The electrophoresis diagram of PCR amplification of Mar28 in 40 carp in the embodiment, wherein NC is a negative control and M is a DL1000 marker;

[0040] Figure 4 This is the sequence comparison result of the Mar28 amplified fragment in male and female horsemouth fish; “-” indicates a missing sequence, red indicates the base difference between male and female sequences, and the blue framed part is the primer position. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the embodiments provided are only for the purpose of illustrating the present invention and should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention all fall within the scope of the present invention.

[0042] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc., used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] Example

[0044] 1. Sample Collection and DNA Extraction

[0045] The black carp used in the experiment was collected from the breeding base of Jiangxi Qili Industrial Co., Ltd. In June 2023, 25 sexually mature male and female fish (body length 8.20±0.89 cm, body weight 7.39±2.80 g) from the same batch were collected for the construction of mixed pools for BSA sequencing. In addition, an artificially cultured population (20 adult fish of each sex: body length 12.38±0.90 cm, body weight 23.60±4.93 g) was collected in September 2023 to verify the universality of sex identification markers. The sex of black carp was identified by phenotypic observation and gonadal anatomical morphology. The muscles and tail fins were cut and immediately placed in liquid nitrogen for quick freezing and then transferred to -80 ℃ for storage until DNA extraction.

[0046] The genomic DNA of the black carp was extracted using the Baypure magnetic bead animal tissue genomic DNA extraction kit (Baypure Biotechnology, Guangzhou).

[0047] The steps are as follows: cut no more than 15 mg of muscle or tail fin tissue, add 200 μL lysis buffer and 20 μL proteinase K and fully homogenize, then add 10 μL RNase A, incubate at 60 °C for 1 h, transfer to the fully automatic nucleic acid extraction and purification instrument (BBEX-32) and run the "BMGB" program. After the operation is completed, the genomic DNA of the horse mouth fish can be obtained. The concentration and purity of the genomic DNA were detected using a NanoDropOne spectrophotometer (Thermo Fisher Scientific), and the integrity of the genomic DNA was detected by 1% agarose gel electrophoresis.

[0048] The extracted genomic DNA of 25 male and female horsemouth fish was detected by NanoDrop spectrophotometer. The results showed that the average DNA concentration was 275.92 ng / μL, and the OD260 / OD280 was between 1.8 and 2.0. The results of 1% agarose gel electrophoresis showed that the DNA bands were clear and there was no serious degradation. Figure 1 This indicates that the genomic DNA is of high quality and can be used for library construction for BSA sequencing.

[0049] 2. Library construction and BSA detection of 5E8F

[0050] The genomic DNA of male and female horsemouth fish that passed the quality inspection was mixed in equal amounts to construct a mixed pool of male and female horsemouth fish genomic DNA. The DNA samples were broken into short fragments of 200-400 bp using a Covaris ultrasonic crusher, and then end-repaired, ployA-tailed and sequenced adapters were added, purified, PCR amplified, and finally the PCR products were circularized to complete the library construction.

[0051] After the library was constructed, the DNA concentration was determined using Qubit 3.0 (Life Technologies, Carlsbad, USA) and the library was preliminarily quantified. The insert fragments of the library were detected using Agilent 2100 Bioanalyzer (Agilent, Germany) to ensure that the insert fragment size was in line with expectations. The effective concentration of the library was accurately quantified using the Q-PCR method to ensure the quality of the library. Finally, the pool was sequenced by BSA based on the DNBSEQ platform, and each pool generated 20 Gb of data.

[0052] 3. Raw data filtering and quality control

[0053] The raw image data files obtained by sequencing were converted into raw sequencing sequences (Raw reads) through base calling. Fastp (0.20.1) was used to perform quality control and filtering on the raw data to obtain valid data (Clean reads). The filtering criteria are as follows: (1) Remove the adapter sequence in the sequence; (2) Remove the polyG and polyX at the end of the reads (the minimum length is 10 bp); (3) Count the bases in the window in a sliding window manner and calculate the average quality value, and trim the sliding window with low quality; (4) Remove reads with N greater than 5; (5) Remove reads with a quality lower than 15 and a base ratio higher than 40%; (6) Remove reads with a length less than 15 bp after filtering.

[0054] Clean reads of all samples were aligned with the annotated female trout reference genome using bwa software (0.7.17). The alignment results were converted from sam files to sorted bam files and duplicates were removed using samtools software. The alignment rate and coverage were calculated using a python script.

[0055] Results: Based on BSA sequencing, the male and female pools of Ocellated octopus generated 21.54 Gb and 28.20 Gb of raw data, respectively. After data filtering and quality control, 20.73 Gb and 26.98 Gb of valid data were obtained, respectively. The Q20 was 98.96% and 99.03%, the Q30 was 95.64% and 95.92%, and the GC content was 38.95 and 38.94%, respectively (Table 1).

[0056] After deduplication of valid data, 142668885 and 186590651 sequences were obtained from male and female mixed pools, respectively. By comparing with the reference genome, 141763631 and 185468646 sequences were aligned to the reference genome, with alignment rates of 99.37% and 99.40%, respectively, and coverage of the reference genome of 96.33% and 96.44%, with average sequencing depths of 24.22X and 31.46X (results are shown in Table 2).

[0057] Table 1: BSA sequencing data statistics

[0058]

[0059] Table 2: Comparison results of male and female mixed pools and reference genomes

[0060]

[0061] 4. Identification and location of sex-related SNPs and Indels

[0062] Based on the alignment result file, GATK software (4.2.5.0) was used to detect variants in the samples, and the identified SNPs and Indels were filtered (sites with sequencing depths lower than 5X and higher than 100X were filtered out). The specific filtering criteria were as follows: the filtering parameters for SNPs were “ QD<2.0 || QUAL<30.0 || SOR>3.0 || FS>60.0 || MQ<40.0 || MQRankSum<-12.5 || ReadPosRankSum<-8.0 ”, and the filtering parameters for Indels were “ QD<2.0 || QUAL<30.0 || FS>200.0 || MQ<40.0 || ReadPosRankSum<-20.0 ”. ANNOVAR software (2020060) was used to annotate the identified SNPs and Indels.

[0063] Sites that are heterozygous in male individuals and homozygous in female individuals and located on exons were selected as sex-related differential sites. Circos (v0.69) was used to plot the distribution of sex-related mutations in the genome. The number of sex-related sites in each sliding window (100 kb) was calculated, and their distribution on the genome was visualized using Circos.

[0064] The results are as follows:

[0065] After identifying and filtering the variants in the samples using GATK software, 12,004,059 SNPs and 3,213,780 Indels were obtained in the male pool, and 12,178,212 SNPs and 3,304,134 Indels were obtained in the female pool. There were 9,079,518 heterozygous sites in the male pool and 7,488,256 homozygous sites in the female pool (see Table 3).

[0066] Based on the variant identification results, 59,637 sex-related (male heterozygous, female homozygous and located in exons) SNPs and 3,503 Indel sites were screened.

[0067] Table 3: Variation statistics in male-female mixed pools

[0068]

[0069] 5. Screening of sex-specific regions

[0070] The chromosome quotient (CQ) method was used to screen female-specific sequence regions in black carp. The CQ value in each sliding window (1 kb) was calculated as follows: CQ = Hm / Hf (Hm is the number of reads from the male pool aligned to the reference genome; Hf is the number of reads from the female pool aligned to the reference genome), and female-specific regions with CQ < 0.2 and Hf > 30 were screened (i.e., regions with reads coverage in the male pool but not in the female pool). The screened female-specific regions were checked and confirmed using the Integrative Genomic Viewer (IGV) software.

[0071] 6. Development and validation of sex identification markers

[0072] The flanking sequences of 1 kb before and after the sex-specific region were extracted, and common PCR amplification primers were designed using Primer Premier (6.0) and sent to Shanghai Sangon Biotech Co., Ltd. for synthesis. Primers for the sex-specific region were screened by two rounds of PCR amplification: first, the genomic DNA of three male and three female croakers was used as a template to preliminarily screen the primers. For primers with unclear amplification bands or no target bands, an annealing gradient was set to find the optimal annealing temperature until a clear and complete target band was obtained; for primers that met expectations, a random population of croakers (20 males and 20 females) was used to further verify the sex specificity of the primers.

[0073] PCR amplification used a 25 μL system, including 12.5 μL of 2× Rapid Taq Master Mix, 1 μL of upstream and downstream primers (10 μmol / L), 1 μL of DNA, and 9.5 μL of ddH2O. At the same time, ddH2O was used as a template as a negative control. The PCR amplification program included: 94 ℃ pre-denaturation for 5 min; 94 ℃ 30 s, 58 ℃ 30 s, 72 ℃ 2 min, 30 cycles; 72 ℃ 10 min, and 4 ℃ storage. PCR amplification products were detected by 1% agarose gel electrophoresis.

[0074] After the primers targeting the sex-specific region amplified the male and female differential bands in the verified population of black carp (20 males and 20 females), the PCR amplification products were purified and connected to the PMD-18 vector, and then transferred into DH5α Escherichia coli competent cells. The expected positive clone colonies were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Finally, SnapGene software (6.0) was used for male and female sequence comparison analysis.

[0075] The results are as follows:

[0076] CQ analysis screened 45 sex-specific regions of black carp (CQ < 0.2). After IGV inspection, 50 primer pairs were designed based on the flanking sequences and 31 sex-specific regions were verified (Table 4). Three black carp of both sexes were randomly selected for preliminary screening of the 50 primer pairs. Among them, four primer pairs (Mar26, 28, 37 and Mar38) amplified bands with male-female differences ( Figure 2 ).

[0077] The four primer pairs were validated separately, and the results showed that Mar28 amplified two bands (509 bp and 814 bp) in all 20 male individuals of the horsemouth fish, and a single band (509 bp) in all 20 female individuals ( Figure 3 ). The comparison results of male and female sequences amplified by Mar28 primer are shown in Figure 4The amplified bands of the other three primer pairs could not reflect the male-female differences in all individuals in the population.

[0078] Table 4: Primer sequences for sex-specific regions of black carp

[0079]

[0080]

[0081]

[0082] The present invention screened 59637 sex-related SNP sites of black carp (male heterozygous, female homozygous) by BSA, and screened 45 sex-specific regions by combining the CQ method, developed black carp sex genetic identification markers based on sex-specific sequences, and established a method for identifying the genetic sex of black carp based on PCR. The PCR-based detection method is an accurate, rapid and economical genetic sex identification tool. For sexually mature black carp, males and females can be distinguished by body surface color during the breeding season. However, it is difficult to distinguish male and female individuals of black carp in the early developmental stage and non-breeding season by body surface morphology. The sex identification marker can accurately and efficiently identify the sex of black carp without being affected by the developmental stage. The method for genetic sex identification of black carp established by the present invention will be helpful for the development of its molecular breeding work, especially in the application of black carp unisexual breeding.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solution requested for protection in this application.

Claims

1. A method for identifying the sex of a mackerel, characterized in that: The identification method comprises: Collect samples to be tested; Extracting DNA from the sample to be tested to obtain DNA of the sample to be tested; The sex identification marker Mar28 is used to perform PCR amplification on the DNA of the sample to be tested; Mar28 is composed of the upstream primer shown in SEQ ID NO: 55 and the downstream primer shown in SEQ ID NO: 56; PCR amplification products were detected by agarose gel electrophoresis; The sex of the black carp is determined by the electrophoresis test results. If the electrophoresis test results amplify two bands of 509 bp and 814 bp, the black carp is determined to be male; if the electrophoresis test results amplify a single band of 509 bp, the black carp is determined to be female.

2. The identification method according to claim 1, characterized in that: The sample to be tested is the muscle or tail fin tissue of the black carp.

3. The identification method according to claim 1, characterized in that: The method for extracting DNA from the sample to be tested is to add the cut muscle or tail fin tissue of the black carp into the lysis solution and proteinase K for thorough homogenization, then add RNase A, incubate, transfer to the fully automatic nucleic acid extraction and purification instrument to run the BMGB program, and after the operation is completed, the genomic DNA of the black carp can be obtained.

4. The identification method according to claim 3, characterized in that: The method also includes using NanoDropOne spectrophotometer to detect the concentration and purity of the genomic DNA, and using agarose gel electrophoresis to detect the integrity of the genomic DNA.

5. The identification method according to claim 1, characterized in that: The PCR amplification system used in the PCR amplification includes: 2×Rapid Taq Master Mix 12.5 μL; 1 μL each of 10 μmol / L upstream and downstream primers; DNA 1 μL; ddHO 9.5 μL.

6. The identification method according to claim 1 or 3, characterized in that: The PCR amplification program used in the PCR amplification is: Pre-denaturation at 94 °C for 5 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min, 30 cycles; 72 ℃ for 10 min.

7. The identification method according to claim 1, characterized in that: The concentration of the agarose gel is 1%.

8. Application of the identification method according to any one of claims 1 to 7 in the parthenogenesis of black carp.

Images