50K liquid phase chip for genetic diversity analysis of platycladus orientalis and application of 50K liquid phase chip

Abstract

The invention provides a 50K liquid phase chip for genetic diversity analysis of platycladus orientalis and application of the 50K liquid phase chip, and belongs to the technical field of molecular genetic breeding. The 50K liquid phase chip for the genetic diversity analysis of the platycladus orientalis, provided by the invention, comprises a nucleotide probe and / or a primer group for detecting an SNP (Single Nucleotide Polymorphism) marker, the SNP marker is composed of 50125 SNP loci in a platycladus orientalis genome, and the SNP loci are distributed on 11 chromosomes. The 50K liquid chip provided by the invention can be used for performing high-throughput typing, genetic diversity evaluation, genetic relationship analysis and the like on platycladus orientalis germplasm resources, and has high accuracy, high repeatability and wide applicability. According to the invention, the blank of platycladus orientalis molecular typing tools is filled, the scientificity and efficiency of platycladus orientalis germplasm resource management can be obviously improved, and the method has important significance for promoting the innovation and genetic improvement of platycladus orientalis germplasm and the development of ecological forestry.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetic breeding, and particularly relates to a 50K liquid phase chip for analyzing the genetic diversity of Platycladus orientalis and an application thereof. Background Art

[0002] Platycladus orientalis is an important ecological afforestation species, particularly crucial for soil and water conservation and greening projects in arid mountainous areas. With rapid environmental and climatic changes, afforestation projects for Platycladus orientalis face long-term challenges. Assessing Platycladus genetic resources, particularly the genetic diversity of parental lines in seed orchards, is a primary prerequisite for promoting improved seed selection and ensuring the sustainable development of afforestation projects. Currently, the utilization of Platycladus breeding resources is still in its infancy, and the genetic background of Platycladus breeding resources is still unclear. Therefore, the development of Platycladus genetic markers is of great significance for genetic evaluation of germplasm resources, identification of kinship relationships, and configuration of seed orchard clones. Systematic research on breeding resources based on Platycladus orientalis seed orchard improvement systems can provide a theoretical foundation for resource collection and high-generation genetic improvement, and offer theoretical guidance for subsequent production seed and afforestation projects. Efficient and reliable molecular markers will facilitate the genetic analysis of complex traits, accelerate the progress of breeding and improvement, and are of great significance for the construction of high-level Platycladus improvement systems and the formulation of sustainable strategies.

[0003] Genetic diversity is an important characteristic of plant germplasm resources and one of the bases for their utilization. It plays a key role in ensuring the long-term adaptability and genetic improvement potential of species. At present, research on the genetic diversity of Platycladus orientalis mostly relies on traditional molecular marker technologies, such as RAPD, ISSR and SSR. However, these methods have limitations such as low throughput, poor repeatability and insufficient resolution, making it difficult to meet the needs of large-scale, high-throughput germplasm resource analysis. In recent years, with the development of high-throughput sequencing technology, large-scale typing technology based on SNP (single nucleotide polymorphism) markers has become an important means of studying plant genetic diversity. However, there are currently no SNP chip products specifically developed for Platycladus orientalis, which limits the in-depth development of systematic identification of the tree's germplasm resources, population genetic structure analysis and molecular-assisted breeding. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a 50K liquid phase chip, a kit and its application for the genetic diversity analysis of Platycladus orientalis, so as to realize the efficient and accurate identification and genetic diversity analysis of Platycladus orientalis germplasm resources, which is of great significance in Platycladus orientalis germplasm innovation, genetic improvement and resource protection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a 50K liquid phase chip for analyzing the genetic diversity of Platycladus orientalis. The 50K liquid phase chip includes nucleotide probes and / or primer sets for detecting SNP markers. The SNP markers are composed of 50,125 SNP sites in the Platycladus orientalis genome. The SNP sites are distributed on 11 chromosomes, which are respectively recorded as chr01, chr02, chr03, chr04, chr05, chr06, chr07, chr08, chr09, chr10 and chr11. The specific position information is shown in Table 1 below. Table 1 uses the form of chromosome: physical position: reference genotype / variant allele type to express:

[0007] Table 1 Location and variation information of 50125 SNP sites

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[0131] Preferably, the nucleotide probe is a probe that places the site marked by the SNP in Table 1 in the middle of the probe, and the probe length is 120 bp.

[0132] Preferably, the GC content of the nucleotide probe is between 20% and 80%, and the number of homology regions of the probe sequence in the whole genome is ≤2.

[0133] The present invention also provides a kit, which includes the above-mentioned 50K liquid phase chip.

[0134] Preferably, the kit further comprises a hybridization reagent.

[0135] The present invention also provides the use of the above-mentioned 50K liquid phase chip or the above-mentioned kit in any of the following items: (1) analysis of genetic diversity of Platycladus orientalis; (2) molecular breeding of Platycladus orientalis; (3) cluster analysis and kinship identification of Platycladus orientalis; (4) construction of Platycladus orientalis genetic map and gene positioning; (5) identification of Platycladus orientalis germplasm.

[0136] Beneficial effects of the present invention:

[0137] The 50K liquid phase chip provided by this invention enables high-throughput typing, genetic diversity assessment, and phylogenetic relationship analysis of Platycladus orientalis germplasm resources, demonstrating high accuracy, high repeatability, and broad applicability. This invention fills a gap in molecular typing tools for Platycladus orientalis, significantly improving the scientific nature and efficiency of Platycladus orientalis germplasm resource management. It is of great significance for promoting Platycladus orientalis germplasm innovation, genetic improvement, and ecological forestry development. This invention is applicable to a variety of fields, including genetic improvement, germplasm identification, and population structure research, and has significant scientific and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 This is a schematic diagram of the distribution of all SNP sites in the Platycladus orientalis whole genome chip on the whole genome;

[0139] Figure 2 This is the principal component analysis (PCA) diagram of Platycladus orientalis accessions;

[0140] Figure 3 A phylogenetic tree of Platycladus orientalis constructed based on the 50K liquid phase chip of the present invention;

[0141] Figure 4 This is the nucleotide polymorphism (Pi) analysis diagram of Platycladus orientalis germplasm resources;

[0142] Figure 5It is the Tajima's D value distribution map of Platycladus orientalis germplasm resources. Specific implementation mode

[0143] The present invention provides a 50K liquid chip for Platycladus orientalis genetic diversity analysis. The 50K liquid chip includes nucleotide probes and / or primer sets for detecting SNP markers. The SNP markers are composed of 50,125 SNP sites in the Platycladus orientalis genome. The SNP sites are distributed on 11 chromosomes, denoted as chr01, chr02, chr03, chr04, chr05, chr06, chr07, chr08, chr09, chr10 and chr11 respectively. The specific position information is shown in Table 1.

[0144] In the 50K liquid chip of the present invention, the fixed nucleotide probes correspond to 50,125 SNP sites respectively. The method for obtaining the SNP sites in the present invention is as follows: Using the resequencing data of 128 excellent clone parents collected from Henan Jiaxian State-owned Forest Farm, a total of 573,880,012 original variant sites (SNP + INDEL) are identified. The variant sites are preliminarily screened and filtered. The filtering conditions are: ① The average sequencing depth DP > 50, ② The site deletion rate miss = 0, ③ The minor allele frequency MAF > 0.05. Probe design is carried out on the preliminarily screened sites, and the sites that cannot be designed are removed (mainly based on GC content and site repeat rate). Then, sliding window site selection is carried out on the variant sites. The screening conditions are as follows: 1. Non-overlapping sliding windows; 2. The window size is 5 kb, and the site with the minimum repeatCount number within the screening window with 30 < GC content < 70 is selected; 3. The sliding window range is: gene regions (exons and introns) and 5 kb upstream and 5 kb downstream thereof; 4. Only the sites on the chromosome are retained, and then the sites within 2 mb at both ends of the chromosome are removed. Sliding window screening is carried out again according to 50k, and finally the number of variant sites obtained is 50,125. The 50,125 SNP molecular markers are shown in Table 1. The physical positions of the SNP molecular marker combinations are analyzed based on the Platycladus orientalis reference genome. The Platycladus orientalis genome can be queried at https: / / figshare.com / s / a8df5d66a796d122f9cb, or queried at the National Bioinformatics https: / / ngdc.cncb.ac.cn / gwh / Assembly / reviewersPage / dVmmHIlHZKrpLRflbGoBTGNcMkSXJnr GDPoLSAOJmZOBIMPvLcXWYlGhBzVFKNkx.

[0145] Established in March 1989, the National Platycladus orientalis Seed Base at the Jiaxian State Forest Farm in Henan Province is the largest such base in China. The base covers 2,175 mu (approximately 1,000 acres) and comprises nine production zones: four zones and 32 plots for mother tree forests; two zones and 13 plots for clonal seed orchards; and three zones and 12 plots for progeny testing forests, with plant spacing of 2 m x 2 m. The primary clonal seed orchard collects 268 high-quality trees from 24 regions across Henan Province. Due to forest farm reconstruction, some parent trees were lost, leaving 213 clonal parents. These 213 clones are arranged in a staggered pattern, with a plant spacing of 2 m x 2 m x 6 m across the 35-hectare area. After planting, the seed orchard undergoes scientific tending twice a year, removing dead plants and replacing them with replacement grafted seedlings. Currently, the orchard remains a primary seed orchard, with the majority of plants in their peak fruiting stage, except for a few in the early stages of fruiting.

[0146] In the present invention, the nucleotide probe preferably places the SNP marker site in Table 1 in the middle position of the probe, and the probe length is 120bp. More preferably, the GC content of the nucleotide probe is between 20% and 80%, and the number of homology regions of the probe sequence in the whole genome is ≤2.

[0147] The present invention also provides a kit, which includes the above-mentioned 50K liquid phase chip. In the kit of the present invention, a hybridization reagent is preferably also included.

[0148] The present invention also provides the use of the above-mentioned 50K liquid phase chip or the above-mentioned kit in any of the following items: (1) analysis of genetic diversity of Platycladus orientalis; (2) molecular breeding of Platycladus orientalis; (3) cluster analysis and kinship identification of Platycladus orientalis; (4) construction of Platycladus orientalis genetic map and gene positioning; (5) identification of Platycladus orientalis germplasm.

[0149] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0150] In the following examples, unless otherwise specified, all methods are conventional.

[0151] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0152] Example 1

[0153] The screening process of 50125 SNP sites of the present invention is as follows:

[0154] 1. Whole-genome resequencing of Platycladus orientalis

[0155] 128 Platycladus orientalis accessions were collected from the state-owned forest farm in Jiaxian County, Henan Province, and whole genome resequencing was performed. The specific steps included:

[0156] (1) Extract the DNA of Platycladus orientalis materials by the magnetic bead method. (2) Adopt the MGI standard library construction method to construct the DNA-seq sequencing library for those with qualified quality inspection. (3) Sequence the libraries with qualified quality inspection using the BGI sequencing platform (MGI), with the sequencing strategy of PE150 and the sequencing depth of 5× to 10×.

[0157] 2. Alignment and variant detection

[0158] Perform alignment and variant detection on 128 resequencing data, and the analysis process is as follows:

[0159] (1) Align the quality-controlled reads to the Platycladus orientalis reference genome, sort the positions, and mark the duplicate reads. (2) Detect the variant sites for each sample to obtain the variant information of each sample. (3) Conduct a joint analysis on the VCF of all samples to obtain the variant results of each individual in the population.

[0160] 3. Development of Platycladus orientalis liquid breeding chip

[0161] a. Obtaining resequencing variant sites: Using the resequencing data of 128 samples from the State-owned Forest Farm in Jiaxian County, Henan Province, a total of 573,880,012 original variant sites (SNP + INDEL) were identified. b. Preliminary screening of candidate sites: Conduct preliminary screening and filtering on the variant sites. The filtering conditions are as follows: ① The average sequencing depth DP > 50, ② The site deletion rate miss = 0, ③ The minor allele frequency MAF > 0.05. Design probes for the preliminarily screened sites, eliminate the sites that cannot be designed (mainly based on the GC content and site repeat rate), and then perform sliding window site selection on the variant sites. c. Sliding window screening, with the following conditions: (1) Non-overlapping sliding windows; (2) The window size is 5 kb, and select the site with the smallest repeatCount number within the window where 30 < GC content < 70; (3) The sliding window range is: gene regions (exons and introns) and 5 kb upstream and 5 kb downstream; (4) Only retain the sites on the chromosome, then eliminate the sites within 2 mb at both ends of the chromosome, and perform sliding window screening again according to 50 k. The final number of variant sites obtained is 50,125. The SNP site distribution map is as Figure 1 shown, and the position information is shown in Table 1.

[0162] Example 2

[0163] Synthesis of 50K liquid chip

[0164] Using NGP liquid chip (a liquid chip product designed and optimized by Novogene using liquid chip technology specifically for the needs of breeding work), probe design is based on the principle of thermodynamic stability, and comprehensive consideration is given to factors such as the complexity of the target species' genome, the location of the target site, and the GC content near the target site to ensure 100% capture of conventional areas. For complex areas with high difficulty, the NGP liquid chip improves the effective coverage of high-difficulty areas by adjusting the probe position and placing multi-layer "shingled" probes. Probe preparation includes template synthesis and probe preparation. The synthesized DNA template (Oligo Pool) undergoes quality control and in vitro amplification experiments before being finally prepared as a finished DNA probe.

[0165] The principle of nucleotide probe design is to place the SNP-marked site in Table 1 in the middle of the probe, the probe length is 120bp, the GC content of the nucleotide probe is between 20% and 80%, and the number of homology regions of the probe sequence in the whole genome is ≤2. When preparing the DNA probe, biotin-labeled NTP and nucleotide analogs are used as synthetic raw materials. Nucleotide analogs can increase the Tm value of the binding between the probe and the library, making the binding between the probe and the library more stable. At the same time, the synthesized probe is labeled with biotin and can be combined with streptavidin-coated magnetic beads in subsequent experiments. The above process was carried out by Beijing Novogene Technology Co., Ltd. to obtain a 50K liquid phase chip.

[0166] Example 3

[0167] The specific application method of the 50K liquid phase chip obtained in Example 2 is as follows:

[0168] (1) Sample DNA extraction and quality control: Sample DNA was extracted using the magnetic bead method. The concentration of the DNA sample was determined using a Qubit fluorescence quantifier. The integrity of the DNA sample was determined using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0169] (2) Library construction and quality control: a. Use fragmentase to digest the DNA sample, repair the digested ends, add A base to the 3' end, and use agarose gel electrophoresis to detect the fragment size. b. Use T4 ligase to connect the sequencing adapter and DNA fragment, and use magnetic beads to purify the ligation product. The concentration of the purified product is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis. c. PCR amplification of the purified ligation product. The total volume of each PCR amplification reaction system is 50μL, containing the following components: 25μL high-fidelity PCR mixture (containing enzyme, buffer, dNTPs), 1μL upstream primer (10μM), 1μL downstream primer (10μM), 50-100ng template DNA, and add nuclease-free water to make up to 50μL. The amplification conditions are: pre-denaturation at 95°C for 3 minutes; followed by 12 to 15 cycles, each cycle including denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; and finally extension at 72°C for 5 minutes. The amplified products are screened for fragments using magnetic beads. The concentration of the products after fragment screening is detected using a Qubit fluorescence quantifier, and the fragment size is detected by agarose gel electrophoresis. d Take 200 ng of the constructed library, add the 50K liquid phase chip obtained in Example 2 and the hybridization reagent (the hybridization reagent uses the pre-mixed reagent provided by NGPHyb&WashKitv2.0), and incubate at 50°C for 16-24 hours to complete the hybridization reaction. Use streptavidin magnetic beads to capture the target segment, use a cleaning solution to wash the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification (amplification conditions and reaction system are the same as above). Use a Qubit fluorescence quantifier to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are qualified, the sequencing library construction is completed. The prepared library was sequenced using a BGI sequencer with a high-throughput sequencing strategy of PE150.

[0170] (3) Bioinformatics analysis: ① Raw data filtering: The raw sequencing sequences (Raw Reads) obtained by sequencing are filtered to obtain high-quality CleanReads, and the data are quality controlled using FASTP software. ② Contamination detection: The sequences are aligned to the NCBINT database using BLAST software for contamination assessment. ③ Reference genome alignment: Sequencing Reads are aligned to the reference genome using BWA software, and position sorting is performed to obtain the bam file after sample sorting. ④ Mutation detection: The bcftools call software is used to detect the variant sites of each sample and obtain the variant result file for each sample and population.

[0171] Example 4

[0172] The 50K liquid phase chip obtained in Example 2 was used to test six additional samples of Platycladus orientalis. The specific detection method was the same as in Example 3, with two replicates. The average reproducibility of the six samples was 99.4267%. This demonstrates that the 50K liquid phase chip of the present invention has a high target site detection rate and accurate and reliable typing results.

[0173] Example 5

[0174] The 50K liquid phase chip obtained in Example 2 was used to detect another 128 Platycladus orientalis materials collected. The specific detection method was the same as that in Example 3.

[0175] The PCA (Principal Component Analysis) components of the test materials were analyzed using Plink software and a PCA scatter plot was constructed. The results are shown in Figure 2 Each site in the scatter plot represents a sample. The farther the distance between two samples in the plot, the greater the difference in their genetic backgrounds. Individuals with similar genetic backgrounds will be clustered into one category in the plot.

[0176] Plink software was used to calculate the genetic distance matrix and cluster analysis was performed on the Platycladus orientalis breeding materials. The phylogenetic tree diagram was constructed to determine the kinship, evolutionary relationship and composition structure of different materials. The 50K liquid phase chip obtained in Example 2 was used to cluster analyze 128 Platycladus orientalis materials from different groups. It was found that the artificially selected groups could not be clustered significantly, and the effect was consistent with the actual grouping. The analysis results are as follows Figure 3 shown.

[0177] Based on high-throughput sequencing data, the present invention uses a sliding window method to calculate the nucleotide polymorphism (Pi value) within the entire genome of Platycladus orientalis breeding materials to assess the level of genetic diversity within the population. The Pi value reflects the average base difference between any two individuals in the population. The larger the value, the higher the genetic diversity in the region. The analysis results are as follows Figure 4 As shown, there was no obvious difference in genetic polymorphisms between different genomic regions.

[0178] Furthermore, in order to explore the potential selection signals or evolutionary events in the Platycladus orientalis population, the present invention conducted Tajima's D value analysis on the samples. Figure 5 As shown, Tajima's D is a statistic that measures the difference between observed nucleotide diversity (π) and theoretical diversity (θ) estimated based on the number of polymorphic sites. Negative D values ​​suggest the presence of population expansion or purifying selection, while positive D values ​​may be associated with balancing selection or bottleneck effects. This study performed a sliding window scan of the D value across the entire Platycladus orientalis genome, identifying regions likely to be under selective pressure. Figure 5 The distribution of Tajima's D values ​​on different chromosomes is shown.

[0179] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A 50K liquid phase chip for analyzing the genetic diversity of Platycladus orientalis, characterized in that: The 50K liquid phase chip includes nucleotide probes and / or primer sets for detecting SNP markers. The SNP markers are composed of 50,125 SNP sites in the Platycladus orientalis genome. The SNP sites are distributed on 11 chromosomes, respectively denoted as chr01, chr02, chr03, chr04, chr05, chr06, chr07, chr08, chr09, chr10 and chr11. The specific location information is shown in the following table:

2. The 50K liquid phase chip according to claim 1, characterized in that: The nucleotide probe is a probe that places the site of the SNP marker according to claim 1 in the middle of the probe, and the probe length is 120 bp.

3. The 50K liquid phase chip according to claim 1, characterized in that: The GC content of the nucleotide probe is between 20% and 80%, and the number of homology regions of the probe sequence on the whole genome is ≤2.

4. A kit, characterized in that The kit comprises the 50K liquid phase chip according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes hybridization reagents.

6. Use of the 50K liquid phase chip according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in the analysis of genetic diversity of Platycladus orientalis.

7. Use of the 50K liquid phase chip according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in molecular breeding of Platycladus orientalis.

8. Use of the 50K liquid phase chip according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in cluster analysis and phylogenetic identification of Platycladus orientalis.

9. Use of the 50K liquid phase chip according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in the construction of a genetic map and gene localization of Platycladus orientalis.

10. Use of the 50K liquid phase chip according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 5 in identifying Platycladus orientalis germplasm.

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