Sorghum whole genome 20k snp chip and application thereof

Abstract

The application relates to the technical field of molecular breeding and genotyping, and provides a whole-genome 20K SNP chip for sorghum and application thereof. The chip contains 23,000 SNP sites, the genotyping site is based on a reference genome HYZ-T2T for sorghum for wine, and the probe length is 110 bp. The SNPs are uniformly distributed on 10 chromosomes of the sorghum, the average density is about 3 SNPs per 100 Kb, and rich polymorphism is shown in all test varieties. The chip can be widely applied to construction of a high-density genetic map of the sorghum, identification of the genetic relationship of germplasm resources, high-throughput genotyping, QTL positioning and whole-genome correlation analysis, and provides important technical support for genetic improvement and molecular breeding of the sorghum.

Description

Technical Field

[0001] This invention relates to the fields of molecular breeding and genotyping technology, and more specifically, to a 20K SNP chip for the whole genome of sorghum and its application. Background Technology

[0002] With the development of molecular breeding and precision agriculture, higher demands are being placed on the efficient detection of crop genetic diversity, germplasm relationships, and candidate genes. However, existing resequencing and gene chip methods are not only costly and involve large amounts of data, but also suffer from insufficient marker density, low coverage, and poor probe specificity in crops such as sorghum, making it difficult to achieve detailed genome-wide genotyping. Although liquid-phase microarrays, as a new generation of high-throughput platforms, have been applied in various crops, sorghum genotyping still mainly relies on traditional phenotypic selection, which is inefficient, costly, and time-consuming. Existing sorghum SNP microarrays have limited sample and marker counts, and their capture accuracy and stability are not ideal, while simply increasing the number of markers significantly increases costs. Therefore, there is an urgent need to develop an economical, high-density, high-capture-efficiency, and high-throughput sorghum genotyping microarray to achieve accurate genotyping and efficient molecular breeding. Summary of the Invention

[0003] The purpose of this invention is to provide a 20K SNP chip for the whole genome of sorghum and its application.

[0004] To achieve the objective of this invention, in a first aspect, this invention provides a sorghum 20K site probe, the probe containing reagents for detecting sorghum SNP molecular marker sites, the information of which is shown in Table 1.

[0005] Table 1. SNP molecular marker site information in sorghum

[0006] In Table 1, Chr01, Chr02, Chr03, Chr04, Chr05, Chr06, Chr07, Chr08, Chr09, and Chr10 in the left column represent the chromosome IDs of the SNP sites. The right column shows the physical location and base polymorphism of each SNP site, with the physical locations based on the HYZ-T2T version of the sorghum reference genome. For example, 17000_T / G represents an SNP site with a base polymorphism of T or G at position 17000 bp on chromosome 1 of sorghum.

[0007] Furthermore, the probe design is centered on each SNP, extending 55 bp to both sides, with a probe length of 110 bp corresponding to each SNP site. The GC content is 30%-65% (preferably 45%), the Tm value is ≥68℃, it does not contain undetermined base N, the sequence complexity is ≥0.9, the number of single-base tandem repeats does not exceed 6, the number of multi-base tandem repeats does not exceed 3, and the number of matching sites in the genome does not exceed 3, in order to ensure specificity.

[0008] Secondly, the present invention provides a 20K SNP chip for the whole genome of sorghum, wherein the 20K SNP chip contains the 20K site probes of sorghum.

[0009] Thirdly, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip in sorghum QTL localization and genome-wide association analysis.

[0010] Fourthly, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip of claim 3 in the genetic diversity analysis of sorghum germplasm resources.

[0011] Fifthly, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip of claim 3 in the identification of kinship among sorghum germplasm resources.

[0012] Sixthly, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip of claim 3 in the construction of sorghum genetic maps.

[0013] In a seventh aspect, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip of claim 3 in sorghum genotyping detection.

[0014] Eighthly, the present invention provides the application of the sorghum 20K site probe or the 20K SNP chip of claim 3 in molecular marker-assisted breeding of sorghum.

[0015] In a ninth aspect, the present invention provides the application of reagents for detecting sorghum SNP molecular marker sites in the preparation of sorghum 20K site probes or sorghum whole genome 20K SNP chips, wherein information on the sorghum SNP molecular marker sites is shown in Table 1.

[0016] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) This invention develops a 20K SNP targeted capture probe set for sorghum based on targeted capture sequencing technology, specifically designed for high-throughput, low-cost genotyping needs. The probe set covers all 10 chromosomes of the sorghum genome, with uniform locus distribution, exhibiting high polymorphism and broad spectral versatility, thus overcoming bottlenecks in existing sorghum molecular marker applications. This technology provides an indispensable and efficient tool for molecular breeding research in sorghum, including genotyping, gene mapping, fingerprinting, and genome-wide selection, significantly advancing the development of sorghum molecular breeding.

[0017] (ii) This chip is currently the only one in China designed using a pan-genome approach. It can guarantee the detection rate of specific fragments for various sorghum varieties.

[0018] (iii) For the first time, an iterative coverage design was used in a sorghum chip, which ensured complete coverage of the chip genome while reducing the use of probes.

[0019] (iv) Using a complete graph genome design provides more complete information.

[0020] (v) Strict SNP quality control and probe design standards ensure high probe specificity and capture efficiency; (vi) SNP markers are evenly distributed throughout the genome, have high information content, and significantly improve the accuracy of trait association analysis; (vii) Repeated testing showed that the stability and repeatability of the chip genotyping exceeded 99%, demonstrating excellent reliability; (viii) The chip has advantages such as high throughput, low cost and easy operation, providing strong technical support for molecular breeding and precision breeding of sorghum. Attached Figure Description

[0021] Figure 1 The uniform distribution of SNP sites of this invention across the entire genome chromosome (probe coverage density calculated with an observation window of 100 kb) was plotted based on the HYZ-T2T version of the sorghum reference genome.

[0022] Figure 2 This is a statistical analysis of the distribution percentage of polymorphic sites in gene functional regions according to the present invention.

[0023] Figure 3 This is a statistical distribution chart of minor allele frequencies (MAF) of this invention.

[0024] Figure 4 The uniform distribution of the SNP sites of this invention across the entire genome chromosome (probe coverage density calculated with an observation window of 100 kb) was plotted based on the sorghum reference genome BTx623-T2T.

[0025] Figure 5 The uniform distribution of the SNP sites of this invention across the entire genome chromosome (probe coverage density calculated with an observation window of 100 kb) was plotted based on the sorghum reference genome HDN-T2T. Detailed Implementation

[0026] The present invention adopts the following technical solution: This invention provides a targeting probe for a sorghum 20K SNP liquid-phase capture chip, comprising a single-stranded DNA probe synthesized based on sorghum 20K SNP molecular markers. The 20K SNP sites include the SNP molecular markers shown in Table 1. These SNP sites are designed based on the HYZ-T2T reference genome of sorghum used for brewing.

[0027] The present invention also provides a 20K SNP chip for the whole genome of sorghum, wherein the 20K SNP chip contains the 20K site probes of sorghum.

[0028] The 20K SNP chip contains 23,000 SNP sites. Its fabrication method includes the following steps: Step 1: Conduct rigorous quality control based on the sorghum whole-genome SNP dataset. Use bioinformatics tools such as PLINK to screen SNP loci with a missing rate ≤ 0.1, a heterozygosity rate ≤ 0.1, and a minor allele frequency (MAF) ≥ 0.05 to ensure the stability, polymorphism, reliability, and representativeness of the selected SNP loci.

[0029] Step 2: Probe design adopts the sliding window method. Taking each SNP site that has passed quality control as the center, it extends 10 bp to both sides to form a probe design window. A sliding strategy with a step size of 5 bp is used to generate multiple potential probe center positions to ensure the optimal probe selection (i.e., candidate probes are generated with a step size of 5 bp, and finally the sequence with the best GC content, specificity and thermodynamic performance is selected).

[0030] Step 3: The probe length for each SNP site is 110 bp. Using each candidate center location as a reference, extend 55 bp to both sides to obtain a total of 110 bp candidate probe sequences. Evaluate multiple candidate probes for each SNP site, selecting sequences that meet the following conditions: GC content 30%-65% (0.3~0.65), annealing temperature (Tm value ≥ 68℃), and absence of N bases (N indicates an undetermined base).

[0031] Step 4: Conduct a comprehensive evaluation of candidate probe sequences. Use bioinformatics tools to calculate GC content, melting temperature (Tm), and sequence complexity (≥0.9), and use BLAST alignment to ensure that the probe sequence has no more than 3 matching positions in the sorghum reference genome.

[0032] Step 5: From the candidate probes, prioritize probes that target gene functional regions and have high MAF to improve the effectiveness of genotype data.

[0033] Step 6: Divide the reference genome into continuous segments at 100 kb physical distances, and select 3 to 4 probes that meet the quality standards in each segment to ensure uniform coverage of the whole genome.

[0034] Step 7: The selected probe sequences are sent to an oligonucleotide synthesis company for synthesis, and the probe specificity and effectiveness are verified through experiments to ensure that they meet the design standards.

[0035] The probes of this invention employ a uniform coverage strategy in genome distribution, dividing the genome into 100 kb segments, selecting 3-4 probes that meet all quality standards for each segment, and prioritizing probes located in functional gene regions or with a high minimum allele frequency to enhance information content and biological value.

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0037] Example 1: Obtaining 20K SNP molecular markers from sorghum and preparing 20K SNP microarrays from the whole sorghum genome. The acquisition of 20K SNP molecular markers in sorghum and the preparation of 20K SNP microarrays of the whole sorghum genome include the following steps: Step 1: Using the reference genome of red sorghum for liquor production, HYZ-T2T (https: / / doi.org / 10.1016 / j.cj.2024.02.011), BWA was used for alignment, and GATK was used to detect SNP variant sites, identifying a total of 796,815 original variant sites.

[0038] Step 2: The above-mentioned variant sites were rigorously screened. The screening criteria included a missing rate of ≤10%, a heterozygosity rate of ≤10%, and a minimum allele frequency (MAF) of ≥0.05 to ensure the reliability and representativeness of the sites. Finally, 109,637 SNP sites were screened out.

[0039] Step 3: In the probe design process, take each SNP site that has passed quality control as the center and extend 10 bp to both sides to form a probe design window. Within this window, a sliding strategy with a step size of 5 bp is used to generate multiple potential probe center positions.

[0040] Step 4: Based on each candidate center position, extend 55 bp to both sides to obtain a total of 110 bp candidate probe sequences. Evaluate the candidate probes and screen the optimal probes that meet the conditions of GC content 30%-65%, annealing temperature (Tm) ≥68℃, and no N bases. Finally, 48,635 SNP sites are retained.

[0041] Step 5: Perform multi-dimensional quality control on all candidate probes to ensure that the GC content is controlled between 30% and 65%, the probe sequence does not contain undetermined bases (N), the sequence complexity is ≥0.9, the maximum length of the homopolymer is no more than 6 bases, the number of tandem repeats is no more than 3, the annealing temperature (Tm) is no less than 68℃, and the number of matching sites across the genome is ensured by BLASTN alignment. After the above rigorous screening, 23,000 SNP sites that meet the criteria were finally obtained. The detailed information of the sites is shown in Table 1. In Table 1, Chr01, Chr02, Chr03, Chr04, Chr05, Chr06, Chr07, Chr08, Chr09, and Chr10 represent the chromosome IDs of the SNP sites, and the numbers after the chromosome IDs indicate the positions of the SNP sites on the chromosomes. The numerical range preceding the chromosome ID indicates the SNP locus number, for example, 1~2915: Chr01 represents the SNP locus number 1~2915 on chromosome 1.

[0042] The 20K SNP loci in sorghum provided by this invention can be used to prepare gene chips for sorghum genotyping. The application of these loci significantly improves the accuracy and level of genotyping while reducing detection costs, providing important technical support for molecular breeding of sorghum.

[0043] Step Six: Using the SNP site obtained in Step Five as the center, select the 110 bp nucleotide sequence with the GC content closest to 45% within a 110 bp range to its left and right as the probe. Synthesize the selected 110 bp nucleotide sequence into a single-stranded DNA nucleotide sequence with a biotinylate group modified at the 5' end, and finally obtain the sorghum 20K SNP site probe (sorghum 20K SNP targeted capture probe).

[0044] The 20K SNP chip of the whole sorghum genome obtained in this embodiment shows the distribution of SNP sites on different chromosomes as follows: Figure 1 As shown. From Figure 1 As can be seen, this invention designs different numbers of probes based on chromosome length to ensure that the capture sites cover the entire genome.

[0045] The uniform distribution map of SNP sites on the 20K SNP chip of the whole genome of sorghum is shown below. Figure 2 As shown. From Figure 1 It can be seen that the 1 Mb window coverage rate of the genome is 86.5%, and the overall coverage effect is good. Except for the centromere region, no large fragments were found to be uncovered.

[0046] Based on the genotype data from the whole sorghum genome 20K SNP chip, the distribution of minor allele frequencies (MAF) is as follows: Figure 3 As shown in the figure. Statistical analysis shows that SNP sites with MAF ≥ 0.05 account for more than 95%, indicating that the SNP sites contained in this chip have high polymorphism in the study population and are suitable for population genetics analysis and molecular breeding research.

[0047] Example 2: Genotyping stability assessment of 20K SNP microarray of whole sorghum genome In this embodiment, the sorghum 20K SNP targeted capture probe (hereinafter referred to as "the chip of this invention") prepared in Example 1 was used to perform liquid-phase capture sequencing on 24 sorghum genomic DNA samples from different sources. The stability and reliability of the chip in actual detection were evaluated by statistically analyzing the number of target sites, the number of deletion sites, the deletion rate and the detection rate.

[0048] Step 1: Genomic DNA was extracted from 24 sorghum leaf samples numbered S1 to S24 using the CTAB method. After passing quality control, 200 ng of DNA was taken from each sample for fragmentation (350 bp master band). Following the standard PE150 sequencing library construction procedure, end repair, A-tailing, adapter ligation, and PCR pre-amplification were completed to obtain 24 libraries to be hybridized.

[0049] Step 2: Take the above 24 libraries and add the sorghum 20K SNP targeting capture probe prepared in Step 6 of Example 1 to each library for liquid-phase hybridization. The hybridization reaction is carried out at 65°C for 16 hours. Subsequently, streptavidin magnetic beads are used to enrich DNA fragments that are complementary to the probe, and non-specifically bound background DNA is washed away.

[0050] Step 3: The captured products were amplified and enriched by PCR, and then precisely quantified using Qubit sequencing before being mixed and sent to the sequencing machine. PE150 sequencing was performed on the BGI T7 sequencing platform. After quality control, the obtained data were aligned to the sorghum reference genome (HYZ-T2T) to statistically analyze the detection of the 23,000 target SNP sites described in Example 1.

[0051] Table 2. Statistical table of detection results of sorghum 20K SNP chip for some samples.

[0052] As shown in Table 2, the average genotyping detection rate of the 23,000 target SNP loci in the 24 samples was >99%, maintaining a high level. This indicates that the probe design of this invention has excellent specificity and affinity, and can stably capture target loci even in different sorghum individuals. The small fluctuations in target acquisition rate and sequencing depth among different samples indicate that the chip system has good system stability when processing samples from different batches or sources. The vast majority of target loci were effectively covered in all samples, demonstrating that the probe's capture performance is balanced across the entire genome, with no obvious capture bias.

[0053] This embodiment demonstrates, through test data from 24 actual samples, that the 20K SNP chip for the whole sorghum genome provided by this invention exhibits excellent genotyping stability. This chip not only efficiently captures target regions but also demonstrates highly consistent detection performance across different samples, meeting the needs of high-throughput, high-precision sorghum molecular breeding and genetic analysis.

[0054] Example 3: Evaluation of the versatility and distribution of the 20K SNP chip for sorghum based on a pan-genome strategy. To verify the universality and stability of the 20K SNP sites screened in this invention under different sorghum genetic backgrounds, this embodiment uses a pan-genome analysis strategy to evaluate the distribution of the microarray probes on three representative sorghum reference genomes with significant differences.

[0055] Step 1: Select three genomes representing different sorghum subgroups as evaluation objects. BTx623-T2T is the internationally used sorghum reference genome, representing the genetic background of American / feed sorghum; HDN-T2T is a local characteristic sorghum genome, representing the specific local germplasm background of Chinese sorghum; HYZ-T2T is the Hongyingzi reference genome used in this invention, representing the genetic background of Chinese liquor sorghum.

[0056] Step 2: The 23,000 SNP probe sequences obtained in Example 1 were aligned to the three reference genomes (BTx623-T2T, HDN-T2T, and HYZ-T2T) using BWA software. To visually demonstrate the probe distribution pattern across the entire genome, a sliding window method was used for statistical analysis: the window size was set to 100 Kb, the step size was 100 Kb, and the number of successfully aligned probes within each window (Coverage Density) was calculated. Based on the statistical results, probe density distribution maps were plotted on the three genomes.

[0057] The 100K-window coverage density distribution of the probe on three different reference genomes are as follows: Figure 4(BTx623-T2T) Figure 5 (HDN-T2T) and Figure 1 (HYZ-T2T, see Example 1). Comparing the three maps, it can be seen that the 23,000 SNP probes of this invention exhibit good coverage across three significantly different genomes. Except for inherently low-density regions such as centromeres, the probes are evenly distributed on chromosome arms, with no large signal loss or coverage blind spots. Although BTx623-T2T, HDN-T2T, and HYZ-T2T show significant differences in genetic background (including structural variations and sequence differences), the density distribution waveforms of the probes of this invention are highly consistent across all three. This indicates that the SNP sites screened by this invention are mainly located in the "core genome" region of the sorghum pan-genome, and these regions are highly conserved across different sorghum germplasms.

[0058] In summary, the probe of this invention can not only accurately detect Chinese sorghum for liquor production (HYZ), but also be highly compatible with international standard strains (BTx623) and other local varieties (HDN). This "core site optimization strategy" effectively eliminates interspecific alignment bias caused by a single reference genome design, giving the chip extremely strong adaptability, enabling it to be stably applied to the detection of breeding populations with complex and diverse genetic backgrounds, and ensuring high detection rate and accuracy of genotyping data.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A sorghum 20K site probe, characterized in that, The probe contains reagents for detecting sorghum SNP molecular marker sites, and the information of the sorghum SNP molecular marker sites is shown in Table 1 of the specification.

2. The sorghum 20K site probe according to claim 1, characterized in that, The probe design is based on each SNP, extending 55 bp to both sides. The probe length corresponding to each SNP site is 110 bp, with a GC content of 30%-65%, a Tm value ≥68℃, no undetermined base N, a sequence complexity ≥0.9, no more than 6 single-base tandem repeats, no more than 3 multi-base tandem repeats, and no more than 3 matching sites in the genome to ensure specificity.

3. A 20K SNP chip for the whole genome of sorghum, characterized in that, The 20K SNP chip contains the sorghum 20K site probe as described in claim 1 or 2.

4. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in sorghum QTL mapping and genome-wide association analysis.

5. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in the genetic diversity analysis of sorghum germplasm resources.

6. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in the identification of kinship of sorghum germplasm resources.

7. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in the construction of sorghum genetic maps.

8. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in sorghum genotyping detection.

9. The application of the sorghum 20K site probe of claim 1 or 2 or the 20K SNP chip of claim 3 in molecular marker-assisted breeding of sorghum.

10. The application of reagents for detecting SNP molecular marker sites in sorghum in the preparation of sorghum 20K site probes or sorghum whole genome 20K SNP chips, characterized in that, Information on the sorghum SNP molecular marker sites is shown in Table 1 of the specification.

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