Sorghum whole genome SNP (Single Nucleotide Polymorphism) molecular marker combination, 10K liquid chip and application thereof

Abstract

The invention belongs to the technical field of molecular biology, bioinformatics, sorghum genetics and whole-genome gene chips, and particularly relates to a sorghum whole-genome SNP molecular marker combination developed based on a targeted sequencing technology, a 10K liquid chip and application of the sorghum whole-genome SNP molecular marker combination and the 10K liquid chip. The SNP molecular marker combination comprises 10,000 SNP loci, and the specific information of the 10,000 SNP loci is as shown in a table 1; the physical positions of the 10000 SNP loci are determined by performing sequence alignment based on a reference genome of the wine red tassel sorghum variety HYZ-T2T with the version number of v1.0. A liquid chip developed based on the SNP molecular marker combination provided by the invention can be effectively applied to genetic diversity analysis, whole genome association analysis, genetic typing, molecular marker-assisted selective breeding, whole genome selective breeding and other applications of sorghum germplasm resources.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology, bioinformatics and sorghum genetics, as well as whole genome gene chip technology. Specifically, it relates to a combination of SNP molecular markers for the whole genome of sorghum developed based on targeted sequencing technology, a 10K liquid phase chip, and their applications. Background Technology

[0002] Sorghum, as an important coarse grain crop, serves multiple purposes, including food, feed, energy, and industrial raw materials, and plays a vital role in national food security and energy strategies. However, current sorghum breeding still relies on traditional phenotypic selection methods, which suffer from limitations such as long cycles, low efficiency, and susceptibility to environmental interference, necessitating a shift towards molecular design breeding. Single nucleotide polymorphism (SNP) markers, due to their high density, high genetic stability, and automated analysis advantages, have become a core tool for genome selection and molecular breeding. Genome-wide SNP genotyping technology is crucial for achieving high-throughput genotyping and accelerating the breeding process, while developing cost-effective, genome-wide SNP liquid-phase chips is the core pathway to overcome the bottlenecks in sorghum molecular breeding.

[0003] In existing technologies, SNP-based genotyping methods mainly include solid-phase microarrays and liquid-phase microarrays. While solid-phase microarrays are widely used in crops, their high development cost, fixed loci, and poor flexibility make them unsuitable for the genetic diversity analysis and genome-wide selection needs of sorghum. Liquid-phase microarrays, based on GBTS (Blockchain-Guided Sequencing-Tracked Genotyping) technology, capture target sequences through liquid-phase probe hybridization, offering advantages such as high throughput, low cost, and strong scalability, and have been successfully applied in crops like maize and wheat. However, SNP genotyping technology for sorghum lags significantly behind: existing SNP microarrays generally suffer from low marker density, uneven genome coverage, and insufficient capture accuracy. Furthermore, in some studies, blindly increasing marker density has not significantly improved genome selection accuracy but has instead led to increased costs. These limitations severely restrict the large-scale application of molecular breeding in sorghum.

[0004] Therefore, it is crucial to develop a high-density, high-capture-efficiency, and low-cost whole-genome SNP liquid-phase chip tailored to the genomic characteristics and breeding needs of sorghum. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of SNP molecular markers for the whole genome of sorghum, a 10K liquid phase chip, and their applications, developed based on targeted sequencing technology.

[0006] According to a first aspect of the present invention, the present invention provides a combination of SNP molecular markers for the whole genome of sorghum, the combination of SNP molecular markers including 10,000 SNP sites, the specific information of the 10,000 SNP sites is shown in Table 1; the physical location of the 10,000 SNP sites is determined by sequence alignment based on the reference genome of the wine-grade red sorghum variety HYZ-T2T with version number v 1.0.

[0007] For SNP liquid phase microarrays used in sorghum whole genome analysis, existing technologies disclose a 50K SNP liquid phase microarray for sorghum whole genome analysis, which is prepared based on 52,074 SNP loci. Compared to this 50K SNP liquid phase microarray, the 10K SNP liquid phase microarray provided by this invention significantly reduces the number of SNP loci used while achieving comparable SNP locus coverage, average SNP locus detection rate, average deletion rate of polymorphic SNP loci, genotype consistency, SNP homozygous locus consistency rate, and genetic locus correlation coefficient. At the same time, compared to the 50K SNP liquid phase microarray, the 10K SNP liquid phase microarray prepared using 10,000 SNP loci provided by this invention significantly reduces costs, data analysis complexity, and hardware dependence, making it more suitable for large-scale breeding applications (the more SNP loci, the greater the computational resources required and the slower the speed), and better meeting the industry demand for high efficiency and low cost in modern molecular breeding.

[0008] According to a second aspect of the invention, the invention also provides a detection kit comprising probes and / or primers for detecting the SNP molecular marker combination as described in claim 1.

[0009] According to a third aspect of the present invention, the present invention also provides a 10K liquid phase chip for the whole genome of sorghum, the liquid phase chip comprising probes and / or primers for detecting the SNP molecular marker combination as described in claim 1.

[0010] In some embodiments of the present invention, the probe has a GC content between 29% and 66%, a sequence complexity greater than 0.9, and no other homologous regions in the genome, while avoiding the inclusion of SSR and GAP regions.

[0011] According to a fourth aspect of the present invention, the present invention also provides the application of SNP molecular marker combinations as described in the first aspect of the present invention and / or detection kits as described in the second aspect of the present invention and / or liquid phase chips as described in the third aspect of the present invention in sorghum breeding or assisted breeding.

[0012] According to a fifth aspect of the present invention, the present invention also provides the application of SNP molecular marker combinations as described in the first aspect of the present invention and / or detection kits as described in the second aspect of the present invention and / or liquid phase chips as described in the third aspect of the present invention in sorghum genome-wide association analysis.

[0013] According to a sixth aspect of the present invention, the present invention also provides the application of SNP molecular marker combinations as described in the first aspect of the present invention and / or detection kits as described in the second aspect of the present invention and / or liquid phase chips as described in the third aspect of the present invention in sorghum genotyping.

[0014] According to a seventh aspect of the present invention, the present invention also provides the application of SNP molecular marker combinations as described in the first aspect of the present invention and / or detection kits as described in the second aspect of the present invention and / or liquid phase chips as described in the third aspect of the present invention in the detection of sorghum DNA samples.

[0015] According to an eighth aspect of the present invention, the present invention also provides the application of SNP molecular marker combinations as described in the first aspect of the present invention and / or detection kits as described in the second aspect of the present invention and / or liquid phase chips as described in the third aspect of the present invention in the analysis of genetic diversity of sorghum germplasm resources, sorghum cluster analysis and / or sorghum kinship identification.

[0016] According to a ninth aspect of the present invention, the present invention also provides a method for detecting sorghum genotypes, comprising the following steps:

[0017] (1) Extract genomic DNA from the sorghum sample to be tested;

[0018] (2) Constructing a DNA library from sorghum samples;

[0019] (3) Hybridize and sequence the sorghum whole genome 10K liquid phase chip described in the third aspect of the present invention with the constructed library;

[0020] (4) The raw sequencing data were quality controlled, compared with the HYZ-T2T reference genome of red tassel sorghum, and SNP screening and filtering were performed to obtain the genomic genotyping results of the sorghum samples to be tested.

[0021] In some embodiments of the present invention, the hybridization and sequencing of the sorghum whole genome 10K liquid phase chip and the constructed library using the third aspect of the present invention includes the following steps: performing DNA quality control on the extracted genomic DNA, constructing a preliminary library (fragmentation and adapter addition), amplifying and purifying the sequencing library, hybridizing using the sorghum whole genome 10K liquid phase chip using the third aspect of the present invention, constructing a hybridization capture library, and performing quality control and sequencing on the hybridization capture library.

[0022] In summary, the present invention has at least one of the following beneficial technical effects:

[0023] (1) This invention provides a combination of SNP molecular markers for the whole genome of sorghum and a 10K SNP liquid phase chip for the whole genome of sorghum developed based on the combination of SNP molecular markers. The 10K SNP liquid phase chip for the whole genome of sorghum can be effectively applied to the analysis of genetic diversity of sorghum germplasm resources, construction of genetic maps, whole genome association analysis, identification of variety authenticity, molecular marker-assisted selection breeding and whole genome selection breeding.

[0024] (2) The 10K SNP liquid phase chip of the whole sorghum genome provided by the present invention has a physical distance window of 100KB and a SNP site coverage rate of 89.4%, with good overall coverage. Except for the centromere region, no large fragments were found to be uncovered. Moreover, the present invention designs different numbers of probes according to chromosome length, thereby ensuring that the capture sites can cover the entire genome. The average SNP site detection rate of 245 sorghum germplasm resources using the 10K SNP liquid phase chip of the whole sorghum genome of the present invention is greater than 99.34%, and the average deletion rate of polymorphic SNP sites is 0.64%, indicating that the site design quality and capture efficiency of the liquid phase chip are high and the specificity is good. The genotyping results of repeated samples showed that the average genotypic consistency was 99.7%, the average SNP homozygous site consistency rate was 99.9%, and the genetic site correlation coefficient was 99.8%, indicating that the genotyping detection stability of the 10K SNP liquid phase chip of the whole sorghum genome provided by the present invention is good.

[0025] (3) The 10K SNP liquid phase chip of the whole genome of sorghum provided by the present invention can be used to analyze the genetic diversity of sorghum germplasm resources. According to the genetic diversity analysis results of 245 sorghum germplasm resources, the liquid phase chip provided by the present invention can accurately classify the varieties of 245 sorghum germplasm resources. Attached Figure Description

[0026] Figure 1 This is a distribution density map of SNP sites on different chromosomes of the 10K liquid phase chip of the whole genome of sorghum in an embodiment of the present invention;

[0027] Figure 2 This is the statistical result of the number of SNP sites on each chromosome of the 10K liquid phase chip of the whole genome of sorghum in this embodiment of the invention;

[0028] Figure 3 This refers to the percentage of polymorphic sites in the 10K liquid phase chip of the whole sorghum genome in the gene region annotation in this embodiment of the invention;

[0029] Figure 4This is a statistical diagram of allele frequency distribution from a 10K liquid-phase chip of the whole genome of sorghum in an embodiment of the present invention;

[0030] Figure 5 This is a diagram showing the phylogenetic tree analysis results of 245 sorghum germplasm resources in an embodiment of the present invention;

[0031] Figure 6 shows the principal component analysis results of 245 sorghum germplasm resources in an embodiment of the present invention.

[0032] Figure 7 This is a diagram showing the population structure analysis results of 245 sorghum germplasm resources in an embodiment of the present invention. Detailed Implementation

[0033] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0034] Example 1: A method for developing a 10K SNP liquid-phase chip for the whole genome of sorghum.

[0035] Step 1: Filtering and obtaining SNP markers:

[0036] To obtain rich genetic variation information across the entire sorghum genome, whole-genome resequencing data of 1145 sorghum germplasm resources from around the world were collected (some data from the National Genome Data Center and NCBI). These sorghum germplasm resources include the following sorghum varieties: 239 from Africa, 94 from North America, 125 from Asia, 243 from Northern China, and 364 from Southern China (including 120 local Guizhou sorghum resources for liquor production). Using the physical map of red-tasseled sorghum for liquor production (version v 1.0, reference genome HYZ-T2T (https: / / ftp.cngb.org / pub / CNSA / data4 / CNP0002968 / CNS0541937 / CNA0105939)) as the reference genome, BWA and GATK software were used to align and detect SNP sites in all sequencing data. After hard filtering, 6,567,428 original variant SNP sites were obtained.

[0037] The 6,567,428 original variant SNP sites obtained above were screened in the following multiple rounds: (1) Preliminary quality control: using bcftools software, SNPs with a miss rate ≤ 0.1, heterozygosity ≤ 0.1, minimum allele frequency (MAF) ≥ 0.05, and not included in the existing 50k microarray background sites were screened, resulting in 1,720,233 high-quality SNP sites; (2) Sequence feature screening and uniqueness verification: taking each site obtained above as the center, 55bp sequences were extracted from the reference genome before and after the site to construct probe sequences. Among them, SNP sites that meet the following conditions were retained: GC content between 0.29 and 0.66 (i.e., 0.29 ≤ GC content ≤ 0.66), sequence complexity (Local Composition) Complexity ≥ 0.9, maximum single nucleotide repeat ≤ 8, and the number of sites matching more than 30 bp is 1 (i.e., using minimap2 software for alignment, retaining the probe sequence that uniquely matches in the genome), and the proportion of repeat regions ≤ 0.3 (i.e., using RepeatMasker to filter probes with a repeat region proportion > 0.3); after the above screening, 85,162 SNP sites that meet the sequence characteristics and uniqueness requirements are retained; (3) Linkage disequilibrium (LD) filtering: LD analysis is performed using PLINK software, with the parameters set as indep-pairwise 50k40.5 (i.e., window size 50kb, moving 4 SNPs each time, r 2 (4) Uniform distribution and supplementation of SNP sites: The entire genome is divided into 100kb continuous intervals, and 1-2 optimal SNPs are selected from each 100kb interval, resulting in a total of 7,066 sites. In each interval, the sites are sorted and screened according to the following priorities: a) Functional annotation priority (CDS>exons>introns>UTR>intergenic regions); b) Secondary allele frequency (closer to 0.5 is better); c) Deletion rate (lower is better). Subsequently, for regions with insufficient coverage (SNP interval > 200kb), 2,934 sites are supplemented from the 85,162 sites screened in the previous step, and finally 10,000 SNP sites with uniform distribution, high specificity and high representativeness are obtained.

[0038] The detailed information of the sorghum genome-wide SNP sites obtained after the final screening is shown in Table 1 below:

[0039] Table 1. Locus information of SNP molecular markers in the whole genome of sorghum.

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[0158] Step 2: Probe design and preparation of 10K SNP liquid-phase chip for the whole sorghum genome:

[0159] (1) Determine the probe design principle: With the target SNP site as the center, design a probe with a length of 110bp, and ensure that the GC content is between 29% and 66%, the sequence complexity is greater than 0.9, and there are no other homologous regions in the genome. At the same time, avoid including SSR and GAP regions to ensure the specificity and balanced distribution of the probe. In this embodiment, the probe design steps are as follows: According to the 10,000 SNP sites obtained in step 1, select the 110bp fragment with the GC content closest to 45% from the nucleotide sequences before and after the corresponding SNP site as the final probe sequence.

[0160] (2) Based on the 110bp nucleotide sequence designed in step (1), a single-stranded DNA probe with a biotinylated group at the 5' end was synthesized. The obtained single-stranded DNA probe was coupled and immobilized on the surface of fluorescent microspheres using C12 molecular arm and amino modification technology. Each fluorescent microsphere was coupled with only one specific probe. Finally, the prepared probe system was added to the targeting capture reagent (provided by Chengdu Tiancheng Future Technology Co., Ltd., the specific product model is Tcuni FastHybridization andWash Kit) to obtain the 10K SNP targeting capture probe of the whole genome of sorghum, namely the 10K SNP liquid phase chip of the whole genome of sorghum.

[0161] The 10K SNP liquid-phase chip of the whole sorghum genome prepared according to the above embodiments is shown in the distribution density map of SNP sites on different chromosomes as follows. Figure 1 As shown, according to Figure 1 As shown, with a physical distance window of 100KB, the SNP site coverage of the genome is 89.4%, which is a good overall coverage. Except for the centromere region, no large segments were found to be uncovered. Figure 2 The statistical results of the number of SNP sites on different chromosomes obtained by the 10K SNP liquid-phase chip prepared in this embodiment are shown. Figure 2 As shown, this invention designs different numbers of probes based on chromosome length, thereby ensuring that the capture sites can cover the entire genome. For example, chr01, with a length of approximately 85.17 Mb, corresponds to 1233 SNP sites; chr02, with a length of approximately 79.41 Mb, corresponds to 1131 SNP sites; chr03, with a length of approximately 80.87 Mb, corresponds to 1124 SNP sites; chr04, with a length of approximately 70.86 Mb, corresponds to 983 SNP sites; and chr05, with a length of approximately [missing information - likely a number], ... The length of chr06 is approximately 77.13 Mb, corresponding to 1081 SNP loci; chr07 is approximately 69.43 Mb, corresponding to 968 SNP loci; chr08 is approximately 65.71 Mb, corresponding to 879 SNP loci; chr09 is approximately 63.63 Mb, corresponding to 857 SNP loci; and chr10 is approximately 64.69 Mb, corresponding to 895 SNP loci. Figure 3 The diagram shows the percentage of SNP sites in the gene region annotated in the 10K SNP liquid chip prepared in this embodiment. Figure 4 The diagram shows the allele frequency distribution in the 10KSNP liquid-phase chip prepared in this embodiment. Figure 4It can be seen that the allele frequencies are basically symmetrically distributed around 0.3, further indicating that SNPs have good polymorphism.

[0162] Example 2: Application of 10K SNP liquid-phase chip for whole genome of sorghum

[0163] This embodiment utilizes a 10K SNP liquid-phase chip to detect sorghum DNA samples, as detailed below:

[0164] Step 1: Sample Preparation: 245 representative sorghum germplasm resources (Table 2) were selected as experimental materials. Young leaves were collected at the third growth stage, treated with liquid nitrogen, and frozen at -80℃ for plant DNA extraction. Three materials, numbered GZ002, GZ176, and P314, underwent three biological replicates to verify the genotyping stability of the sorghum 10K SNP liquid phase chip.

[0165] Step 2: Extraction of plant DNA: Plant DNA was extracted from the selected experimental materials using the TIANGEN DP305 plant genomic DNA extraction kit.

[0166] Step 3: Follow the experimental technical manual from Chengdu Tiancheng Future Technology Co., Ltd. to complete the following operations: DNA quality control, preliminary library construction (fragmentation and adapter ligation), sequencing library amplification and purification, hybridization capture pre-library construction, and hybridization capture library quality control; as follows:

[0167] DNA quality control: The extracted DNA undergoes strict quality control, including assessing DNA purity, integrity, and contamination using 1% agarose gel electrophoresis, accurately quantifying DNA concentration using a Qubit fluorescence quantitative analyzer, and precisely detecting DNA integrity using an Agilent 2100 bioanalyzer. The quality control standard is that the total DNA amount is not less than 4 μg, the concentration is not less than 40 ng / μl, the integrity is good, and there is no impurity contamination.

[0168] Preliminary library construction (fragmentation and adapter addition): The quality-tested DNA samples are randomly fragmented using an ultrasonic disruptor. DNA fragments of a specific target length are recovered by electrophoresis, and sequencing adapters are then added to their ends to complete the construction of the preliminary library.

[0169] Sequencing library amplification and purification: The preliminary library is amplified using adapter-mediated PCR (LM-PCR), and the amplification product is then purified. The resulting product is the final sequencing library, which can be used for subsequent probe hybridization and capture experiments.

[0170] Hybridization capture preliminaries were constructed as follows: 100 ng of genomic DNA (gDNA) was fragmented using enzyme digestion; the fragmented DNA was end-repaired, including end-completion, 5' end phosphorylation, and 3' end dA tail addition; hybridization capture adapters were then ligated into the end-repaired products, and the ligation products were purified using magnetic beads; the purified ligation products were then subjected to PCR amplification, and the PCR amplification products were purified again using magnetic beads. The purified products were the final hybridization capture preliminaries.

[0171] Hybrid capture library quality control: The constructed hybrid capture preliminaries are subjected to quality control. First, the concentration is initially determined using a Qubit 4.0 fluorescence quantitative PCR instrument. Then, the effective concentration of the library (i.e., the effective molecular concentration containing complete adapters) is accurately quantified using quantitative PCR (qPCR) to ensure that the library quality meets the requirements of subsequent high-throughput sequencing.

[0172] Step 4: Sequencing: Sequencing is performed on the BGI T7 high-throughput sequencer platform to obtain raw sequencing data.

[0173] Step 5: Analysis: The raw sequencing data was quality controlled using FastQC software to obtain clean data. BWA software was then used to align the clean data with the HYZ-T2T reference genome of *Sorghum sorghum*. GTAK software was used for SNP screening and filtering to extract genotyping information from probe-captured sequencing, thus obtaining genotyping data for all samples. Finally, bedtools software was used to analyze the coverage of each probe site sequence in different samples.

[0174] Based on the data obtained above, the detection rate, genotyping stability, and genetic diversity of the 10K SNP liquid phase microarray of the whole sorghum genome were calculated, evaluated, and analyzed, as follows:

[0175] (1) Detection rate calculation: The product detection rate is an important indicator for evaluating the quality of liquid phase microarrays. In plants, the detection rate is generally measured by the ratio of the number of detected sites to the number of developed sites. Calculations show that the average SNP site detection rate of the 245 sorghum germplasm resources in this embodiment is greater than 99.34%, and the average deletion rate of polymorphic SNP sites is 0.64% (Table 3). This indicates that the sorghum whole genome 10K SNP liquid phase microarray provided in this embodiment has high site design quality, high capture efficiency, and good specificity.

[0176] (2) Genotyping stability assessment: Stability is generally assessed by the consistency and correlation coefficient of genotyping results from repeated samples. Three sets of repeated samples were used to test the detection stability of the 10K SNP liquid-phase chip for the whole sorghum genome provided in this embodiment of the invention. The results showed that the average genotypic consistency was 99.7%, the average homozygous SNP consistency rate was 99.9%, and the genetic locus correlation coefficient was 99.8%, indicating that the genotyping detection stability of the 10K SNP liquid-phase chip for the whole sorghum genome provided in this embodiment of the invention is good.

[0177] Table 2. Source information of 245 sorghum germplasm resources

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[0183] Table 3. Polymorphic SNP deletion sites

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[0188] Example 3: Application of 10K SNP liquid-phase chip for whole genome of sorghum

[0189] This embodiment utilizes a 10K SNP liquid-phase chip based on the whole genome of sorghum to analyze the genetic diversity of sorghum samples, as detailed below:

[0190] Using the same method as in Example 2, genotyping of 245 sorghum germplasm resources was performed using a 10K SNP liquid-phase chip for the whole sorghum genome. Genotyping data for all samples were obtained, yielding a total of 10,000 polymorphic SNP loci. Downstream analysis was then performed based on these genetic loci, as detailed below:

[0191] Clustering and principal component analysis were performed using VCF2Dis and Ming PCACluster software to construct NJ cluster diagrams of sorghum natural population germplasm resources and calculate PCA values. Population structure analysis was performed using Structure software.

[0192] The phylogenetic tree analysis results and principal component analysis results of the 245 sorghum germplasm resources obtained by the above methods are as follows: Figure 5 As shown in Figure 6, according to Figure 5 The results show that the 245 sorghum germplasm resources can be divided into four major groups: sorghum germplasm resources from foreign regions, sorghum germplasm resources for brewing, sorghum germplasm resources from southern China, and sorghum germplasm resources from northern China. This indicates that the analysis results are largely consistent with the actual situation of the 245 sorghum germplasm resources. Furthermore, through phylogenetic tree analysis combined with principal component analysis, it can be further found that the overall genetic diversity level of Chinese sorghum germplasm resources is lower than that of foreign sorghum germplasm resources. Figure 7 The population structure analysis results further indicate that when K=2, the 245 sorghum germplasm resources are mainly divided into two groups: foreign sorghum and Chinese sorghum germplasm. When K=3, they are mainly divided into foreign sorghum germplasm, sorghum for brewing in Guizhou / Sichuan, and sorghum germplasm from northern China. When K=4 (the optimal grouping value), they are divided into foreign sorghum germplasm, sorghum for brewing in Guizhou / Sichuan, sorghum germplasm from northern China, and sorghum germplasm from southern China. These results are largely consistent with the findings of studies analyzing sorghum genetic diversity using resequencing technology (see the literature: "GWAS of grain color and tannincontent in Chinese sorghum based on whole-genome sequencing").

[0193] It is understood that this invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of this invention. Furthermore, under the teachings of this invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of this invention. Therefore, this invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this invention are within the protection scope of this invention.

Claims

1. A combination of SNP molecular markers for the entire sorghum genome, characterized in that, The SNP molecular marker combination includes 10,000 SNP sites, and the specific information of the 10,000 SNP sites is shown in Table 1. The physical location of the 10,000 SNP sites was determined by sequence alignment based on the reference genome of the HYZ-T2T red sorghum variety for winemaking, version v 1.

0.

2. A test kit, characterized in that, The detection kit contains probes and / or primers for detecting the SNP molecular marker combination as described in claim 1.

3. A 10K liquid-phase chip for the whole genome of sorghum, characterized in that, The liquid-phase chip includes probes and / or primers for detecting the SNP molecular marker combination as described in claim 1.

4. The detection kit according to claim 2 and / or the liquid phase chip according to claim 3, characterized in that, The probe has a GC content between 29% and 66%, a sequence complexity greater than 0.9, and no other homologous regions in the genome, while avoiding the inclusion of SSR and GAP regions.

5. The application of the SNP molecular marker combination as described in claim 1 and / or the detection kit as described in claim 2 and / or the liquid phase chip as described in any one of claims 3-4 in sorghum breeding or assisted breeding.

6. The application of the SNP molecular marker combination as described in claim 1 and / or the detection kit as described in claim 2 and / or the liquid phase chip as described in any one of claims 3-4 in the genome-wide association analysis of sorghum.

7. The application of the SNP molecular marker combination as described in claim 1 and / or the detection kit as described in claim 2 and / or the liquid phase chip as described in any one of claims 3-4 in sorghum genotyping.

8. The application of the SNP molecular marker combination as described in claim 1 and / or the detection kit as described in claim 2 and / or the liquid phase chip as described in any one of claims 3-4 in the detection of sorghum DNA samples.

9. The application of the SNP molecular marker combination as described in claim 1 and / or the detection kit as described in claim 2 and / or the liquid phase chip as described in any one of claims 3-4 in the genetic diversity analysis, sorghum cluster analysis and / or sorghum kinship identification of sorghum germplasm resources.

10. A method for detecting sorghum genotype, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sorghum sample to be tested; (2) Constructing a DNA library from sorghum samples; (3) Hybridize and sequence the constructed library with the 10K liquid phase chip of the whole sorghum genome as described in claim 3; (4) The raw sequencing data were quality controlled, compared with the HYZ-T2T reference genome of red tassel sorghum, and SNP screening and filtering were performed to obtain the genomic genotyping results of the sorghum samples to be tested.

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