A 50K liquid-phase chip for the whole genome of Leymus chinensis and its applications

Abstract

The present invention belongs to the technical field of whole-genome gene chip, and particularly relates to a 50K liquid-phase chip of the whole genome of Leymus chinensis and its application. The 50K liquid-phase chip of the whole genome of Leymus chinensis in the present invention utilizes the whole-genome resequencing technology to determine the SNP molecular marker combination of the Leymus chinensis, and then develops the 50K liquid-phase chip of the whole genome of Leymus chinensis. This chip combines high-density SNP probes, realizes high-throughput and high-precision genotyping of the Leymus chinensis genome, and has broad application prospects particularly in the field of molecular breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of whole-genome gene chip, and particularly relates to a 50K liquid-phase chip of the whole genome of Leymus chinensis and its application. Background Art

[0002] Leymus chinensis ( Leymus chinensis (Trin.) Tzvel.) belongs to the genus Leymus of the Gramineae family and is one of the important constructive species in the meadow steppe and arid steppe in the eastern part of the Eurasian steppe region. The Songnen Plain in the northeast of China and the eastern part of Inner Mongolia are also the main distribution centers of Leymus chinensis. At the same time, it is widely distributed in Hebei, Shanxi, Henan, Shaanxi, Ningxia, Gansu, Qinghai, Xinjiang and other regions; in addition, it is also sporadically distributed in Russia, Mongolia, North Korea, Japan and other places. As an important native forage grass in northern China, Leymus chinensis not only has high nutritional value, stable yield and good palatability, and is liked by various livestock, but also has excellent stress resistance and can adapt to extreme climates and diverse soil environments. Moreover, the developed horizontal rhizomes of Leymus chinensis endow it with strong penetration and invasion ability, and by forming a strong root network, it has a significant role in soil and water conservation. Therefore, Leymus chinensis plays an irreplaceable role in the ecological construction, vegetation restoration, and livestock husbandry development of northern grasslands.

[0003] However, at present, the cultivated varieties of Leymus chinensis are relatively scarce, and there is a lack of breakthrough varieties, which cannot effectively meet the needs of ecological restoration and forage production. It is urgent to promote the development of the Leymus chinensis industry and ecological construction by accelerating the cultivation of excellent new varieties. However, as an allopolyploid perennial plant, Leymus chinensis has a large and complex genome, is self-incompatible, difficult to genetically transform, and has complex and diverse phenotypic traits. These factors greatly limit the development of basic research and molecular breeding technology of Leymus chinensis. At present, Leymus chinensis breeding still mainly relies on traditional breeding methods, but traditional methods still have the defects of low selection accuracy, low breeding efficiency, long cycle, and extremely high difficulty in cultivating breakthrough varieties. These technical bottlenecks seriously restrict the innovative development of Leymus chinensis molecular breeding.

[0004] With the rapid development of genomics and sequencing technologies, high-throughput SNP liquid chips developed based on big data and omics sequencing technologies have been widely used in various crops for molecular breeding research fields such as high-throughput genotyping, germplasm identification, QTL mapping, genome-wide association study (GWAS), and genomic selection (GS). Compared with traditional genotyping technologies, the new SNP liquid chip technology has advantages such as high flexibility, high detection efficiency, low cost, high sensitivity, large throughput, and support for parallel detection, which can significantly improve the efficiency and accuracy of molecular breeding. At present, although liquid chip technology has been widely applied in many crops, there has been no relevant research or application in Leymus chinensis. Therefore, there is an expectation in this field to develop a SNP liquid chip specifically for Leymus chinensis, which can fill the technical gap in this field and provide an efficient, accurate, and economical technical tool for molecular breeding of Leymus chinensis, solve the "stuck neck" technical bottleneck in current breeding, and provide important technical support for promoting the development of the Leymus chinensis industry and ecological construction. Summary of the Invention

[0005] To this end, the first object of the present invention is to provide a 50K liquid chip for the whole genome of Leymus chinensis. Based on the target capture technology, this chip has characteristics such as strong representativeness, high polymorphism, strong specificity, and strong expansibility, and can be widely applied to molecular breeding fields such as genetic diversity assessment of Leymus chinensis germplasm resources, genetic map construction, gene mapping, molecular marker-assisted selection, variety identification, and genome-wide selection breeding;

[0006] The second object of the present invention is to provide the application of the above-mentioned 50K liquid chip for the whole genome of Leymus chinensis.

[0007] To solve the above technical problems, for a 50K liquid chip for the whole genome of Leymus chinensis described in the present invention, the loci of the liquid chip include multiple SNP loci located on the Leymus_chinensis_Lc6-5 reference genome of Leymus chinensis or its updated version, covering gene regions and their related non-gene regions;

[0008] The specific information of the core SNP loci of the liquid chip is shown in Table 1 in the specification.

[0009] The position and variation information of the SNP loci are represented in the form of chromosome / physical position / reference genotype / variant allele genotype, and the physical position of the SNP molecular marker combination is located and analyzed based on the Leymus chinensis reference genome.

[0010] Specifically, for the 50K liquid chip for the whole genome of Leymus chinensis, the liquid chip includes a primer set and / or probe for detecting the SNP loci, especially the core SNP loci.

[0011] Specifically, the 50K liquid-phase chip for the whole genome of Leymus chinensis includes a 50K probe mixture for SNP capture and reagents for liquid-phase hybridization capture; among them,

[0012] The 50K probe mixture contains probes with high sequence specificity, which can effectively identify and detect the target SNP sites, especially the core SNP sites; and / or,

[0013] The reagents applicable to liquid-phase hybridization capture can efficiently and stably capture the target SNP sites, especially the core SNP sites.

[0014] As an exemplary selection, the reagents for liquid-phase hybridization capture are such as the Hyb&Wash kit, for example, the Novogene NGP Kit.

[0015] Specifically, for the 50K liquid-phase chip of the whole genome of Leymus chinensis, the 50K liquid-phase targeted capture probes are designed based on the positive strand of the Leymus chinensis reference genome;

[0016] Preferably, the length range of the probes is 55 - 120 nt, and the average length is about 100 nt.

[0017] Specifically, for the 50K liquid-phase chip of the whole genome of Leymus chinensis, the 50K liquid-phase targeted capture probes are designed based on the principle of thermodynamic stability to ensure 99% capture of the conventional regions;

[0018] Preferably, for the difficult and complex regions, the 50K liquid-phase targeted capture probes improve the effective coverage of the difficult regions through the scheme of adjusting the probe positions and placing the multi-layer "overlapping" probes.

[0019] Furthermore, the probe design of this liquid-phase chip is based on the principle of thermodynamic stability, including factors such as melting temperature (Tm), GC content, and sequence specificity, and comprehensively considers the genomic complexity of the target species, the position of the target site, and the GC content around it to ensure 99% capture of the conventional regions. For the complex regions with too high or too low GC content, the coverage of the difficult regions is improved by adjusting the probe positions and adopting the multi-layer 'overlapping' probe design. This scheme improves the capture efficiency in the extreme GC regions by increasing the complementarity and stability between the probes. The probe design is based on the positive strand of the latest version of the Leymus chinensis reference genome, the length range of the probes is 55 - 120 nt, and the average length is about 100 nt, which is optimized to ensure efficient SNP capture.

[0020] Furthermore, this chip uses the principle of nucleic acid hybridization for targeted capture. When two nucleic acid strands are complementary, a stable double-stranded structure will form between them. Through the denaturation and renaturation processes, the target region library is first denatured to remove repetitive sequences and adapter sequences, and then probes complementary to the bases of the target region are added for renaturation. Non-specific bindings are washed away, and finally a specific probe-library conjugate is obtained, thus completing the capture of the target region.

[0021] Furthermore, the preparation of the liquid-phase targeted capture probe includes template synthesis and RNA preparation. The probe template consists of single-stranded DNA (oligo) synthesized organically. After quality control (QC) and pooling, the qualified templates are used to prepare RNA analog probes. In the synthesis of RNA probes, biotin-labeled nucleotide analogs (such as LNA, PNA, etc.) are used to improve the binding stability between the probe and the library. The biotin-labeled probe can bind to streptavidin-coated magnetic beads, further improving the capture efficiency.

[0022] The present invention also discloses a kit, characterized in that the kit includes a primer set and / or a probe capable of detecting the SNP sites, especially the core SNP sites, in the liquid-phase chip.

[0023] The present invention also discloses the applications of the Leymus chinensis whole-genome 50K liquid-phase chip and the kit in the fields of variety identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene mapping, marker-assisted selection, genotyping, genome-wide association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Leymus chinensis.

[0024] The present invention also discloses a Leymus chinensis SNP molecular marker combination, wherein the physical positions of the SNP molecular marker sites are determined by sequence alignment based on the Leymus chinensis reference genome Leymus_chinensis_Lc6-5;

[0025] The SNP molecular marker sites are as shown in Table 1 in the specification.

[0026] The present invention also discloses a primer set and / or a probe for detecting the Leymus chinensis SNP molecular marker.

[0027] The present invention also discloses the applications of the Leymus chinensis SNP molecular marker or the primer set and / or the probe in the fields of variety identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene mapping, marker-assisted selection, genotyping, genome-wide association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Leymus chinensis.

[0028] The present invention determines the SNP molecular marker combination of the said Leymus chinensis by using whole-genome sequencing, and screens out SNP molecular marker sites based on the Leymus chinensis reference genome Leymus_chinensis_Lc6-5, which can comprehensively evaluate the gene characteristics of Leymus chinensis.

[0029] Based on the target capture sequencing technology, the present invention develops a 50K liquid-phase chip for the whole genome of Leymus chinensis. By combining high-density SNP site probes, it realizes high-throughput and high-precision genotyping of the Leymus chinensis genome, and is widely applicable to fields such as genetic diversity assessment, genetic map construction, QTL mapping, genome-wide association analysis (GWAS), marker-assisted selection breeding (MAS), and genome-wide selection breeding (GS) of Leymus chinensis.

[0030] For the 50K liquid-phase chip of the whole genome of Leymus chinensis of the present invention, in addition to using the Leymus chinensis reference genome, homology alignment analysis of the de novo transcriptome is also adopted for probe design, optimizing the selection of SNP sites and genome coverage, ensuring the uniform distribution of SNP sites in the Leymus chinensis genome and good polymorphism. The probe sites cover the key functional gene regions of Leymus chinensis, including genes for important traits such as growth, development, and resistance, and can provide important bases for the molecular marker identification of agronomic traits, quality traits, and stress resistance traits of Leymus chinensis.

[0031] For the 50K liquid-phase chip of the whole genome of Leymus chinensis of the present invention, for difficult complex regions, by adjusting the probe positions and adopting a multi-layer "tiling" probe placement scheme, the effective coverage of difficult regions is improved, while ensuring the uniform distribution of SNP sites in the genome and the coverage of key functional regions, improving the coverage of functional regions and supporting the research on agronomic traits and stress resistance traits.

[0032] The 50K liquid-phase chip of the whole genome of Leymus chinensis of the present invention can provide high-quality genotyping data. Compared with other genotyping technologies (such as GBS), the data consistency is higher, and it has stronger data alignment and accumulation capabilities. The high-throughput and data consistency of this chip provide a strong guarantee for long-term data storage and comprehensive utilization, greatly improving the long-term availability of data and the simplicity of analysis.

[0033] For the 50K liquid-phase chip of the whole genome of Leymus chinensis of the present invention, the liquid-phase chip technology of the present invention has higher flexibility compared with traditional solid-phase chips. It can adjust the SNP density of the chip according to needs, provide multiple versions such as 50K, 40K, and 30K, and can capture the sequence information of 100 bp upstream and downstream of the target SNP site, providing more data for functional gene research and adapting to different scales of research and application needs. The liquid-phase chip can complete high-throughput detection, has high detection flexibility, can effectively shorten the sample detection cycle, and improve scientific research efficiency.

[0034] The 50K liquid-phase chip of the whole genome of Leymus chinensis in the present invention is based on the targeted capture sequencing technology. When designing the probes, the tolerance of the flanking sequences is fully considered. When the variation of the flanking sequences does not exceed 10%, the target sequences can still be stably captured. This technical characteristic ensures the high reliability of the data. At the same time, in addition to obtaining the target SNP information, this chip can also obtain the sequence information of 100 bp upstream and downstream of each target SNP site, providing more supporting data for the functional gene research of Leymus chinensis.

[0035] The 50K liquid-phase chip of the whole genome of Leymus chinensis in the present invention provides strong technical support for the molecular breeding of Leymus chinensis by introducing the advanced targeted capture sequencing technology. It can effectively support the transition from traditional breeding to molecular breeding, effectively improve the breeding efficiency, promote the improvement of Leymus chinensis varieties, fill the technical gap in the field of molecular breeding of Leymus chinensis in China, provide an important technical platform for the basic research, genetic improvement and variety innovation of Leymus chinensis, and further promote the modern development of the Leymus chinensis industry.

[0036] The 50K liquid-phase chip of the whole genome of Leymus chinensis in the present invention realizes the completely domestic synthesis of probes, sample detection and reagent use, avoiding the high costs brought by imported equipment and reagents. By reducing the material costs and reducing the technical dependence, the autonomy and controllability of production and application are realized, the risk of core data leakage is reduced, and the inconvenience brought by external trade frictions is avoided, meeting the domestic demand for the autonomy and controllability of agricultural science and technology. Brief Description of the Drawings

[0037] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the attached drawings, wherein,

[0038] Figure 1 is the flow chart of SNP site screening in Example 1 of the present invention;

[0039] Figure 2 is the distribution map of 51,696 SNP site probes on chromosomes in Example 3 of the present invention;

[0040] Figure 3 is the statistical chart of the distribution of SNP markers in the gene structure in Example 3 of the present invention;

[0041] Figure 4 is the radar chart of the expected performance and actual test performance of the 50K liquid-phase chip of Leymus chinensis in Example 4 of the present invention;

[0042] Figure 5 is the phylogenetic tree analysis of 148 materials of Leymus chinensis in Example 5 of the present invention;

[0043] Figure 6PCA scatter plot of 148 Leymus chinensis materials in Example 5 of the present invention;

[0044] Figure 7 Genome-wide association analysis result diagram of 148 Leymus chinensis materials in Example 5 of the present invention. Detailed implementation manners

[0045] In the following examples of the present invention, the 50K liquid-phase chip of the Leymus chinensis genome is developed based on the target capture technology. Its working principle is to design probes for the target region sequences, target and capture the target fragments, and then use a sequencing platform for high-throughput sequencing to target detect the target genes and loci, quickly detect a large number of samples, so as to achieve the purpose of detecting and genotyping the genes in the target region.

[0046] In the following examples of the present invention, the 50K liquid-phase chip of the Leymus chinensis genome contains a set of 50K SNP probe sites of Leymus chinensis.

[0047] In the following examples of the present invention, the 50K liquid-phase chip of the Leymus chinensis genome includes an independently packaged 50K probe mixture and a Hyb&Wash kit suitable for liquid-phase hybridization capture; wherein,

[0048] The probe mixture contains SNP molecular marker sites, a total of 50K liquid-phase target capture probes.

[0049] In some embodiment solutions, the liquid-phase chip of the present invention uses the principle of nucleic acid hybridization for target capture. When two nucleic acid strands are complementary, a stable double-stranded structure will be formed between them. Through the denaturation and renaturation processes, first denature the target region library to remove repetitive sequences and adapter sequences, and then add probes complementary to the bases of the target region for renaturation; the non-specific binding is washed away, and finally a specific probe-library conjugate is obtained, thus completing the capture of the target region.

[0050] In some embodiment solutions, the preparation of the liquid-phase target capture probes of the liquid-phase chip of the present invention includes template synthesis and RNA preparation. The probe template is composed of synthetic single-stranded DNA (oligo). After quality control (QC) and pooling, the qualified template is used to prepare RNA analog probes. In the synthesis of RNA probes, biotin-labeled nucleotide analogs (such as LNA, PNA, etc.) are used to improve the binding stability between the probes and the library. The biotin-labeled probes can bind to streptavidin-coated magnetic beads to further improve the capture efficiency.

[0051] In some embodiment solutions, for the 50K liquid-phase chip of the Leymus chinensis genome, the 50K liquid-phase target capture probes are designed based on the positive strand of the Leymus chinensis reference genome;

[0052] Preferably, the length range of the probes is 55 - 120 nt, and the average length is about 100 nt.

[0053] Specifically, for the 50K liquid-phase chip of the whole genome of Leymus chinensis, the probe design of this liquid-phase chip is based on the principle of thermodynamic stability, including factors such as melting temperature (Tm), GC content, and sequence specificity, and comprehensively considers the genomic complexity of the target species, the position of the target site, and the GC content around it to ensure 99% capture of the conventional region. For complex regions with too high or too low GC content, by adjusting the probe position, a multi-layer 'tiling' type probe design is adopted to improve the coverage of difficult regions. This scheme improves the capture efficiency in extreme GC regions by increasing the complementarity and stability between probes. The probe design is based on the positive strand of the latest version of the Leymus chinensis reference genome, the length range of the probes is 55 - 120 nt, and the average length is about 100 nt, which is optimized to ensure efficient SNP capture.

[0054] Example 1

[0055] This example provides a 50K liquid-phase chip for the whole genome of Leymus chinensis, and the specific screening process is as Figure 1 shown, and the specific screening process is as follows.

[0056] (I) Preliminary filtering and screening of SNPs

[0057] In this example, 100 Leymus chinensis samples were re-sequenced, and SNP variant detection was performed on these sequencing data. The reference genome version used was Leymus_chinensis_Lc6-5. The preliminary SNP filtering and screening process is as follows:

[0058] (1) Depth filtering: On the basis that the average sequencing depth of the samples reaches 10X, SNP sites with a depth of not less than 3X are selected to ensure the reliability of the data, and low-depth sites are filtered out;

[0059] (2) Integrity filtering: SNP markers with a genotype coverage of at least 50% of all sample individuals were screened; specifically, for each polymorphic marker locus, it was required that at least 50 out of 100 samples had a determined genotype, so as to filter out markers with poor genotype integrity;

[0060] (3) Minor allele frequency (MAF) filtering: Sites with an MAF value lower than 0.05 were filtered out to ensure that the selected sites have sufficient variation information;

[0061] (4) After the above preliminary filtering, 40M basic SNP sites were retained.

[0062] (II) Deep filtering of SNPs

[0063] Linkage disequilibrium (LD)-based filtering: Linkage disequilibrium analysis was performed using the PLINK software with two parameter settings: one was a 50-kb window, 10-bp step size, and R2 < 0.2; the other was a 100-kb window, 1-bp step size, and R2 < 0.2. These filtering parameters referred to the reported locus screening criteria for allopolyploid plants.

[0064] Hardy-Weinberg equilibrium (HWE) filtering: The p-values of the Hardy-Weinberg equilibrium for all SNP loci were calculated and filtered at 1e-6.

[0065] Gene region screening: SNP loci located in gene regions and their upstream and downstream regions were selected, and other loci within 100 bp upstream and downstream of the SNP loci were filtered out. For exon regions, if the distance between a certain SNP and the previous SNP was less than 100 bp and it was a non-synonymous mutation, then this SNP was retained.

[0066] Non-exon region filtering: In non-exon regions, variant loci of the A / T and G / C types were filtered out.

[0067] After deep filtering of SNPs, a total of 95,954 background loci, namely VIP SNP loci, were finally retained.

[0068] (III) Screening of trait-associated loci

[0069] In this example, using the resequencing data of 100 Leymus chinensis samples, a genome-wide association study (GWAS) was performed on 8 agronomic traits of Leymus chinensis using the mixed linear model (MLM) of the GEMMA software. In the analysis, default parameters were used, and the significance threshold was set to 6 to identify the associated loci for each trait. The locus retention strategy was as follows:

[0070] (1) For traits with fewer than 450 associated loci, all associated loci were retained;

[0071] (2) For traits with more than 450 associated loci, loci less than 100K from the gene were screened, sorted by P-value, and the top 450 loci were selected;

[0072] (3) Among the screened loci, if the distance between two loci was less than 1K, only the locus closest to the gene was retained;

[0073] (4) Through these steps, a total of 1,667 loci associated with the agronomic traits of Leymus chinensis, namely VVIP SNP loci, were finally selected.

[0074] (IV) Determination of SNP loci on 50K liquid-phase chips

[0075] Merge the SNPs of basic, VIP, and VVIP obtained in the above analysis, remove duplicates, and then screen the locus dataset according to the sliding window method. In each window, SNPs of VIP and VVIP are preferentially selected, and background loci are used to supplement the remaining positions. 51,696 SNP loci can be screened out as the locus set of the 50K liquid chip. The specific information of the core SNP loci included is recorded in Table 1 below. Among them, the position and variation information of the SNP loci are represented in the form of chromosome: physical position: reference genotype / variant allele genotype, and the physical position of the SNP molecular marker combination is located and analyzed based on the Leymus chinensis reference genome.

[0076] Table 1 Core SNP Locus Information

[0077]

[0078] Example 2

[0079] This example designs liquid chip probes based on the SNP locus information screened in Example 1.

[0080] The chip designed in this example is based on the principle of thermodynamic stability, including factors such as melting temperature (Tm), GC content, and sequence specificity, and comprehensively considers the genomic complexity of the target species, the position of the target locus, and the GC content around it to ensure 99% capture in the conventional region. The probe is based on the positive strand of the Leymus chinensis reference genome, with a length range of 55 - 120 nt and an average length of about 100 nt.

[0081] For complex regions with too high or too low GC content, by adjusting the probe position, a multi-layer 'tile' type probe design is adopted to improve the coverage of difficult regions. This scheme improves the capture efficiency in extreme GC regions by increasing the complementarity and stability between probes. In this example, the key design parameters of the probe include:

[0082] (1) GC content: 30% - 80%; the number of sites near a single target site is less than 10; site specificity is greater than 50%; based on these criteria, the chip selects the set of sites with the lowest scores according to the scoring.

[0083] (2) 1,667 GWAS-related sites are retained without considering the scoring values.

[0084] Based on the above criteria, a liquid-phase chip probe was designed and synthesized in this example.

[0085] Example 3

[0086] In this example, the distribution and annotation of SNP sites on the chromosomes of the liquid-phase chip were carried out based on the fact.

[0087] In this example, the distribution of the liquid-phase chip screened in Example 1 on 14 chromosomes of Leymus chinensis was counted. On average, each chromosome contains about 3,689 SNPs, and the average distance between adjacent sites is 145 kb. The 51,697 SNPs screened show an almost uniform distribution, and the site distribution map is as Figure 2 shown. The above SNP annotation results show that among the 51,697 sites, 34% of the sites are located in the gene region and 2% are located in the UTR region. In addition, 34% of the sites may have a significant impact on gene function, potentially leading to changes in the function of protein-coding genes. The annotation information of SNP sites is as Figure 3 shown. The SNP combination of Leymus chinensis screened by this chip significantly improves the detection success rate of candidate gene sites, providing strong support for subsequent functional gene research.

[0088] Example 4

[0089] In this example, based on the Leymus chinensis liquid-phase chip developed in the previous Example 2, 12 samples of Leymus chinensis were selected to test the performance of the chip, and the specific operation steps are as follows.

[0090] (I) Probe preparation

[0091] The preparation of the Leymus chinensis liquid-phase targeted capture probe described in this example includes template synthesis and RNA preparation. The probe template is composed of single-stranded DNA (oligo) synthesized organically. After quality control (QC) and pooling, the qualified template is used to prepare RNA analog probes. In the synthesis of RNA probes, biotin-labeled nucleotide analogs (such as LNA, PNA, etc.) are used to improve the binding stability between the probe and the library. The biotin-labeled probe can bind to streptavidin-coated magnetic beads to further improve the capture efficiency.

[0092] (II) Library construction and capture

[0093] The DNA samples are sheared by ultrasound or enzyme digestion, end repair and 3' end A addition, adapter ligation and purification, and Pre-PCR library amplification to obtain the library required for hybrid capture. The capture method is carried out according to the SOP for hybrid capture, and after library and probe hybridization (12-18h), probe and magnetic bead binding, non-specific binding library rinsing, post-capture PCR amplification, library quantification and quality inspection, it is sequenced on the machine.

[0094] (III) Library quality control and quantification

[0095] Take 1 μL of library and use Qubit dsDNA HS Assay Kit reagent to detect the library concentration on Qubit 4.0 Fluorometer. The library concentration before capture >25 ng / μL is considered a qualified library.

[0096] Take 1 μL sample and use Qsep 100 for detection. The main peak of the library should be around 270-450 bp, with no impurity peaks before and after the main peak.

[0097] (IV) Sequencing on the machine

[0098] Take 1 μL of library and use Qubit dsDNA HS Assay Kit reagent to detect the library concentration on Qubit 4.0 Fluorometer and record the library concentration. The concentration of the library after capture is about 1-20 ng / μL.

[0099] Take 1 μL sample and use Qsep 100 to measure the length of library fragments. The length of the library is approximately between 270-450 bp.

[0100] Sequencing was performed using a high-throughput sequencing platform.

[0101] (V) Data analysis

[0102] In this embodiment, the raw image data obtained by high-throughput sequencing (Nova platform, MGI platform, etc.) is identified by a specific program, converted into raw sequencing data in Fastq format, and then compared with the reference genome for bioinformatics analysis, including the following steps:

[0103] (1) Quality control: Remove low-quality sequences, adapter sequences, ployG and other abnormal sequences in the original sequencing data to obtain clean data;

[0104] (2) Alignment: Align, sort, and remove duplicates of the Clean data with the reference genome to obtain a bam file;

[0105] (3)Statistics: Statistical analysis is carried out for indicators such as alignment rate, coverage rate, capture rate, and uniformity.

[0106] (VI) Test results

[0107] In this embodiment, through the above test analysis, the test results include various indicators of the target region, various indicators of the probe region, the coverage of different regions, etc., comprehensively and detailedly demonstrating the various performances of the chip. The product site detection rate is high, and the average site detection rate is 98.9%; the product has good stability and high genotype accuracy, and the average consistency rate of genotypes of repeated samples is 99%.

[0108] In this embodiment, in order to visually display the expected performance and actual test performance of the chip, the average values of indicators such as the QC rate, alignment rate, coverage, capture rate, and uniformity of 2 samples are calculated and plotted as a radar chart. The results are as Figure 4 shown. It can be seen that all indicators are good and meet the expectations.

[0109] Example 5

[0110] This embodiment further verifies the application of the Leymus chinensis liquid-phase chip in the population structure analysis and genome-wide association analysis of Leymus chinensis germplasm resources.

[0111] In this embodiment, the above-mentioned Leymus chinensis liquid-phase chip is used to detect 148 Leymus chinensis materials, extract the genotype typing of the target sites, and annotate the SNP detection results using the ANNOVAR software.

[0112] In this embodiment, the TreeBeST (http: / / treesoft.sourceforge.net / treebest.shtml) software is used to calculate the distance matrix. Based on this, a phylogenetic tree is constructed by the neighbor-joining method. The bootstrap values are obtained through 1000 calculations. The phylogenetic tree analysis results are as Figure 5 shown.

[0113] In this embodiment, the GCTA software is used to analyze the PCA (principal component analysis) composition of the test materials and construct a PCA scatter plot. Each site in the scatter plot represents a sample. The farther the distance between two samples in the figure, the greater the genetic background difference between the two samples, and individuals with similar genetic backgrounds will cluster into one category in the figure. It is found that the 148 Leymus chinensis materials are basically scattered and there is no obvious clustering. The results are shown in Figure 6 .

[0114] In this example, the GEMMA (http: / / www.xzlab.org / software.html) analysis software was used to perform genome-wide association studies (GWAS) using a mixed linear model, with population genetic structure as a fixed effect and individual kinship as a random effect to correct for the effects of population structure and individual kinship while reducing computation time. Potential candidate SNPs were selected based on the significance level (P-value) of the association. The results are as Figure 7 shown. Only partial results of the trait association analysis are presented in the figure, Figure 7 where (A) is a Manhattan plot, Figure 7 and (B) is a Q-Q plot (Quantile-quantile plot).

[0115] In summary, the present invention developed the described 50K liquid-phase chip for the whole genome of Leymus chinensis based on targeted capture sequencing technology, and by combining high-density SNP locus probes, achieved high-throughput and high-precision genotyping of the Leymus chinensis genome, which is widely applicable to fields such as genetic diversity assessment, genetic map construction, QTL mapping, genome-wide association studies (GWAS), marker-assisted selection breeding (MAS), and genome-wide selection breeding (GS) of Leymus chinensis.

[0116] Obviously, the above examples are merely illustrations for clear explanation and not limitations on the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementations here. The obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A 50K liquid-phase chip for the whole genome of Leymus chinensis, characterized in that, The loci of the liquid-phase chip include SNP loci located on the Leymus chinensis reference genome Leymus_chinensis_Lc6-5, covering gene regions and related non-gene regions; The core SNP locus information of the liquid-phase chip is shown in the following table: The liquid-phase chip includes a primer set and / or probe for detecting the SNP locus, a 50K probe mixture for SNP capture, and a reagent for liquid-phase hybridization capture; wherein, The 50K probe mixture contains probes with high sequence specificity for identifying and detecting target SNP loci; The reagent for liquid-phase hybridization capture is used to capture target SNP loci; The 50K liquid-phase targeted capture probe is designed based on the positive strand of the Leymus chinensis reference genome; The length range of the probe is 55 - 120 nt, and its average length is about 100 nt; Among the 50K liquid-phase targeted capture probes, the conventional region is covered by 1X probes, and the high-difficulty complex region is covered by multi-layer overlapping probes; Among them, the conventional region is a region with low sequence complexity and high homogeneity; the high-difficulty complex region is a region containing high GC content, repetitive sequences or other complex structures.

2. A kit, characterized in that, The kit includes a primer set and / or probe for detecting the SNP locus in the liquid-phase chip described in claim 1.

3. Application of the 50K liquid-phase chip of the whole genome of Leymus chinensis described in claim 1 and the kit described in claim 2 in the fields of variety identification, whole-genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene mapping, marker-assisted selection, genotyping, genome-wide association analysis, cluster analysis or kinship identification, germplasm resource improvement and protection of Leymus chinensis.

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