Pinus massoniana SNP molecular marker combination and application thereof

Abstract

The invention relates to the technical field of gene chips, in particular to a masson pine SNP molecular marker combination and application thereof. The SNP molecular marker combination is composed of 113 SNP molecular markers and 709 SNP molecular markers, wherein the physical positions of the SNP molecular markers are determined by carrying out sequence alignment on the basis of a reference genome Pinus.tabuliformes V1.0 of the Chinese pines. According to the embodiment of the invention, the masson pine whole genome chip containing 113 and 709 SNP sites is provided for the first time, and has the advantages of high efficiency, low cost, good hereditary stability and the like; the method can be widely applied to different application scenes such as pinus massoniana germplasm resource identification, breeding material genetic background analysis, gene localization, whole-genome association analysis, whole-genome selective breeding and intelligent design breeding.

Description

Technical Field

[0001] The present application relates to the technical field of gene chips, and in particular to a combination of Masson pine SNP molecular markers and applications thereof. Background Art

[0002] Masson pine is the most widely distributed tree species in the genus Pinus in my country. Its wood and rosin can be used in many industries such as pulp and paper, wood processing, forest chemical industry and rosin production. It has high comprehensive utilization value and broad prospects for promotion and application. However, after being infected by pine wood nematodes, Masson pine shows symptoms of acute dehydration, discoloration and even death, causing immeasurable losses to forest ecology and forestry economy. However, the traditional breeding method of improving the target traits of Masson pine through phenotypic selection has a long breeding cycle, low efficiency and poor controllability, and can no longer meet the current development needs of the Masson pine industry.

[0003] For important traits such as yield, quality, and resistance of Masson pine, molecular marker-assisted selection and whole genome selection can quickly and accurately analyze the genetic composition of individuals, thereby achieving direct selection of genotypes and conducting molecular breeding. Available related technologies include traditional marker-assisted selection methods based on restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) and methods based on whole genome sequencing technology. However, the former is expensive, inefficient, and time-consuming, and cannot be applied on a large scale to molecular breeding of Masson pine; although the latter can obtain a higher density of molecular markers, it is also expensive and cannot be applied on a large scale due to the large reference genome.

[0004] Therefore, it is urgent to develop a whole-genome SNP chip for Masson pine to provide technical support for the efficient breeding of high-quality and disease-resistant Masson pine varieties. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0006] To this end, the first embodiment of the present invention proposes a Masson pine SNP molecular marker combination, which consists of 113,709 SNP molecular markers as shown in Table 1, wherein the physical positions of the SNP molecular markers are determined by sequence alignment based on the reference genome of Pinus tabuliformis V1.0.

[0007] The second aspect of the present invention provides a Masson pine whole genome chip, which comprises probes or primers for detecting the SNP molecular marker combination as described in any embodiment of the first aspect.

[0008] The third aspect of the present invention provides a kit, which comprises probes or primers for detecting the SNP molecular marker combination as described in any embodiment of the first aspect.

[0009] The fourth aspect of the present invention provides a probe for detecting the Masson pine SNP molecular marker combination as described in any embodiment of the first aspect, wherein the SNP molecular marker combination is 113,709 SNP molecular markers as shown in Table 1.

[0010] The fifth aspect of the present invention provides a use of the probe described in the fourth aspect of the present invention in preparing a Masson pine whole genome chip.

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

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

[0013] In some embodiments, the genetic breeding analysis includes germplasm resource identification analysis, purity identification analysis, genetic background analysis of breeding materials, gene positioning 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 invention are as follows:

[0015] (1) The 113,709 SNP molecular marker combinations provided in the embodiments of the present invention are selected from the rich diversity of Masson pine germplasm resource data, and also additionally cover 25,119 gene function markers related to the resistance to pine wood nematode disease of Masson pine and 4,536 common markers for the chip of slash pine and loblolly pine. The gene region sites are rich (the gene region sites account for 49.40%), with strong representativeness, high polymorphism, good versatility, high coverage rate on the genome (average coverage rate 99.90%) and uniform distribution, which is particularly suitable for applications such as chip preparation, molecular marker-assisted selection, directional improvement, important trait gene mining and identification, and functional analysis.

[0016] (2) The embodiment of the present invention provides a 100K Masson pine whole genome chip comprising probes for detecting the above-mentioned sites. The chip has the characteristics of high efficiency, low cost, good genetic stability, simple typing, high throughput, etc. It can output a large amount of data at one time and can cover the detection of nearly a thousand materials at the same time. It is also suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, and has wide platform adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the screening process of molecular markers for the Masson pine whole genome chip in the embodiment of the present invention.

[0018] Figure 2 It is a schematic diagram of the annotation results of molecular markers used for the Masson pine whole genome chip in the embodiment of the present invention.

[0019] Figure 3 It is a schematic diagram of the statistical results of chromosome distribution of molecular markers used in the Masson pine whole genome chip in the embodiment of the present invention.

[0020] Figure 4 It is a schematic diagram of the statistical results of the distribution of minor allele values ​​of molecular markers used in the Masson pine whole genome chip according to the embodiment of the present invention.

[0021] Figure 5 It is a schematic diagram of the principle of the liquid-phase probe capture sequencing typing technology used in the Masson pine whole genome chip of the embodiment of the present invention.

[0022] Figure 6 It is a schematic diagram of the genotype detection rate results of the Masson pine whole genome chip in the sample according to the embodiment of the present invention.

[0023] Figure 7 It is a schematic diagram of the results of cluster analysis of the genotyping results of the Masson pine whole genome chip in the samples according to the embodiment of the present invention. DETAILED DESCRIPTION

[0024] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0025] For easier understanding, the terms in this document are explained and illustrated below. Those skilled in the art should understand that these explanations and illustrations should not be construed as limiting the scope of protection of the present invention.

[0026] In this article, the term "polymorphism" refers to genetic polymorphism, which is used to describe the diversity of species (such as humans) genomes, and it 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 allele states in the same population. Polymorphism relates to one of two or more variants of a specific DNA sequence. Single nucleotide polymorphism (SNP) is the modal type of polymorphism, which is a DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.

[0027] The liquid phase probe pinpoint sequencing technology (cGPS) liquid phase chip technology described in this article is based on the optimized thermodynamic stability algorithm model to design specific probes for target interval sequences, and uses synthetic specific probes to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic locations, and then constructs libraries and performs second-generation sequencing on the captured and enriched target intervals to obtain the genotype of the SNP / InDel site in the target area. The principle and process of cGPS liquid phase chip technology are as follows Figure 5 shown.

[0028] The first aspect of the present invention proposes a Masson pine 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 specifically shown in Table 1. The Masson pine SNP molecular markers proposed in the embodiment of the present invention are highly representative, highly polymorphic, and have good versatility, high coverage on the genome (average coverage 99.90%), and are evenly distributed, and are particularly suitable for applications such as chip preparation, molecular marker-assisted selection, directional improvement, important trait gene mining and identification, and functional analysis.

[0029] In some embodiments, the molecular markers used in the Masson pine whole genome chip of the present invention are as follows Figure 1 The screening process shown in the figure was obtained. Specifically, 1) Masson pine sample resequencing data, of which 84,209 initial sites were obtained through quality control; 2) transcriptome analysis of Masson pine resistance to pine wood nematode disease (disease resistance / susceptibility) was performed, and 25,119 gene function markers located on significantly differential genes were obtained; 3) 4,536 markers common to the chips of slash pine and loblolly pine. After integration and deduplication, a total of 113,709 SNP molecular markers were obtained.

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

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

[0032] Table 1

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

[0169] The second aspect of the present invention provides a whole genome chip of Masson pine, which comprises a probe or primer for detecting the combination of SNP molecular markers as described in any embodiment of the first aspect. The chip has the characteristics of high efficiency, low cost, good genetic stability, simple typing, high throughput, etc., and can output a large amount of data at one time, and can cover the detection of nearly a thousand materials at the same time. It is also suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, and has wide platform adaptability.

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

[0171] The fourth aspect of the present invention provides a probe for detecting the Masson pine SNP molecular marker combination as described in any embodiment of the first aspect, wherein the SNP molecular marker combination is 113,709 SNP molecular markers as shown in Table 1.

[0172] The fifth aspect of the present invention provides a use of the probe described in the fourth aspect of the present invention in preparing a Masson pine whole genome chip.

[0173] The sixth aspect of the present invention provides a genotyping method, comprising using the SNP molecular marker combination according to any embodiment of the first aspect, the chip according to any embodiment of the second aspect, the kit according to any embodiment of the third aspect, and / or the probe according to any embodiment of the fourth aspect to perform the genotyping on the sample. In a specific embodiment, the method comprises: (1) Extraction and quality control of genomic DNA: extracting sample DNA by magnetic bead method and performing quality detection on the DNA sample. The quality detection comprises determining the DNA concentration by Qubit fluorescence quantitative instrument and detecting the integrity of DNA by 1% agarose gel electrophoresis. Samples that pass the quality inspection are used for library preparation. (2) Construction and quality control of cGPS library: a. digesting the DNA sample with fragmentase, repairing the digestion end, adding A base to the 3' end, and detecting the fragment size by agarose gel electrophoresis. b. connecting the sequencing adapter and the DNA fragment using T4 ligase, and purifying the ligation product using magnetic beads. The concentration of the purified product is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis. c. Perform PCR amplification on the purified ligation product, and use magnetic beads to screen the fragments of the amplified product. The concentration of the product after fragment screening is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. d. Take 200ng of the constructed library, add probes and hybridization reagents, and incubate at 50°C for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to clean the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. Use Qubit fluorescence quantification instrument to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are qualified, the construction of cGPS sequencing library is completed. The prepared library is sequenced by high-throughput sequencing using BGI sequencer, and the sequencing strategy is PE150; (3) Data analysis: The raw data after high-throughput sequencing is subjected to quality control filtering and other processing, and the adapter fragments and low-quality reads are removed using FASTP software to obtain high-quality Clean Reads. The obtained Clean reads were compared with the reference genome using BWA software, and the positions were sorted to obtain the sample sorted bam file. The sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target sites. It should be noted that the method described in this embodiment is only an exemplary step and is not intended to limit the specific operation process of genotyping.

[0174] The seventh aspect of the present invention proposes the use of the SNP molecular marker combination according to any embodiment of the first aspect, the chip according to any embodiment of the second aspect, the kit according to any embodiment of the third aspect and / or the probe according to any embodiment of the fourth aspect in genetic breeding analysis.

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

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

[0177] Example 1: Design and preparation of Masson pine 100K whole genome chip

[0178] 1.1 Screening of SNPs for Masson pine whole genome array

[0179] 1.1.1 SNPs-whole genome background sites screened based on the results of resequencing of Masson pine samples

[0180] (1) Thirty diverse Masson pine individuals from seven provinces were selected for resequencing. The original sequencing data of the resequencing were quality controlled using fastp with default parameters, and then aligned to the reference genome of Pinus tabuliformis V1.0 (Niu S et al., 2022; https: / / doi.org / 10.1016 / j.cell.2021.12.006) using Sentieon for analysis to obtain the variation file of the resequencing data.

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

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

[0183] (4) Use plink software to perform linkage disequilibrium analysis on the initial loci set for the third step of screening. The screening conditions are: using the LD parameter R 2 ≥0.2 was filtered to remove highly linked sites.

[0184] (5) Based on the above conditions and in accordance with the principle of uniform distribution of sites on chromosomes, 84,209 SNP sites evenly distributed on Masson pine chromosomes were selected as whole-genome background sites.

[0185] 1.1.2 SNPs screened based on transcriptome analysis of Masson pine samples - functional loci for resistance to pine wood nematode disease

[0186] (1) Using an inoculation experiment, 33 Masson pine samples were divided into a high resistance group and a low resistance group according to their resistance levels. 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 differentially expressed genes were aligned to the reference genome of Pinus tabulaeformis, and the physical location intervals of the differentially expressed gene sequences aligned to the reference genome of Pinus tabulaeformis were obtained based on the sequence length alignment rate > 70% and sequence similarity > 90%.

[0188] (3) Based on the physical position intervals, interval loci were extracted from the initial loci 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 Masson pine.

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

[0190] (1) Ten samples of Masson pine were tested using the Pinus elliottii and Pinus taeda 51K chip (Diao S et al., 2024; https: / / doi.org / 10.1016 / j.indcrop.2024.118777) and probes with a 100% detection rate in the Masson pine samples were screened.

[0191] (2) The probe sequence was aligned to the Pinus tabulaeformis reference genome, and the physical position interval aligned to the Pinus tabulaeformis reference genome was obtained by screening based on sequence length alignment rate > 70% and sequence similarity > 90%.

[0192] (3) Based on the physical location intervals, interval site extraction was performed from the initial site set in 1.1.1 above, and a total of 4,536 SNPs were obtained.

[0193] 1.1.4 SNPs used in whole genome array of Masson pine

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

[0195] The average coverage of the Masson pine 100K whole genome chip loci on chromosomes is 99.90%, the average spacing is 214.5Kb, and the loci are evenly distributed on each chromosome. 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. Figure 4 shown.

[0196] 1.2 Preparation of liquid phase chip

[0197] The 113,709 molecular markers screened out were developed into a Masson pine 100K whole genome liquid phase chip through liquid phase probe precise positioning sequencing typing technology.

[0198] Example 2

[0199] In order to verify the genotyping effect of the Masson pine 100K whole genome chip, the Masson pine 100K whole genome chip designed in Example 1 was used to perform genotyping detection on 16 Masson pine samples.

[0200] 2.1 Extraction and quality control of genomic DNA

[0201] The magnetic bead method was used to extract DNA from 16 Masson pine samples, and the DNA samples were quality tested. The quality test included measuring the DNA concentration with a Qubit fluorescence quantifier and testing the integrity of the DNA with 1% agarose gel electrophoresis. The samples that passed the quality test were used for library preparation.

[0202] 2.2cGPS library construction and quality control

[0203] a. Use fragmentase to digest the DNA sample, repair the digested ends, add A base to the 3' end, and detect the fragment size by agarose gel electrophoresis.

[0204] 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 quantification instrument, and the fragment size is detected by agarose gel electrophoresis.

[0205] c. Perform PCR amplification on the purified ligation product, and use magnetic beads to screen the amplified product. The concentration of the product after fragment screening is detected by Qubit fluorescence quantitative instrument, and the fragment size is detected by agarose gel electrophoresis.

[0206] d. Take 200 ng of the constructed library, add probes and hybridization reagents, and incubate at 50°C for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to wash the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification.

[0207] e. The library concentration was detected by Qubit fluorescence quantification instrument, and the fragment size was detected by agarose gel electrophoresis. Once the concentration and fragment size were qualified, the construction of cGPS sequencing library was completed.

[0208] f. The prepared library was sequenced using a BGI sequencer with a high-throughput sequencing strategy of PE150;

[0209] 2.3 Data Analysis

[0210] The raw data after high-throughput sequencing was processed by quality control filtering, and the adapter fragments and low-quality reads were removed using FASTP software to obtain high-quality Clean Reads. The obtained Cleanreads were aligned with the reference genome using BWA software, and the positions were sorted to obtain the bam file after the sample sorting. The sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target sites.

[0211] After sequencing and data analysis, the detection rates of 16 Masson pine samples ranged from 97.28% to 99.16%, with an average detection rate of 98.82%. Figure 6 The heterozygosity rate ranged from 29.28% to 33.06%, with an average heterozygosity rate of 32.07%. The above data show that the Masson pine 100K whole genome chip can achieve genotyping of Masson pine samples, with a high detection rate of target sites.

[0212] Example 3 Application of Masson pine 100K whole genome chip in cluster analysis

[0213] (1) The Masson pine 100K whole genome chip prepared in Example 1 was used to perform genotyping on 33 Masson pine samples. The specific operation process is shown in Example 2.

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

[0215] The results are as follows Figure 7As shown in the figure, it can be seen that the 33 Masson pine samples are mainly divided into 2 subgroups, which is consistent with the known results. The results show that the Masson pine 100K whole genome chip of the present invention can effectively distinguish Masson pine samples from different sources and can perform cluster analysis and identification on Masson pine.

[0216] Example 4 Application of the Masson Pine 100K Whole Genome Chip in the Differential Analysis of Masson Pine Samples

[0217] The 12 Masson pine samples were genotyped using the Masson pine 100K whole genome chip prepared in Example 1. The specific operation process is shown in Example 2. The 12 samples came from 6 families, and 2 samples with indistinguishable phenotypes were selected from each family. Based on the genotype data of the 12 materials, the differences in samples within the family were analyzed.

[0218] The results are shown in Table 2. The genotype consistency rates of the samples in the 6 pairs of families were between 65.3% and 74.36%, and the average consistency rate was 71.47%. The results show that the Masson pine 100K whole genome chip of the present invention can accurately and effectively distinguish different samples with similar phenotypes in a family, and realize the difference analysis of its samples at the genotype level.

[0219] Table 2 Statistics of genotype consistency rate of samples within the family

[0220]

[0221] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0222] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Masson pine SNP molecular marker combination, characterized in that: The SNP molecular marker combination consists of 113,709 SNP molecular markers as shown in Table 1, wherein the physical positions of the SNP molecular markers are determined by sequence alignment based on the reference genome of Pinus tabuliformis V1.

0.

2. A whole genome chip of Masson pine, characterized in that: The chip comprises probes or primers for detecting the SNP molecular marker combination as claimed in claim 1.

3. A kit, characterized in that: The kit comprises probes or primers for detecting the SNP molecular marker combination as described in claim 1 or 2.

4. A probe for detecting the Masson pine SNP molecular marker combination as claimed in claim 1, characterized in that: The SNP molecular marker combination is 113,709 SNP molecular markers as shown in Table 1.

5. Use of the probe according to claim 4 in preparing a Masson pine whole genome chip.

6. A genotyping method, characterized in that: The method comprises performing the genotyping on the sample using the SNP molecular marker combination according to claim 1, the chip according to claim 2, the kit according to claim 3 and / or the probe according to claim 4.

7. Use of the SNP molecular marker combination according to claim 1, the chip according to claim 2, the kit according to claim 3 and / or the probe according to claim 4 in genetic breeding analysis.

8. The use according to claim 7, characterized in that: The genetic breeding analysis includes germplasm resource identification analysis, purity identification analysis, genetic background analysis of breeding materials, gene positioning analysis, whole genome association analysis, whole genome selection breeding analysis and intelligent design breeding analysis.

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