Pinus massoniana snp molecular marker combination and application thereof

Abstract

The application relates to the technical field of gene chips, in particular to a SNP molecular marker combination of Pinus massoniana and application thereof. The SNP molecular marker combination is composed of 113,709 SNP molecular markers, wherein the physical position of the SNP molecular markers is determined by sequence alignment based on a reference genome Pinus.tabuliformis V1.0 of Pinus tabulaeformis. The application provides a whole genome chip of Pinus massoniana containing 113,709 SNP sites for the first time, and the chip has the advantages of high efficiency, low cost, good genetic stability and the like, and can be widely applied to different application scenarios such as identification of Pinus massoniana germplasm resources, genetic background analysis of breeding materials, gene positioning, whole genome association analysis, whole genome selection breeding and intelligent design breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene chip, in particular to a SNP molecular marker combination of Pinus massoniana and application thereof. BACKGROUND

[0002] Pinus massoniana is the tree species with the widest distribution in Pinus in China. The wood and turpentine thereof can be used for pulp and paper making, wood processing, forest chemical industry and rosin extraction, etc. Pinus massoniana has high comprehensive utilization value and wide application prospect. However, Pinus massoniana shows symptoms of acute dehydration, discoloration and even death after being infected by pine wood nematode, which causes immeasurable huge loss to forest ecology and forestry economy. However, the traditional breeding method of improving target traits of Pinus massoniana through phenotype selection has long breeding cycle, low efficiency and poor controllability, and cannot meet the current demand of development of Pinus massoniana industry.

[0003] For important traits of Pinus massoniana such as yield, quality and resistance, molecular marker assisted selection and whole genome selection can quickly and accurately analyze the genetic composition of individuals, so as to realize direct selection of genotypes and molecular breeding. Available related technologies include traditional marker assisted selection methods based on restriction fragment length polymorphism (RFLP), simple sequence repeats (SSR) and methods based on whole genome sequencing technology. However, the former has high cost, low efficiency and long time, and cannot be applied to large-scale molecular breeding of Pinus massoniana; the latter can obtain high-density molecular markers, but due to the large reference genome, the cost is high, and it also cannot be applied on a large scale.

[0004] Therefore, it is urgent to develop a whole genome SNP chip of Pinus massoniana to provide technical support for efficient breeding of high-quality and disease-resistant Pinus massoniana varieties. SUMMARY

[0005] The present application aims to at least partly solve one of the problems in the related art.

[0006] To this end, a first aspect of the present application provides a SNP molecular marker combination of Pinus massoniana, which is composed of 113,709 SNP molecular markers as shown in Table 1, wherein the physical position of the SNP molecular marker is determined by sequence alignment based on the reference genome of Pinus tabuliformis V1.0.

[0007] A second aspect of the present application provides a whole genome chip of Pinus massoniana, which comprises probes or primers for detecting the SNP molecular marker combination according to any one of the first aspect.

[0008] The third aspect of the present application provides a kit comprising probes or primers for detecting the SNP molecular marker combination according to any one of the first aspect.

[0009] The fourth aspect of the present application provides a probe for detecting the SNP molecular marker combination of Pinus massoniana according to any one of the first aspect, which is 113,709 SNP molecular markers as shown in Table 1.

[0010] The fifth aspect of the present application provides a use of the probe according to any one of the fourth aspect in the preparation of a Pinus massoniana whole genome chip.

[0011] The sixth aspect of the present application provides a genotyping method comprising using the SNP molecular marker combination according to any one of the first aspect, the chip according to any one of the second aspect, the kit according to any one of the third aspect and / or the probe according to any one of the fourth aspect to perform the genotyping on a sample.

[0012] The seventh aspect of the present application provides the use of the SNP molecular marker combination according to any one of the first aspect, the chip according to any one of the second aspect, the kit according to any one of the third aspect and / or the probe according to any one of the fourth aspect in genetic breeding analysis.

[0013] In some embodiments, the genetic breeding analysis comprises germplasm resource identification analysis, purity identification analysis, genetic background analysis of breeding materials, gene mapping analysis, whole genome association analysis, whole genome selection breeding analysis and intelligent design breeding analysis.

[0014] The advantages and technical effects brought by the independent claims according to the embodiments of the present application are as follows:

[0015] (1) The 113,709 SNP molecular marker combination provided by the embodiments of the present application is selected from a diversity-rich Pinus massoniana germplasm resource data, additionally covers 25,119 gene function markers related to Pinus massoniana resistance to pine wood nematode disease and 4,536 universal markers of Pinus elliottii and Pinus taeda chips, has rich gene region sites (gene region sites account for 49.40%), is strong in representation, high in polymorphism, good in universality, high in coverage on the genome (average coverage 99.90%) and evenly distributed, and is particularly suitable for applications such as chip preparation, molecular marker assisted selection, directional improvement, important trait gene mining and identification and function analysis.

[0016] (2) The embodiment of the present application provides a 100K Pinus massoniana whole genome chip containing probes for detecting the above-mentioned sites, and the chip has the characteristics of high efficiency, low cost, good genetic stability, simple typing, high throughput and the like, can produce a large amount of data at one time, can simultaneously cover detection of nearly a thousand materials, is suitable for mainstream platforms such as illumina and MGI, and has platform wide adaptability. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a screening process schematic diagram of molecular markers for the Pinus massoniana whole genome chip of the embodiment of the present application.

[0018] Figure 2 It is an annotation result schematic diagram of molecular markers for the Pinus massoniana whole genome chip of the embodiment of the present application.

[0019] Figure 3 It is a statistical result schematic diagram of chromosome distribution of molecular markers for the Pinus massoniana whole genome chip of the embodiment of the present application.

[0020] Figure 4 It is a statistical result schematic diagram of allelic value distribution of molecular markers for the Pinus massoniana whole genome chip of the embodiment of the present application.

[0021] Figure 5 It is a principle schematic diagram of a liquid phase probe capture sequencing typing technology for the Pinus massoniana whole genome chip of the embodiment of the present application.

[0022] Figure 6 It is a genotype detection rate result schematic diagram of the Pinus massoniana whole genome chip of the embodiment of the present application in samples.

[0023] Figure 7 It is a result schematic diagram of clustering analysis of genotyping results of the Pinus massoniana whole genome chip of the embodiment of the present application in samples. DETAILED DESCRIPTION

[0024] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0025] In order to facilitate understanding, the terms in the present application are explained and described below, and those skilled in the art should understand that the explanations and descriptions should not be understood as a limitation of the protection scope of the present application.

[0026] In this paper, the term "polymorphism" refers to genetic polymorphism, which is used to describe the diversity of the genome of a species (such as humans), which essentially refers to the inter-individual differences in the DNA sequence unique to an individual. In other words, genetic polymorphism is the occurrence of multiple discrete allelic states in the same population. Polymorphism involves one of two or more variants of a specific DNA sequence. Single nucleotide polymorphism (SNP) is the most common type of polymorphism, which is a DNA sequence polymorphism caused by a single nucleotide variation at the genome level.

[0027] The liquid probe pinpoint sequencing genotyping technology (cGPS) liquid chip technology described herein is based on an optimized thermodynamic stability algorithm model for specific probe design of target interval sequences, uses synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions by liquid hybridization, and then constructs a library and performs second-generation sequencing on the captured and enriched target interval, thereby obtaining the genotype of the SNP / InDel site in the target region. The principle and process of the cGPS liquid chip technology are shown in Figure 5 .

[0028] The first aspect embodiment of the present application proposes a Pinus massoniana SNP molecular marker combination, which is composed of 113,709 SNP molecular markers as shown in Table 1, wherein the physical position of the SNP molecular marker is determined by sequence alignment based on the reference genome of Pinus tabuliformis V1.0. The site information is shown in Table 1. The Pinus massoniana SNP molecular marker proposed in the embodiments of the present application is representative, has high polymorphism, good universality, high coverage on the genome (average coverage 99.90%) and uniform distribution, and is particularly suitable for chip preparation, molecular marker assisted selection, directional improvement, important trait gene mining and identification and functional analysis and the like.

[0029] In some embodiments, the molecular markers used in the Pinus massoniana whole genome chip of the embodiments of the present application are obtained by the screening process as shown in Figure 1 . Specifically, 1) Pinus massoniana sample resequencing data, wherein 84,209 initial sites are obtained by quality control; 2) transcriptome analysis for Pinus massoniana resistance to pine wood nematode disease (resistant / susceptible), obtaining 25,119 gene function markers located on significantly different genes; 3) 4,536 universal markers of Pinus elliottii and Pinus taeda chips. After integration and deduplication, a total of 113,709 SNP molecular markers are obtained.

[0030] In some embodiments, the combination of SNP molecular markers can consist of one or more of the 113,709 SNP molecular markers as shown in Table 1.

[0031] In some embodiments, the combination of SNP molecular markers consists of the 113,709 SNP molecular markers as shown in Table 1.

[0032] Table 1

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[0168] Note: the information of the SNP molecular markers in Table 1 is in turn the position of the site in the corresponding chromosome genome (POSITION) / the reference genotype of the site (REF) / the variation genotype of the site (ALT).

[0169] The second aspect embodiment of the present application provides a Pinus massoniana whole genome chip, wherein the chip comprises probes or primers for detecting the SNP molecular marker combination as described in any one of the first aspect embodiments.

[0170] The third aspect embodiment of the present application provides a kit, wherein the kit comprises probes or primers for detecting the SNP molecular marker combination as described in any one of the first aspect embodiments.

[0171] The fourth aspect embodiment of the present application provides a probe for detecting the SNP molecular marker combination of Pinus massoniana as described in any one of the first aspect embodiments, and the SNP molecular marker combination is 113,709 SNP molecular markers as shown in Table 1.

[0172] The fifth aspect embodiment of the present application provides a use of the probe as described in the fourth aspect embodiment in the preparation of a Pinus massoniana whole genome chip.

[0173] In a sixth aspect, the present application provides a method for genotyping, comprising using the SNP marker combination according to any one of the first aspect, the chip according to any one of the second aspect, the kit according to any one of the third aspect and / or the probe according to any one of the fourth aspect to perform the genotyping on a sample. In specific embodiments, the method comprises: (1) extraction and quality control of genomic DNA: the sample DNA is extracted by magnetic bead method, and the DNA sample is subjected to quality detection. The quality detection comprises determining the DNA concentration by Qubit fluorescence quantifier, and detecting the integrity of the DNA by 1% agarose gel electrophoresis. The sample that passes the quality control is used for library preparation. (2) cGPS library construction and quality control: a. the DNA sample is subjected to enzyme cutting by using a fragmentation enzyme, the enzyme cutting end is repaired, and A base is added to the 3' end, and the fragment size is detected by agarose gel electrophoresis. b. The sequencing adapter is connected to the DNA fragment by using T4 ligase, and the connection product is purified by using magnetic beads. The concentration of the purified product is detected by Qubit fluorescence quantifier, and the fragment size is detected by agarose gel electrophoresis. c. The purified connection product is subjected to PCR amplification, and the amplification product is subjected to fragment screening by using magnetic beads. The concentration of the product after fragment screening is detected by Qubit fluorescence quantifier, and the fragment size is detected by agarose gel electrophoresis. d. 200 ng of the constructed library is taken, the probe and hybridization reagent are added, and the hybridization reaction is completed at 50 DEG C for 16-24 hours. The target segment is captured by using magnetic beads, the capture product is washed by using washing solution to remove non-specific binding fragments, and then a round of PCR amplification is performed. The library concentration is detected by Qubit fluorescence quantifier, and the fragment size is detected by agarose gel electrophoresis. After the concentration and fragment size determination are qualified, the cGPS sequencing library construction is completed. The prepared library is subjected to high-throughput sequencing by using a Huada sequencer, and the sequencing strategy is PE150; (3) data analysis: the raw data after high-throughput sequencing is subjected to quality control filtering and the like, the adapter fragments and low-quality reads are removed by using FASTP software to obtain high-quality Clean Reads. The obtained Clean reads are aligned with the reference genome by using BWA software, and the position is sorted to obtain a sample sorted bam file. The sequencing results are analyzed by using GATK software to obtain the genotyping results of the target site. It should be noted that the method described in the present embodiment is only an exemplary step, and is not intended to limit the specific operation process of genotyping.

[0174] In a seventh aspect, the present application provides the SNP marker combination according to any one of the first aspect, the chip according to any one of the second aspect, the kit according to any one of the third aspect and / or the probe according to any one of the fourth aspect for use in genetic breeding analysis.

[0175] In some embodiments, the genetic breeding analysis includes germplasm identification analysis, purity identification analysis, genetic background analysis of breeding materials, gene mapping analysis, whole genome association analysis, whole genome selection breeding analysis, and intelligent design breeding analysis.

[0176] The experimental methods in the following examples are all conventional methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified.

[0177] Example 1: Design and preparation method of Pinus massoniana 100K whole genome chip

[0178] 1.1 Screening SNPs for Pinus massoniana whole genome chip

[0179] 1.1.1 SNPs screened based on resequencing results of Pinus massoniana samples - whole genome background sites

[0180] (1) Select 30 Pinus massoniana individuals with rich diversity from 7 provinces for resequencing. Perform quality control on the raw sequencing data of resequencing using fastp with default parameters, and then use Sentieon to align to the Pinus tabuliformis reference genome Pinus.tabuliformis V1.0 (Niu S et al., 2022; https: / / doi.org / 10.1016 / j.cell.2021.12.006) for analysis to obtain the variation file of resequencing data.

[0181] (2) Based on the obtained resequencing data, perform the first step of screening of whole genome background sites, with the following screening criteria: remove contigs, filter SNP sites within 10bp indel; screen polymorphic SNP sites with minimum allele frequency MAF>0.05, deletion rate <0.1, heterozygosity <0.2, and sequencing depth <40x.

[0182] (3) Based on the data of the preliminary screening, further remove sites not suitable for probe design to perform the second step of screening to obtain the initial site set, with the following screening criteria: for each site, design probes within 100bp upstream and downstream thereof; the probe length is 100bp, the GC content is 20%-80%, the whole genome copy number is 1-3, and the probes containing repetitive sequences and N bases are removed.

[0183] (4) Perform linkage disequilibrium analysis on the initial site set using plink software to perform the third step of screening, with the following screening conditions: use LD parameter R 2 ≥0.2 to filter to remove high linkage sites.

[0184] (5) Based on the above conditions, 84,209 SNPs evenly distributed on the chromosomes of Pinus massoniana were selected as the whole genome background sites according to the principle of uniform distribution of sites on chromosomes.

[0185] 1.1.2 SNPs-Functional sites against P. wilt disease screened based on transcriptome analysis of Pinus massoniana samples

[0186] (1) Using inoculation tests, 33 Pinus massoniana samples were divided into high and low groups according to resistance levels. The samples were extracted for transcriptome sequencing and differential gene expression analysis. The differential gene screening criteria were as follows: padj < 0.05 & log2FoldChange > 1, padj < 0.05 & log2FoldChange < -1.

[0187] (2) The sequences of the differential genes were aligned to the Pinus tabulaeformis reference genome. According to the sequence length alignment rate > 70% and sequence similarity > 90%, the physical location interval of the differential gene sequence alignment to the Pinus tabulaeformis reference genome was obtained.

[0188] (3) According to the physical location interval, interval sites were extracted from the initial site set in 1.1.1 above, and a total of 25,119 SNPs were obtained. These markers can be used for disease resistance breeding of Pinus massoniana.

[0189] 1.1.3 Universal sites of Pinus elliottii and Pinus taeda chips

[0190] (1) The 51K chip of Pinus elliottii and Pinus taeda (Diao S et al., 2024; https: / / doi.org / 10.1016 / j.indcrop.2024.118777) was used to detect 10 Pinus massoniana samples, and probes with a detection rate of 100% in Pinus massoniana samples were screened.

[0191] (2) The probe sequences were aligned to the Pinus tabulaeformis reference genome. According to the sequence length alignment rate > 70% and sequence similarity > 90%, the physical location interval of the alignment to the Pinus tabulaeformis reference genome was obtained.

[0192] (3) According to the physical location interval, interval sites were extracted from the initial site set in 1.1.1 above, and a total of 4,536 SNPs were obtained.

[0193] 1.1.4 SNPs for Pinus massoniana whole genome chip

[0194] All marker sites were obtained by integrating examples 1.1.1-1.1.3, and screened and filtered according to the quality indicators of these sites, and finally 113,709 molecular markers with strong representation, high polymorphism, good universality and uniform distribution on chromosomes were obtained, including 25,119 functional markers related to the resistance of Pinus massoniana to pine wood nematode disease, 1195 universal markers for Pinus elliottii and Pinus taeda chips, 56,176 gene region sites (accounting for 49.40%), see Figure 2 .

[0195] The average coverage of the 100K whole genome chip sites of Pinus massoniana on the chromosomes was 99.90%, the average interval was 214.5Kb, and the sites were uniformly distributed on each chromosome, and the density distribution diagram is shown in Figure 3 . The site polymorphism is good, and the MAF of all sites is greater than 0.05, as shown in Figure 4 .

[0196] 1.2 Preparation of liquid phase chip

[0197] The 113,709 molecular markers screened out are developed into a 100K whole genome liquid phase chip of Pinus massoniana by liquid phase probe precise positioning sequencing typing technology.

[0198] Example 2

[0199] In order to verify the genotyping effect of the 100K whole genome chip of Pinus massoniana, the 100K whole genome chip of Pinus massoniana designed in example 1 was used to genotype and detect 16 samples of Pinus massoniana.

[0200] 2.1 Extraction and quality control of genomic DNA

[0201] The DNA of 16 samples of Pinus massoniana was extracted by magnetic bead method, and the quality of the DNA sample was detected. The quality detection includes measuring the DNA concentration by Qubit fluorescence quantifier and detecting the integrity of the DNA by 1% agarose gel electrophoresis. The qualified samples are used for library preparation.

[0202] 2.2 cGPS library construction and quality control

[0203] a. The DNA sample was cut by fragmentation enzyme, the cut end was repaired, and A base was added to the 3' end, and the fragment size was detected by agarose gel electrophoresis.

[0204] b. The sequencing adapter was connected with the DNA fragment using T4 ligase, and the connection product was purified by magnetic beads. The concentration of the purified product was detected by Qubit fluorescence quantifier, and the fragment size was detected by agarose gel electrophoresis.

[0205] c. The purified ligation product was subjected to PCR amplification, and the amplified product was subjected to fragment screening using magnetic beads. The product after fragment screening was detected for concentration using a Qubit fluorescence quantifier, and the fragment size was detected by agarose gel electrophoresis.

[0206] d. 200 ng of the completed library was taken, and after adding probes and hybridization reagents, 50°C incubation was performed for 16-24 hours to complete the hybridization reaction. Target segment capture was performed using magnetic beads, the capture product was washed using a washing solution to remove non-specific binding fragments, and then a round of PCR amplification was performed.

[0207] e. The library concentration was detected using a Qubit fluorescence quantifier, and the fragment size was detected by agarose gel electrophoresis. After the concentration and fragment size determination were qualified, the cGPS sequencing library construction was completed.

[0208] f. The prepared library was subjected to high-throughput sequencing using a Huada sequencer, and the sequencing strategy was PE150.

[0209] 2.3 Data analysis

[0210] The raw data after high-throughput sequencing was subjected to quality control filtering and the like, and the FASTP software was used to remove adapter fragments and low-quality reads to obtain high-quality Clean Reads. The obtained Clean reads were aligned with the reference genome using the BWA software, and the position was sorted to obtain the sample sorted bam file. The GATK software was used for variant site analysis of the sequencing results to obtain the genotyping results of the target sites.

[0211] After sequencing and data analysis, the detection rate of the data of 16 Pinus massoniana samples was between 97.28% and 99.16%, and the average detection rate was 98.82%, as shown in Table 1. Figure 6 The heterozygosity rate was between 29.28% and 33.06%, and the average heterozygosity rate was 32.07%. The above data show that the Pinus massoniana 100K whole genome chip can realize genotyping of Pinus massoniana samples, and the target site detection rate is high.

[0212] Example 3 Application of Pinus massoniana 100K whole genome chip in cluster analysis

[0213] (1) The Pinus massoniana 100K whole genome chip prepared in Example 1 was used for genotyping of 33 Pinus massoniana samples, and the specific operation process was referred to Example 2.

[0214] (2) The Plink software was used to calculate the genetic distance matrix of 33 Pinus massoniana materials and draw a phylogenetic tree.

[0215] The results are shown in Table 2. Figure 7As shown in the figure, 33 samples of Pinus massoniana were mainly divided into two subgroups, which was consistent with the known results. The results showed that the Pinus massoniana 100K whole genome chip of the application can effectively distinguish the samples of Pinus massoniana from different sources, and can perform cluster analysis and identification on Pinus massoniana.

[0216] Example 4 Application of Pinus massoniana 100K whole genome chip in difference analysis of Pinus massoniana samples

[0217] The Pinus massoniana 100K whole genome chip prepared in Example 1 was used for genotyping of 12 samples of Pinus massoniana, and the specific operation process was referred to Example 2. Among them, the 12 samples were from 6 families, and 2 samples with indistinguishable phenotypes were selected from each family. According to the genotype data of the 12 materials, the difference of samples in the family was analyzed.

[0218] The results are shown in Table 2, and the genotype consistency rate of the 6 pairs of samples in the family is between 65.3% and 74.36%, and the average consistency rate is 71.47%. The results show that the Pinus massoniana 100K whole genome chip of the application can accurately and effectively distinguish different samples with similar phenotypes in the family, and realize the difference analysis of the samples at the genotype level.

[0219] Table 2 Genotype consistency rate statistics table of samples in the family

[0220]

[0221] In the application, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the application. In the specification, the illustrative representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0222] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

1. A Pinus massoniana full genome chip, characterized in that, The chip comprises probes for detecting the SNP molecular marker combination of Pinus massoniana, wherein the SNP molecular marker combination of Pinus massoniana consists of 113,709 SNP molecular markers as shown in Table 1, wherein the physical position of the SNP molecular markers of Pinus massoniana is determined by sequence alignment based on the reference genome of Pinus tabuliformis V1.

0.

2. A kit characterized in that, The kit comprises probes for detecting the SNP molecular marker combination of Pinus massoniana as claimed in claim 1.

3. A probe for detecting the molecular marker combination of Pinus massoniana SNP according to claim 1, characterized in that, The SNP molecular marker combination is 113,709 SNP molecular markers as shown in Table 1.

4. Use of the probes of claim 3 in the preparation of a whole genome chip of Pinus massoniana.

5. A method of genotyping, characterized by, Genotyping of a sample of Pinus massoniana using the chip of claim 1, the kit of claim 2 and / or the probes of claim 3.

6. Use of the chip of claim 1, the kit of claim 2 and / or the probes of claim 3 in genetic breeding analysis of Pinus massoniana.

7. Use according to claim 6, characterized in that, The genetic breeding analysis comprises germplasm resource identification analysis, purity identification analysis, genetic background analysis of breeding materials, gene mapping analysis, whole genome association analysis and whole genome selection breeding analysis.

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