A mitochondrial molecular marker combination for identifying the maternal origin of large yellow croaker and application thereof

Abstract

This invention discloses a mitochondrial molecular marker combination for identifying the maternal origin of large yellow croaker and its application, belonging to the field of molecular detection technology. This invention mines 256 SNP molecular markers from the mitochondrial genomic DNA of large yellow croaker and develops 11 haplotype markers based on these markers, enabling accurate differentiation and identification of maternal lines in large yellow croaker. The molecular marker combination of this invention features maternal genetic stability, high resolution, strong specificity, and ease of operation. It can accurately distinguish different maternal origins of large yellow croaker and spring and autumn / winter breeding populations, and can be widely applied in fields such as large yellow croaker germplasm resource identification, maternal origin identification, genetic lineage construction, seedling tracing, improved breeding, and stock enhancement management, possessing significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a combination of mitochondrial molecular markers for identifying the maternal origin of large yellow croaker and its application. Background Technology

[0002] Large yellow croaker ( Larimichthys crocea Large yellow croaker (Cyprinus macrantha) is an important economic fish species unique to my country's marine aquaculture industry and a core leading species in marine aquaculture along the southeast coast. It plays a vital role in ensuring the supply of high-quality aquatic products and promoting the development of the coastal fishery economy. However, due to the long-term accumulation of problems such as overfishing of wild large yellow croaker germplasm resources and inbreeding depression during artificial breeding, my country's large yellow croaker industry faces a germplasm degradation crisis. This is specifically manifested in decreased growth performance of farmed individuals and weakened resistance to stress and disease, which greatly restricts the protection of large yellow croaker germplasm resources, the process of breeding improved varieties, and the high-quality and sustainable development of the aquaculture industry.

[0003] In the process of breeding and seedling propagation of large yellow croaker, determining the source of the maternal parent and clarifying the maternal pedigree of the selected population are the core foundations for ensuring the genetic purity of the selected population, preserving excellent economic traits, and achieving the purification and rejuvenation of the breed. Therefore, developing a molecular marker technology that is stable, highly specific, easy to operate, and low in cost to rapidly identify the maternal source of large yellow croaker is of great practical significance and industrial application value for promoting the breeding process of large yellow croaker, standardizing seedling production, protecting germplasm resources, and building a standardized breeding system.

[0004] Fish mitochondrial genomes follow maternal inheritance patterns, exhibiting characteristics such as no gene recombination, moderate evolutionary rate, high sequence specificity, and high genetic stability. They can completely preserve the specific genetic information of the maternal parent and accurately reflect the maternal differentiation and pedigree evolutionary relationship of a species. Therefore, they are ideal molecular carriers for tracing maternal origins, identifying maternal germplasm, and classifying pedigrees in fish. Compared to nuclear gene markers, mitochondrial molecular markers have irreplaceable technical advantages in identifying maternal origins and tracing maternal lineages.

[0005] Currently, germplasm identification techniques for large yellow croaker largely rely on traditional morphological identification or nuclear gene molecular markers such as microsatellite (SSR) and single nucleotide polymorphism (SNP). However, morphological identification results are greatly affected by the farming environment and individual developmental stage, resulting in unstable accuracy. Nuclear gene markers simultaneously inherit genetic information from both parents, making it difficult to effectively distinguish maternal-specific genetic characteristics. Furthermore, existing technologies generally suffer from drawbacks such as cumbersome operating procedures, high testing costs, and poor result reproducibility, failing to meet the industry demand for rapid maternal lineage tracing in large-scale seedling production. How to develop stable and specific haplotype molecular markers using mitochondrial DNA sequences to achieve rapid and accurate identification of the maternal origin of large yellow croaker is a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a combination of mitochondrial molecular markers for identifying the maternal origin of large yellow croaker and its applications, thereby addressing the problems existing in the prior art. This invention mines 256 SNP molecular markers from the mitochondrial genomic DNA of large yellow croaker and develops 11 haplotype markers based on these markers, enabling accurate differentiation of maternal lines in large yellow croaker. The molecular marker combination of this invention features maternal genetic stability, high resolution, strong specificity, and ease of operation. It can accurately distinguish different maternal origins of large yellow croaker and spring and autumn / winter breeding populations, and can be widely applied in fields such as large yellow croaker germplasm resource identification, maternal origin identification, genetic pedigree construction, seedling tracing, breeding selection, and stock enhancement management, possessing significant industrial application value.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a mitochondrial molecular marker array for identifying the maternal origin of large yellow croaker, comprising 256 SNP molecular markers; the SNP site information corresponding to the SNP molecular markers is as follows, with the position information being relative to the reference sequence SEQ ID NO.1: .

[0008] This invention also provides a mitochondrial haplotype marker combination for identifying the maternal origin of large yellow croaker, comprising 11 haplotype markers; the SNP site information corresponding to the haplotype markers is as follows: .

[0009] The present invention also provides a product for detecting the above-mentioned combination of mitochondrial molecular markers or the above-mentioned combination of mitochondrial haplotype markers, the product comprising reagents, kits, probes or chips.

[0010] This invention also provides the use of the above-described mitochondrial molecular marker combination, the above-described mitochondrial haplotype marker combination, or the above-described product in any of the following: (1) Identification and protection of germplasm resources of large yellow croaker; (2) Identification of spring and autumn / winter breeding populations of large yellow croaker; (3) Identification of the maternal parentage of individual large yellow croakers; (4) Construction of the maternal genetic lineage of large yellow croaker; (5) Assessment of genetic diversity in large yellow croaker; (6) Select high-quality large yellow croaker broodstock; (7) Pedigree certification and traceability of large yellow croaker fry; (8) Monitoring of genetic background during the breeding of superior varieties of large yellow croaker; (9) Molecular marker-assisted breeding and targeted improvement of superior traits in large yellow croaker; (10) Hybrid breeding of large yellow croaker from different maternal sources; (11) Tracing the source of the maternal parent of the released individuals and evaluating the effect.

[0011] This invention also provides a method for identifying the parent stock of large yellow croaker, comprising the following steps: Genomic DNA was extracted from the offspring of the large yellow croaker to be tested, and a sequencing library was constructed based on the genomic DNA. Using SEQ ID NO.1 as a reference sequence, the genotypes of the above-mentioned SNP sites were obtained by sequencing and compared with the above-mentioned haplotype markers to determine the mitochondrial haplotypes of the offspring large yellow croaker to be tested; The mitochondrial haplotypes of the offspring large yellow croaker to be tested were compared with those of the maternal large yellow croaker to determine the maternal origin of the offspring large yellow croaker to be tested.

[0012] Optionally, the sequencing method includes high-throughput sequencing using a second-generation sequencing platform or a third-generation sequencing platform.

[0013] The present invention discloses the following technical effects: 1. High resolution and accuracy: This invention relies on second-generation WGS sequencing and third-generation long-read sequencing technologies, overcoming the limitations of relying only on a few polymorphic sites such as the mitochondrial D-loop region. At the same time, it can eliminate the interference of nuclear mitochondrial pseudogenes and construct a combination of 256 SNP molecular markers covering the mitochondrial genome of large yellow croaker. This combination of markers can classify the maternal origin of large yellow croaker into 11 major haplotypes with high identification resolution.

[0014] 2. High specificity and stability: The SNP sites selected in this invention are located on the mitochondrial genome and are not affected by nuclear genome recombination or paternal gene flow, resulting in stable and reproducible detection results.

[0015] 3. Wide range of applications: The molecular marker combination and detection method provided by this invention are not only applicable to the tracing of parents and seedlings, population genetics research and germplasm resource assessment, but also to key links in the seed industry such as breeding of superior varieties of large yellow croaker, pedigree certification and stock enhancement management.

[0016] 4. Easy to operate: Combined with high-throughput sequencing technology, it can complete SNP typing of multiple samples in one test, which greatly reduces the testing cost and time per sample. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the combination of 11 haplotype mitochondrial SNP molecular markers. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0025] Example 1: Screening of mitochondrial-specific SNP sites 1. Materials and Methods 1.1 Sample Collection and DNA Extraction Large yellow croaker samples were collected from Zhoushan City and Ningbo City (including Xiangshan County) in Zhejiang Province, and Ningde City and Fuding City in Fujian Province, during November-December 2024 and November-December 2025. Adult samples were dissected and their gonadal development stages identified before sampling; samples from autumn / winter breeding were 3-month-old larvae, and sample information is detailed in Table 1. Approximately 500 mg of dorsal muscle tissue was extracted from each fish after anesthesia and immediately stored in dry ice before being transported back to the laboratory for genomic DNA extraction.

[0026] Using EZNA ® The Tissue DNA Kit (Omega Bio-Tek) was used to extract total genomic DNA according to the instructions. The DNA concentration and purity were then determined by 1% agarose gel electrophoresis, Nanodrop 2000, and Qubit 3.0. Qualified samples were stored at -80℃ for later use.

[0027] 1.2 Genome sequencing Take 1 μg of qualified DNA sample and use a Covaris S220 ultrasonic disruptor to randomly break the DNA into fragments of about 450 bp. Construct sequencing libraries using the Illumina TruSeq™ Nano DNA LT Sample Prep Kit and use a TBS380 (Picogreen) to detect the fragment size distribution of the library. Qualified libraries are subjected to high-throughput sequencing of paired ends (PE150) on the MGI DNBSEQ-T7 next-generation sequencing platform, with a sequencing depth of 30× for each sample.

[0028] Take 8 μg of qualified DNA and centrifuge it at 6000 rpm for 60 seconds using a Covaris g-TUBE (Covaris, Massachusetts) in an Eppendorf 5424 centrifuge (Eppendorf, New York) to break the genomic DNA into long fragments of 6K-20K in length. Then, repair the ends of the DNA fragments, ligate them with MGI Cyclone Nanopore sequencing adapters, and finally purify the sequencing library with Agencourt AMPure XP magnetic beads before sequencing on the MGI Cyclone Nanopore third-generation sequencing platform.

[0029] To ensure the accuracy of the high-throughput sequencing results, primers were used to amplify the target fragment by PCR, and the amplified products were then subjected to Sanger sequencing to verify the high-throughput sequencing results.

[0030] 1.3 Obtaining Public Data The raw whole-genome sequencing data of large yellow croaker were downloaded from the database of the National Center for Biotechnology Information (NCBI) in the United States. A total of 45 sequencing data that met the requirements were obtained: 43 second-generation WGS sequencing samples with a sequencing depth of not less than 20× and 2 third-generation WGS sequencing samples. All sample information is detailed in Table 1.

[0031] Table 1. Basic information of 55 large yellow croaker samples 1.4 Assembly of mitochondrial genome sequence Before assembling the second-generation sequencing data, the raw data was subjected to quality trimming. Trimmomatic v0.39 software was used to remove adapter sequences from reads, remove non-AGCT bases at the 5' end, trim low-quality bases at the ends of reads (sequencing quality value less than Q20), remove reads containing N up to 10%, and discard short fragments less than 75bp after trimming.

[0032] The obtained high-quality reads (clean data) were used to assemble the mitochondrial genome using GetOrganelle v1.7.7 software. Except for sample 1, all other samples were assembled using SEQ ID NO.1 as the seed sequence. The assembly diagram files generated by GetOrganelle were visualized and checked using Bandage software to confirm whether the mitochondrial genome formed a complete circular structure and to assess the integrity and accuracy of the assembly. The obtained complete assembly sequence was used as a reference genome with SEQ ID NO.1 to determine the starting position and orientation, resulting in the corrected mitochondrial genome sequence. Third-generation sequencing data were directly assembled using MitoHiFi software.

[0033] 1.5 SNP Analysis The obtained large yellow croaker mitochondrial genome sequence was imported into MEGA v12.0 software, and multiple sequence alignment was performed using the ClustalW algorithm with the parameters set to default. After alignment, the alignment file in FASTA format was exported, and single nucleotide polymorphism (SNP) sites were extracted and counted using Snipit alignment file. Sites with gaps or missing data were removed, and only SNP sites that could be stably detected in all individuals and were biallelic were retained.

[0034] 2. Results 2.1 Mitochondrial genome sequence characteristics of large yellow croaker De novo assembly of the high-throughput sequencing data yielded 55 complete mitochondrial genome sequences from large yellow croaker. These mitochondrial genomes are all typical closed circular double-stranded molecules, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a D-loop control region.

[0035] 2.2 Distribution of Single Nucleotide Polymorphism (SNP) Sites Based on multiple sequence alignment results of 55 mitochondrial genome sequences, a total of 319 SNP sites were detected in the entire mitochondrial genome. Among them, the D-loop region contained 49 SNP sites; the coding region contained 270 SNP sites; protein-coding genes (PCGs) contained 241 SNP sites; transfer RNA (tRNA) genes contained 16 SNP sites; and ribosomal RNA (rRNA) genes contained 9 SNP sites.

[0036] Of the 319 SNP loci identified, 3 were biallelic loci, and the rest were diallelelic loci. After removing gap loci, 256 diallele SNP loci that were consistently detectable in all individuals were finally identified, as detailed in Table 2. Based on these 256 SNP loci, the 55 samples were classified into 11 major haplotypes, namely Hap1 to Hap11. The reference sequence for Hap1 is SEQ ID NO.1. The specific base variation combinations for the other haplotypes are shown in Table 3 and [Table 4]. Figure 1 As shown in Table 4, the haplotype typing results of 55 large yellow croaker samples are presented. These results demonstrate that using the 256 SNP sites selected in this invention as molecular markers can accurately type the haplotypes of large yellow croaker.

[0037] Table 2 Information on 256 SNP sites Table 3. Haplotype characteristics of 11 mitochondria in large yellow croaker Table 4. Statistical results of haplotype typing of 55 large yellow croaker samples SEQ ID NO.1 (Hap1 mitochondrial genome sequence):

[0038] Example 2 Functional verification of mitochondrial SNP molecular marker assemblages To verify the accuracy of the 256 mitochondrial SNP molecular marker combinations and their corresponding haplotype classification methods screened in Example 1, this example uses independent samples from known maternal sources for verification.

[0039] 1. Experimental Materials Twelve independent large yellow croaker samples (offspring) that were not involved in the SNP site screening process of Example 1 were collected from three hatcheries in Ningbo and Zhoushan, Zhejiang Province, and Fujian Province. The maternal parents of these 12 offspring samples were confirmed to be cultured parents of the Hap1, Hap3, and Hap11 haplotypes through Sanger sequencing, second-generation WGS sequencing, and third-generation WGS sequencing. The offspring samples were used to conduct verification experiments.

[0040] 2. Experimental Methods 2.1 Genomic DNA Extraction DNA was extracted from the back muscle tissue of each sample using the method described in Example 1. DNA A 260 / A 280 Samples with a ratio between 1.8 and 2.0 and a concentration of not less than 50 ng / μL are considered qualified samples. Qualified samples should be stored at -80℃ for future use.

[0041] 2.2 Library Construction and High-Throughput Sequencing Libraries were constructed according to the method in Example 1. Qualified libraries were subjected to high-throughput pairwise (PE150) sequencing on the MGI DNBSEQ-T7 next-generation sequencing platform, with each sample generating at least 5 G of sequencing data.

[0042] 2.3 Mitochondrial genome assembly and SNP typing The raw reads obtained from sequencing were quality-cut using Trimmomatic v0.39 software, following the same procedure as in Example 1. The obtained high-quality clean reads were then used to perform de novo mitochondrial genome assembly using GetOrganelle v1.7.7 software, with SEQ ID NO.1 as the reference sequence. The assembled mitochondrial genome sequence was then aligned with SEQ ID NO.1 using MEGAv12.0 software (ClustalW algorithm, default parameters) to extract the genotypes of target SNP sites, obtaining the genotypes of the samples at the sites listed in Table 2. By comparing the SNP site combinations of each sample with the characteristic variant sites of each haplotype in Table 3, the mitochondrial haplotype of each sample can be determined.

[0043] 3. Experimental Results Twelve offspring samples with known maternal parents who were haplotypes Hap1, Hap3, and Hap11 were identified using the above method. The SNP locus genotype combinations were completely consistent with the characteristics of the corresponding haplotypes in Table 3, and all were accurately identified as haplotypes Hap1, Hap3, and Hap11, with an identification accuracy of 100%. This result demonstrates that the molecular marker combinations and genotyping method obtained by this invention have good reliability and accuracy.

[0044] Table 5 Sequencing information and identification accuracy of different haplotypes in large yellow croaker Example 3: Directed hybridization breeding experiment of Hap11 and Hap1 large yellow croaker Large yellow croaker bred in spring and those bred in autumn and winter show highly similar body size, color, and external morphology. Relying solely on appearance is insufficient for accurate parentage differentiation. Conventional artificial selection easily leads to the misidentification of spring-bred individuals as autumn-bred individuals, resulting in wasted scarce autumn-bred parentage resources and failing to achieve the breeding goal of directionally aggregating the superior traits of both populations. This invention proposes a directional hybridization breeding scheme based on haplotype molecular marker-assisted identification.

[0045] The parent lines were pre-typed using the method described in Example 2 and divided into two groups: (1) Hap1 parent (spring breeding population): haplotype is Hap1, mitochondrial sequence completely matches SEQ ID NO.1, spawns naturally in spring, and is a healthy adult fish of 2.5 years old; (2) Hap11 parent (autumn breeding population): haplotype Hap11, carrying 14 characteristic variation sites shown in Table 3, spawning naturally in autumn and winter, and becoming 2-year-old healthy adult fish; Healthy large yellow croaker parent fish were selected from late November to late December of that year and cultured in separate ponds until their gonads matured. Then, the two hybrid parent fish were placed in separate spawning ponds: female fish from the spring and summer breeding group × male fish from the autumn breeding group (♀Hap1×♂Hap11, i.e., ♀Spring×♂Autumn) and female fish from the autumn breeding group × male fish from the spring and summer breeding group (♀Hap11×♂Hap1, i.e., ♀Autumn×♂Spring). Fertilized eggs were obtained after they naturally spawned and released sperm.

[0046] Within 4 hours after fertilization, collect the fertilized eggs floating on the surface or upper layer of the pond water using an 80-mesh net and transfer them to a disinfected hatching pond for incubation. During incubation, maintain the water temperature at 23℃ and the salinity at 22-28‰, using a micro-flow aeration incubation mode. Clean the dead eggs that sink to the bottom every 6 hours, and maintain the dissolved oxygen content of the incubation water at ≥7mg / L. Newly hatched fry can be obtained after 32-35 hours of incubation.

[0047] Newly hatched fry were raised to 3 days old and then began feeding on rotifers and artichoke nauplii in sequence. After 30 days, they were gradually introduced to formulated feed. When the fry reached 60 days old, 5 fry were randomly selected from the two hybrid combinations, and DNA was extracted from muscle tissue for molecular identification of the hybrid offspring's haplotype, using the same method as in Example 2. The results showed that all hybrid individuals carried the characteristic markers of the parents Hap1 and Hap11, and the mitochondrial haplotypes were consistent with the corresponding maternal haplotypes.

[0048] In summary, this invention, through the early typing and screening of haplotype molecular markers selected in Example 1, fundamentally solves the problems of difficulty in distinguishing the appearance of large yellow croakers bred in spring and autumn, and the scarcity of parent fish for large yellow croakers bred in autumn. It enables controllable directional hybridization between the two breeding populations, which not only increases the genetic diversity of large yellow croaker populations but also effectively improves the current problem of germplasm degradation in farmed large yellow croakers, providing a replicable technical path for cultivating superior new varieties of large yellow croakers.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A mitochondrial molecular marker combination for identifying the maternal origin of Pseudosciaena crocea, characterized in that, It includes 256 SNP molecular markers; the SNP site information corresponding to the SNP molecular markers is as follows, and the position information is relative to the reference sequence SEQ ID NO.1: 。 2. A mitochondrial haplotype marker combination for identifying the maternal origin of Pseudosciaena crocea, characterized in that, It includes 11 haplotype markers; the SNP site information corresponding to the haplotype markers is as follows: 。 3. A product of detecting the combination of mitochondrial molecular markers of claim 1 or the combination of mitochondrial haplotype markers of claim 2, characterized in that, The products include reagents, kits, probes, or chips.

4. The use of the mitochondrial molecular marker combination of claim 1, the mitochondrial haplotype marker combination of claim 2, or the product of claim 3 in any of the following: (1) Identification and protection of germplasm resources of large yellow croaker; (2) Identification of spring and autumn / winter breeding populations of large yellow croaker; (3) Identification of the maternal parentage of individual large yellow croakers; (4) Construction of the maternal genetic lineage of large yellow croaker; (5) Assessment of genetic diversity in large yellow croaker; (6) Select high-quality large yellow croaker broodstock; (7) Pedigree certification and traceability of large yellow croaker fry; (8) Monitoring of genetic background during the breeding of superior varieties of large yellow croaker; (9) Molecular marker-assisted breeding and targeted improvement of superior traits in large yellow croaker; (10) Hybrid breeding of large yellow croaker from different maternal sources; (11) Tracing the source of the maternal parent of the released individuals and evaluating the effect.

5. A method for identifying the parent species of large yellow croaker, characterized in that, Includes the following steps: Genomic DNA was extracted from the offspring of the large yellow croaker to be tested, and a sequencing library was constructed based on the genomic DNA. Using SEQ ID NO.1 as a reference sequence, the genotype of the SNP site described in claim 1 was obtained by sequencing and compared with the haplotype marker described in claim 2 to determine the mitochondrial haplotype of the offspring large yellow croaker to be tested; The mitochondrial haplotypes of the offspring large yellow croaker to be tested were compared with those of the maternal large yellow croaker to determine the maternal origin of the offspring large yellow croaker to be tested.

6. The method according to claim 5, characterized in that, The sequencing methods include high-throughput sequencing using second-generation sequencing platforms or third-generation sequencing platforms.

Images