Armillaria galbana 5K SNP liquid phase chip and its application

Abstract

This application discloses a 5K SNP liquid phase chip for *Armillaria galbana* and its application, relating to the field of biotechnology. The *Armillaria galbana* 5K SNP liquid phase chip includes probes for capturing a combination of *Armillaria galbana* SNP molecular markers; wherein the combination of *Armillaria galbana* SNP molecular markers includes 5510 SNP molecular markers, and the physical locations of the 5510 SNP molecular markers are based on *Armillaria galbana*. French Armillaria The site information, determined by genome sequence alignment, is shown in Table 1 of the specification. This invention provides the first SNP liquid-phase chip for Armillaria mellea, filling the gap in products applicable to molecular-assisted breeding and genome-wide selection breeding of Armillaria mellea, and providing an effective tool for accelerating basic research and new variety creation in Armillaria mellea.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a 5K SNP liquid phase chip of Armillaria galbana and its application. Background Technology

[0002] Currently, molecular research on Armillaria mellea mainly employs simple sequence repeat (SSR) molecular marker technology. Due to its high polymorphism and co-dominance, SSR markers are widely used in research on genetic mapping, fingerprinting, germplasm identification, and population genetic structure analysis of eukaryotes.

[0003] However, traditional molecular marker technologies such as SSR have significant limitations. These technologies suffer from low throughput, high development costs, long development cycles, and low efficiency, making it difficult to meet the needs of large-scale commercial identification. With the development of sequencing technology, molecular marker technologies based on single nucleotide polymorphisms (SNPs) have gradually become a research hotspot. SNP markers are widely distributed in the genome, numerous, and highly representative, and have advantages such as good genetic stability and simple genotyping, enabling them to more comprehensively reflect the genetic information of a species.

[0004] Whole-genome SNP microarrays, as a breeding tool based on high-throughput molecular markers, can cover SNP loci distributed throughout the entire genome, featuring wide coverage, high sensitivity, high throughput, and high detection accuracy. Compared to traditional molecular markers, whole-genome microarrays containing a large number of SNP markers can efficiently and cost-effectively acquire genetic information from Armillaria mellea, providing an ideal high-throughput platform for genotyping of large numbers of samples.

[0005] Accurate genotyping is a crucial component of germplasm resource identification. Conventional genotyping methods primarily include molecular markers, SNP microarrays, and genome sequencing. Among these, molecular markers based on PCR (polymerase chain reaction) amplification have limited throughput, and the cost increases with the number of markers detected. For species with large genomes, whole-genome sequencing is expensive and data analysis is complex. Liquid-phase microarray technology based on SNPs, as an innovative method enabling efficient, rapid, and large-scale SNP genotyping, has demonstrated significant potential in livestock and poultry breeding genetic research. Compared to traditional solid-phase microarray technology, liquid-phase microarrays are not only simpler to manufacture but also offer significant improvements in cost control and detection accuracy. In particular, liquid-phase microarray technologies based on multiplex amplification capture sequencing and probe hybridization capture sequencing, with their advantages of high throughput, high accuracy, and low cost, have become indispensable tools for biological germplasm resource assessment and breeding research.

[0006] Therefore, developing a high-throughput, low-cost SNP detection tool suitable for Armillaria mellea is of great significance for promoting genetic research and breeding applications of Armillaria mellea. Summary of the Invention

[0007] To address or partially address the problems existing in related technologies, this application provides a 5K SNP liquid phase chip of Armillaria galbana and its application.

[0008] This application discloses a 5K SNP liquid phase chip for *Armillaria galbana*, the chip comprising probes for detecting SNP molecular marker combinations of *Armillaria galbana*; wherein the *Armillaria galbana* SNP molecular marker combinations comprise 5510 SNP molecular markers, the physical locations of the 5510 SNP molecular markers being based on *Armillaria galbana*. Armillaria gallica The site information determined by genome sequence alignment is shown in Table 1 of the instruction manual.

[0009] This application also provides a probe for detecting Armillaria mellea SNP molecular marker combinations, the probe comprising oligonucleotide probes capable of specifically capturing the 5510 SNP molecular markers described in Table 1.

[0010] This application also provides a kit comprising the probes described above.

[0011] This application also provides the use of the probe described above in the preparation of Armillaria galbana 5K SNP liquid phase chips.

[0012] This application also provides a genotyping method, including genotyping Armillaria galbana samples using the chip, probe and / or kit described above.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The first SNP liquid phase chip for Armillaria mellea provided by this invention fills the gap in applicable products for molecular-assisted breeding and genome-wide selection breeding of Armillaria mellea. The application of this chip significantly reduces the cost of scientific research applications such as genetic diversity analysis, QTL mapping, and GWAS analysis of Armillaria mellea, and provides an effective tool for accelerating basic research and the creation of new varieties of Armillaria mellea.

[0014] 2. Based on 238 diverse Armillaria mellea resequencing data, this invention screened 5510 SNP molecular markers that are highly representative, polymorphic, universal, and evenly distributed throughout the genome. Validation showed that the microarray markers had an average MAF value of 0.32, a genome coverage of 99.40%, a target site detection rate of 97.15%, and a repeatability rate of 98.73%, demonstrating high throughput, high accuracy, high repeatability, low cost, and multifunctionality, meeting the needs of various application scenarios.

[0015] 3. Compared to traditional solid-phase chips, the liquid-phase chip involved in this invention offers greater flexibility, allowing for the addition of marker sites as needed. Based on a next-generation sequencing platform and utilizing targeted capture sequencing technology, this chip achieves high-throughput detection while significantly reducing genotyping costs, covering nearly a thousand samples in a single test. Furthermore, this technology not only accurately genotypes target sites but also captures genetic variation information within a certain range surrounding the target site, obtaining richer SNP genotyping data than the expected marker sites, providing more comprehensive site information support for Armillaria mellea molecular breeding research. Attached Figure Description

[0016] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0017] Figure 1 This is a schematic diagram of the 5K SNP liquid-phase chip site annotation results of Armillaria galbana in an embodiment of the present invention; Figure 2 This is a distribution map of the Armillaria galbana 5K SNP liquid-phase chip sites on the chromosome in an embodiment of the present invention; Figure 3 This is a MAF distribution map of the 5K SNP liquid-phase chip sites of Armillaria mellea in this embodiment of the invention; Figure 4 This is an embodiment of the present invention. c A schematic diagram illustrating the principles and process of GPS library construction; Figure 5 This is a graph showing the genotype detection rate of Armillaria mellea 5K SNP liquid-phase chip sites in samples in an embodiment of the present invention; Figure 6 This is a graph showing the genotypic consistency rate of repeat samples of Armillaria mellea 5K SNP liquid-phase chip in an embodiment of the present invention. Figure 7 This is a cluster analysis result diagram of 93 Armillaria mellea samples in an embodiment of the present invention. Detailed Implementation

[0018] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0019] Example 1 This embodiment 1 provides a combination of SNP molecular markers for Armillaria galbana, including 5510 SNP molecular markers, the physical locations of which are based on Armillaria galbana. Armillaria gallica The specific site information determined by genome sequence alignment is shown in Table 1.

[0020] Table 1 Specifically, the screening process is as follows: 1. Genome-wide background loci To obtain a rich variety of whole-genome SNP sites, whole-genome sequencing data of 238 Armillaria mellea materials from different regions and varieties were collected. The sequencing data of 238 Armillaria mellea germplasm resources and varietal materials were analyzed using the GATK analysis pipeline to detect SNPs. Population quality indicators of all SNP loci were calculated and statistically analyzed. Polymorphic SNP loci with minimum allele frequency (MAF) ≥ 0.05, deletion rate < 0.1, heterozygosity ≤ 0.3, and sequencing depth > 5× were selected as initial candidate loci.

[0021] All the candidate sites mentioned above were used for probe design. The probe design method involved designing probes within a 100 bp range upstream and downstream of each site. Probes that could not be uniquely aligned on the genome or contained repetitive sequences in their flanking sequences were removed. Following the principle of uniform distribution of sites on the chromosome, SNP sites uniformly distributed on the Armillaria mellea chromosome were selected as background sites for the entire genome.

[0022] 2. Gene region loci Based on the impact of SNP mutation locations on gene function, snpEff annotation software was used to perform site function annotation on all candidate sites. Candidate sites were screened according to the location of SNP mutations and the degree of their impact on gene function, with the priority for SNP site screening being: gene region > upstream, downstream > intergenic region. Sites were screened from high to low priority based on their annotation. A total of 3214 gene region sites were ultimately obtained, accounting for 58.30% (see details). Figure 1 ).

[0023] All SNP loci were integrated, and based on the quality indicators of these SNP loci, 5510 SNP loci that were highly representative, highly polymorphic, universal, and evenly distributed on chromosomes were finally selected.

[0024] Furthermore, this embodiment 1 also provides a 5K SNP liquid phase chip for Armillaria galbana.

[0025] The 5510 SNP loci obtained through screening were then used, based on the probe design method mentioned above, and Huazhi's independently developed liquid-phase probe precise localization sequencing and genotyping technology (Genotyping by Pinpoint Sequencing of liguidcaptured target). c GPS was used to develop a liquid-phase microarray of Armillaria galbana 5K SNPs. The average coverage of the Armillaria galbana 5K SNP loci on the chromosome was 99.40%, with an average spacing of 15.52 kb. The 5K loci were evenly distributed on each chromosome, as shown in the density distribution diagram. Figure 2 As shown in the figure. The 5K SNP liquid-phase microarray loci of *Armillaria galbana* exhibit high polymorphism, with a mean minor allele frequency (MAF value) of 0.32. The MAF value distribution is shown in the figure. Figure 3 As shown.

[0026] Example 2 Genotyping Procedure for Armillaria galbana 5K SNP Liquid Microarray This embodiment provides a method for genotyping Armillaria mellea samples using the Armillaria mellea 5K SNP liquid phase chip from Embodiment 1. The steps are as follows: (1) Extraction and quality control of genomic DNA DNA was extracted from the samples using a magnetic bead method, and the DNA samples underwent quality testing. Quality testing included determining DNA concentration using a Qubit real-time fluorescence analyzer and assessing DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0027] (2) c GPS Library Construction and Quality Control a. The DNA sample was digested with a fragmentation enzyme, the enzyme ends were repaired, and an A base was added to the 3' end. The fragment size was then detected by agarose gel electrophoresis.

[0028] b. The sequencing adapter and DNA fragment were ligated using T4 ligase, and the ligation product was purified using magnetic beads. The concentration of the purified product was detected using a Qubit quantitative PCR instrument, and the fragment size was detected by agarose gel electrophoresis.

[0029] c. Perform PCR amplification on the purified ligation products, and use magnetic beads to screen the amplified products for fragments. The concentration of the screened products is detected using a Qubit real-time fluorescence instrument, and the fragment size is detected by agarose gel electrophoresis.

[0030] d. Take 200 ng of the constructed library, add the probe and hybridization reagent, and incubate at 54℃ for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target region, wash the captured product with washing buffer to remove non-specific binding fragments, and then perform one round of PCR amplification. Use a Qubit real-time fluorescence instrument to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. Once the concentration and fragment size are within acceptable limits, the PCR is complete. c GPS sequencing library construction. The prepared library was subjected to high-throughput sequencing using the BGI sequencer with the PE150 sequencing strategy.

[0031] (3) Data Analysis The raw data from high-throughput sequencing underwent quality control filtering, and the FASTP software was used to remove adapter fragments and low-quality reads, resulting in high-quality Clean Reads. The obtained Clean Reads were then aligned with a reference genome using BWA software, and their positions were sorted to obtain the sorted BAM file. Finally, GATK software was used to analyze the sequencing results for variant sites, obtaining the genotyping results for the target loci. c The principles and processes for constructing a GPS library are detailed in [link / documentation]. Figure 4 As shown.

[0032] Example 3 Evaluation of the genotyping effect of Armillaria glomerata 5K SNP liquid phase chip To verify the genotyping effect of the Armillaria mellea 5K SNP liquid chromatography chip, the Armillaria mellea 5K SNP liquid chromatography chip designed in Example 1 was used to perform genotyping detection on 99 Armillaria mellea samples, including 6 technical replicates. The specific usage method is shown in Example 2.

[0033] Sequencing and data analysis revealed that the locus detection rate of Armillaria mellea samples ranged from 93.12% to 98.20%, with an average detection rate of 97.15%. (See details below.) Figure 5 The genotypic consistency rate of the technically replicated samples ranged from 98.57% to 98.99%, with an average consistency rate of 98.73%. See details... Figure 6 .

[0034] The above data demonstrate that the Armillaria mellea 5K SNP liquid phase chip exhibits high target site detection rate, good stability, and accurate and reliable typing results when used for genotyping of the tested materials.

[0035] Example 4 Application of Armillaria galbana 5K SNP liquid phase chip in cluster analysis Genotyping of 93 *Armillaria mellea* samples was performed using the *Armillaria mellea* 5K SNP liquid phase chip prepared in Example 1 and the genotyping procedure in Example 2. The source information of the 93 *Armillaria mellea* samples is shown in Table 2. Population genetic structure analysis was performed using PLINK software. Effective genetic loci with MAF ≥ 0.05 were screened from the 93 *Armillaria mellea* 5K genotype files. Cluster analysis was performed on the samples using the cluster parameter, and the genetic distance matrix between samples was calculated and output using the distance-matrix parameter. Visualization was also performed using the itol website. Finally, the distance matrix and clustering results used for constructing phylogenetic trees and analyzing population genetic differentiation relationships were obtained. The results are shown in Table 2. Figure 7 As shown in the figure, the 93 Armillaria mellea samples were mainly divided into three subgroups, which are basically consistent with the material sources in Table 2. The results indicate that the Armillaria mellea 5K SNP liquid phase chip of this invention can effectively distinguish Armillaria mellea samples from different sources, and can be applied to cluster analysis and identification of Armillaria mellea.

[0036] Table 2 The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A 5K SNP liquid phase chip for Armillaria galbana, characterized in that, The chip includes probes for detecting Armillaria mellea SNP molecular marker combinations; wherein the Armillaria mellea SNP molecular marker combinations include 5510 SNP molecular markers, and the physical locations of the 5510 SNP molecular markers are based on Armillaria mellea. Armillaria gallica The site information determined by genome sequence alignment is shown in Table 1 of the instruction manual.

2. A probe for detecting the Armillaria mellea SNP molecular marker combination as described in claim 1, characterized in that, The probes comprise oligonucleotide probes capable of specifically capturing the 5510 SNP molecular markers described in Table 1.

3. A reagent kit, characterized in that, The kit contains the probe as described in claim 2.

4. The use of the probe according to claim 2 in the preparation of Armillaria galbana 5K SNP liquid phase chip.

5. A genotyping method, characterized in that, This includes genotyping Armillaria galbana samples using the chip described in claim 1, the probe described in claim 2, and / or the kit described in claim 3.

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