Leymus chinensis whole genome 50K liquid phase chip and application thereof

Abstract

The invention belongs to the technical field of whole-genome gene chips, and particularly relates to a leymus chinensis whole-genome 50K liquid chip and application thereof. According to the leymus chinensis whole genome 50K liquid phase chip, a whole genome re-sequencing technology is utilized, an SNP molecular marker combination of leymus chinensis is determined, and then the leymus chinensis whole genome 50K liquid phase chip is developed. The chip is combined with a high-density SNP probe, realizes high-throughput and high-precision genetic typing of leymus chinensis genomes, and particularly has wide application prospects in the field of molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of whole genome gene chips, and specifically relates to a 50K liquid phase chip of the whole genome of Leymus chinensis and an application thereof. Background Art

[0002] Leymus chinensis Leymus chinensis (Trin.) Tzvel.) is a perennial forage grass belonging to the genus Leymus in the Poaceae family. It is one of the important community-building species in the meadow steppe and arid steppe in the eastern steppe region of Eurasia. The Songnen Plain in northeastern my country and eastern Inner Mongolia are also the main distribution centers of Leymus chinensis. It is also widely distributed in Hebei, Shanxi, Henan, Shaanxi, Ningxia, Gansu, Qinghai, Xinjiang and other regions; in addition, it is also scattered in Russia, Mongolia, North Korea, Japan and other places. As an important native forage grass in northern my country, Leymus chinensis not only has high nutritional value, stable yield, and good palatability, and is a favorite food for all kinds of livestock, but also has superior stress resistance and can adapt to extreme climates and diverse soil environments. Moreover, the developed horizontal rhizomes of Leymus chinensis give it a strong ability to penetrate and invade, and by forming a strong root network, it has a significant role in consolidating soil and retaining water. Therefore, Leymus chinensis has played an irreplaceable role in the ecological construction of northern grasslands, vegetation restoration, and animal husbandry development.

[0003] However, the current bred 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 allotetraploid perennial plant, Leymus chinensis has a large and complex genome, self-incompatibility, and difficulty in genetic transformation. In addition, the phenotypic traits are complex and diverse. These factors have greatly limited the 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 have seriously restricted the innovative development of Leymus chinensis molecular breeding.

[0004] With the rapid development of genomics and sequencing technology, high-throughput SNP liquid phase chips developed based on big data and omics sequencing technology have been widely used in molecular breeding research fields such as high-throughput genotyping, germplasm identification, QTL positioning, genome-wide association analysis (GWAS), and genomic selection (GS) in a variety of crops. Compared with traditional typing technology, the new SNP liquid phase chip technology has the advantages of high flexibility, high detection efficiency, low cost, high sensitivity, high throughput, and support for parallel detection, which can significantly improve the efficiency and accuracy of molecular breeding. At present, although liquid phase chip technology has been widely used in many crops, there has been no related research or application in Leymus chinensis. Therefore, this field expects to develop a SNP liquid phase chip specifically for Leymus chinensis, which can fill the technical gap in this field and provide efficient, accurate, and economical technical tools for Leymus chinensis molecular breeding, solve the current technical bottlenecks in breeding, and provide important technical support for promoting the development of 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 phase chip of the whole genome of Leymus chinensis, which is based on targeted capture technology and has the characteristics of strong representativeness, high polymorphism, strong specificity and strong expansibility, and can be widely used in molecular breeding fields such as genetic diversity assessment of Leymus chinensis germplasm resources, genetic map construction, gene positioning, molecular marker-assisted selection, variety identification, and whole genome selection breeding; The second object of the present invention is to provide an application of the above-mentioned Leymus chinensis whole genome 50K liquid phase chip.

[0006] In order to solve the above technical problems, the present invention provides a 50K liquid phase chip of the whole genome of Leymus chinensis, wherein the sites of the liquid phase chip include multiple SNP sites located on the reference genome of Leymus chinensis Leymus_chinensis_Lc6-5 or its updated version, covering gene regions and related non-gene regions; The core SNP site information of the liquid phase chip is specifically shown in Table 1 in the specification.

[0007] The position and variation information of the SNP site are expressed in the form of chromosome / physical position / reference genotype / variant allele type, and the physical position of the SNP molecular marker combination is located and analyzed based on the reference genome of Leymus chinensis.

[0008] Specifically, the Leymus chinensis whole genome 50K liquid phase chip comprises a primer set and / or a probe for detecting the SNP sites, especially the core SNP sites.

[0009] Specifically, the Leymus chinensis whole genome 50K liquid phase chip includes 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, which can effectively identify and detect the target SNP sites, especially the core SNP sites; and / or, The reagent suitable for liquid phase hybridization capture can efficiently and stably capture the target SNP site, especially the core SNP site.

[0010] As an exemplary selection scheme, the liquid phase hybridization capture reagent is such as a Hyb&Wash kit, for example, Novogene NGP Kit.

[0011] Specifically, the Leymus chinensis whole genome 50K liquid phase chip, the 50K liquid phase targeted capture probe is designed based on the positive strand of the Leymus chinensis reference genome; Preferably, the probe length ranges from 55 to 120 nt, with an average length of about 100 nt.

[0012] Specifically, the 50K liquid phase chip of the whole genome of Leymus chinensis, the 50K liquid phase targeted capture probe is designed based on the principle of thermodynamic stability, ensuring 99% capture of the conventional area; Preferably, for complex areas with high difficulty, the 50K liquid phase targeted capture probe improves the effective coverage of the complex areas by adjusting the probe position and placing multiple layers of "shingled" probes.

[0013] Furthermore, the probe design of the liquid 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 location of the target site and the GC content around it to ensure 99% capture of conventional areas. For complex areas with high or low GC content, the probe position is adjusted and a multi-layer 'shingled' probe design is used to increase the coverage of high-difficulty areas. This scheme improves the capture efficiency in GC extreme areas by increasing the complementarity and stability between probes. The probe design is based on the positive strand of the latest version of the reference genome of Leymus chinensis. The probe length ranges from 55-120nt, with an average length of about 100nt, and is optimized to ensure efficient SNP capture.

[0014] Furthermore, the chip uses the principle of nucleic acid hybridization for targeted capture. When two nucleic acid chains are complementary, a stable double-stranded structure will be formed between them. Through the denaturation and renaturation process, the target region library is first denatured to remove the repetitive sequence and the adapter sequence, and then a probe complementary to the base of the target region is added for renaturation. Non-specific binding is washed away, and finally a specific probe-library conjugate is obtained, thereby completing the capture of the target region.

[0015] Furthermore, the preparation of liquid-phase targeted capture probes includes template synthesis and RNA preparation. The probe template is composed of organically synthesized single-stranded DNA (oligo). After quality control (QC) and pooling, 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 be combined with streptavidin-coated magnetic beads to further improve the capture efficiency.

[0016] The present invention also discloses a kit, characterized in that the kit comprises a primer set and / or a probe capable of detecting the SNP sites, especially the core SNP sites, in the liquid phase chip.

[0017] The present invention also discloses the application of the 50K liquid phase chip of the whole genome of Leymus chinensis and the kit in the fields of variety identification, whole genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene positioning, molecular marker-assisted selection, genotyping, whole genome association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Leymus chinensis.

[0018] The present invention also discloses a combination of SNP molecular markers of Leymus chinensis, wherein the physical position of the SNP molecular marker site is determined by sequence comparison based on a reference genome of Leymus chinensis, Leymus_chinensis_Lc6-5; The SNP molecular marker sites are shown in Table 1 in the specification.

[0019] The invention also discloses a primer set and / or a probe for detecting the Leymus chinensis SNP molecular marker.

[0020] The present invention also discloses the application of the SNP molecular marker of Leymus chinensis or the primer set and / or probe in the field of variety identification, whole genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene positioning, molecular marker-assisted selection, genotyping, whole genome association analysis, cluster analysis or kinship identification, germplasm resource improvement and protection of Leymus chinensis.

[0021] The present invention determines the SNP molecular marker combination of the sheepgrass by whole genome sequencing, and screens out the SNP molecular marker sites based on the sheepgrass reference genome Leymus_chinensis_Lc6-5, so as to comprehensively evaluate the genetic characteristics of the sheepgrass.

[0022] The present invention develops the Leymus chinensis whole genome 50K liquid phase chip based on targeted capture sequencing technology, and realizes high-throughput and high-precision genotyping of the Leymus chinensis genome by combining high-density SNP site probes. It is widely applicable to the fields of genetic diversity assessment of Leymus chinensis, genetic map construction, QTL positioning, genome-wide association study (GWAS), molecular marker-assisted selection breeding (MAS), and genome-wide selection breeding (GS).

[0023] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention not only uses the reference genome of Leymus chinensis, but also adopts the homology comparison analysis of the transcriptome without reference to design probes, optimizes the selection of SNP sites and the coverage of the genome, and ensures the uniform distribution and good polymorphism of SNP sites in the genome of Leymus chinensis. The probe sites cover the key functional gene regions of Leymus chinensis, including important trait genes such as growth, development, and resistance, and can provide an important basis for the molecular marker identification of agronomic traits, quality traits, and stress resistance traits of Leymus chinensis.

[0024] The sheep fescue whole genome 50K liquid phase chip described in the present invention improves the effective coverage of difficult complex areas by adjusting the probe positions and placing multi-layer "shingled" probes, while ensuring the uniform distribution of SNP sites in the genome and the coverage of key functional areas, thereby improving the coverage of functional areas and supporting the research on agronomic traits and stress resistance traits.

[0025] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention can provide high-quality genotyping data, and compared with other typing technologies (such as GBS), the data consistency is higher, and it has stronger data comparison and accumulation capabilities. The high throughput and data consistency of the 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.

[0026] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention has higher flexibility than the traditional solid phase chip, and can adjust the SNP density of the chip according to the needs, provide multiple versions such as 50K, 40K, 30K, etc., 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 research and application needs of different scales. 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.

[0027] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention is based on targeted capture sequencing technology. The tolerance of flanking sequences is fully considered in the probe design. When the flanking sequence variation does not exceed 10%, the target sequence can still be stably captured. This technical feature ensures the high reliability of the data. At the same time, in addition to obtaining the target SNP information, the chip can also obtain the sequence information of 100bp upstream and downstream of the target SNP site, providing more supporting data for the functional gene research of Leymus chinensis.

[0028] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention provides strong technical support for the molecular breeding of Leymus chinensis by introducing advanced targeted capture sequencing technology, can effectively support the transition from traditional breeding to molecular breeding, can effectively improve breeding efficiency, promote the improvement of Leymus chinensis varieties, fill the technical gap in the field of molecular breeding of Leymus chinensis in China, and provide an important technical platform for basic research, genetic improvement and variety innovation of Leymus chinensis, further promoting the modernization of the Leymus chinensis industry.

[0029] The 50K liquid phase chip of the whole genome of Leymus chinensis described in the present invention realizes the complete localization of probe synthesis, sample detection and reagent use, avoiding the high cost of imported equipment and reagents. By reducing material costs and reducing technology dependence, it realizes independent control of production and application, reduces the risk of core data leakage, and avoids the inconvenience caused by external trade frictions, which meets the domestic demand for independent control of agricultural technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein: Figure 1 This is a flow chart of SNP site screening in Example 1 of the present invention; Figure 2 The distribution diagram of 51696 SNP site probes on chromosomes in Example 3 of the present invention; Figure 3 This is a distribution statistical diagram of SNP markers in the gene structure in Example 3 of the present invention; Figure 4 The radar chart of the expected performance and actual test performance of the Aneurolepidium chinense 50K liquid phase chip in Example 4 of the present invention; Figure 5 This is a phylogenetic tree analysis of 148 Leymus chinensis materials in Example 5 of the present invention; Figure 6 The PCA scatter plot of 148 Leymus chinensis materials in Example 5 of the present invention is shown; Figure 7 This is a graph showing the results of the whole genome association analysis of 148 Leymus chinensis materials in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] In the following embodiment of the present invention, the Leymus chinensis whole genome 50K liquid phase chip is developed based on targeted capture technology, and its working principle is based on probe design for the target region sequence, targeted capture of the target fragment and then high-throughput sequencing using a sequencing platform, targeted detection of target genes and sites, and rapid detection of large quantities of samples, thereby achieving the purpose of target region gene detection and typing.

[0032] In the following embodiments of the present invention, the Leymus chinensis whole genome 50K liquid phase chip comprises a set of Leymus chinensis 50K SNP probe sites.

[0033] In the following embodiments of the present invention, the Leymus chinensis whole genome 50K liquid phase chip comprises an independently packaged 50K probe mixture and a Hyb&Wash kit suitable for liquid phase hybridization capture; wherein, The probe mixture contains a total of 50K liquid phase targeted capture probes related to the SNP molecular marker sites.

[0034] In some embodiments, the liquid phase chip of the present invention adopts the principle of nucleic acid hybridization for targeted capture. When two nucleic acid chains are complementary, a stable double-stranded structure will be formed between them. Through the denaturation and renaturation process, the target region library is first denatured to remove the repeated sequence and the adapter sequence, and then a probe complementary to the base of the target region is added for renaturation; the non-specific binding is washed away, and finally a specific probe-library conjugate is obtained, thereby completing the capture of the target region.

[0035] In some embodiments, the preparation of the liquid-phase targeted capture probe of the liquid-phase chip of the present invention includes template synthesis and RNA preparation. The probe template is composed of organically synthesized single-stranded DNA (oligo). After quality control (QC) and pooling, 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 be combined with magnetic beads coated with streptavidin to further improve the capture efficiency.

[0036] In some embodiments, the Leymus chinensis whole genome 50K liquid phase chip, the 50K liquid phase targeted capture probe is designed based on the positive strand of the Leymus chinensis reference genome; Preferably, the probe length ranges from 55 to 120 nt, with an average length of about 100 nt.

[0037] Specifically, the probe design of the 50K liquid phase chip of the whole genome of Leymus chinensis is based on the principle of thermodynamic stability, including factors such as melting temperature (Tm), GC content and sequence specificity, and comprehensively considers the complexity of the target species genome, the position of the target site and the GC content around it to ensure 99% capture of conventional areas. For complex areas with high or low GC content, by adjusting the probe position, a multi-layer 'shingled' probe design is used to increase the coverage of high-difficulty areas. This scheme increases the complementarity and stability between probes, thereby improving the capture efficiency in GC extreme areas. The probe design is based on the positive chain of the latest version of the Leymus chinensis reference genome, with a probe length range of 55-120nt, an average length of about 100nt, and is optimized to ensure efficient SNP capture.

[0038] Example 1 This embodiment provides a 50K liquid phase chip of the whole genome of Leymus chinensis. The specific screening process is as follows: Figure 1 The specific screening process is as follows.

[0039] 1. Preliminary SNP filtration and screening In this example, 100 samples of Leymus chinensis were resequenced and SNP variation detection was performed on these sequencing data. The reference genome version used was Leymus_chinensis_Lc6-5. The preliminary SNP filtering and screening process was as follows: (1) Depth filtering: On the basis of the average sequencing depth of the sample reaching 10X, select SNP sites with a depth of no less than 3X to ensure the reliability of the data and filter out sites with low depth; (2) Completeness filtering: SNP markers whose genotypes cover at least 50% of all sample individuals were screened; specifically, for each polymorphic marker site, at least 50 samples out of 100 samples were required to have a definite genotype, thereby filtering out markers with poor genotype integrity; (3) Minimum allele frequency (MAF) filtering: sites with MAF values ​​lower than 0.05 were filtered out to ensure that the selected sites had sufficient variation information; (4) After the above preliminary filtering, 40M basic SNP sites were retained.

[0040] 2. SNP depth filtering Filtering based on linkage disequilibrium (LD): PLINK software was used for linkage disequilibrium analysis with two parameter settings: one was 50 kb window, 10 bp step, R2<0.2R2<0.2; the other was 100 kb window, 1 bp step, R2<0.2R2<0.2. These filtering parameters refer to the reported screening criteria for allopolyploid plant loci.

[0041] Hardy-Weinberg equilibrium (HWE) filtering: Calculate the p-value of Hardy-Weinberg equilibrium for all SNP loci and filter according to 1e-6.

[0042] Gene region screening: Select SNP sites located in the gene region and its upstream and downstream, and filter out other sites within 100bp upstream and downstream of the SNP site. For the exon region, if the distance between a SNP and the previous SNP is less than 100bp and it is a non-synonymous mutation, then keep the SNP.

[0043] Non-exon region filtering: In non-exon regions, A / T and G / C type variant sites are filtered out.

[0044] After deep filtering of SNPs, 95,954 background sites, namely VIP SNP sites, were finally retained.

[0045] 3. Screening of trait-associated loci This example uses the resequencing data of 100 samples of Leymus chinensis to perform a genome-wide association study (GWAS) on eight agronomic traits of Leymus chinensis using a mixed linear model (MLM) using GEMMA software. In the analysis, the default parameters were used and the significance threshold was set to 6 to identify the associated sites for each trait. The strategy for site retention is as follows: (1) For traits associated with less than 450 loci, all associated loci were retained; (2) For traits associated with more than 450 loci, we screened loci that were less than 100K away from the gene and sorted them by P-value to select the top 450 loci; (3) Among the selected sites, if the distance between two sites is less than 1K, only the site closest to the gene is retained; (4) Through these steps, 1,667 loci associated with the agronomic traits of Leymus chinensis were finally selected, namely the VVIP SNP loci.

[0046] 4. Determination of SNP sites on 50K liquid phase array The basic, VIP and VVIP SNPs obtained in the above analysis were merged, and after removing duplicates, the site data set was screened according to the sliding window method. In each window, VIP and VVIP SNPs were given priority, and the remaining positions were supplemented with background sites. 51,696 SNP sites can be screened as a 50K liquid chip site set. The specific information of the core SNP sites contained therein is recorded in Table 1 below. Among them, the position and variation information of the SNP site are expressed in the form of chromosome: physical position: reference genotype / variant allele type, and the physical position of the SNP molecular marker combination is based on the reference genome of sheep grass for positioning analysis.

[0047] Table 1 Core SNP locus information

[0048] Example 2 In this example, liquid phase chip probes were designed based on the SNP site information screened in Example 1.

[0049] The chip design in this embodiment is based on the principle of thermodynamic stability, including factors such as melting temperature (Tm), GC content and sequence specificity, and comprehensively considers the complexity of the target species genome, the location of the target site and the GC content around it to ensure 99% capture of the conventional area. The probe is based on the positive strand of the reference genome of Leymus chinensis, with a length range of 55-120nt and an average length of about 100nt.

[0050] For complex areas with high or low GC content, the probe position is adjusted and a multi-layer 'shingled' probe design is used to improve the coverage of difficult areas. This solution increases the complementarity and stability between probes, thereby improving the capture efficiency in GC extreme areas. In this embodiment, the key design parameters of the probe include: (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 will select the lowest quantile site set according to the score; (2) All 1,667 GWAS-related loci were retained regardless of their scoring values.

[0051] Based on the above standards, this embodiment designed and synthesized a liquid chip probe.

[0052] Example 3 This embodiment is based on the distribution and annotation of chromosomes of SNP sites in liquid phase microarray.

[0053] This example statistically analyzed the distribution of the liquid phase chip screened in Example 1 on 14 chromosomes of Leymus chinensis. On average, each chromosome contained about 3,689 SNPs, and the average spacing between adjacent sites was 145 kb. The 51,697 SNPs screened showed a nearly uniform distribution. The site distribution diagram is shown in Figure 2 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 protein-coding gene function. The annotation information of the SNP sites is shown in Figure 3 The combination of SNPs screened by this chip significantly improved the detection success rate of candidate gene loci, providing strong support for subsequent functional gene research.

[0054] Example 4 This example is based on the Leymus chinensis liquid phase chip developed in the aforementioned Example 2. Twelve Leymus chinensis samples were selected to test the performance of the chip, which specifically involves the following steps.

[0055] 1. Probe preparation The preparation of the liquid phase targeted capture probe of Leymus chinensis described in this embodiment includes template synthesis and RNA preparation. The probe template is composed of organically synthesized single-stranded DNA (oligo). After quality control (QC) and pooling, 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 be combined with magnetic beads coated with streptavidin to further improve the capture efficiency.

[0056] 2. Library construction and capture 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.

[0057] (III) Library quality inspection and quantification 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.

[0058] 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.

[0059] 4. Sequencing 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.

[0060] 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.

[0061] Sequencing was performed using a high-throughput sequencing platform.

[0062] 5. Data Analysis 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: (1) Quality control: Remove low-quality sequences, adapter sequences, ployG and other abnormal sequences in the original sequencing data to obtain clean data; (2) Alignment: Align, sort, and remove duplicates of the Clean data with the reference genome to obtain a bam file; (3) Statistics: Statistical analysis of indicators such as alignment rate, coverage rate, capture rate, and uniformity.

[0063] (VI) Test results In this embodiment, after the above test analysis, the test results include various indicators of the target area, various indicators of the probe area, coverage of different areas, etc., which comprehensively and in detail demonstrate the various performances of the chip. The product has a high site detection rate, with an average site detection rate of 98.9%; the product has good stability and high genotype accuracy, and the average consistency rate of genotypes of repeated samples is 99%.

[0064] In this example, in order to intuitively display the expected performance and actual test performance of the chip, the QC rate, comparison rate, coverage, capture rate, uniformity and other indicators of two samples were taken to calculate the average value and draw a radar chart. The results are shown in the figure. Figure 4 As shown, it can be seen that all indicators are good and in line with expectations.

[0065] Example 5 This example further verifies the application of the Leymus chinensis liquid phase chip in population structure analysis and whole-genome association analysis of Leymus chinensis germplasm resources.

[0066] In this example, the above-mentioned Leymus chinensis liquid phase chip was used to detect 148 samples of Leymus chinensis, extract the genotype of the target site, and annotate the SNP detection results using ANNOVAR software.

[0067] In this example, TreeBeST (http: / / treesoft.sourceforge.net / treebest.shtml) software was used to calculate the distance matrix, and based on this, a phylogenetic tree was constructed using the neighbor-joining method. The bootstrap values ​​were calculated 1000 times, and the phylogenetic tree analysis results are shown in Figure 1. Figure 5 shown.

[0068] This example uses GCTA software to analyze the PCA (principal component analysis) components 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 plot, the greater the difference in the genetic background of the two samples. Individuals with similar genetic backgrounds will be clustered into one category in the plot. It was found that the 148 samples of Leymus chinensis were basically scattered, with no obvious grouping. The results are shown in Figure 6 .

[0069] This example uses GEMMA (http: / / www.xzlab.org / software.html) analysis software and a mixed linear model to perform genome-wide association analysis (GWAS), 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 calculation time. Potential candidate SNPs were screened out by the significance of the association (P-value). The results are shown in Figure 7As shown in the figure, only some of the trait association analysis results are shown. Figure 7 The middle (A) is the Manhattan map. Figure 7 Middle (B) is the QQ plot (Quantile-quantileplot).

[0070] In summary, the present invention develops the Leymus chinensis whole genome 50K liquid phase chip based on targeted capture sequencing technology, and realizes high-throughput and high-precision genotyping of the Leymus chinensis genome by combining high-density SNP site probes. It is widely used in the fields of genetic diversity assessment of Leymus chinensis, genetic map construction, QTL positioning, genome-wide association study (GWAS), molecular marker-assisted selection breeding (MAS), and whole genome selection breeding (GS).

[0071] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled 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 list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A 50K liquid phase chip of the whole genome of Leymus chinensis, characterized in that: The sites of the liquid phase chip include SNP sites located on the reference genome of Leymus chinensis_Lc6-5 or its updated version, covering gene regions and related non-gene regions; The core SNP site information of the liquid phase chip is shown in the following table:

2. The whole genome 50K liquid phase chip of Leymus chinensis according to claim 1, characterized in that: The liquid phase chip includes a primer set and / or a probe for detecting the SNP site.

3. The whole genome 50K liquid phase chip of Leymus chinensis according to claim 2, characterized in that: It includes 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 the target SNP site; The reagent for liquid phase hybridization capture is used to capture the target SNP site.

4. The Leymus chinensis whole genome 50K liquid phase chip according to claim 3, characterized in that: The 50K liquid phase targeted capture probe is designed based on the positive strand of the Leymus chinensis reference genome; The probe length ranged from 55 to 120 nt, and the average length was about 100 nt.

5. The whole genome 50K liquid phase chip of Leymus chinensis according to claim 3 or 4, characterized in that: In the 50K liquid phase targeted capture probe, conventional areas are covered with 1X probes, and difficult and complex areas are covered with multi-layer shingled probes; Among them, the conventional region is a region with low sequence complexity and high uniformity; the high-difficulty complex region is a region containing high GC content, repetitive sequences or other complex structures.

6. A kit, characterized in that: The kit comprises a primer set and / or a probe for detecting the SNP site in the liquid phase chip according to any one of claims 1 to 5.

7. Application of the whole genome 50K liquid phase chip of Leymus chinensis according to any one of claims 1 to 5 and the kit according to claim 6 in the fields of variety identification, whole genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene positioning, molecular marker-assisted selection, genotyping, whole genome association analysis, cluster analysis or kinship identification, and germplasm resource improvement and protection of Leymus chinensis.

8. A combination of SNP molecular markers of Leymus chinensis, characterized in that: The physical location of the SNP molecular marker site is determined by sequence alignment based on the reference genome of Leymus_chinensis_Lc6-5; The SNP molecular marker sites are shown in Table 1 in the specification.

9. A primer set and / or probe for detecting the Leymus chinensis SNP molecular marker according to claim 8.

10. Application of the SNP molecular marker of Leymus chinensis according to claim 8 or the primer set and / or probe according to claim 9 in variety identification, whole genome breeding or assisted breeding, genetic diversity assessment, genetic map construction, gene positioning, molecular marker-assisted selection, genotyping, whole genome association analysis, cluster analysis or kinship identification, germplasm resource improvement and protection of Leymus chinensis.

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