Pine whole genome 50K liquid phase chip and application thereof

Abstract

The invention belongs to the technical field of gene chips, and discloses a pine whole genome 50K liquid chip and application thereof. The liquid phase chip comprises a pine 50K site probe mixed solution and a hybridization capture reagent, and the pine tree 50K chip comprises information of 49693 sites. According to the present invention, the liquid phase chip design gives consideration to species specificity and transferability, is not only suitable for the target species, but also can be expanded to other related pine tree class groups, fills the blank in the tropical and subtropical pine tree genetic typing in the prior art, and provides the efficient and accurate tool for pine tree genetic resource protection, molecular breeding and genomics research; the method solves the problem of low conversion rate of pine tree molecular assisted breeding and whole genome selective breeding products, overcomes the defect of high application cost of pine trees in scientific research and practical application, and provides technical support for pine tree germplasm resource utilization and molecular breeding research.

Description

Technical Field

[0001] The present invention relates to the technical field of gene chips, and relates to a 50K liquid-phase chip for pine trees and its applications. Background Art

[0002] The genus Pinus L. contains more than 100 species, which are widely distributed in the Northern Hemisphere. Among them, China and the Mexico-Central America region are two major biodiversity hotspots of the genus Pinus. As a key component of temperate and boreal forest ecosystems, Pinus species play an important role in the global carbon cycle, nutrient and atmospheric cycles, and have significant ecological value. At the same time, most Pinus species grow rapidly and have strong adaptability. They are important afforestation tree species and wood sources, and their economic value is reflected in multiple fields such as pulp and solid wood products.

[0003] Tropical and subtropical pine trees (such as Pinus yunnanensis, Pinus kesiya, etc.) are usually distributed in areas with high ecological diversity and variable environments, making them important indicator species for climate change. Under the influence of frequent hybridization and complex gene flow, these pine trees have formed unique genetic adaptability and have great potential for cultivating stress-resistant varieties. Pinus yunnanensis and Pinus kesiya are important timber tree species in China and are widely planted in Yunnan Province. Pinus kesiya distributed in the tropics has a wider distribution, covering the entire highly variable climate region of tropical Southeast Asia and being affected by unique evolutionary drivers such as frequent hybridization and complex gene flow. Studying its genome and population dynamics can not only provide a scientific basis for the protection of wild pine resources in China, but also provide sustainable management strategies for coping with climate change. At the same time, current pine tree breeding still mainly relies on traditional methods, which have limitations such as long cycles (6-10 years) and high costs, especially the evaluation of offspring traits consumes a large amount of resources. The application of genome-assisted breeding technology can significantly improve breeding efficiency. However, the genomes of Pinus species are huge (18-35 Gb), about 180-350 times that of the Arabidopsis thaliana genome, and are rich in repetitive sequences and pseudogenes, resulting in difficulties in genome assembly, annotation, and SNP screening. This characteristic makes the genomic research of Pinus species far behind that of crops, and the development of whole-genome SNP chips faces problems such as complex site screening strategies, high computing power requirements, and expensive sequencing costs. Therefore, there is an urgent need to establish low-cost, high-throughput, and repeatable genotyping technologies to break through the bottlenecks in pine tree genetics research and breeding.

[0004] The existing pine SNP chips are mainly developed based on North American pines (such as the Axiom_PineGAP chip of Thermo Fisher), and their conversion rate in several other pine species is only 42%, with insufficient applicability. In addition, the patent with the patent number CN202410922435.5 is mainly designed for a single species of cold temperate Larix olgensis, which is distantly related to the pines distributed in tropical and subtropical regions of China. Its cross-species transferability is limited and it is difficult to meet the genotyping needs of tropical and subtropical pines. As a country rich in pine resources, especially Pinus yunnanensis, Pinus kesiya var. langbianensis and other species with important ecological and economic values, China urgently needs to develop genotyping tools suitable for native pines to support germplasm resource screening, variety identification and molecular breeding research. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a 50K liquid-phase chip for the whole genome of pine and its application. The technical solution of the present invention is as follows:

[0006] A 50K liquid-phase chip for the whole genome of pine, the liquid-phase chip includes a 50K site probe mixture of the whole genome of pine and a hybridization capture reagent; the site probes include 49,693 SNP sites, which are distributed on 12 chromosomes. The SNP site information is shown in Table 1, where the first row is the chromosome position information where the SNPs are located, and the numbers on both sides of the "-" after that are the start and end positions of the SNPs on this chromosome respectively.

[0007] Further, the design steps of the 50K site probe of the whole genome of pine include:

[0008] S1 The probe length is 100bp, and the probe GC content is between 50%. The selected region maximally does not contain SSR and GAP regions;

[0009] S2 Design a nucleotide sequence with 60% - 70% overlap and covering the SNP sites according to the screened SNP sites;

[0010] S3 Perform single-stranded nucleotide synthesis according to the designed nucleotide sequence, synthesize site probes with a length of 100bp modified with a biotin group at the 5' end, and increase the amount of site probes by PCR amplification after synthesis;

[0011] S4 Mix the above synthesized site probes in equimolar mass, and use a mixed solution of EDTA and Tris HCl to make a probe mixture with a volume of 3 pmol / mL.

[0012] Further, the method for obtaining the 50K SNP sites of pine is as follows:

[0013] Genotyping of S11 mutation sites: Whole-genome resequencing was performed on pine individuals. Fastp v0.23.2 was used to perform quality control filtering on the original sequencing data. The filtered data was aligned to the Pinus tabuliformis reference genome GCA_031772625.1 using BWA. After sorting by samtools, insertion rearrangement was performed using GATK v3.8-1-0, where only reads with base quality value > 20 and mapping quality > 30 were retained, and PCR duplicates and secondary alignments were excluded. Then, freebayes was used for SNP calling with the parameter settings of --report-monomorphic --standard-filters --min-repeat-entropy --ploidy 2 --haplotype-length 0 --use-best-n-alleles 6 --genotype-qualities to obtain the genotyping results of all sites including polymorphic sites and non-polymorphic sites.

[0014] Screening of S12 mutation sites: The following filtering conditions were sequentially executed: Only single nucleotide base mutations were retained, and insertion mutations were removed; Sites with allele number > 2 were excluded; Sites with genotype quality (GQ) < 20 or sequencing depth < 3 were marked as missing; Sites with a missing rate > 30% were removed; Sites with an average depth > 28× were excluded; Finally, non-variant sites were removed to obtain 129.6M SNPs.

[0015] Screening of S13 50K SNP sites: According to individual information including genotype consistency of repeated sample sites, haploid heterozygosity rate, MAF, and PCA population structure analysis, the genotype data was subjected to a second quality control, and the MAF of the remaining SNP sites was statistically analyzed. Based on the rare allele frequency and whether it covered the genomic coding region as the criteria, SNPs with different rare allele frequencies and evenly covering the genome were initially screened. After screening 20M SNPs, high-level structure quality control was performed, and sites with complex high-level structures that might affect probe hybridization efficiency were filtered out to obtain 6.34M SNPs after preliminary filtering. Finally, SNP sites with a smaller Repeatsequence count, a GC content greater than 30 and less than 70, and preferentially covering the gene coding region were selected, and finally 49,693 SNP sites were screened out, which were distributed on 12 chromosomes.

[0016] Furthermore, the pine trees are Pinus yunnanensis and Pinus kesiya var. langbianensis.

[0017] The present invention also protects the application of the 50K liquid-phase chip of the pine tree whole genome in pine genetic diversity analysis, molecular genetic map construction, genome-wide association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and genome-wide selection breeding.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The liquid-phase chip of the present invention is based on the whole-genome resequencing data of individuals selected from populations with a large regional distribution of tropical pine Pinus kesiya and subtropical pine Pinus yunnanensis, and can comprehensively reflect the extensive genetic variations of the two pine species, revealing adaptive evolution and species differentiation. By integrating multi-species data, the chip of the present invention can accurately capture the genetic differences between species and populations; in addition, the probe design of this chip takes into account both species specificity and transferability, and is applicable not only to the target species, but also can be extended to other related pine groups, filling the blank in the genotyping of tropical and subtropical pines in the prior art, and providing an efficient and accurate tool for pine genetic resource protection, molecular breeding and genomics research.

[0020] The SNP molecular marker loci screened from the whole genome of pine in the present invention are evenly distributed on the genome and rich in diversity information. The whole-genome liquid-phase chip prepared by the present invention can realize the evaluation of genetic diversity of pine species, the identification of germplasm resources and genetic relationships, genome-wide association analysis and genomic selection breeding, and has high application value in the fields of forestry breeding, conservation biology and evolutionary genetics of pine.

[0021] For the 50K whole-genome liquid-phase chip of pine in the present invention, compared with whole-genome resequencing, the present invention can perform large-scale and low-cost genotyping of pine, greatly reducing the cost; in addition, based on the flexible characteristics of the liquid-phase chip loci, new marker loci can be added later for more application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the flow chart for screening 50K SNP loci of pine,

[0023] Figure 2 is the PCA plot of population structure based on whole-genome SNPs of the screened individuals

[0024] Figure 3 is the distribution plot of minor allele frequency (MAF) of 50K SNPs,

[0025] Figure 4 is the distribution plot of 50K SNPs on the whole-genome chromosomes,

[0026] Figure 5 is the PCA plot of all samples of the pine individuals to be tested and known pine species,

[0027] Figure 6 is the Maximum Likelihood (ML) tree plot of all samples of the pine individuals to be tested and known pine species,

[0028] Figure 7 It is an admixture component diagram of parental species individuals and hybrid individuals. Detailed implementation manners

[0029] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions. For those not specified in the embodiments, the operations are carried out under conventional conditions or conditions recommended by the manufacturers. All reagents or instruments, unless otherwise specified, are commercially available.

[0030] Example 1 A 50K liquid-phase chip for pine trees and its development

[0031] A 50K liquid-phase chip for pine trees includes a 50K site probe mixture for pine trees and a hybridization capture reagent; the 50K site probe mixture for pine trees contains 49,683 probes, and each probe hybridizes with one or more SNP site regions in the SNP site combination. Information on 49,693 sites among the 50K SNP sites of pine trees is shown below; the first row is the chromosomal position information of the SNPs, and the numbers on both sides of the subsequent "-" are the starting and ending positions on the chromosome, respectively.

[0032] Table 1 Information table of 49,693 SNP sites of pine trees

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[0184] Example 2 Development of a 50K liquid chip for pine trees

[0185] For the specific process of liquid chip development, see Figure 1 .

[0186] 2.1 Screening of SNP sites in pine trees

[0187] Tropical pine trees are usually distributed in habitats with high ecological diversity and marginalization, making them sensitive indicator species of environmental change. Their genetic resources have great potential in cultivating climate-adaptable varieties. In addition, given the accelerating climate change in the tropical region, studying the genomes and population dynamics of tropical pine tree populations can provide guidance for effective and sustainable management strategies for the conservation of wild pine tree resources in China. Using chips to conduct genetic evaluations of individuals can effectively capture minor genes, accurately predict complex traits of forest trees, and more accurately predict breeding values at an early stage, which is an important direction for the future development of forest tree breeding. Pinus yunnanensis is an important timber tree species in China and is widely planted in the Yunnan region. And Pinus kesiya, which is distributed in the tropics, is distributed in areas with highly variable climates and is affected by unique evolutionary forces such as frequent hybridization and complex gene flow. Therefore, a total of 57 individuals from two varieties, Pinus yunnanensis and Pinus kesiya, including 4 individual replicates and 5 haploid individuals taken from endosperm, were selected for whole-genome resequencing, with 200 - 300G of data sequenced for each individual. The individual information is shown in Table 2.

[0188] The specific steps for screening SNPs are as follows:

[0189] (1) Variant site genotyping: The original resequencing data of 57 pine individuals was filtered using fastp v0.23.2 (parameters: --cut_front --cut_tail -c -l 36, other parameters default), and then the clean data was aligned to the Pinus tabuliformis reference genome (GCA_031772625.1 https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_031772625.1 / ) using BWA. The results were sorted using samtools, and then indel realignment was performed using GATK v3.8-1-0; Bases with base quality value > 20 and reads with mapping quality > 30 were considered, and PCR duplicates, secondary alignments, etc. were not considered to reduce errors; Finally, freebayes was used for SNP calling with the parameters (--report-monomorphic --standard-filters --min-repeat-entropy --ploidy 2 --haplotype-length 0

[0190] --use-best-n-alleles 6 --genotype-qualities), and the genotyping results of all sites including polymorphic sites and non-polymorphic sites were obtained.

[0191] (2) Variant site filtering: Quality control was performed on all the sites (SNPs) obtained above, and only high-confidence SNPs were retained for the next step of analysis. The filtering criteria are as follows: All insertion mutations were removed, and only single nucleotide mutations were retained; Sites with allele numbers greater than 2 were filtered out; SNPs with genotype quality (GQ) lower than 20 or depth lower than 3 were redefined as missing; Sites with a missing rate greater than 30% were filtered out; Sites with an average depth greater than 28× were filtered out, and non-variant sites were removed, obtaining 129,630,494 high-confidence SNPs covering the entire genome.

[0192] Table 2 List of pine varieties used

[0193]

[0194] (3) Screening of 50K SNP sites: According to individual information including genotype consistency of duplicate sample sites and haploid heterozygosity rate, MAF (minor allele frequency), and PCA population structure ( Figure 2) Analyze to perform a second quality control on the genotype data, then calculate the MAF of the remaining SNP sites. Based on the rare allele frequency and whether it covers the genomic coding region as criteria, select SNP sites with different rare allele frequencies and evenly cover the genome. After initially screening out 20M SNP sites, perform quality control on the high-level structure, filter out sites with complex high-level structures that may affect the probe hybridization efficiency, and obtain 6.34M SNPs after preliminary filtering. Finally, select SNP sites with a smaller Repeat sequence count, a GC content greater than 30 and less than 70, and preferably covering the gene coding region. Ultimately, 49,693 SNP sites are screened out, which are distributed on 12 chromosomes for chip probe design. Figures 3-4 )

[0195] 2.2 Preparation of Pinus 50K liquid chip

[0196] Based on the 49,693 sites obtained by screening, synthesize liquid capture probes through Novogene Co., Ltd. The probe design steps are as follows:

[0197] (1) The probe length is 100bp, the probe GC content is between 50%, and try to ensure the specificity of the probe in the genomic range. Select regions that do not contain SSR and GAP regions to the greatest extent;

[0198] (2) Design a nucleotide sequence with 60% - 70% overlap and covering the SNP sites according to the screened SNP sites;

[0199] (3) Perform single-stranded nucleotide synthesis according to the designed nucleotide sequence, and synthesize a DNA nucleotide sequence with a length of 100bp modified with a biotin group at the 5' end, that is, the site probe. A total of 49,683 site probes are obtained. After synthesis, PCR amplification is used to increase the probe amount;

[0200] (4) Mix the amplified site probes mentioned above in equimolar mass, and use a mixture of EDTA and Tris HCl to make up a probe mixture with a volume of 3pmol / mL.

[0201] Example 3 Application of Pinus 50K liquid chip

[0202] 3.1 Identification of Pinus species

[0203] Use the Pinus 50K liquid chip of the present invention for the identification of Pinus species and the assessment of genetic diversity. The specific steps are as follows:

[0204] (1) DNA extraction and quality inspection: DNA samples of 22 species to be identified and samples of several known pine species were used as reference samples to extract DNA for chip detection: DNA extraction kit (Tiangen Plant Total DNA Kit) was used to extract DNA from the samples. 1% agarose gel electrophoresis was used to evaluate the purity and integrity of the DNA, and the Qubit fluorescence quantification instrument was used to accurately measure the DNA concentration to ensure that the sample quality met the requirements of subsequent experiments.

[0205] (2) Targeted gene capture technology based on liquid phase probe hybridization

[0206] (http: / / www.molbreeding.com / index.php / Technology / GenoBaits.html) Standard process for constructing liquid phase DNA chips

[0207] Hybrid capture library, library sequencing relies on Illumina NovaSeq TM mX Plus sequencing platform.

[0208] (3) After obtaining the captured sequencing data based on the 50K pine liquid phase chip, the sequencing data of the pine species to be analyzed were first aligned to the aforementioned Pinus tabulaeformis reference genome using the BWA software. Subsequently, the GATK tool was used to identify the genotype of the population SNPs, and the SNP site typing result set of the chip was extracted. After that, the SNP typing result set containing all samples was subjected to principal component analysis (PCA) and phylogenetic tree construction. According to the clustering results of the phylogenetic tree, it was analyzed to determine which known pine samples the sample to be identified was closer to, and the species to which the pine sample to be tested belonged could be determined. On this basis, the genetic diversity parameters of each pine were calculated, including nucleotide diversity (π), expected heterozygosity (He), and observed heterozygosity (Ho), to evaluate the genetic diversity of different pine species or populations.

[0209] The results are shown in Table 3. According to the sample individual information and test results, the chip loci have a high success rate in several pine trees. The success rate of SNP detection in Yunnan pine and Pinus cassiae is 92.19%-98.49%, in alpine pine it is 92.75%-95.82%, in Pinus tabulaeformis it is 93.45%-93.94%, in Pinus sutchuenensis it is 93.68%-98.23%, and in Pinus sutchuenensis it is 69.10%-76%. Figure 6 As shown in the figure, the phylogenetic tree also shows that the samples to be tested (labeled as sample01-22) are clustered with the known pine trees, and the samples can be accurately identified as species. As shown in Table 4 below, it can be seen that the pine whole genome 50K liquid phase chip of the present invention can also be used to evaluate the genetic diversity level of the pine species to be tested.

[0210] It can be seen that the 50K pine sap phase chip developed by the present invention has achieved accurate identification of pine species through phylogenetic analysis and principal component analysis, and has shown excellent SNP typing success rates (92.19%-98.49%) in Yunnan pine, Cassia pine, alpine pine, Chinese pine and ground pine, and has maintained operability (69.10%-76.00%) even in the distantly related South Asian pine. The chip can not only clearly distinguish different pine species through phylogenetic trees, but also simultaneously evaluate genetic diversity parameters such as nucleotide diversity, expected heterozygosity and observed heterozygosity, realizing efficient integration of species identification and genetic analysis. The experimental results show that the technology has the characteristics of broad spectrum applicability, high stability and good repeatability, and provides a reliable integrated solution for the taxonomic research, germplasm resource protection and genetic evolution analysis of pine plants.

[0211] Table 3 Sample individual information and test results

[0212]

[0213]

[0214] Note: Pk is Pinus cusiae, Py is Pinus yunnanensis, Pm is Pinus sylvestris, Pd is Pinus alpine, Pt is Pinus tabulaeformis, and Pyv is Pinus terrestris.

[0215] Table 4 Observed heterozygosity (hardyH) of six pine species based on site polymorphism (π) o ), and based on the observed heterozygosity of individuals (H o ) and expected heterozygosity (H e )Test results table

[0216]

[0217]

[0218] Note: Pk is Pinus cusiae, Py is Pinus yunnanensis, Pm is Pinus sylvestris, Pd is Pinus alpine, Pt is Pinus tabulaeformis, and Pyv is Pinus terrestris.

[0219] 3.2 Identification of pine hybrid individuals

[0220] Individuals of the parent species Yunnan pine and Cassia pine and the individuals to be identified were selected, DNA was extracted, and after obtaining the capture sequencing data based on the 50K liquid phase chip, the sequencing data of the parents and the samples to be identified were firstly obtained using the BWA software according to the data acquisition and analysis method in Example 3.1 to obtain the vcf file of the SNP variation site of the sample to be tested, and then the admixture software was used to analyze the sample parent components, where 0.1 <Q值<0.9的个体鉴定为杂交个体。

[0221] The results are as Figure 7 shown in Table 5. By analyzing both known parental individuals and the hybrid individuals to be tested together, the proportion of the genetic components of the parental species contained in each hybrid individual can be obtained, thereby determining the origin and composition of the genetic components of the hybrid individuals. Among them, individuals with 0.1 < Q value < 0.9 can be identified as hybrid individuals. This application is not only used for the identification of hybrid individuals of Pinus yunnanensis and Pinus kesiya, but also applicable to the identification of hybrid individuals of other closely related pine species using the same method.

[0222] Table 5 Q-component table of parental species individuals and hybrid individuals based on admixture analysis

[0223]

[0224]

[0225] Note: Py is Pinus yunnanensis, Pk is Pinus kesiya, and hybrid represents hybrid individuals.

Claims

1. A 50K liquid-phase chip for the whole genome of pine trees, characterized in that: The liquid phase chip includes a 50K site probe mixture of the pine genome and a hybrid capture reagent; the site probe includes 49693 SNP sites distributed on 12 chromosomes, and the SNP site information is as follows: The first line contains the chromosome location information of the SNPs, and the numbers on both sides of the "-" are the starting and ending positions of the SNPs on the chromosome.

2. The 50K chip for the whole genome of pine as described in claim 1, characterized in that The design steps of the pine whole genome 50K site probe include: The S1 probe length was 100 bp, the probe GC content was between 50%, and the selected region did not contain SSR and GAP regions to the greatest extent possible; S2 designs a nucleotide sequence with 60% to 70% overlap and covering the SNP site based on the SNP site obtained by screening; S3 synthesizes single-stranded nucleotides according to the designed nucleotide sequence, and synthesizes a site probe with a length of 100 bp modified with a biotin group at the 5' end. After synthesis, PCR amplification is performed to increase the amount of the site probe; S4: Mix the synthesized site probes in equal molar mass and adjust the volume to 3 pmol / mL of the probe mixture using a mixture of EDTA and Tris HCl.

3. The pine whole-genome 50K chip according to claim 2, characterized in that, The method for obtaining the pine 50K SNP loci is as follows: S11 variant locus typing Whole genome resequencing was performed on pine individuals, and fastp v0.23.2 was used to perform quality control filtering on the raw sequencing data. The filtered data were aligned to the reference genome of Pinus tabulaeformis GCA_031772625.1 using BWA. After sorting by samtools, insertion rearrangement was performed using GATK v3.8-1-0, and only reads with base quality values ​​> 20 and mapping quality > 30 were retained, and PCR duplications and secondary alignments were excluded. Freebayes was then used for SNP calling, with the parameters set to --report-monomorphic --standard-filters --min-repeat-entropy --ploidy 2 --haplotype-length0 --use-best-n-alleles 6 --genotype-qualities, to obtain typing results for all sites including polymorphic sites and non-polymorphic sites. S12 mutation site screening The following filtering conditions were performed in sequence: only single nucleotide base mutations were retained and insertion mutations were removed; sites with the number of alleles > 2 were excluded; sites with genotype quality (GQ) < 20 or sequencing depth < 3 were marked as missing, and sites with a missing rate > 30% were eliminated; sites with an average depth > 28× were excluded; and finally, non-variable sites were removed, resulting in 129.6M SNP sites; S1350K SNP site screening According to individual information including the genotype consistency of repeated sample sites and the haploid heterozygosity rate, MAF, and PCA population structure analysis, the genotype data is subjected to a second quality control, and the MAF of the remaining SNP sites is statistically analyzed. Based on the rare allele frequency and whether it covers the genomic coding region, SNP sites with different rare allele frequencies and evenly covering the genome are initially screened for 20M SNP sites, and then high-level structure quality control is performed to filter out sites with complex high-level structures that may affect probe hybridization efficiency, obtaining 6.34M SNPs after preliminary filtering; finally, SNP sites with a smaller Repeat sequence count, a GC content greater than 30 and less than 70, and preferably covering the gene coding region are selected, and finally 49,693 SNP sites are screened out, which are distributed on 12 chromosomes.

4. The pine whole genome 50K chip according to claim 1, wherein The pine trees described are Pinus yunnanensis and Pinus kesiya.

5. The 50K liquid-phase chip of the pine tree whole genome according to any one of claims 1-4 is applied to pine tree genetic diversity analysis, molecular genetic map construction, genome-wide association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and genome-wide selection breeding.

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