Poplar whole genome SNP (Single Nucleotide Polymorphism) molecular marker combination, liquid phase chip prepared from same and application of liquid phase chip

Abstract

The invention provides a poplar whole genome SNP molecular marker combination, a liquid phase chip prepared from the poplar whole genome SNP molecular marker combination and application of the poplar whole genome SNP molecular marker combination, the whole genome liquid phase chip comprises a probe used for detecting the poplar SNP molecular marker combination, and the SNP molecular marker combination comprises 6549 SNP molecular markers. The physical positions of the 6549 SNP loci are determined based on comparison of a populus simonii haplotype A reference genome sequence, and the specific SNP molecular marker condition is shown in the specification table 1. According to the invention, the probe formed by using a few SNP marker combinations can efficiently and accurately distinguish five branch poplar varieties, the detection cost is reduced, the detection time is shortened, and the SNP marker combinations can provide theoretical and data support for the identification of new poplar varieties, group division and other work, and have important theoretical value and application significance.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to a whole genome SNP molecular marker combination of poplar, its preparation in liquid phase chip and its application. Background Technology

[0002] Poplar is a general term for plants in the genus *Populus* of the family Salicaceae. The genus is generally divided into five major groups: White Poplar (Sophora Populus), Black Poplar (Aigeiros), Common Poplar (Tacamahaca), Large-leaved Poplar (Leucoides), and Eurasian Poplar (Turanga). Poplars are characterized by rapid growth, strong adaptability, and easy propagation, making them one of the world's major afforestation species and playing a vital role in forestry production and ecological protection forest system construction. In my country, there are over 60 species of poplars, which can be planted in three-quarters of the country's land area. China has the world's largest number of planted poplar forests, providing approximately 30% of the country's timber annually and contributing significantly to the economy and society.

[0003] Due to the vast variety of poplar species and relatively outdated breeding methods, existing microarray technology suffers from low versatility and utilization, resulting in low breeding efficiency. Currently, poplar population classification primarily relies on morphological indicators. However, morphological indicator collection is time-consuming and easily affected by environmental factors. With the continuous increase in poplar varieties, the differences in morphological indicators between varieties are diminishing, increasing the difficulty of distinguishing poplar varieties using morphological indicators. The increasing import of germplasm resources from abroad also places higher demands on population classification.

[0004] Currently, molecular markers such as SSR and InDel are used for germplasm resource screening, variety identification, and variety protection. However, their experimental procedures are complex, and only a few dozen molecules can be detected from a single poplar material. SNP markers, on the other hand, are evenly distributed across the genome and exhibit high polymorphism. Liquid-phase probe chips based on SNP markers can simultaneously capture thousands of markers, offering high automation and throughput, which is more conducive to establishing poplar population classification systems. Chinese patent CN118895377B discloses a combination of poplar SNP molecular markers and its preparation of a whole-genome liquid-phase chip, containing 60,944 SNP molecular markers. However, due to the large number of SNP molecular markers, the preparation of the liquid-phase chip is costly and time-consuming. Therefore, there is an urgent need for a method that ensures high accuracy while reducing detection costs and time. Summary of the Invention

[0005] In view of this, the present invention provides a combination of SNP molecular markers for the whole genome of poplar, a liquid phase chip prepared therefrom, and its application. The combination of molecular markers consists of only 6549 molecules, but it can still accurately classify poplar cultivars, achieving the effect of low detection cost and short detection time.

[0006] One of the objectives of this invention is to provide a whole-genome liquid-phase chip, which includes probes for detecting combinations of SNP molecular markers in poplar trees. The combinations of SNP molecular markers contain 6549 SNP molecular markers. The physical locations of the 6549 SNP sites are determined based on the comparison of the reference genome sequence of poplar haplotype A. The specific details of the SNP molecular markers are shown in Table 1 of the specification.

[0007] The second objective of this invention is to provide a kit comprising the aforementioned whole-genome liquid-phase chip.

[0008] The third objective of this invention is to provide the application of the above-mentioned whole genome liquid phase chip or the above-mentioned kit in the detection of poplar variety population classification.

[0009] Furthermore, the poplar cultivar group includes black poplar, large-leaf poplar, green poplar, white poplar, and desert poplar.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned whole genome liquid phase chip or the above-mentioned kit in poplar breeding.

[0011] Furthermore, the application includes using the aforementioned whole-genome liquid phase chip or the aforementioned kit to detect the DNA of the poplar trees to be tested, and after determining the poplar population, selecting poplar trees for subsequent breeding.

[0012] The fifth objective of this invention is to provide the application of the above-mentioned whole-genome liquid chip or the above-mentioned kit in any of the following:

[0013] Poplar genome-wide association analysis;

[0014] Poplar germplasm resource identification and variety authenticity identification;

[0015] Poplar cluster analysis and phylogenetic identification;

[0016] Poplar genetic mapping and gene localization;

[0017] Identification of genetic diversity in poplar trees.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention screened 6549 SNP marker combinations, capable of distinguishing five poplar varieties. The liquid-phase probe prepared using these SNP marker combinations exhibits high detection throughput and can rapidly identify the population divisions of the five poplar varieties. Specifically, the capture efficiency was 94.93-95.21% for the black poplar variety, 85.52-93.67% for the white poplar variety, 94.8-99.92% for the green poplar variety, 92.04-97.71% for the large-leaved poplar variety, and 95.11-97.14% for the euphratica variety; all capture efficiencies were high and fully met the detection requirements.

[0020] Furthermore, this SNP marker combination can provide theoretical and data support for the identification of new poplar varieties and population division. At the same time, since there are relatively few SNP markers, it can greatly reduce detection costs and time, and has important theoretical value and application significance. Attached Figure Description

[0021] Figure 1 This is a distribution map of poplar SNP markers in Embodiment 1 of the present invention.

[0022] Figure 2 The results show the capture efficiency of different poplar trees in Example 3 of the present invention.

[0023] Figure 3 This is the phylogenetic tree analysis diagram in Embodiment 3 of the present invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0025] Example 1

[0026] This embodiment provides a screening process for poplar SNP marker combinations and a method for designing liquid phase probes, specifically including the following process:

[0027] (1) Collected 207 natural germplasm samples of Populus simonii from 10 provinces including Inner Mongolia, Ningxia, Heilongjiang, Jilin, Liaoning, Shanxi, Shaanxi, Shandong, Hebei and Qinghai.

[0028] (2) Genomic DNA was extracted from 207 poplar leaf tissues using the magnetic bead method and whole-genome resequencing was performed, yielding 3.048.175 Gb of data.

[0029] (3) The clean data obtained from sequencing in step (2) was aligned to the reference genome of Populus simonii haplotype A using bwa, with a size of 412.23 Mb.

[0030] (4) Use samtools and picard to convert file formats and remove redundancy to generate bam files. Based on the bam files, GATK generates whole genome SNP variant vcf files for 207 materials, totaling 297,538,998 SNP sites.

[0031] (5) The SNP sites selected in step (3) were filtered out, and those with a sequencing depth of less than 3, allele variation of more than 2, sample missing rate of more than 20%, and allele frequency of less than 0.05 were removed, resulting in a total of 1,753,369 SNP sites. The software and parameters were: "vcftools minDP 3min alleles 2max alleles 2max missing 0.8maf 0.05".

[0032] (6) The SNP sites selected in step (4) were further filtered by extracting 200bp sequences upstream and downstream of the sites using bcftools. The filtering criteria were: single-copy sequences, sequences without insertions or deletions of more than 5bp, sequences with a GC content between 40% and 60%, and sequences without SSRs. A total of 654,253 SNP sites were obtained.

[0033] (7) Using a Perl script, SNP marker combinations were screened according to the principle of uniform chromosome distribution, ultimately obtaining 6549 SNP marker combinations. Marker details are shown in Table 1, and the SNP marker distribution map is shown in […]. Figure 1 .

[0034] (8) Combine the 6549 SNP markers obtained in step (6), select 100bp sequences upstream and downstream of the site to design liquid phase probes for subsequent capture sequencing.

[0035] Table 1. Details of 6549 SNP markers identified through whole-genome screening of poplar.

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Due to confidentiality requirements and database review criteria, the reference genome "Populus simonii haplotype A reference genome" in this application, although assigned the number (SAMC4027246), cannot be publicly disclosed at this time and will not be released until October 2026. Therefore, this application has corrected the data for the publicly disclosed reference gene "GCA_015852605.2". Each data point in Table 1 is followed by a parenthetical data point, which represents the SNP locus data obtained after comparison with the reference gene "GCA_015852605.2". The specific correction steps are as follows:

[0088] Analysis Procedure: For the Populus simonii genome, 6549 100bp sequences upstream and downstream of the markers were extracted using bedtools (v2.30.0), totaling 201bp sequences. The 201bp marked sequences were aligned to the “GCA_015852605.2” genome using blastn (v2.11.0). The chromosome position and location at the last alignment position were used as the corrected position on the “GCA_015852605.2” genome. Alignment parameters: -max_target_seqs 30 -evalue 1e-4 -word_size 30. A few sites showed NA (Not Associated) results due to genetic differences between the two materials used in the genome assembly, which is normal.

[0089] Example 2

[0090] This embodiment provides a workflow for using a liquid-phase probe designed to detect poplar trees based on the 6549 SNP markers obtained in Example 1, as detailed below:

[0091] (1) Genomic DNA extraction

[0092] Poplar leaves were collected, preserved with ice packs, and transported back to the laboratory. Total DNA was extracted from the poplar using the magnetic bead method. Poplar material samples with a quantification value greater than 200 ng were then analyzed by liquid chromatography-particle dual chromatography.

[0093] (2) Construction of whole genome library: fragmentation and end repair of poplar genomic DNA, ligation of adapters and indexing of samples, library amplification and purification. Magnetic beads were used to screen DNA libraries in the 350-500 bp range for hybridization capture.

[0094] (3) Hybridization capture: The constructed DNA library was hybridized with probes prepared by combining poplar SNP markers and incubated overnight.

[0095] (4) Capture of the target region DNA library: Adsorption magnetic beads containing streptomycin are used to adsorb the DNA fragment library that hybridizes with the liquid phase probe. The adsorbed DNA fragment library is amplified by PCR, and the total amount of DNA fragment library is finally above 200 ng.

[0096] (5) High-throughput sequencing: The DNA library obtained after capture and amplification was sequenced using the DNBSEQ-T7 sequencing platform to obtain the sequencing results of poplar genomic DNA, and the obtained data was subjected to basic filtering.

[0097] (6) Evaluation of liquid phase probe capture rate

[0098] 1. The cleaned sequencing data were compared with the reference genome sequence of haplotype A of Populus simonii using bwa software, and the SNP genotyping data of poplar varieties were obtained using GATK software;

[0099] 2. Use SNP genotyping data to evaluate the capture efficiency of different poplar materials.

[0100] 3. Analyze population structure using SNP genotyping data, generate phylogenetic tree results using TreeBest software, and generate principal component analysis results using Plink software.

[0101] Example 3

[0102] This embodiment further verifies the detection effect of the liquid phase probe designed with 6549 SNP markers obtained in Example 1, based on the method provided in Example 2, as follows:

[0103] 1. Capture efficiency verification

[0104] Material Collection: Four samples of black poplar, four samples of white poplar, 22 samples of green poplar (2 large green poplar, 3 green poplar, and 17 small-leaved poplar), four samples of large-leaved poplar, and four samples of desert poplar were collected, totaling 38 poplar samples. Testing was conducted according to the procedure in Example 2, and the results are shown in Table 2. Figure 2 .

[0105] Table 2 shows the information of the 38 selected samples and their capture efficiency.

[0106]

[0107]

[0108] Depend on Figure 2 Table 2 shows that the capture efficiency of the black poplar species is 94.93-95.21%, that of the white poplar species is 85.52-93.67%, that of the green poplar species is 94.8-99.92%, that of the large-leaved poplar species is 92.04-97.71%, and that of the euphratica species is 95.11-97.14%. Among them, the small-leaved poplar of the green poplar species has the highest capture efficiency, and the capture efficiency of other poplar species is also relatively high, which can meet the detection requirements.

[0109] 2. Population structure analysis verification

[0110] Material Collection: A total of 222 poplar samples were collected, including 54 samples of black poplar, 51 samples of white poplar, 59 samples of green poplar, 30 samples of large-leaved poplar, and 28 samples of desert poplar. Testing was conducted according to the procedure in Example 2. Details of the 222 poplar samples are shown in Table 3, and the test results are as follows. Figure 3 .

[0111] Table 3. Source information of 222 selected poplar germplasm accessions

[0112]

[0113]

[0114]

[0115] Depend on Figure 3 It can be seen that the poplar phylogenetic tree and principal component analysis results based on 6549 SNPs show that the five poplar lineages can be significantly separated.

[0116] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0117] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A whole-genome liquid-phase chip, characterized in that, The whole-genome liquid-phase chip includes probes for detecting SNP molecular marker combinations in poplar trees. The SNP molecular marker combinations contain 6,549 SNP molecular markers. The physical locations of the 6,549 SNP sites are determined based on the comparison of the reference genome sequence of poplar haplotype A. The specific SNP molecular marker information is shown in Table 1 of the instruction manual.

2. A reagent kit, characterized in that, The kit includes the whole genome liquid phase chip of claim 1.

3. The application of the whole genome liquid phase chip of claim 1 or the kit of claim 2 in the detection of poplar variety population classification.

4. The application according to claim 3, characterized in that, The poplar cultivar group includes black poplar, large-leaf poplar, green poplar, white poplar, and desert poplar.

5. The application of the whole genome liquid phase chip of claim 1 or the kit of claim 2 in poplar breeding.

6. The application according to claim 5, characterized in that, The application includes using the whole genome liquid phase chip of claim 1 or the kit of claim 2 to detect the DNA of the poplar trees to be tested, and after determining the poplar population, selecting poplar trees for subsequent breeding.

7. The use of the whole genome liquid array of claim 1 or the kit of claim 2 in any of the following: Poplar genome-wide association analysis; Poplar germplasm resource identification and variety authenticity identification; Poplar cluster analysis and phylogenetic identification; Poplar genetic mapping and gene localization; Identification of genetic diversity in poplar trees.

Images