Lateolabrax japonicus liquid phase chip based on targeted genotype detection technology and application thereof

Abstract

The invention discloses a lateolabrax japonicus liquid-phase chip based on a targeted genotype detection technology and application of the lateolabrax japonicus liquid-phase chip, the lateolabrax japonicus liquid-phase chip can detect genotypes of 45 and 459 target sites, and the lateolabrax japonicus liquid-phase chip comprises 44 and 927 background SNPs sites uniformly distributed in a whole genome, 454 INDELs sites located in a coding region and capable of causing frame shift mutation and 78 functional sites related to lateolabrax japonicus growth. The chip is based on a targeted sequencing genotype detection technology (GBTS) of liquid-phase probe hybridization, and compared with an existing lateolabrax japonicus genotyping method, the liquid-phase chip provided by the scheme has great advantages in the aspects of repeatability and accuracy of target site detection, experiment standardization degree, data analysis simplicity degree, required computing resources and the like. The invention fills up the blank that the lateolabrax japonicus lacks genetic typing chips, and is suitable for genome selective breeding, whole genome association analysis, germplasm identification and genetic diversity analysis of lateolabrax japonicus.

Description

Technical Field

[0001] The invention relates to the technical fields of fish genetic breeding and molecular biology, and in particular to a Lateolabrax liquid phase chip based on targeted genotype detection technology and an application thereof. Background Art

[0002] The japonica seabass (Lateolabrax maculatus) is widely distributed in coastal and estuarine areas of my country. In 2023, the production of seabass in my country reached 247,000 tons, ranking among the top three in marine aquaculture fish. With the gradual expansion of the scale of japonica seabass farming, the increase in farming density and inbreeding, the problems of germplasm degradation, frequent diseases and lack of improved varieties have seriously restricted the sustainable development of the japonica seabass farming industry. Breeding new germplasm of japonica seabass with high resistance and high quality is an important way to solve these problems. However, it takes 3 to 4 years for japonica seabass to grow from juveniles to sexual maturity, and it takes at least 10 years to breed new varieties using traditional breeding techniques. Genomic selection (GS) breeding is a method of selective breeding using high-density molecular markers covering the entire genome. Genomic selection breeding can significantly shorten the generation interval, improve breeding accuracy and reduce breeding costs. Genomic selection breeding requires whole genome typing of the constructed reference population and candidate population to construct a genomic selection model and calculate the estimated breeding value of the candidate population genome. At present, the main challenge of genomic selection breeding of Lateolabrax japonicus is how to establish low-cost, high-throughput whole-genome typing technology and accurately obtain individual genotype data.

[0003] Genotyping, also known as genotyping, refers to the method of obtaining individual genotypes by detecting individual DNA sequences and sequence comparisons. Establishing low-cost, high-throughput whole genome typing technology and accurately obtaining individual genotype data are essential processes for whole genome selection breeding, whole genome association analysis, germplasm identification or genetic diversity analysis, and are also the main challenges they face. At present, the methods of whole genome typing mainly include simplified genome sequencing, whole genome low-depth resequencing and gene chips. Compared with simplified genome sequencing and whole genome low-depth resequencing, gene chips have great advantages in the repeatability and accuracy of site detection, the degree of experimental standardization, the simplicity of data analysis and the required computing resources. Many aquaculture species such as carp, channel catfish, large yellow croaker, tilapia, rainbow trout, Atlantic salmon, flounder, oysters, etc. have developed corresponding chips, which have greatly promoted the analysis of the genetic mechanism of related breeding traits and the genetic breeding process. However, there is currently no gene chip for Lateolabrax japonicus.

[0004] Gene chips are divided into solid-phase chips and liquid-phase chips. The mainstream chips on the market are solid-state chips. However, solid-state chips have defects such as poor flexibility, strict sample quantity requirements, and high costs, which limit their large-scale application in breeding. Compared with solid-state chips, liquid-phase chips have the advantages of convenient marker addition and removal, no sample quantity requirements, and low cost. In view of this, the patent applicant has developed a liquid-phase breeding chip for striped seabass, which will be widely used in the analysis of the genetic mechanism of striped seabass traits and genetic breeding practices. Summary of the invention

[0005] In view of this, the object of the present invention is to propose a Lateolabrax liquid phase chip based on targeted genotype detection technology and its application which is reliable and has good detection effect.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A marker combination for genotyping of striped seabass, the marker combination comprising background sites and functional sites; the background sites comprising 44,927 SNPs sites as shown in Table 1 and / or 454 INDELs sites as shown in Table 2; the functional sites comprising 68 SNPs sites as shown in Table 3 and / or 10 INDELs sites as shown in Table 4.

[0008] Among them, the SNPs in the background sites are evenly distributed in the genome, and the INDELs are located in the gene coding region and can cause frameshift mutations of the gene.

[0009] Based on the above, this solution also provides a Lateolabrax liquid phase chip based on targeted genotype detection technology, which includes probes and / or primers for detecting the above-mentioned marker combination; the Lateolabrax liquid phase chip is based on targeted genotype detection technology based on liquid phase probe hybridization.

[0010] The target sites detected by the striped seabass liquid phase chip totaled 45,459, including 45,381 background sites and 78 functional sites related to the growth of striped seabass; wherein, the background sites included 44,927 SNPs sites as described in Table 1 and 454 INDELs sites as described in Table 2, and the functional sites included 68 SNPs sites as described in Table 3 and 10 INDELs sites as described in Table 4.

[0011] This solution also provides a method for preparing the above-mentioned Leptothorax liquid phase chip, which comprises the following steps:

[0012] 1) Sample collection, sequencing and data analysis: Collect samples of wild and farmed Leptospermum japonicum from my country, extract their genomic DNA, use next-generation sequencing technology to resequence the whole genome of the samples, perform quality control on the sequencing results, compare them with the reference genome, and use bioinformatics to identify and screen SNPs and INDELs sites;

[0013] 2) Target site screening and probe design: Preliminary screening of SNPs sites based on one or more criteria including sequencing depth, minor allele frequency, deletion rate, heterozygosity and chromosome distribution;

[0014] INDELs sites were initially screened based on sequencing depth, MAF, deletion rate, heterozygosity, and number of insertion and deletion base pairs;

[0015] By searching the literature, we searched for the variation sites related to the growth of Lateolabrax japonicus and used them to screen the functional sites.

[0016] The SNPs and INDELs sites are combined to obtain 45,459 target sites including background sites and functional sites, and then a probe is designed for each target site;

[0017] 3) Chip preparation and quality assessment: synthesize probes and modify them with biotin to prepare Lateolabrax japonicus liquid phase chips;

[0018] The prepared Lateolabrax liquid phase chip was used to detect target sites, calculate the detection rate, evaluate the chip quality, and obtain a Lateolabrax liquid phase chip whose quality met the preset requirements.

[0019] Specifically, it may include the following:

[0020] S1: 90 wild japonica seabass from the Yellow Sea, Bohai Sea, East China Sea, and Beibu Gulf of my country, as well as 31 farmed japonica seabass fins provided by Fujian Minwei Industrial Co., Ltd. were collected and preserved in anhydrous ethanol. The whole genome was resequenced using the second-generation double-end PE150 method with a sequencing depth of 10×. From the public data provided by the published articles, the raw data of 60 japonica seabass from 6 locations were selected for subsequent processing and analysis. The chip sample population covers the main sea areas and populations in my country, ensuring the wide applicability and effectiveness of this chip.

[0021] S2: Fastp software was used to perform quality control on the raw sequencing data to obtain high-quality clean reads. The quality-controlled data were aligned with the Lateol abrax maculatus reference genome (https: / / figshare.com / articles / dataset / Draft_genome_of_the_Chinese_seabass_Lateol abrax_maculatus_ / 7405694) using the mem algorithm of BWA software. Subsequently, samtools software was used for file conversion, and variant detection was performed using the GATK software best practice recommendations, ultimately obtaining 22,222,554 variant sites.

[0022] Wherein, step (2) includes the following process:

[0023] S1: All sites were initially filtered according to the following criteria:

[0024] The sequencing depth was ≥5×, the deletion rate was <90%, the minor allele frequency (MAF) was ≥0.05, and non-biallelic (i.e., multi-allelic) SNP sites were removed.

[0025] S2: In the filtered data set, select SNP variant sites according to the following conditions:

[0026] Heterozygosity rate ≤ 0.5, deletion rate ≤ 0.1, MAF ≥ 0.35, and ensure uniform distribution on chromosomes ( Figure 2 ).

[0027] S3: The screening criteria for INDELs are: located in the gene coding region and can cause frameshift mutations (the number of inserted or deleted bases is not 3 or a multiple of 3), sequencing depth ≥5×, deletion rate ≤0.2, heterozygosity rate ≤0.5, and MAF ≥0.2.

[0028] After the above series of filtering and screening, the number of background sites was reduced to 5,312,663.

[0029] S4: Search the literature and screen the loci related to the important economic traits of L. japonica. A total of 85 functional loci related to the growth traits of L. japonica were collected from the following two publicly published papers:

[0030] (1)https: / / www.sciencedirect.com / science / article / pii / S0044848622013126;

[0031] Its index information is:

[0032] Zhang, C.; Wen, H.; Zhang, Y.; Zhang, K.; Qi, X.; Li, Y. First genome-wide association study and genomic prediction for growth traits in spotted seabass (Lateolabrax maculatus) using whole-genome resequencing. Aquaculture 2023,566,739194,doi:https: / / doi.org / 10.1016 / j.aquaculture.2022.739194.

[0033] (2)https: / / www.mdpi.com / 2076-2615 / 14 / 20 / 2995;

[0034] Its index information is:

[0035] Zhou, Z.; Shao, G.; Shen, Y.; He, F.; Tu, X.; Ji, J.; Ao, J.; Chen,

[0037] S5: Design probes for the background sites described in S3 and the functional sites described in S4 and further screen the sites. The probe length is 110 bases (bp), covering the sites to be tested. The GC content of the probe is between 30% and 70%, and the number of homologous regions is ≤5 to reduce the possibility of cross-hybridization. Finally, 45,459 sites were retained, including 45,381 background sites and 78 functional sites related to the growth of L. japonicus; the background sites include 44,927 SNP sites and 454 INDELs sites, and the functional sites include 68 SNPs sites and 10 INDELs sites.

[0038] Specifically, step (3) includes the following process:

[0039] S1. synthesizing the probe described in step (1) and labeling it with biotin;

[0040] S2. Randomly select Lateolabranch samples, extract their DNA and construct a DNA library, mix the obtained DNA library with the probe prepared in step S2 in a solution, and based on the principle of DNA base complementary pairing, the probe will be complementary to the target region of the genome to form a double strand;

[0041] S3. Using streptavidin-coated magnetic beads, the DNA fragments carrying biotin are adsorbed and enriched, thereby capturing the target sites hybridized with the probe;

[0042] S4. The target DNA fragment captured in step S3 is eluted and amplified, and the amplified product is sequenced by high-throughput sequencing and then compared with the reference genome of L. japonica to obtain the genotype data of the tested L. japonica;

[0043] S5. Use the genotyping results obtained in step S4 to compare with the site data of the prepared probe to obtain the site detection rate of the Lateolabrax liquid phase chip.

[0044] Based on the above, this solution also provides the use of the Lateolabrax liquid phase chip in the preparation of a product for detecting single nucleotide polymorphisms SNPs and / or insertion and deletion INDELs genetic variation.

[0045] Based on the above, this solution also provides a method for detecting the genotype of Lateolabrax japonicus, which uses the Lateolabrax japonicus liquid phase chip described above or the Lateolabrax japonicus liquid phase chip prepared by the method described above, and the method comprises the following steps:

[0046] 1) extracting genomic DNA of the Lateolabrax japonicus sample to be tested and constructing a DNA library;

[0047] 2) hybridizing the DNA library with the probe of the liquid phase chip in the solution to form a probe-DNA complex;

[0048] 3) Capturing the complex using magnetic beads coated with streptavidin;

[0049] 4) Performing PCR amplification and high-throughput sequencing on the captured target DNA fragments, and obtaining the genotyping of the target site through bioinformatics analysis (e.g., obtaining the genotype data of the target SNPs and INDELs through bioinformatics analysis).

[0050] Based on the above, this solution also provides the application of the above-mentioned method for detecting the genotype of Lateolabrax japonicus in one of the following:

[0051] 1) Used for genomic selection breeding of Lateolabrax japonicus;

[0052] 2) used for genome-wide association analysis of Lateolabrax japonicus;

[0053] 3) Used for identification of Lateolabrax japonicus germplasm;

[0054] 4) Used for genetic diversity analysis of Lateolabrax japonicus populations / groups.

[0055] By adopting the above-mentioned technical scheme, the present invention has the following beneficial effects compared with the prior art: This scheme provides a liquid phase chip for striped seabass, which is based on the targeted genotype detection technology of liquid phase probe hybridization, and can detect the genotypes of 45,459 target sites evenly distributed on the striped seabass genome, including 45,381 background sites and 78 functional sites related to the growth of striped seabass; the background sites include 44,927 SNP sites and 454 INDELs sites, and the functional sites include 68 SNPs sites and 10 INDELs sites. Compared with the currently available genome-scale genotyping methods such as high-throughput sequencing and simplified genome sequencing, the liquid phase chip provided by this scheme has great advantages in the repeatability and accuracy of target site detection, as well as the degree of experimental standardization, the simplicity of data analysis and the required computing resources. This scheme has developed a liquid phase genotyping chip for striped seabass for the first time, which will effectively promote the analysis of the genetic mechanism of striped seabass traits and genetic breeding practices. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0057] Figure 1 This is the principal component analysis diagram of the Lateolabrax japonicus samples used for the development of liquid phase chips; the labels represent: TJ: Tianjin; YT: Yantai; WD: Wendeng; QD: Qingdao; ZS: Zhoushan; MW: Fujian Minwei Industrial Co., Ltd.; ND: Ningde; ST: Shantou; TS: Tieshan Port; BH: Beihai; FC: Fangchenggang Port.

[0058] Figure 2 This is the heat map of the distribution of the target sites of the Lateolabrax liquid phase microarray on chromosomes;

[0059] Figure 3 This is the target site detection rate diagram of the Lateolabrax japonicus liquid phase chip. DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0061] Example 1: Preparation of Lateolabrax liquid phase chip

[0062] The preparation of L. japonica liquid phase microarray includes L. japonica sample collection, whole genome resequencing of samples, collection of L. japonica resequencing data in the database, detection of variant sites, target site screening and probe synthesis. The specific steps are as follows:

[0063] 1. Collection of Lateolabrax samples

[0064] A total of 121 samples of L. japonicum were collected, including 90 wild samples and 31 farmed samples. Wild samples were collected from the Yellow Sea, Bohai Sea, East China Sea and Beibu Gulf of my country, including 15 from Tianjin, 15 from Qingdao, 15 from Zhoushan, 15 from Ningde, 15 from Shantou and 15 from Beihai. The sampling points and their corresponding group numbers for the development of liquid phase chips are as follows: TJ: Tianjin; YT: Yantai; WD: Wendeng; QD: Qingdao; ZS: Zhoushan; MW: Fujian Minwei Industrial Co., Ltd.; ND: Ningde; ST: Shantou; TS: Tieshan Port; BH: Beihai; FC: Fangchenggang; The samples cover three populations of L. japonicum ( Figure 1 ), the cultured samples were provided by Fujian Minwei Industrial Co., Ltd., and all sample fin rays were cut and preserved in anhydrous ethanol.

[0065] 2. Whole genome resequencing of Lateolabrax japonicus samples

[0066] The whole genome of L. japonicus was resequenced based on the double-end PE150 mode of the Illumina or MGI-2000 / MGI-T7 sequencing platform to obtain the raw sequencing data.

[0067] 3. Collection of Lateolabrax japonicus resequencing data in the database

[0068] A total of 60 resequencing data of spotted seabass were collected from existing literature. These spotted seabass came from six locations including Tianjin, Wendeng, Yantai, Fangchenggang, Haikang Port, and Tieshan Port, with 10 fish in each location. These data were published by Chen et al. in the journal Evolutionary Applications in 2023 (https: / / onlinelibrary.wiley.com / doi / 10.1111 / eva.13551).

[0069] 4. Processing of resequencing data and detection of variant sites

[0070] The resequencing data of 121 japonica seabass obtained in step 2 and the resequencing data of 60 japonica seabass obtained in step 3 were processed, including the following steps:

[0071] 4.1 Raw data quality control

[0072] Fastp software was used to perform quality control on the raw sequencing data, remove low-quality bases and adapter contamination, and obtain high-quality clean reads.

[0073] 4.2 Sequence alignment with reference genome

[0074] The quality-controlled data were compared with the high-quality Lateolabrax japonicus reference genome sequence using the mem algorithm of the BWA software.

[0075] (https: / / figshare.com / articles / dataset / Draft_genome_of_the_Chinese_seabass_Late olabrax_maculatus_ / 7405694) was used for comparison and a SAM file was generated.

[0076] 4.3 Convert file formats

[0077] Use samtools software to convert SAM files to BAM files for subsequent analysis.

[0078] 4.4 Sorting BAM files

[0079] Use the sort command of the samtools software to sort the BAM files to improve the efficiency of subsequent operations.

[0080] 4.5 Marking duplicate reads and creating indexes

[0081] The MarkDuplicates tool of GATK software was used to identify and mark duplicate reads. Subsequently, the index command of samtools software was used to create an index for the BAM file.

[0082] 4.6 Variant Detection Using GATK

[0083] The HaplotypeCaller tool of GATK was used to detect SNPs and Indels for each sample and generate individual g.vcf files. The processed g.vcf files of 181 spotted sea bass were merged for joint analysis of variant sites, and 22,222,554 variant sites were initially obtained.

[0084] 5. Selection of target sites

[0085] 5.1 Preliminary Filtering

[0086] All sites were initially filtered according to the following criteria: sequencing depth ≥5×, missing rate <90%, minor allele frequency (MAF) ≥0.05, and non-biallelic SNP sites were removed.

[0087] 5.2SNP site selection:

[0088] In the filtered data set, SNP variant sites were selected according to the following conditions: heterozygosity rate ≤ 0.5, missing rate ≤ 0.1, MAF ≥ 0.35, and ensuring uniform distribution on the chromosome.

[0089] 5.3 Indel Selection

[0090] The screening conditions for Indel are: located in the gene coding region and can cause frameshift mutation (the number of inserted or deleted bases is not 3 or a multiple of 3), sequencing depth ≥5×, deletion rate ≤0.2, heterozygosity rate ≤0.5, and MAF ≥0.2.

[0091] 5.4 Collection of functional sites

[0092] Literature was searched and loci related to important economic traits of L. japonicus were screened. A total of 85 loci related to growth traits of L. japonicus were collected from the following two publicly published papers:

[0093] (1)https: / / www.sciencedirect.com / science / article / pii / S0044848622013126;

[0094] Its index information is:

[0095] Zhang, C.; Wen, H.; Zhang, Y.; Zhang, K.; Qi, X.; Li, Y. First genome-wide association study and genomic prediction for growth traits in spotted seabass (Lateolabrax maculatus) using whole-genome resequencing. Aquaculture 2023,566,739194,doi:https: / / doi.org / 10.1016 / j.aquaculture.2022.739194.

[0096] (2)https: / / www.mdpi.com / 2076-2615 / 14 / 20 / 2995;

[0097] Its index information is:

[0098] Zhou, Z.; Shao, G.; Shen, Y.; He, F.; Tu, X.; Ji, J.; Ao, J.; Chen,

[0100] 5.5 Probe design and target site determination

[0101] Probes were designed for the background sites and functional sites obtained in steps 5.2-5.4. The probe length was 110 bases (bp), covering the sites to be tested. The GC content of the probe was between 30% and 70%, and the number of homologous regions was ≤5 to reduce the possibility of cross-hybridization. Finally, 45,459 sites were retained, including 45,381 background sites and 78 functional sites related to the growth of L. japonicus; the background sites included 44,927 SNP sites and 454 INDELs sites, and the functional sites included 68 SNPs sites and 10 INDELs sites.

[0102] 5.6 Probe Synthesis

[0103] Synthesize the probes targeting 45,459 sites as described in 5.5, and label them with biotin;

[0104] Example 2: Quality evaluation of Lateolabrax liquid phase chip

[0105] 12 samples of Lateolabrax japonicus were selected, and the liquid phase chip prepared in Example 1 was used to detect the genotype of the target site of this proposal. The specific steps are as follows:

[0106] 1. Extraction of genomic DNA from Lateolabrax japonicus and construction of its library

[0107] The genomic DNA of the tested Lateolabrax was fragmented by ultrasonic shearing, and the Lateolabrax DNA library was obtained by steps such as end filling, A-tailing, and ligating sequencing adapters.

[0108] 2. Acquisition of target DNA fragments

[0109] The Lateolabranch DNA library and biotin-labeled chip probes are mixed in a solution. Based on the principle of DNA base complementary pairing, the probes will complement the genomic target region of the obtained DNA sample to form a double strand. The DNA fragments carrying biotin are adsorbed and enriched using streptavidin-coated magnetic beads to obtain the target DNA fragments.

[0110] 3. Acquisition of Lateolabrax japonicus genotyping data

[0111] The captured DNA fragments were amplified by PCR and sequenced with high throughput, and the sequencing data were compared with the reference genome of the Lateolabrax japonicus to obtain the genotyping data of the Lateolabrax japonicus.

[0112] In this case, the detection rate of the target sites of 12 Lateolabrax Lateolabrax by liquid phase microarray was between 97.90% and 99.06%, and the average site detection rate was 98.427% (Table 5-7 and Figure 3 ).

[0113] Table 5. SNP detection results of Lateolabrax HPLC microarray in 12 Lateolabrax samples

[0114] Sample No. Number of SNPs detected SNP detection rate % Number of missing SNPs SNP missing rate % 1 44572 99.06％ 423 0.94％ 2 44217 98.27％ 778 1.73％ 3 44047 97.89％ 948 2.11％ 4 44302 98.46％ 693 1.54％ 5 44186 98.20％ 809 1.80％ 6 44245 98.33％ 750 1.67％ 7 44184 98.20％ 811 1.80％ 8 44278 98.41％ 717 1.59％ 9 44421 98.72％ 574 1.28％ 10 44306 98.47％ 689 1.53％ 11 44303 98.46％ 692 1.54％ 12 44368 98.61％ 627 1.39％

[0115] Table 6. Indel detection results of L. japonicus liquid phase microarray in 12 L. japonicus samples

[0116] Sample No. Indel number detected Indel detection rate % Indel number Indel deletion rate % 1 459 98.92％ 5 1.08％ 2 460 99.14％ 4 0.86％ 3 458 98.71％ 6 1.29％ 4 459 98.92％ 5 1.08％ 5 460 99.14％ 4 0.86％ 6 459 98.92％ 5 1.08％ 7 458 98.71％ 6 1.29％ 8 456 98.28％ 8 1.72％ 9 458 98.71％ 6 1.29％ 10 457 98.49％ 7 1.51％ 11 457 98.49％ 7 1.51％ 12 457 98.49％ 7 1.51％

[0117] Table 7. Total site detection results of L. japonicus liquid phase microarray in 12 L. japonicus samples

[0118]

[0119]

[0120] The above descriptions are only some embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A marker combination for genotyping of Lateolabrax japonicus, characterized in that: The marker combination includes background sites and functional sites; The background sites include 44,927 SNPs sites as shown in Table 1 and / or 454 INDELs sites as shown in Table 2; The functional sites include 68 SNPs sites as shown in Table 3 and / or 10 INDELs sites as shown in Table 4; Table 1. Location of background SNPs in Lateolabrax HPLC array Table 2. Location information of Lateolabrax japonicus liquid phase array background INDELs in the reference genome Table 3. Location of functional SNPs in Lateolabrax japonicus liquid phase array in the reference genome Table 4. Location of functional INDELs in the Lateolabrax chromatin array in the reference genome 。 2. The marker combination for genotyping of Lateolabrax japonicus according to claim 1, characterized in that: The SNPs in the background sites are evenly distributed in the genome, and the INDELs are located in the gene coding region and can cause frameshift mutations of the gene.

3. A liquid phase chip for Lateolabrax japonicus based on targeted genotype detection technology, characterized in that: It comprises a probe and / or a primer for detecting the marker combination according to claim 1 or 2.

4. The method for preparing the Lateolabrax liquid phase chip according to claim 3, characterized in that: It includes the following steps: 1) Collect samples of wild and cultured Leptospermum japonicum and extract their genomic DNA; 2) Use the next-generation sequencing technology to resequence the whole genome of the sample and use bioinformatics to identify SNPs and INDELs sites; 3) Preliminary screening of SNPs sites based on one or more criteria including sequencing depth, minor allele frequency, deletion rate, heterozygosity and chromosome distribution; INDELs sites were initially screened based on sequencing depth, MAF, deletion rate, heterozygosity, and number of insertion and deletion base pairs; By searching the literature, we searched for the variation sites related to the growth of Lateolabrax japonicus and used them to screen the functional sites. 4) The SNPs and INDELs sites are combined to obtain 45,459 target sites including background sites and functional sites, and then a probe is designed for each target site; 5) synthesizing probes and modifying the probes with biotin to prepare a Lateolabrax japonicus liquid phase chip; 6) Use the prepared Lateolabrax liquid phase chip to detect target sites, calculate the detection rate, evaluate the chip quality, and obtain a Lateolabrax liquid phase chip whose quality meets the preset requirements.

5. Use of the Lateolabrax liquid phase chip as described in claim 3 in the preparation of a product for detecting single nucleotide polymorphisms (SNPs) and / or insertion and deletion (INDELs) genetic variation.

6. A method for detecting the genotype of Lateolabrax japonicus, characterized in that: The method uses the Lateolabrax liquid phase chip according to claim 3 or the Lateolabrax liquid phase chip prepared by the method according to claim 4, and the method comprises the following steps: 1) extracting genomic DNA of the Lateolabrax japonicus sample to be tested and constructing a DNA library; 2) hybridizing the DNA library with the probe of the liquid phase chip in the solution to form a probe-DNA complex; 3) Capturing the complex using magnetic beads coated with streptavidin; 4) The captured target DNA fragments are amplified by PCR and sequenced by high-throughput sequencing, and the genotyping of the target site is obtained through bioinformatics analysis.

7. Use of the method for detecting the genotype of Lateolabrax japonicus as claimed in claim 6 in one of the following: 1) Used for genomic selection breeding of Lateolabrax japonicus; 2) used for genome-wide association analysis of Lateolabrax japonicus; 3) Used for identification of Lateolabrax japonicus germplasm; 4) Used for genetic diversity analysis of Lateolabrax japonicus populations / groups.

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