Chrysanthemum cherax quadricarinatus whole genome liquid phase chip and application thereof

Abstract

The invention discloses a cherax quadricarinatus whole genome liquid phase chip and application thereof, and belongs to the technical field of gene chips. The SNP molecular marker combination for detecting the whole genome of the cherax quadricarinatus is obtained through screening, the SNP molecular marker combination comprises 10028 SNP molecular markers, the physical positions and site information of the 10028 SNP molecular markers on the reference genome of the cherax quadricarinatus are shown in an SNP ID column and an Allele column in a specification table 1 respectively, and the version of the reference genome of the cherax quadricarinatus is ASM2687515v2. On the basis, a liquid chip for genetic typing of cherax quadricarinatus is developed, rapid and accurate genetic typing of cherax quadricarinatus samples is achieved, and technical support is provided for molecular marker-assisted breeding and whole-genome selective breeding of cherax quadricarinatus.

Description

Technical Field

[0001] This invention relates to the field of gene chip technology, and in particular to a liquid phase chip of the whole genome of the red swamp crayfish and its application. Background Technology

[0002] Redclaw crayfish ( Cherax quadricarinatus The red swamp crayfish (Procambarus clarkii), also known as the Australian freshwater crayfish, is native to Oceania and is one of the largest known freshwater crayfish species. Compared to the red swamp crayfish (Procambarus clarkii), it is larger. Procambarus clarkii Red claw crayfish have significant advantages such as fast growth rate, large size, tender meat, high edibility, and good transportability. In recent years, their aquaculture scale has been expanding in my country and other countries and regions, showing good prospects for industrial development.

[0003] With the expansion of redclaw crayfish farming, the need for genetic improvement of important economic traits such as growth rate, body weight, reproductive capacity, survival rate, and stress resistance is becoming increasingly urgent in production practices. Traditional phenotypic selection breeding methods are greatly affected by the environment and have long breeding cycles, making it difficult to achieve simultaneous improvement of multiple complex quantitative traits. Utilizing modern molecular breeding technologies such as genome-wide association study (GWAS) and genomic selection (GS) to perform genotyping on a large scale of individuals is an important technical approach to achieve precise and efficient breeding of redclaw crayfish. However, the implementation of the above molecular breeding strategies is highly dependent on stable, economical genotyping tools that cover the entire genome.

[0004] Current research largely relies on techniques such as solid-phase microarrays and simplified genome sequencing for genotyping. Solid-phase microarrays typically detect genotyping based on fluorescence signals generated after probes immobilized on the microarray surface hybridize with sample DNA. While offering some stability, this method is costly to develop, dependent on specific detection platforms, and its fixed probe sites make it difficult to flexibly add or remove detection sites according to breeding needs. Simplified genome sequencing uses restriction endonucleases to randomly fragment and sequence the genome. However, its detection sites are non-targeted, exhibiting poor consistency and uneven coverage across different samples, making it difficult to achieve stable detection of specific functional sites and unsuitable for large-scale commercial molecular breeding. Single nucleotide polymorphisms (SNPs) are the most widely distributed and genetically stable type of genetic variation. Genotyping based on SNP markers is fundamental to modern molecular breeding, and liquid-phase microarrays, as an important technique for targeted detection of a large number of SNP sites, have become an effective approach for achieving high-throughput, low-cost genotyping. Currently, in the fields of crop and livestock breeding, genotyping chips are widely used for precise identification of germplasm resources, analysis of population genetic structure, screening of molecular markers for superior traits, and whole-genome selection breeding. However, there are no publicly reported reports on dedicated genotyping liquid-phase chips for redclaw crayfish. Summary of the Invention

[0005] The purpose of this invention is to provide a liquid-phase chip for the whole genome of redclaw crayfish and its application, in order to solve the problems existing in the prior art. By constructing a high-quality SNP molecular marker combination covering the whole genome of redclaw crayfish, and developing a liquid-phase genotyping chip based on this, rapid and accurate genotyping of redclaw crayfish samples can be achieved, providing technical support for molecular marker-assisted breeding and whole-genome selection breeding of redclaw crayfish.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a combination of SNP molecular markers for detecting the whole genome of redclaw crayfish. The combination of SNP molecular markers includes 10,028 SNP molecular markers. The physical location and site information of the 10,028 SNP molecular markers on the redclaw crayfish reference genome are shown in the “SNP ID” and “Allele” columns of Table 1 in the specification, respectively. The version of the redclaw crayfish reference genome is ASM2687515v2.

[0007] The present invention also provides a probe combination targeting the SNP molecular marker combination; the probe combination has a probe length of 110 bp, a GC content of 30%-70%, and a number of homologous matching regions ≤5.

[0008] The present invention also provides a chip for detecting the whole genome of redclaw crayfish, including the probe combination described above.

[0009] Preferably, the chip is a liquid phase chip.

[0010] The present invention also provides a kit for detecting the whole genome of redclaw crayfish, including the aforementioned chip.

[0011] The present invention also provides the application of the SNP molecular marker combination or the probe combination described herein in the preparation of a liquid-phase chip for detecting the whole genome of redclaw crayfish.

[0012] The present invention also provides the application of the reagents, probe combinations, chips, or kits for detecting the SNP molecular marker combinations, in the genotyping of redclaw crayfish samples.

[0013] The present invention also provides the application of the SNP molecular marker combination, the probe combination, the chip, or the kit in the trait association analysis of redclaw crayfish breeding materials.

[0014] The present invention also provides the application of the SNP molecular marker combination, the probe combination, the chip, or the kit in the breeding of redclaw crayfish.

[0015] The present invention discloses the following technical effects: This invention constructs a high-quality SNP molecular marker set covering the entire genome of the redclaw crayfish (including 10,028 SNP molecular markers), and develops a liquid-phase genotyping chip based on this set. This invention achieves accurate and high-throughput detection of target SNP loci across the entire redclaw crayfish genome, providing key technical support for rapid and accurate genotyping, precise identification of germplasm resources, marker-assisted breeding, and whole-genome selection breeding of redclaw crayfish. This further shortens the breeding cycle, improves the efficiency of new variety breeding, and enhances the economic benefits of aquaculture production. Attached Figure Description

[0016] 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.

[0017] Figure 1 A distribution map of SNP chromosomes on a liquid phase typing chip for red claw crayfish; Figure 2 Correlation matrix of major phenotypic traits of redclaw crayfish; Figure 3 QQ plot for significance test of genome-wide association analysis; Figure 4 Manhattan plot of genome-wide association analysis results. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Example 1: Design and Development of SNP Liquid Phase Chip for Red Claw Crawfish 1. Testing Group To ensure the representativeness of the redclaw crayfish data and the universality of the liquid phase chip, 50 redclaw crayfish diversity data were collected from three major aquaculture areas in China: Hainan, Zhejiang, and Guangdong.

[0024] 2. Whole-genome resequencing and SNP site screening of redclaw crayfish The collected redclaw crayfish samples underwent whole-genome resequencing and SNP site screening. Specific steps included: (1) Genomic DNA was extracted from the muscle tissue of redclaw crayfish using a DNA extraction kit. After extraction, the DNA concentration was measured, and agarose gel electrophoresis was performed to check the DNA integrity, ensuring that the DNA concentration was above 50 ng / μL and that there was no significant degradation. Samples that passed the test were used to construct sequencing libraries using a DNA sequencing library preparation kit.

[0025] (2) The libraries that passed quality inspection were sequenced, and the obtained sequencing data were aligned to the redclaw crayfish reference genome (GenBank: ASM2687515v2). The initial loci were filtered according to the following conditions: minimum sequencing depth ≥ 5X, deletion rate < 10%, minimum allele frequency (MAF) > 0.05, and non-diaryl SNP loci and loci with insertion / deletion variations were removed. Then, loci were selected according to the following principles: heterozygosity < 50%, loci evenly distributed on chromosomes, probe length of 110 bp, probe GC content between 30-70%, and no more than 5 homologous regions (similar fragments). This can effectively improve the hybridization stability of the sequence containing each SNP and improve the efficiency of fragment capture. At the same time, the specificity of the flanking sequences of the loci on the genome was fully considered to ensure the locus-specific capture rate and reduce sequencing costs.

[0026] (3) After testing and evaluating the capture stability, a total of 10,028 superior loci (including trait-related loci) were finally screened. The physical location of each SNP locus on the reference genome of red claw crayfish is shown in Table 1. The SNP ID represents the chromosome and physical location information of the SNP locus, such as "1:4927" which means the physical location at position 4927 on chromosome 1; Allele represents the gene polymorphism of the locus.

[0027] Table 1. Location and genotype information of 10,028 SNP loci in redclaw crayfish. 3. Development of SNP liquid phase chip for red claw crayfish Based on the location of the SNP sites and the sequence information on both sides in Table 1, we commissioned Bored Biotechnology Co., Ltd. to design primers and synthesize probes using targeted capture sequencing technology. The primers consisted of an 80bp upstream primer and an 80bp downstream primer; the probes were designed to contain the sites, with 50-60bp flanking sequences, thus preparing a liquid-phase chip of redclaw crayfish SNPs. The main steps are as follows: (1) Chip probe design and synthesis Based on the principle of DNA complementarity, one or more probes covering the target SNP are designed for each test site. The probes are 110 bp in length and have a GC content between 30% and 70%. The probes are labeled with biotin and hybridized with the target region in the denatured resequencing library to form a stable double strand. Then, streptavidin-coated magnetic beads that have been pre-balanced and fully resuspended are added to capture the hybrid molecules carrying the biotin probes. The molecules are then subjected to 3-4 rigorous washes at 60-65℃ to remove non-specific binding fragments. After elution and recovery, PCR amplification is performed for 8-14 cycles using universal adapter primers. After purification and quality control, the molecules are sequenced. Finally, the genotype of the target SNP is obtained based on the sequencing depth of the target site and allele readings.

[0028] (2) Data Analysis The raw sequencing data was filtered using FastQC software to remove low-quality reads and adapter contamination sequences. The high-quality data after quality control was then aligned to the redclaw crayfish reference genome using BWA software to obtain SNP sequencing information. GATK software was used to perform SNP genotyping analysis on the sequencing data, calculating the allele frequency of each SNP locus in different populations and assessing SNP polymorphism. The distribution density of the 10028 detected SNP loci on the chromosome is shown below. Figure 1 As shown in the figure. The results show that the minimum MAF for all sites is greater than 0.05, and the average MAF is 0.42, which is much greater than 0.25. This effectively avoids the problems of uneven marker density and poor polymorphism that may be caused by simplified genome sequencing genotyping technology.

[0029] Example 2: Genotyping of Red Claw Crawfish using SNP Liquid Chromatography Chip 1. Test Sample Three hundred samples of red claw crayfish cultured in Huzhou, Zhejiang Province, were collected for testing.

[0030] 2. DNA library construction and targeted capture sequencing Genomic DNA was extracted from the muscle tissue of redclaw crayfish using a genomic extraction kit. The extracted genomic DNA was fragmented by sonication to construct a high-throughput sequencing library. A-tails were added to the ends of the fragmented DNA, sequencing adapters were ligated, and PCR amplification was performed. The amplified products were purified to obtain the sequencing library. The constructed sequencing library was mixed with biotin-labeled SNP probes from the liquid-phase chip in Example 1. A magnetic bead capture system was used to capture DNA fragments containing the target SNPs, utilizing the high affinity between biotin and streptavidin. The captured fragments were then amplified by PCR and purified, and finally, high-throughput sequencing was performed on a sequencing platform to obtain genotyping data for the target region.

[0031] 3. Data Analysis and Results The raw sequencing data was filtered using FastQC software to remove low-quality reads and adapter contamination sequences. The high-quality data after quality control was aligned to the redclaw crayfish reference genome using BWA software to obtain SNP sequencing information. SNP genotyping analysis was performed on the sequencing data using GATK software, and the SNP data and genotyping results of all samples were merged. After sequencing and data analysis, the sample locus detection rate ranged from 97.66% to 99.07%, with an average detection rate of 98.41%. Therefore, the described SNP liquid-phase chip can be used to detect the genotype of the redclaw crayfish.

[0032] Example 3: Genome-wide association analysis using SNP liquid phase chip of red swamp crayfish To further evaluate the value of this chip in redclaw crayfish breeding, a genome-wide association analysis (GWAS) was conducted on the growth traits of redclaw crayfish.

[0033] Phenotypic correlation analysis was performed on eight key growth traits of the redclaw crayfish, including body weight, body length, total length, claw length, propodus length, carapace length, carapace width, and abdomen length. The results are as follows: Figure 2 As shown, highly significant positive correlations existed among all traits (P<0.001). In particular, the correlations among core economic traits such as body weight, body length, and total length were extremely strong, indicating a trend of synergistic phenotypic changes in these traits. These high phenotypic correlations suggest the possible existence of shared genetic regulatory mechanisms.

[0034] Genotyping of 1145 redclaw crayfish samples was performed using the aforementioned redclaw crayfish SNP liquid chromatography genotyping chip. Plink2 was used to quality control the SNP loci of these 1145 redclaw crayfish samples, ensuring that the retention rate of retained loci did not exceed 5%, the MAF was not less than 0.05, and the HWE P value was not less than 1 e. -6 SNP loci were collected. Samples underwent quality control, retaining samples with a missing rate not exceeding 5%, a heterozygosity within three standard deviations of the mean, and no missing claws. A total of 1065 red claw crayfish samples were collected for GWAS analysis. Genome-wide association analysis (GWAS) was performed on genotype data and three growth traits (body weight, body length, and total length) using rMVP software. Simple linear model (GLM), mixed linear model (MLM), and fixed-random model with unified cycle probability (FarmCPU) were used, with sex and age included as covariates. A total of 43 significantly associated SNP loci were identified (see [link to analysis]). Figure 3 These sites are distributed across multiple chromosomes throughout the genome (see...). Figure 4 This indicates that the method can effectively detect phenotypic variations.

[0035] In summary, the results show that the liquid-phase chip for redclaw crayfish SNPs constructed in Example 1 can stably obtain high-quality genotyping data, meeting the technical requirements for genetic analysis of the target population and providing a reliable data foundation for subsequent molecular breeding research. This example further demonstrates that genome-wide association analysis (GWAS) based on the 10028 SNP loci provided by this invention, combined with the liquid-phase chip described in Example 1, not only improves analysis efficiency but also effectively locates genomic regions significantly associated with the target trait, relying on high coverage and high accuracy genotyping results, thus providing a scientific basis for the formulation and optimization of breeding strategies.

[0036] 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 combination of SNP molecular markers for detecting the whole genome of the redclaw crayfish, characterized in that, The SNP molecular marker combination includes 10,028 SNP molecular markers. The physical location and site information of the 10,028 SNP molecular markers on the redclaw crayfish reference genome are shown in the "SNP ID" column and "Allele" column of Table 1 in the specification, respectively. The version of the redclaw crayfish reference genome is ASM2687515v2.

2. A probe array targeting the SNP molecular marker array of claim 1.

3. A chip for detecting the whole genome of the redclaw crayfish, characterized in that, Includes the probe assembly as described in claim 2.

4. The chip as described in claim 3, characterized in that, The chip is a liquid phase chip.

5. A kit for detecting the whole genome of the redclaw crayfish, characterized in that, Includes the chip described in claim 3 or 4.

6. The application of the SNP molecular marker combination as described in claim 1 or the probe combination as described in claim 2 in the preparation of a liquid-phase chip for detecting the whole genome of redclaw crayfish.

7. The application of the reagent for detecting the SNP molecular marker combination of claim 1, the probe combination of claim 2, the chip of claim 3 or 4, or the kit of claim 5 in the genotyping of redclaw crayfish samples.

8. The application of the SNP molecular marker combination as described in claim 1, the probe combination as described in claim 2, the chip as described in claim 3 or 4, or the kit as described in claim 5 in the trait association analysis of redclaw crayfish breeding materials.

9. The application of the SNP molecular marker combination as described in claim 1, the probe combination as described in claim 2, the chip as described in claim 3 or 4, or the kit as described in claim 5 in the breeding of redclaw crayfish.

Images