Specific molecular markers for gender identification of three subpopulations of blue gourami (Radiichthys argenteus) and application thereof

Abstract

The application discloses specific molecular markers for gender identification of a three-subspecies group of Tanakia himantegus and application thereof. The application develops specific molecular markers capable of identifying the genetic gender of the three-subspecies Tanakia himantegus through whole genome resequencing and related bioinformatics analysis, designs specific primers, and performs identification. It is verified that the genetic gender is completely consistent with the physiological gender, and the accuracy rate reaches 100%. Further, the primer pair Marker1 and / or Marker2 capable of specifically amplifying the specific molecular markers of the three-subspecies Tanakia himantegus is screened. It is shown that only one DNA band is obtained in wild male fish of the three-subspecies group through amplification, and no DNA band is obtained in female fish. The PCR product can be clearly distinguished through electrophoresis. The amplification results of a child group are consistent with those of the parent, which proves that the primer pair Marker1 and Marker2 can both be used for genetic gender identification of the three-subspecies Tanakia himantegus, and both have stability and universality in the child group, and can be widely used for genetic gender identification of the three-subspecies Tanakia himantegus.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular technology. More specifically, it relates to specific molecular markers for sex identification in Sanya populations of the medaka (a type of medaka bird). Background Technology

[0002] In vertebrates, sex determination and differentiation are highly plastic processes, influenced by both genetics and environment. Sex determination in bony fishes is primarily determined by two mechanisms: genetic sex determination (GSD), such as the XX / XY and ZZ / ZW sex determination systems, and environmental sex determination (ESD). Medaka, due to its small size, high reproductive rate, short reproductive cycle, and sensitivity to water quality and environmental changes, is an important model organism for developmental biology, environmental toxicology, and evolutionary biology. The Hainan medaka (Oryzias curvinotus), also known as the Hainan medaka, is mainly distributed in the southern waters of China, exhibiting a wide salinity tolerance, rapid growth, and early sexual maturity. Screening for sex-related molecular markers in fish allows for sex identification early in their growth and development, which is crucial for sex control in fish. As a model species, determining the genetic sex of the Hainan medaka is essential for environmental monitoring; therefore, developing relevant sex-identifying molecular markers is necessary and can provide a theoretical basis for sex control breeding in other economically important fish species.

[0003] Sex control technology is indispensable in aquaculture, and elucidating the molecular mechanisms of sex determination and differentiation in fish is a major focus in fish genetics and breeding. Current research generally considers sex-determining genes to be the most rapid and accurate specific molecular markers for sex identification. Most fish in the genus *Oryzias* possess the male sex-determining gene *dmy*. However, our research group previously confirmed through morphological and mitochondrial phylogenetic analysis of *Oryzias* collected from the Sanya estuary in Hainan that this group belongs to a new population of *Oryzias serratus*, referred to as Sanya *Oryzias* (SY-medaka). Through genetic sex identification, genome resequencing, and gonadal transcriptome analysis, we preliminarily confirmed that *Oryzias serratus* does not contain the male sex-determining gene *dmy*. Therefore, the existing Chinese patent CN107385095A, "A Primer for Rapid Identification of the Genetic Sex of *Oryzias serratus* and Its Application," is for genetic sex identification of *Oryzias serratus* populations containing the *dmy* gene and cannot be used to identify *Oryzias serratus* populations that do not contain the *dmy* gene.

[0004] In fish testing, traditional methods such as AFLP, SSR, and RAPD have been successfully used to develop sex-specific markers. However, these methods require cumbersome primer selection, and most of the obtained marker sequences are relatively short for primer design, making it difficult to amplify the target sequence. Currently, there is a lack of molecular markers for rapid identification of the genetic sex of the Sanya medaka. Summary of the Invention

[0005] This invention provides specific molecular markers and their applications for sex identification of the Sanya population of medaka, establishing a rapid, accurate, economical and reliable method for genetic sex identification of Sanya medaka.

[0006] The first objective of this invention is to provide a specific molecular marker for sex identification of the Sanya population of the medaka.

[0007] A second objective of this invention is to provide the application of specific molecular markers for sex identification in Sanya populations of the medaka.

[0008] The third objective of this invention is to provide a primer pair for identifying the sex of a Sanya population of the medaka.

[0009] The fourth objective of this invention is to provide applications for specific amplification primer pairs.

[0010] The fifth objective of this invention is to provide a kit for sex identification of the Sanya population of the medaka.

[0011] The sixth objective of this invention is to provide a method for rapidly identifying the sex of a Sanya population of medaka.

[0012] The seventh objective of this invention is to provide an application of a rapid method for identifying the sex of the Sanya population of the medaka.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention develops a specific molecular marker for identifying the genetic sex of Sanya medaka through whole-genome resequencing and related bioinformatics analysis. The molecular marker is a male-specific sequence fragment in the Sanya medaka population, and its nucleotide sequence is shown in SEQ ID NO: 1~2. Further, primers were designed and synthesized based on the sex-specific molecular marker sequence of Sanya medaka, genomic DNA was extracted, and conventional PCR amplification was performed to identify the sex of Sanya medaka. The results showed that the genetic sex and physiological sex were completely consistent, with an accuracy rate of 100%.

[0015] Therefore, the present invention provides the application of the above-mentioned specific molecular markers in identifying the sex of the Sanya population of the medaka or in preparing primers for identifying the sex of the Sanya population of the medaka.

[0016] This invention further screened and identified primer pairs capable of specifically amplifying sex-specific molecular markers in Sanya medaka, obtaining two sex-specific primer pairs, Marker1 and Marker2. Amplification using these primer pairs yielded a DNA band in wild Sanya male populations, while no DNA band was observed in females. The PCR products were clearly distinguishable by electrophoresis, and both primer pairs demonstrated stability and universality in their offspring. Furthermore, these primer pairs could only specifically identify the genetic sex of the Sanya population of *Meretrix meretrix*, and could not identify the genetic sex of other populations of *Meretrix meretrix*. This indicates that both primer pairs, Marker1 and Marker2, can rapidly and accurately identify the genetic sex of Sanya medaka and can be widely used for genetic sex identification in Sanya medaka.

[0017] This invention provides a primer pair for identifying the sex of a Sanya population of medaka (a type of medaka bird), wherein the primer pair is Marker1 and / or Marker2, and the sequences of the upstream and downstream primers of the primer pair are as follows:

[0018] Marker1-ocF: 5′-GGGATCGTCTTTAAAGATCACCT-3′;

[0019] Marker1-ocR: 5′-CACCATTATTTGAGAAGGACTGC-3′;

[0020] Marker2-ocF: 5′-AGTGACACTGGGCAAGATTATAG-3′;

[0021] Marker2-ocR: 5′-TCATAGGTGGGCATAGTTCATTC-3′.

[0022] The present invention also provides the application of the above primer pairs in identifying the sex of the Sanya population of the medaka or in preparing a kit for identifying the sex of the Sanya population of the medaka.

[0023] The present invention also provides a kit for sex identification of a Sanya population of medaka, the kit containing the aforementioned primer pair Marker1 and / or Marker2.

[0024] Preferably, the kit also includes DNA extraction reagents and reagents commonly used in PCR technology.

[0025] In addition, the present invention also provides a method for rapidly identifying the sex of a Sanya population of medaka, comprising the following steps:

[0026] S1. Extract genomic DNA from the Sanya population sample of the medaka (a type of medaka) to be tested;

[0027] S2. Using the genomic DNA of S1 as a template, PCR amplification was performed using primer pairs for identifying the sex of the Sanya population of the medaka pheasant, and the PCR amplification products were obtained.

[0028] S3. Perform agarose gel electrophoresis on the PCR amplification products of S2; if the electrophoresis result is a single band, the sample to be tested is male; if the electrophoresis result is no band, the sample to be tested is female.

[0029] Preferably, the PCR amplification system is as follows: 10.0 μL of 2×PCR Mix Buffer, 0.3 μL each of upstream and downstream primers, 0.3 μL of template DNA, and ddH2O to make up to 20.0 μL.

[0030] Preferably, the PCR amplification program is as follows: 94℃ for 3 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 50 s, 30 cycles; 72℃ for 5 min, and storage at 4℃.

[0031] The present invention also provides the application of the above method in identifying the sex of the Sanya population of the medaka.

[0032] The present invention has the following beneficial effects:

[0033] This invention develops specific molecular markers for identifying the genetic sex of Sanya medaka through whole-genome resequencing and related bioinformatics analysis. Primers were designed for PCR amplification targeting these markers to identify the genetic sex of Sanya medaka. Verification showed that the genetic sex completely matched the physiological sex with an accuracy of 100%. Further screening revealed primers Marker1 and Marker2 that specifically amplify the molecular markers specific to the Sanya population of *Meretrix meretrix*. Amplification and identification showed that only one DNA band was obtained in the males of the wild Sanya population, while no DNA band was found in the females. The PCR products were clearly distinguishable by electrophoresis. The amplification results of the offspring population were consistent with those of the parents, proving that both marker primers Marker1 and Marker2 can be used for genetic sex identification in Sanya medaka and are stable and universal in their offspring, making them widely applicable for genetic sex identification in Sanya medaka.

[0034] Compared with traditional methods, this invention establishes a rapid, accurate, economical, and reliable method for genetic sex determination of the Sanya medaka. It can determine the genetic sex of the Sanya medaka solely through genomic DNA extraction and routine PCR amplification and electrophoresis, and is suitable for sex determination of large numbers of samples. This invention lays the foundation for further exploration of sex-determining genes in the Sanya medaka population, and provides a certain technical and theoretical basis for better developing the medaka into a model species and for the protection of its germplasm resources. Attached Figure Description

[0035] Figure 1 Figure 1 shows the PCR amplification results of wild Sanya medaka (3♀, 3♂) with different primer pairs (A. Positive control amplified with Ocsex primers, B. Amplification products of primers Marker1 and Marker2; M: Trans2K DNA Marker).

[0036] Figure 2 The results of the second round of PCR amplification of genomic DNA from a wild population of Sanya medaka and its offspring (30 males and 30 females) are shown in the figure (M: Trans2K DNA Marker; A: Primer Maker1, B: Primer Marker2; Octex-control: Octex primer control).

[0037] Figure 3 The image shows the PCR amplification results (M: Trans2K DNA Marker) of 94 individuals from the F2 generation of Sanya medaka full-sib family (45♀, 49♂). Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] The wild Sanya medaka individuals used in the following examples were collected from the coastal waters of Sanya (SY), Hainan, my country (E109.50°N18.25°). The F1 and F2 generations of Sanya medaka were domesticated and bred in our laboratory.

[0041] Example 1: Whole genome resequencing and related bioinformatics analysis of Sanya medaka

[0042] 1. Extraction of genomic DNA and whole-genome resequencing

[0043] Genomic DNA was extracted from the muscle of 10 female and 10 male medaka fish from Sanya using the phenol-chloroform method. The quality of the genomic DNA was then checked using a NanoDrop 2000 spectrophotometer and 1% agarose gel electrophoresis. The DNA from each fish was mixed in equal amounts and used for library construction and sequencing. The sequencing library fragment size was 350 bp, and paired-end sequencing was performed using the Illumina HiSeq Xten sequencing platform, with a sequencing depth of 30× for each sample.

[0044] Before alignment, the raw data obtained after sequencing was rigorously filtered to obtain high-quality clean data. The filtering criteria are as follows:

[0045] 1) Filter out reads containing connector sequences;

[0046] 2) If the proportion of N in a single-end sequencing read exceeds 10%, remove the paired reads.

[0047] 3) When the proportion of low-quality bases in a single-end sequencing read exceeds 50%, remove the paired read.

[0048] After obtaining high-quality data, the sequencing results were aligned to the reference genome of *Meretrix sanyaensis* using BWA software with alignment parameters of mem-T 4-K 32-M. PCR duplicates were removed using GATK4 (MarkDuplicates) software to obtain whole-genome resequencing data for male and female *Meretrix sanyaensis*.

[0049] 2. Screening of sex-specific regions in the genome of Sanya medaka

[0050] The chromosome quotient (CQ) screening method primarily involves comparing the number of reads aligned to a reference genome after resequencing data from female and male fish to determine whether a specific region is linked to the Y chromosome. The specific analysis method is as follows: After aligning the sequencing data to the reference genome using BWA software, the number of reads within each medaka window is counted using a sliding window method (500bp step size, 1000bp window). Simultaneously, the sequencing depth is normalized using the TPM normalization method. Then, the CQ for each window is calculated as CQ = Hf / Hm. Here, Hf represents the number of reads aligned to the female sample within the window; Hm represents the number of reads aligned to the male sample within the window. The approach of taking CQ values ​​of 0 completely misses a large number of segments close to 0, and some segments related to sex determination will be ignored. Therefore, the windows were finally screened by the criteria of CQ < 0.1 and Hm > 30, and overlapping windows were merged to finally obtain two male sex-specific regions, whose nucleotide sequences are shown in SEQ ID NO: 1~2 below, and were used for subsequent experiments.

[0051]

[0052]

[0053] Example 2: Development and Validation of Sex-Specific Markers

[0054] 1. Primer design and synthesis

[0055] Primers were designed in batches using Primer3 software for the two male-specific regions (SEQ ID NO: 1~2) with CQ<0.1 selected in Example 1. 30 pairs of primers were designed based on the male-specific sequence regions, and some primers were randomly selected for synthesis and verification. The specific primer sequences are shown in Table 1 below.

[0056] Table 1 Specific Primer Table

[0057]

[0058] 2. Genomic DNA extraction and PCR verification

[0059] Physiological sex was determined by observing the shape of the anal fin and the anatomical gonads. Thirty sexually mature individuals of each sex (12 wild females and 12 wild males; 10 females and 10 males of F1 generation; and 8 females and 8 males of F2 generation) were randomly selected from the Sanya population of *Phytophthora spp. Genomic DNA was extracted using the phenol-chloroform method. The DNA quality and concentration were detected using a Nanodrop 2000 spectrophotometer. The DNA quality was further assessed by 1% agarose gel electrophoresis and then stored at -20℃ for later use.

[0060] PCR amplification was performed, and the sex-specific primers Ocsex-F / Ocsex-R (Ocsex-F: 5′-ATGGTAACGCAGCCTTTCC-3′, Ocsex-R: 5′-GCCACATTCTTCTCAGGCA-3′) developed by Dong Zhongdian et al. were used as positive controls (related literature: Dong Zhongdian, et al. "A rapid method for identifying the genetic sex of the medaka." Journal of Guangdong Ocean University 38.03(2018):25-29.); Since the Sanya medaka population lacks the dmy gene, only one DNA band was amplified in both male and female fish, such as Figure 1 (As shown in A).

[0061] PCR amplification system: Total volume 20.0 μL, including 10.0 μL 2×PCR Mix Buffer, 0.3 μL each of forward and reverse primers, 0.3 μL template DNA, and 9.1 μL ddH2O. Reaction program: 94℃ for 3 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 50 s, 30 cycles; 72℃ for 5 min, then stored at 4℃. PCR products were then detected by 1.5% agarose gel electrophoresis.

[0062] First, based on male-specific sequence regions, 30 primer pairs were designed. After the first round of PCR validation on 3 female and 3 male samples of wild Sanya medaka, only 2 primer pairs (Maker1 and Maker2) were effective in distinguishing sex. Figure 1 As shown in Figure B, both primer pairs Maker1 and Maker2 amplified a single DNA band in male medaka individuals, while no band was observed in female individuals. However, using other primer pairs resulted in either bands being detected in both males and females, or none at all, indicating no difference in sex and rendering them unsuitable for sex determination.

[0063] Furthermore, primers Marker1 and Marker2 were used to perform a second round of PCR amplification on the genomic DNA of the wild population of Sanya medaka and its offspring (30 males and 30 females), under the same amplification conditions as above.

[0064] The results are as follows Figure 2 As shown, the genetic sex identified by Marker1 and Marker2 primers matched the phenotypic sex 100% of the time, indicating that Marker1 and Marker2 primers are also stable and universal in offspring and can be widely used for genetic sex identification of Sanya medaka.

[0065] In addition, sex-specific primers Marker1 and Marker2 were used to assess the natural sex ratio of full-sib families in the F2 generation of *Meretrix meringue* from Sanya. Electrophoresis results are shown below. Figure 3 The genetic sex ratio (female to male) of 94 F2 offspring from a full-sib family of Sanya medaka identified using markers was 45:49 (1:1.09), close to the natural sex ratio (1:1). This indicates that the genetic sex identification of this population of Sanya medaka using Marker1 and Marker2 primers is reliable, and that the sex ratio of the fish population has a certain impact on its reproductive output.

[0066] Example 3: Verification of sex specificity in different medaka populations

[0067] This embodiment uses a wild population of *Phyllostachys edulis* from Zhanjiang, with both male and female fish collected from Gaoqiao (E109.74°N21.55°). Three females and three males were selected from the Zhanjiang population, and genomic DNA was extracted using the phenol-chloroform method. The DNA quality and concentration were detected using a Nanodrop 2000 spectrophotometer, and the DNA quality was further assessed by 1% agarose gel electrophoresis before storage at -20°C for later use.

[0068] The Zhanjiang population of *Gnaphalium argus* was amplified using specific primer pairs Marker1 and Marker2. The PCR amplification methods and conditions were the same as in Example 2. The positive control used primers Ocsex-F / Ocsex-R. Electrophoresis results showed that the genetic sex of the Zhanjiang population of *Gnaphalium argus* could not be determined.

[0069] The results showed that using primer pairs Marker1 and Marker2, specifically designed for identifying the genetic sex of the Sanya killifish population, to amplify other populations (the Zhanjiang population) yielded no bands, regardless of sex. There was no difference between males and females, making it impossible to use primer pairs Marker1 and Marker2 to identify the genetic sex of the Zhanjiang killifish population. This is because the sub-killifish population differs significantly from other populations, and the specific sequences of the Sanya killifish cannot be used to identify the genetic sex of other populations. In other words, these male-specific sequences do not exist in other populations, therefore the designed primers are unsuitable for other populations.

[0070] In summary, this invention, through the development of specific molecular markers for the genetic characteristics of Sanya medaka populations, screened out two pairs of specific molecular marker primers, Marker1 and Marker2, capable of specifically amplifying the genetic characteristics of Sanya medaka populations. When these primer pairs were used for genetic sex identification in wild Sanya populations, a DNA band was amplified in male fish, while no DNA band was observed in female fish. The PCR products were clearly distinguishable by electrophoresis, and the amplification results in the offspring populations were consistent with those of the parents. This demonstrates that both Marker1 and Marker2 can be used for genetic sex identification in Sanya medaka, and both exhibit stability and universality in their offspring, making them widely applicable for genetic sex identification in Sanya medaka.

[0071] Compared with traditional methods, this invention establishes a rapid, accurate, economical, and reliable method for genetic sex determination of the Sanya medaka. Genetic sex determination can be achieved solely through extraction of genomic DNA from the Sanya medaka, followed by routine PCR amplification and electrophoresis. This invention lays the foundation for further exploration of sex-determining genes in the Sanya medaka population and provides a certain technical and theoretical basis for better developing the Sanya medaka into a model species and for the protection of its germplasm resources.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A specific molecular marker for gender identification of a three-subpopulation of Oryzias latipes, characterized in that, The molecular marker is a male-specific sequence fragment in the Oryzias latipes trihybrid population, and the nucleotide sequence is shown as SEQ ID NO: 1-2.

2. A primer pair for identifying the gender of a three sub-population of Oryzias latipes, characterized in that, The primer pair is Marker1 and / or Marker2, and the sequences of the upstream and downstream primers of the primer pair are as follows: Marker1-ocF: 5'-GGGATCGTCTTTAAAGATCACCT-3'; Marker1-ocR: 5'-CACCATTATTTGAGAAGGACTGC-3'; Marker2-ocF: 5'-AGTGACACTGGGCAAGATTATAG-3'; Marker2-ocR: 5'-TCATAGGTGGGCATAGTTCATTC-3'.

3. The primer pair of claim 2 is used for identifying the gender of the Oryzias latipes trihybrid population or for preparing a kit for identifying the gender of the Oryzias latipes trihybrid population.

4. A kit for gender identification of a three-subpopulation of Oryzias latipes, characterized by, The kit comprises the primer pair of claim 2.

5. The kit of claim 4, wherein The kit further comprises DNA extraction reagents and reagents commonly used in PCR technology.

6. A method for rapid identification of gender of a three subpopulation of Oryzias latipes, characterized by, The kit comprises the following steps: S1. Extracting genomic DNA of the sample of the Oryzias latipes trihybrid population to be tested; S2. Using the primer pair of claim 2 to perform PCR amplification on the genomic DNA of S1 as a template to obtain a PCR amplification product; S3. Performing agarose gel electrophoresis on the PCR amplification product of S2; if the electrophoresis detection result is a band, the sample to be tested is male; if the electrophoresis detection result is no band, the sample to be tested is female.

7. The method of claim 6, wherein, The PCR amplification system is as follows: 2x PCR Mix Buffer 10.0 μL, 0.3 μL of each of the upstream and downstream primers, 0.3 μL of template DNA, and ddH2O to make up 20.0 μL.

8. The method of claim 6, wherein, The PCR amplification program is as follows: 94℃ for 3 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 50 s, 30 cycles; 72℃ for 5 min, and 4℃ storage.

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