A spot-fin catfish SNP molecular marker combination, probe, 50K liquid chip, kit and application

Abstract

The application provides a spotted seal catfish SNP molecular marker combination, a probe, a 50K liquid phase chip, a kit and application, and belongs to the technical field of gene chips.The GWAS site probe, the window method site probe, the gender site probe and the GWAS site sequence which cannot be designed are integrated to construct a complete initial probe library, and optimization is carried out, so that a spotted seal catfish SNP molecular marker combination including 53407 core target points is obtained.The 50K liquid phase chip prepared based on the core target points has high reliability (the core site detection rate reaches 98.21%) in actual application.The 50K liquid phase chip not only includes 53,407 core sites which are high in polymorphism and rich in function annotation, but also can capture a large number of flanking sites, and the total assessable sites are more than 126,000, so that the information flux and cost performance of detection are significantly improved, and a powerful tool is provided for molecular design breeding.

Description

Technical Field

[0001] This invention relates to the field of gene chip technology, and in particular to a combination of SNP molecular markers for spotted catfish, probes, a 50K liquid phase chip, a kit, and applications. Background Technology

[0002] Gene chips, especially high-density gene chips based on single nucleotide polymorphisms (SNPs), have become a core tool for modern molecular breeding, germplasm resource identification, and genetic analysis of complex traits. They significantly improve the efficiency and accuracy of breeding selection by performing high-throughput genotyping on tens of thousands of predetermined genetic loci in a single operation.

[0003] In the breeding practices of important aquatic economic species such as the channel catfish, the demand for high-performance gene chips is becoming increasingly urgent. However, the current field mainly faces the following technical bottlenecks: the site representativeness and functionality of existing commercial general-purpose chips are insufficient; there is a lack of systematic site screening and integration design methods for specific species; and probe design libraries have not been experimentally verified in target populations, resulting in uncertainty in their actual performance.

[0004] Therefore, there is an urgent need for a highly reliable custom gene chip and its design method that is specifically designed for specific species such as the spotted catfish, can integrate multi-dimensional functional evidence, and has been experimentally verified and optimized, in order to overcome the shortcomings of existing general-purpose chips in terms of specificity, functionality and reliability, and meet the needs of precision molecular breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker combinatorial array of channel catfish SNPs, probes, a 50K liquid phase chip, a kit, and applications. A high-quality SNP combinatorial array with high polymorphism and rich functional annotation was obtained. The final 50K liquid phase chip product achieved synergistic improvements in site functional quality, genome coverage balance, detection reliability, and data throughput. This invention solves the technical problem of lacking efficient and dedicated molecular typing tools in channel catfish breeding and has a significant positive effect on promoting molecular design breeding of this species.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a group of SNP molecular markers for the spotted catfish, which includes 53,407 SNP molecular markers. The location information of the SNP molecular markers on the T2T genome is shown in Table 3.

[0007] The present invention also provides a probe that targets the above-mentioned combination of SNP molecular markers in the spotted catfish.

[0008] Preferably, the probe design method is as follows: For each SNP site, 120 bp sequences upstream and downstream are extracted. Using these sequences as templates, oligonucleotide probes with a length of 120 bp, a Tm value of 55-65℃, and a GC content of 40%-60% are designed.

[0009] The present invention also provides a 50K liquid phase chip for the T2T genome of the spotted catfish, wherein the 50K liquid phase chip for the T2T genome of the spotted catfish includes the above-mentioned probe.

[0010] The present invention also provides a SNP kit for the T2T genome of the spotted catfish, the kit comprising the above-mentioned probe or the above-mentioned 50K liquid phase chip for the T2T genome of the spotted catfish.

[0011] This invention also provides an application of the above-mentioned SNP molecular marker combination, probe, 50K liquid phase chip, or SNP kit in genetic diversity, genetic distance assessment, germplasm resource fingerprinting construction and identification, breeding material assisted selection, molecular design breeding, or trait genetic regulatory site discovery.

[0012] The beneficial effects of this invention compared to the prior art are as follows: This invention integrates GWAS site probes, windowing site probes, sex-specific site probes, and GWAS site sequences for which probes cannot be designed, constructing a complete initial probe library. This library is then optimized to obtain a molecular marker combinatorial library of 53,407 core target sites for channel catfish SNPs. Site-specific capture can be performed based on the principle of complementary base pairing between probes and all or part of the target genomic region. Specific DNA probes are designed for each target site and biotinylated through in vitro transcription; subsequently, the biotinylated probes are hybridized with fragmented genomic DNA; finally, streptavidin-coated magnetic beads are used to enrich the biotinylated hybridization products, followed by elution, amplification, and high-throughput sequencing to obtain genomic sequence information of the target site and its vicinity (e.g., Figure 1 (As shown). The 50K liquid-phase chip prepared based on these core targets demonstrates high reliability in practical applications (core site detection rate reaches 98.21%). This 50K liquid-phase chip not only contains 53,407 highly polymorphic and functionally annotated core sites, but also captures a large number of flanking sites, bringing the total number of evaluable sites to over 126,000. This significantly improves the information throughput and cost-effectiveness of detection, providing a powerful tool for molecular design breeding. Attached Figure Description

[0013] 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.

[0014] Figure 1 This is a flowchart of the hybridization capture sequencing experiment in this invention; Figure 2 This is a density distribution diagram of the chip core sites in Embodiment 1 of the present invention; Figure 3 This is a MAF histogram of the core sites of the chip in Embodiment 1 of the present invention; Figure 4 This is a bar chart annotating the core site features of the chip in Embodiment 1 of the present invention, where RNA-seq: variants are located within differentially expressed genes; UTR: variants are located in the UTR region of a gene; exonic: variants are located in the coding region of an exon; Splicing: variants are located in the splicing region; ATAC: variants are located in open chromatin regions; intergenic: variants are located in the intergenic region; intronic: variants are located in the intron region; GWAS: trait-associated functional region; upstream, downstream: variants are located in upstream and downstream regions. Figure 5 This is the PCA diagram of the core sites of the chip before optimization in Embodiment 1 of the present invention; Figure 6 This is the optimized PCA diagram of the core target sites of the chip in Embodiment 1 of the present invention; Figure 7 This is an upset diagram of the core target site in Embodiment 1 of the present invention; Figure 8 This is a bar chart showing the detection rate of the core sites on the chip for each sample in Example 2 of the present invention. Detailed Implementation

[0015] 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.

[0016] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Example 1

[0021] Embodiment 1 of the present invention provides a method for constructing a 50K liquid-phase gene chip of the T2T genome of the spotted catfish, the specific steps of which are as follows: 1. Initial probe library design (1) GWAS analysis 1) Phenotypic data for seven traits (body weight, total length, head length, body height, caudal peduncle length, caudal peduncle height, and weight gain) were obtained from 303 channel catfish individuals with high-depth sequencing (10×). Genome-wide association studies (GWAS) were performed using rMVP and EMMAX software, employing FarmCPU and MLM models. Significant association loci obtained from both software analyses were combined, yielding a total of 8,012 significant core loci. Of these, 5,455 loci originated from the EMMAX analysis, and 2,576 loci originated from the rMVP analysis.

[0022] 2) Phenotypic data for two traits (body weight and total length) were obtained from 403 channel catfish individuals with low-depth sequencing (1×). GWAS analysis was performed using the rMVP FarmCPU and MLM models, yielding 269 significant loci. By combining the sites obtained in the two steps above, a core target site set of 8277 sites for probe design was obtained. Probe design was performed on these 8277 sites (with each SNP site as the center, 120 bp sequences upstream and downstream were extracted, and specificity was evaluated by comparison using the k-mer algorithm and the capture efficiency was predicted by a deep learning model; oligonucleotide probes with a length of 120 bp, a Tm value of 55-65℃, and a GC content of 40%-60% were selected, and dual probes were designed for some sites). 5572 probes were successfully designed for 3441 sites (dual probes were designed for some sites suitable for dual probe design to ensure detection rate).

[0023] (2) Functional site selection and probe design using the windowing method Based on the channel catfish reference genome (approximately 862,751,499 bp in total length), the entire genome was divided into 61,625 planned windows with a fixed window length of 14 kb. For each window, the site with the highest weight was selected for probe design according to the weight table shown in Table 1. If a site with the highest weight could not be designed with a probe, the next highest weight linked site was selected, and so on, until a probe was successfully designed. Of the 61,625 windows ultimately defined, 54,960 probes were successfully designed for 54,960 windows, covering 54,960 core sites.

[0024] Table 1. Weights of SNP Types type Weight Known functional sites 30 Stopgain (exon region) 6 Nonsynonymous / Stoploss (exon region) 5 Synonymous (exon region) 2 Nonsynonymous / Stopgain / Stoploss (shearing region) 5 Other (cut-off region) 4 Downstream / UTR region 3 Intergenic regions / intronic regions 1 MAF MAF * 2 ATAC 5 RNAseq differential gene regions 3 (3) Design of probes for sex determination sites in older versions of the genome In previous studies, 38 key candidate loci associated with growth, salt tolerance, and sex determination were identified based on the old reference genome Coco_2.0 (assembly number: GCF_001660625.3) of the spotted catfish. Using homology sequence alignment software, the old Coco_2.0 reference genome sequence was systematically aligned with the new T2T reference genome sequence to map it onto the T2T reference genome of this invention. The alignment results were converted into a coordinate mapping library file, successfully converting the 38 old growth, salt tolerance, and sex determination related loci into homologous loci of the T2T reference genome. The results are shown in Table 2.

[0025] Table 2. Homologous site correspondence between Coco_2.0 and T2T

[0026] Chip design was performed on the 38 sites after the above conversion, and 73 probes were successfully designed, covering 38 sites related to growth, salt and alkali tolerance, and sex determination.

[0027] (4) Probe sites cannot be designed to forcibly cover significant GWAS sites. During the probe design process for GWAS significant sites, probes could not be designed for 4,836 sites. These designed sites were referred to as sequences, not probes. For the GWAS sites that could not be designed, the preceding and following sequences were forcibly obtained to supplement the design results, resulting in 4,836 supplementary sequences. However, the actual effectiveness of these supplementary sequences cannot be guaranteed.

[0028] The probes obtained through GWAS screening, genome-wide windowing, and design of important trait loci (sex determination) were integrated with sequences supplemented for difficult-to-design GWAS loci to construct a complete initial probe library.

[0029] The library contains a total of 65,441 oligonucleotide sequences that can specifically target 63,239 predefined core target sites.

[0030] Furthermore, based on the principle of liquid-phase hybridization capture sequencing, the probes immobilized on the chip capture the DNA fragment containing the target core site, while simultaneously capturing and sequencing genomic fragments in adjacent regions non-specifically. Therefore, this invention, in addition to detecting the target core site, can also simultaneously acquire genetic variation information in the flanking regions of the core site. Such genetic variation sites that can be detected simultaneously but are not pre-designed targets are defined as flanking sites in this invention. According to insilico (computer simulation) evaluation, this design is expected to cover approximately 76,561 additional flanking sites. This feature significantly improves the single-detection throughput and application value of the chip.

[0031] 2. Probe optimization Hybridization capture sequencing was performed on the initial probe library using 20 representative channel catfish samples. The deletion rate of loci across all test samples was used as the optimization metric, and loci with a deletion rate exceeding 30% were removed. After optimization, the final gene chip can stably detect a total of 126,686 genetic loci, including 53,407 core target loci (as shown in Table 3) and 73,279 flanking loci.

[0032] Table 3 Core target sites and their chromosomal locations

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[0116] 3. Genomic distribution of core target sites (1) Genome distribution: Using 1 Mb as a fixed window, the genomic characteristics of the core target sites were statistically analyzed, and the results are as follows: Figure 2 As shown.

[0117] Figure 2 The results show that the core target sites are relatively evenly distributed across all chromosomes, indicating that the screening strategy of this invention (especially the windowing method) effectively avoids site clustering, ensures balanced coverage of the whole genome by the chip, and improves its representativeness and statistical power in applications such as genetic mapping and genome-wide association analysis.

[0118] (2) Polymorphism Genotyping data from 303 high-depth individuals and 403 low-depth individuals (a total of 706 individuals) used for GWAS analysis were integrated. For 53,407 core target loci, the minimum allele frequency of each locus in this mixed population was calculated, and the results are as follows: Figure 3 As shown.

[0119] Figure 3 The results showed that 62.18% of the core target loci had a MAF greater than 0.1. This indicates that over 60% of the loci exhibited high polymorphism in the baseline population. Highly polymorphic loci are fundamental for effective population genetic analysis, parentage testing, and breed identification, demonstrating the high practical value and information content of the loci screened in this invention.

[0120] (3) Functional annotations The coordinates of the core target site were compared with the structural annotation file of the channel catfish T2T genome to determine its location in intergenic regions, introns, exons, UTRs, splicing sites, etc. The results are as follows: Figure 4 As shown.

[0121] Figure 4 The results show that the core target sites are widely distributed across various functional regions, including a significant proportion of exon regions, regulatory regions (upstream and downstream / UTR), and regions associated with epigenetic activation (ATAC-seq regions). This directly demonstrates the successful realization of the "functional site customization" design concept of this invention. The chip sites not only exhibit polymorphism but are also enriched in biologically important regions that may affect gene function or regulation, enhancing their potential for functional marker development and trait analysis in molecular breeding.

[0122] (4) Consistency verification of group structure analysis Experimental / Analysis Procedure: Principal component analysis was performed on 706 individuals using core target locus genotyping data before optimization (63,239 loci) and after optimization (53,407 loci), respectively. The first two principal components were extracted, and scatter plots were generated. The results are shown below. Figure 5 , 6 As shown.

[0123] contrast Figure 5 (Before optimization) and Figure 6 The optimized PCA results show that the relative clustering relationships and population structure of individuals in both datasets are completely consistent. This result indicates that the optimization process, based on 20 test samples and categorized by detection rate, removes sites with technically unstable capture efficiency, rather than sites carrying population-specific genetic signals. Therefore, the optimization operation did not introduce bias and preserved the chip's ability to resolve the true genetic structure of the population.

[0124] (5) An intersection analysis was performed on the classifications to which the core target sites belong, and the results are as follows: Figure 7 As shown.

[0125] Figure 7 The results show that this core target combination covers different functional regions of the genome. By integrating genetic, transcriptomic, and epigenetic evidence, it significantly enhances the likelihood of association between microarray sites and potential biological functions, providing a high-quality, high-information data foundation for subsequent gene function analysis and molecular marker-assisted breeding.

[0126] Example 2

[0127] The specific steps of Embodiment 2 of the present invention are as follows: (1) Probe design and synthesis Based on probe sequences targeting 53,407 sites in Table 3 designed in Example 1, a 50K liquid-phase microarray of the T2T genome of the spotted catfish was prepared. (2) Genotyping success rate (detection rate) test Twenty representative channel catfish individuals were randomly selected, and standard hybridization capture sequencing experiments were performed using a 50K liquid-phase microarray of the channel catfish T2T genome. The procedure included: genomic DNA extraction, fragmentation, library construction, hybridization with microarray probes, capture and washing, PCR amplification, and high-throughput sequencing. After quality control and alignment (to the T2T genome), SNP calling was performed using a standard genotyping procedure (such as GATK Best Practices). The proportion of samples in the 20 samples that successfully genotyped (i.e., not deleted) each core target locus was calculated, and the average detection rate was determined. The results are shown below. Figure 8 As shown. Figure 8The results showed that the average genotyping success rate for 53,407 core target sites across 20 test samples reached 98.21%. This result provides direct validation of the chip design, probe synthesis, and experimental procedures. The extremely high detection rate indicates that the chip possesses excellent reliability, stability, and repeatability, meeting the stringent requirements for data integrity and accuracy in large-scale breeding population testing, thus ensuring the quality of subsequent data analysis.

[0128] In summary, the 50K functional locus gene chip for spotted catfish designed in this invention exhibits excellent performance in terms of genome coverage balance, locus polymorphism and functionality, population analysis reliability, and technical stability, fully achieving the design goals and being suitable for various complex molecular breeding and genetic research applications.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of SNP molecular markers for the spotted catfish, characterized in that, The spotted catfish SNP molecular marker assemblage includes 53,407 SNP molecular markers, and the location information of the SNP molecular markers on the T2T genome is shown in Table 3.

2. A probe targeting the SNP molecular marker combination of the spotted catfish as described in claim 1.

3. The probe targeting the SNP molecular marker combination of spotted catfish according to claim 2, characterized in that, The probe design method is as follows: For each SNP site, 120 bp sequences upstream and downstream are extracted. Using these sequences as templates, oligonucleotide probes with a length of 120 bp, a Tm value of 55-65℃, and a GC content of 40%-60% are designed.

4. A 50K liquid-phase chip for the T2T genome of the spotted catfish, characterized in that, The spotted catfish T2T genome 50K liquid phase chip includes the probe described in claim 2 or 3.

5. A T2T genome SNP kit for channel catfish, characterized in that, The kit includes the probe of any one of claims 2 to 3 or the 50K liquid phase chip of the spotted catfish T2T genome of claim 4.

6. A combination of SNP molecular markers as described in claim 1, or a probe as described in any one of claims 2-3, or a 50K liquid phase chip as described in claim 4, or a SNP kit as described in claim 5, for the assessment of genetic diversity, genetic distance, Applications include the construction and identification of germplasm resource fingerprinting, assisted selection of breeding materials, molecular design breeding, and the discovery of genetic regulatory sites for traits.

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