SNP (Single Nucleotide Polymorphism) site combination for grassiness genotyping, whole genome liquid phase chip and application of SNP site combination and whole genome liquid phase chip
By developing the Xiangcao full genome liquid phase chip, including 11,319 SNP sites, the problem of lack of high-density and low-cost genotyping tools in Xiangcao breeding is solved, and efficient genotyping and breeding tools are achieved, supporting application scenarios of multiple sample volumes.
Patent Information
- Application Number
- CN202510801371.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-08-12
AI Technical Summary
The lack of high-density and low-cost genotyping tools in the breeding of elephant grasses results in low breeding selection efficiency, unclear goals, and targeted capture sequencing technology has not yet been applied in elephant grasses.
A whole genome liquid phase chip for Elephant Grass is developed, containing 11,319 SNP sites, and a probe for Elephant Grass reference genome is designed to efficiently detect these sites and support the genotyping needs of multiple sample volumes.
It provides an efficient genotyping tool for genetic diversity analysis, variety identification, gene localization and molecular marker-assisted breeding of sylla grass, which improves breeding selection efficiency, reduces costs and supports flexible application scenario requirements.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of gene chips, in particular to a SNP site combination for elephant grass genotyping, a whole genome liquid phase chip and applications thereof. Background Art
[0002] Elephant grass (Pennisetum Schumach.) is a C4 plant of the genus Pennisetum in the Poaceae family, native to tropical and subtropical regions. This plant has both forage and industrial value, characterized by rapid growth, high yield, and excellent nutritional value, making it a high-quality green fodder and silage. In recent years, with the advancement of sequencing technology, denove sequencing has been completed for elephant grass, and some resequencing data have been published, providing a wealth of genetic variation information. However, this genetic variation information is difficult to directly apply to practical elephant grass breeding. Currently, there are many varieties of Pennisetum, some of which are morphologically similar. This, coupled with asexual reproduction and human influence, has led to a confusion of germplasm materials and irregular nomenclature. Although SSR and SRAP markers have been developed for elephant grass germplasm identification, the lack of basic research on elephant grass and the limited number of markers have resulted in low selection efficiency and unclear targets, limiting their application in elephant grass breeding. Therefore, there is an urgent need for new genotyping technologies to improve selection efficiency in elephant grass breeding and promote the development of molecular breeding for elephant grass.
[0003] Single nucleotide polymorphisms (SNPs) are considered one of the most ideal genetic markers due to their widespread distribution across the genome, their large number, and their ease of screening and genotyping. They are therefore of great significance in biological research. Currently, SNP markers have become essential tools for population identification, genetic structure analysis, gene mapping, and genomic selection. With the advancement of high-throughput sequencing technology, SNP genotyping based on whole-genome sequencing has become mainstream for species with available reference genomes. However, this technology places high demands on data management and analysis, and is relatively expensive, a particularly significant issue in species such as elephant grass. Targeted capture sequencing offers a more cost-effective alternative. This technology involves designing probes targeting known sequences or developing microarrays. Randomly fragmented genomic DNA fragments are then mixed with the microarray to capture DNA fragments containing the target locus, which are then amplified, purified, and sequenced at high throughput. This approach reduces library complexity, enabling deep sequencing of a large number of target loci. This not only simplifies data analysis and management, but also significantly reduces the cost of genotyping. However, to date, this technology has not been applied in elephant grass.
[0004] Liquid-phase capture chip technology uses DNA capture for sequencing, enabling repeated sequencing of target sites, significantly improving data accuracy. This technology is also low-cost, and the density of the SNP chip can be flexibly adjusted according to demand, allowing for the addition of new sites or technical upgrades at any time. Furthermore, in the future, different chip types could be designed and combined to achieve efficient detection based on different application scenarios and specific needs. However, this technology has not yet been applied to elephant grass. Once a specialized chip for elephant grass is developed, it is expected to promote the development of the elephant grass breeding industry and provide important support for related research and applications. Summary of the Invention
[0005] The present invention aims to provide a SNP locus combination, a whole-genome liquid phase chip, and its application for elephant grass genotyping, to address the aforementioned problems of the prior art. The present whole-genome liquid phase chip contains 11,319 SNP loci and can simultaneously detect all 11,319 loci with high numbers, density, and efficiency. It can be applied to basic research and molecular breeding of elephant grass, addressing the technical problem of the lack of high-density, low-cost genotyping tools in molecular breeding of tropical forage grasses.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The present invention provides a SNP site combination for elephant grass genotyping, wherein the SNP site combination is 11319 SNP sites located on the elephant grass reference genome version GCA_028749085.1, and the position information of the SNP sites is shown in Table 1.
[0008] The present invention also provides a liquid phase chip of the whole genome of elephant grass, wherein the genotyping sites of the liquid phase chip include the SNP site combination.
[0009] Optionally, it also includes a probe designed based on the gene sequence covering the SNP site.
[0010] Optionally, the GC content of the probe is between 30% and 70%, and the number of homology regions of the probe sequence in the whole genome is ≤5.
[0011] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of elephant grass in the genetic diversity analysis of elephant grass.
[0012] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of the elephant grass in the analysis of the elephant grass population structure.
[0013] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of the elephant grass in identifying the kinship of the elephant grass.
[0014] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of the elephant grass in the improvement and protection of the elephant grass germplasm resources.
[0015] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of elephant grass in background selection based on molecular markers of elephant grass.
[0016] The present invention also provides an application of the SNP site combination or the whole-genome liquid phase chip of elephant grass in molecular marker-assisted breeding of elephant grass.
[0017] The present invention discloses the following technical effects:
[0018] The present invention provides a whole-genome liquid phase chip of elephant grass, which contains 11,319 SNP sites located on the elephant grass reference genome version GCA_028749085.1. The chip can simultaneously detect all 11,319 sites with a high number, density and efficiency, and can be applied to basic research and molecular breeding of elephant grass.
[0019] The whole-genome liquid phase chip of elephant grass provided by the present invention provides an efficient genome typing tool for genetic diversity analysis, variety identification, gene positioning, background screening and molecular marker-assisted breeding of elephant grass; at the same time, the present invention can design new probes according to new sites at any time, and has no high requirements for sample volume, so as to meet the different usage scenarios and usage needs of users, and has high application value in elephant grass molecular breeding.
[0020] The invention solves the technical problem of lack of high-density and low-cost genotyping tools in tropical forage molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0022] Figure 1 The chromosome distribution map of SNP markers was drawn based on 11,319 SNP loci;
[0023] Figure 2 The figure shows the distribution of SNP numbers on each chromosome based on 11,319 SNP loci.
[0024] Figure 3 To draw MAF distribution statistics based on 11,319 SNP sites;
[0025] Figure 4 To draw a statistical map of SNP marker types based on 11,319 SNP loci;
[0026] Figure 5 This is a phylogenetic tree of 17 elephant grass materials constructed based on the unweighted group average method (UPGMA);
[0027] Figure 6 This is a quantitative map of allele frequencies (MAF) of 17 elephant grass materials;
[0028] Figure 7 The Nei's SNP number map of genetic diversity of 17 elephant grass materials;
[0029] Figure 8 The figure shows the distribution of the number of polymorphic information content (PIC) SNPs of 17 elephant grass species;
[0030] Figure 9 This is a phylogenetic tree diagram of the population structure analysis of the genotypes of 450 elephant grass samples using the liquid phase chip in Example 2 of the present invention. DETAILED DESCRIPTION
[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.
[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0036] Example 1 Screening of SNP site combinations in the whole genome of elephant grass
[0037] This example uses whole-genome resequencing data from 450 samples of elephant grass to perform alignment analysis based on the purple elephant grass reference genome CpPurple, detecting a series of single-nucleotide polymorphisms (SNPs) marker combinations (SNPs dataset). After rigorous quality control and screening, a high-quality, non-redundant SNPs dataset of 11,319 was obtained. The specific steps are as follows:
[0038] (1) Based on 450 resequencing data of elephant grass uploaded to The European Nucleotide Archive (ENA), high-quality clean data were obtained. The clean data were aligned to the reference genome of purple elephant grass (CpPurple) (version number: GCA_028749085.1) using the bwa tool, and variant detection was performed using GATK. After strict SNP filtering parameters (QD < 2.0, QUAL < 30.0, SOR > 3.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, ReadPosRankSum < -8.0), 1,039,605 high-quality SNP sites were finally obtained.
[0039] (2) To further screen SNP sites suitable for liquid phase array development, a Perl script was used to screen the above SNP sites in combination with the following principles: ensure that SNP markers are evenly distributed on the chromosome, and retain one marker every 4 kb; use bedtools software to extract the SNP site and its upstream and downstream 100 bp sequence, and screen out SNP sites with a GC content between 35% and 65% and no N bases in the sequence; use blastn software to align the extracted sequences with the purple elephant grass reference genome CpPurple, and only retain single-copy SNP sites. In addition, SNP sites that meet the following conditions were further screened: maximum deletion rate ≤ 0.5, minor allele frequency (MAF) ≥ 0.05, linkage disequilibrium (LD) ≤ 0.2, and final screening was performed according to the principle of retaining one marker every 40 kb. After the above multi-step screening, 11,319 high-quality SNP marker combinations were finally obtained, and their detailed information is shown in Table 1.
[0040] Table 1
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[0101] The chromosome distribution map of SNP markers based on the 11,319 SNP sites obtained by screening is shown in the figure below. Figure 1 The distribution of the number of SNPs on each chromosome is shown in Figure 2 As shown, the MAF distribution statistics are drawn as follows Figure 3 As shown, the statistical diagram of SNP marker types is as follows Figure 4 shown.
[0102] The SNP marker combination obtained by the above screening can be used for population structure analysis and genotyping of elephant grass.
[0103] Example 2: Use of SNP site combinations from the whole genome of elephant grass to prepare liquid phase chips
[0104] For the 11,319 SNP markers screened in Example 1, liquid phase probes were designed according to conventional liquid phase chip design principles. A 110 bp sequence that can cover the SNP site region was selected. The probe length was designed to be 110 bp, the GC content of the probe was between 30% and 70%, and the number of homology regions of the probe sequence across the entire genome was ≤5.
[0105] The liquid phase probe containing the above-mentioned 11,319 SNP marker combinations was developed according to the conventional design principles of liquid phase chips to obtain the Elephant Grass 10K liquid phase chip based on targeted capture sequencing.
[0106] Example 3 Application of the Elephant Grass 10K Liquid Phase Chip in the Detection of Elephant Grass DNA Samples
[0107] 1. Extraction of genomic DNA from elephant grass
[0108] Fresh leaves were placed in 2.0 mL Eppendorf centrifuge tubes. One steel ball (5 mm in diameter) was placed in each tube. 1000 μL of 2% CTAB extraction buffer was then added. The leaves were disrupted in a tissue disruptor (model: Retsch MM400, Germany) at a frequency of 30 times / second for 1 min. Incubate at 65°C for 1 hour, add 500 μL of 24:1 chloroform / isoamyl alcohol extract after cooling, shake up and down 30 times, let stand to separate, centrifuge (12000 r / min) for 10 minutes, aspirate 400 μL of supernatant into a new 1.5 mL centrifuge tube, then add 400 μL of isopropanol to precipitate DNA, mix up and down, centrifuge (12000 r / min) for 5 minutes, discard the supernatant, and then add 750 μL of 70% ethanol to wash the DNA precipitate, centrifuge (12000 r / min) for 2 minutes, discard the supernatant, dry the DNA at room temperature, add 100 μL of ddH2O to dissolve the DNA, and place in a -20°C refrigerator for use.
[0109] 2. DNA sample quality testing
[0110] Use 1%-1.5% agarose gel electrophoresis for detection, and use a gel imaging system to evaluate the electrophoresis results to ensure genomic integrity. Measure the genomic DNA concentration using a micro-volume UV spectrophotometer (Q5000, Quawell, USA) or a similar nucleic acid protein analyzer and adjust the DNA concentration to a working concentration of 10-50 ng / μL.
[0111] 3. Elephant Grass Liquid Chip Detection
[0112] 1) The DNA sample is sheared by ultrasound or enzyme digestion, end-repaired and 3'-end "A" added, adapter-ligated and purified, and pre-PCR library amplified to obtain the library required for hybridization capture.
[0113] 2) Capture method: Hybridization capture was performed according to "NGPHyb&WashKit v2.0". After library and probe hybridization, probe and magnetic bead binding, non-specific binding library washing, post-capture PCR amplification, library quantification and quality control, sequencing was performed.
[0114] Take 1 μL of library and use NGP Qubit dsDNA HS Assay Kit reagent to detect the library concentration on Qubit 4.0 Fluorometer, and sequence it using a high-throughput sequencing platform.
[0115] 4. Data Analysis
[0116] Raw image data generated by high-throughput sequencing (such as the Illumina platform and MGI platform) is converted into Fastq format raw sequencing data (called raw data) through a specific program. Fastq files contain sequence information and sequencing quality information. The data is organized into four lines, and the information in each line is described as follows:
[0117] 1) Starts with "@", followed by sequence identifiers and description text (optional part);
[0118] 2) base sequence;
[0119] 3) Starts with "+", followed by a sequencing identifier (optional part);
[0120] 4) Sequencing quality corresponding to the base sequence;
[0121] After obtaining the raw data, bioinformatics analysis is performed with a reference genome (hg19, hg38, or others). The analysis steps mainly include the following:
[0122] 1) Quality control: Remove low-quality sequences, adapter sequences, ployG and other abnormal sequences from the original sequencing data to obtain clean data;
[0123] 2) Alignment: Align, sort, and remove duplicates of the clean data with the reference genome to obtain a bam file;
[0124] 3) Statistics: SNPs are detected using statistical standard procedures based on indicators such as alignment rate, coverage, and uniformity to obtain SNP data for each site in each sample.
[0125] The 10K liquid phase chip of elephant grass prepared in this example was used to detect 17 samples. All samples were from the Grassland Science and Technology College of Sichuan Agricultural University. One replicate was taken for each of giant fungus grass and Reyan No. 4. The test results are shown in Table 2.
[0126] Table 2 Detection results of 17 test samples
[0127] Sample name Total number of sites Number of Snp deletion sites Number of Snp detection sites Snp detection rate Chuanyu No. 1 11319 353 11133 96.93％ Giant Fungus Grass-1 11319 302 11184 97.37％ Giant Fungus Grass-2 11319 299 11187 97.40％ SA03 11319 236 11250 97.95％ Dwarf elephant grass 11319 391 11095 96.60％ Bond No. 1 11319 385 11101 96.65％ Xucao No. 2 11319 247 11239 97.85％ Sichuan Livestock No. 4 11319 222 11264 98.07％ Dehong elephant grass 11319 276 11210 97.60％ Gui Minyin 11319 271 11215 97.64％ Guimu No. 1 11319 223 11263 98.06％ South China elephant grass 11319 276 11210 97.60％ Reyan No. 4-1 11319 278 11208 97.58％ Reyan No. 4-2 11319 256 11230 97.77％ Minmu No. 6 11319 277 11209 97.59％ Su Mu No. 2 11319 235 11251 97.95％ Purple elephant grass 11319 40 11446 99.65％
[0128] Among the 17 test samples in Table 2, the detection rates ranged from 96.6% to 99.65%, with an average detection rate of 97.31%, indicating that the detection accuracy of the Elephant Grass 10K liquid phase chip was high.
[0129] Example 4 Application of the 10K Liquid Chip of Elephant Grass in the Analysis of Elephant Grass Genetic Diversity
[0130] Using the SNP data generated by the 10K liquid phase microarray of the elephant grass, phylogenetic analysis was performed on the 17 elephant grass materials listed in Table 2. The phylogenetic tree constructed based on the unweighted group average method (UPGMA) shows the following: Figure 5 All accessions can be divided into two major clades, with genetic distances ranging from 0.0018 to 0.1301. The first clade (red) includes accessions "Jujuncao-1," "Jujuncao-2," "Reyan 4-1," and "Reyan 4-2," which have highly similar genetic backgrounds (genetic distance <0.03). The use of replicate samples demonstrates that this liquid chromatography array can yield highly accurate results. The second clade (blue) encompasses accessions such as "Guiminyin," "Huanan Xiangcao," and "Chuanyu 2," demonstrating a high degree of internal differentiation. Among them, "Jujuncao," "Xucao 2," "Chuanxu 4," and "SA03" are new elephant grass lines. Notably, accessions "SA03," "Xucao 2," "Chuanxu 4," and "Guimu 1" cluster together in a subgroup, suggesting possible genetic differentiation due to introgression or selective pressure. This study provides important insights for the utilization of elephant grass genetic resources and recommends selecting accessions from different clades as hybrid parents to broaden the genetic base.
[0131] The present invention used 10K liquid phase microarray to analyze the genetic diversity of 17 individuals of elephant grass. The results showed that:
[0132] (1) Allele frequency (MAF): There are 304 sites with high major allele frequency, which accounts for the highest proportion. This indicates that there are some high-frequency dominant alleles in this population, which may be closely related to artificial selection or domestication process (see Figure 6 ).
[0133] (2) Nei's genetic diversity: The genetic diversity index ranged from 0.24 to 0.75, with 260 loci showing a diversity range of 0.34 to 0.44, reflecting a moderate level of overall genetic diversity. It is noteworthy that 147 loci had a genetic diversity index higher than 0.5, indicating that these loci had a high degree of genetic variation and could serve as potential targets for the development of molecular markers (see Figure 7 ).
[0134] (3) Polymorphic Information Content (PIC): The PIC value distribution results show that 441 sites have a PIC value greater than 0.3, which is a high proportion and belongs to highly polymorphic sites, suitable for genetic map construction and association analysis. At the same time, the PIC values of 276 sites are low (≤0.2), which suggests that these sites may be affected by selection pressure or functional constraints (see Figure 8 ).
[0135] Example 5 Application of the Elephant Grass 10K Liquid Phase Chip in Elephant Grass Population Structure Analysis
[0136] Population structure analysis is an important method for studying the genetic structure of a population and the genetic relationships between individuals. By analyzing genotype data, this method can divide individuals into different subpopulations or populations and assess the genetic similarities and differences between individuals. Common population structure analysis methods include cluster analysis, which uses clustering algorithms to group individuals, thereby revealing genetic differences and population structure characteristics.
[0137] In the present invention, the 10K liquid microarray prepared in Example 2 was used to obtain genotype data for 450 elephant grass samples. These samples came from a wide range of sources, including 62 from the International Livestock Research Institute (ILRI) gene bank, 131 from the Brazilian Agricultural Research Corporation (Embrapa), 23 from the United States Department of Agriculture (USDA), 6 from China, and 118 from the Kenya Agriculture and Livestock Research Organization (KALRO). In addition, 109 elephant grass hybrids and one purple elephant grass strain were included for reference genome analysis.
[0138] like Figure 9 As shown, by clustering and labeling individuals from different geographical origins, the phylogenetic tree results showed that there were significant genetic differences between groups from different geographical origins.
[0139] In summary, the 10K liquid phase microarray prepared in Example 2 successfully analyzed the population structure of the 450 samples of elephant grass. The analysis results showed that the elephant grass population could be roughly divided into four subpopulations, providing important evidence for further research on its genetic diversity and evolutionary history.
[0140] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A SNP locus combination for elephant grass genotyping, characterized in that: The SNP site combination is 11,319 SNP sites located on the elephant grass reference genome version GCA_028749085.
1. The position information of the SNP sites is shown in the following table:
2. A whole genome liquid phase chip of elephant grass, characterized in that: The liquid chip genotyping sites include the SNP site combination according to claim 1.
3. The whole genome liquid phase chip of elephant grass according to claim 2, characterized in that: Also included are probes designed based on the gene sequence covering the SNP site.
4. The whole genome liquid phase chip of elephant grass according to claim 3, characterized in that: The GC content of the probe is between 30% and 70%, and the number of homology regions of the probe sequence on the whole genome is ≤5.
5. Use of the SNP locus combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in analyzing the genetic diversity of elephant grass.
6. Use of the SNP locus combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in analyzing the population structure of elephant grass.
7. Use of the SNP locus combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in identifying the genetic relationship of elephant grass.
8. Use of the SNP locus combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in the improvement and protection of elephant grass germplasm resources.
9. Use of the SNP site combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in molecular marker-based background selection of elephant grass.
10. Use of the SNP locus combination according to claim 1 or the whole-genome liquid phase chip of elephant grass according to any one of claims 2 to 4 in molecular marker-assisted breeding of elephant grass.