SNP (Single Nucleotide Polymorphism) molecular marker of millettia specisoa champ germplasm

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying a millettia specisoa champ germplasm resource, which comprises any one or a combination of any more of 384 SNP molecular markers, the physical positions of the 384 SNP molecular markers are determined by comparison based on a reference genome of millettia specisoa champ, the sites of the 384 SNP molecular markers are uniformly distributed on a millettia specisoa champ chromosome, and the number of the sites of the SNP molecular markers is MilSNP001-MilSNP384. The invention further discloses a specific probe corresponding to the SNP site, a detection method of the molecular marker, application of the molecular marker and a millettia speciosa champ breeding method. According to the 384 SNP markers provided by the invention, 1152 allelic variations are detected in 469 parts of germplasm resource materials in China, the average MAF value is 0.45, and the SNP markers have relatively high resolution on the germplasm resources of the beautiful millettia roots.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection technology, specifically relating to SNP molecular markers for identifying *Smilax glabra* germplasm resources. This invention also relates to specific probes corresponding to the above-mentioned SNP sites, detection methods for molecular markers, applications of molecular markers, and breeding methods for *Smilax glabra*. Background Technology

[0002] SNP molecular markers, as third-generation molecular markers, have advantages such as simplicity, economy, efficiency, and short detection cycle. They can also be used for large-scale and automated detection, making them an effective means of identifying germplasm resources. SNP marker analysis techniques can be divided into two categories according to the research object: SNP marker development and SNP marker detection. SNP marker development mainly involves finding and analyzing unknown SNPs, while SNP marker detection involves detecting the genetic diversity of SNPs in different materials and performing genotyping.

[0003] *Millettia speciosa*, a plant belonging to the genus *Millettia* in the legume family, is a traditional medicinal and edible plant, with its root used in medicine. It has extremely high economic and medicinal value. In traditional Chinese medicine, *Millettia speciosa* is neutral in nature and sweet in taste, possessing the effects of tonifying deficiency and moistening the lungs, strengthening muscles and tendons, and promoting blood circulation. It is commonly used for lumbar muscle strain, rheumatoid arthritis, tuberculosis, chronic bronchitis, chronic hepatitis, spermatorrhea, and leukorrhea. Currently, the demand for *Millettia speciosa* is increasing daily, and artificial cultivation is increasing year by year. However, the mixed germplasm resources seriously restrict the development of the *Millettia speciosa* industry. Germplasm resource identification of *Millettia speciosa* is a urgently needed technology; however, there is currently no technology specifically for identifying *Millettia speciosa* germplasm resources. Summary of the Invention

[0004] The first objective of this invention is to provide SNP molecular markers for identifying *Smilax glabra* germplasm resources, thereby solving the problem of mixed and difficult-to-distinguish artificially cultivated *Smilax glabra* germplasm resources.

[0005] A second objective of this invention is to provide specific probes for detecting the genotypes of the aforementioned 384 SNP loci.

[0006] The third objective of this invention is to provide a detection method for SNP molecular markers used to identify *Smilax glabra* germplasm resources, as described above.

[0007] The fourth objective of this invention is to provide the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the analysis of genetic diversity of *Smilax glabra* varieties.

[0008] The fifth objective of this invention is to provide the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the identification of *Smilax glabra* varieties and phylogenetic relationships.

[0009] The sixth objective of this invention is to provide the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the evaluation and improvement of *Smilax glabra* germplasm resources.

[0010] The seventh objective of this invention is to provide the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in genome-wide association analysis and genome-wide selection breeding of *Smilax glabra*.

[0011] The eighth objective of this invention is to provide the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in *Smilax glabra* breeding or assisted breeding.

[0012] The ninth objective of this invention is to provide a method for breeding *Smilax glabra*.

[0013] The first technical solution adopted in this invention is to identify SNP molecular markers for the germplasm resources of *Smilax glabra*, including any one or any combination of 384 SNP molecular markers. The physical locations of the 384 SNP molecular markers are determined based on the alignment of the *Smilax glabra* reference genome. The 384 SNP molecular marker loci are evenly distributed on the *Smilax glabra* chromosome. The reference genome is a haplotype genome, i.e., 2n=16. The 384 SNP molecular marker loci are numbered MilSNP001-MilSNP384, as shown in Table 1.

[0014] The second technical solution adopted in this invention is the genotype-specific probes for the above 384 SNP sites. The specific probe sequences corresponding to each SNP site are shown in Table 2.

[0015] The third technical solution adopted in this invention is to apply the above-mentioned detection method for SNP molecular markers used to identify *Smilax glabra* germplasm resources, specifically implemented according to the following steps: Step 1: Extract genomic DNA from the leaf tissue of the submitted sample and store it for later use; Step 2: After the DNA from Step 1 is broken, end-repaired, adapter-ligated, and the adapter product is purified, a sequencing library DNA is obtained. Step 3: After hybridizing the library DNA obtained in Step 2 with the above-mentioned 384 SNP probes, the unbound DNA is removed by elution to obtain the hybridized DNA. Step 4: Perform PCR amplification on the DNA obtained after hybridization in Step 3; Step 5: Sequencing the product obtained in Step 4 to obtain sequencing data; Step 6: Compare the sequencing data from Step 5 with the *Echinocactus grusonii* reference genome to obtain genotyping data.

[0016] The fourth technical solution adopted in this invention is the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the analysis of genetic diversity of *Smilax glabra* varieties.

[0017] The fifth technical solution adopted in this invention is the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the identification of *Smilax glabra* varieties and kinship.

[0018] The sixth technical solution adopted in this invention is the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the evaluation and improvement of *Smilax glabra* germplasm resources.

[0019] The seventh technical solution adopted in this invention is the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in genome-wide association analysis and genome-wide selection breeding of *Smilax glabra*.

[0020] The eighth technical solution adopted in this invention is the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in *Smilax glabra* breeding or assisted breeding.

[0021] The eighth technical solution adopted in this invention is further characterized by: Furthermore, breeding or assisted breeding includes any one or more of the following: assisted selection of major genes, molecular assisted breeding, whole-genome selection breeding, identification of *Euphorbia hirta* varieties, construction of genetic maps, gene mapping, species evolution analysis, and identification of germplasm resources.

[0022] The ninth technical solution adopted in this invention is a method for breeding *Smilax glabra*, which includes the following steps: using the above-mentioned *Smilax glabra* SNP molecular marker to detect the DNA of the sample to be tested, and selecting suitable *Smilax glabra* for subsequent breeding.

[0023] The beneficial effects of this invention are: 1) The 384 SNP markers provided by this invention detected 1152 allelic variations in 469 germplasm resources in my country, with an average MAF value of 0.45, which has a high resolution for *Smilax glabra* germplasm resources; 2) The SNP markers provided by this invention exhibit relatively uniform allelic variation distribution frequencies at each locus in the collected *Smilax glabra* germplasm resources. 3) The SNP markers provided by this invention have clear positioning information and physical location information; 4) The 384 SNP probes and their combinations provided by this invention have stable, repeatable, and specific capture capabilities, making them easy to promote and apply. 5) The 384 SNP loci screened in this invention were finally screened and determined after genotyping the most complete collection of *Smilax glabra* germplasm materials collected in my country; 6) There is no linkage relationship among the 384 SNP sites provided in this invention; 7) The 384 SNP sites provided by this invention are evenly distributed on the chromosome of *Euphorbia lathyris*. 8) The 384 SNPs provided by this invention are suitable for the Borui GBTS liquid phase chip platform; 9) The SNP labeling and probe combination of the present invention is low in cost and easy to scale up for detection. 5,000 samples can be identified in the laboratory in 2 weeks. Attached Figure Description

[0024] Figure 1 This is the chromosome distribution of the 384 SNP loci screened in this invention; Figure 2 This is a MAF statistical chart of the 384 SNP sites screened in this invention; Figure 3 This is a cluster analysis diagram of 469 *Smilax glabra* germplasm resources with 384 SNP markers selected in this invention. Figure 4 This is a chromosome distribution map of SNP marker sites in Embodiment 2 of the present invention; Figure 5 This is the MAF statistical graph of the SNP marker sites in Example 2. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] This invention provides SNP molecular markers for identifying *Smilax glabra* germplasm resources, solving the problem of mixed germplasm resources in artificially cultivated *Smilax glabra*. The chromosome distribution of the 384 SNP loci screened in this invention is as follows: Figure 1 As shown, this includes any one or several combinations of 384 SNP molecular markers. The physical locations of these 384 SNP molecular markers were determined based on alignment with the reference genome of *NiuDaLi* (NiuDaLi_final). The 384 SNP molecular marker sites are evenly distributed on the *NiuDaLi* chromosome and are numbered MilSNP001-MilSNP384. The SNP molecular marker site numbers, the chromosomes on which the SNPs are located, their specific physical locations, and deoxynucleotide information are shown in Table 1. Table 1. 384 SNP molecular markers

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[0031] This invention also provides specific probes for detecting the genotypes of the aforementioned 384 SNP loci. The specific probe sequences corresponding to each SNP locus are shown in Table 2. Table 2. Specific probe sequences corresponding to SNP sites

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[0060] This invention also provides a detection method for the SNP molecular markers used to identify *Smilax glabra* germplasm resources, specifically implemented according to the following steps: Step 1: Extract genomic DNA from the leaf tissue of the submitted sample and store it for later use; Step 2: After the DNA from Step 1 is broken, end-repaired, adapter-ligated, and the adapter product is purified, a sequencing library DNA is obtained. Step 3: After hybridizing and capturing the library DNA obtained in Step 2 with the 384 SNP probes of the present invention, the unbound DNA is eluted to obtain the hybridized DNA. Step 4: Perform PCR amplification on the DNA obtained after hybridization in Step 3; Step 5: Sequencing the product obtained in Step 4 to obtain sequencing data; Step 6: Compare the sequencing data from Step 5 with the *Echinocactus grusonii* reference genome to obtain genotyping data.

[0061] This invention also provides the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the analysis of genetic diversity of *Smilax glabra* varieties.

[0062] This invention also provides the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the identification of *Smilax glabra* varieties and phylogenetic relationships.

[0063] This invention also provides the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in the evaluation and improvement of *Smilax glabra* germplasm resources.

[0064] This invention also provides the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in genome-wide association analysis and genome-wide selection breeding of *Smilax glabra*.

[0065] This invention also provides the application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in *Smilax glabra* breeding or assisted breeding.

[0066] Furthermore, breeding or assisted breeding includes any one or more of the following: assisted selection of major genes, molecular assisted breeding, whole-genome selection breeding, identification of *Euphorbia hirta* varieties, construction of genetic maps, gene mapping, species evolution analysis, and identification of germplasm resources.

[0067] This invention also provides a method for breeding *Smilax glabra*, comprising the following steps: using the above-mentioned *Smilax glabra* SNP molecular marker to detect the DNA of the sample to be tested, and selecting suitable *Smilax glabra* for subsequent breeding.

[0068] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings.

[0069] Example 1: 1) Extract genomic DNA from the leaf tissue of the submitted sample.

[0070] 2) DNA quality requirements: The total amount of DNA measured by Qubit® 2.0 should be greater than or equal to 500 ng, with no RNA contamination, and the main band of agarose gel electrophoresis should be clear and undegraded. Adjust the DNA concentration to 10 ng / ul and store at 4℃ for later use.

[0071] 3) The DNA from step 2) is broken, end-repaired, adapter-ligated, and the adapter product is purified to obtain a sequencing library.

[0072] 4) After hybridizing the library DNA obtained in step 3) with the 384 SNP probes of the present invention, the unbound DNA is eluted to obtain the hybridized DNA.

[0073] 5) Perform PCR amplification on the DNA obtained from hybridization in step 4) using a conventional PCR instrument.

[0074] 6) Sequencing the product obtained in step 5) using the BGI sequencing platform to obtain sequencing data.

[0075] 7) Compare the sequencing data from step 6) with the reference genome of *Dendrobium nobile* to obtain genotyping data.

[0076] Example 2: 1) Screening of extended sites for the identification of *Smilax glabra* germplasm resources Fifty-two resource material samples were scanned using resequencing, and genotypic data from 141,098 SNP loci in these 52 samples were analyzed. A total of 40,728 SNPs with a deletion rate of less than 10%, a heterozygosity rate of less than 15%, a MAF > 0.3, and even chromosome distribution were selected to form extended loci for the identification of *Euphorbia hirta* germplasm resources. Figure 2 and Figure 3 As shown. The distribution of the 40,728 extended loci on chromosomes is shown in the figure. Figure 4 The distribution of MAF (minimum allelic frequency) values ​​for the 40,728 extended sites is shown in the figure. Figure 5 The average MAF is higher than 0.2, which can better reflect the genetic diversity among germplasm materials.

[0077] 2) Screening of SNP sites suitable for identification of *Smilax glabra* germplasm resources Flanking sequences of 40,728 SNP loci were obtained, probes were designed and synthesized, and 469 germplasm samples were used for evaluation. The optimal number of locus combinations was analyzed, and 384 loci were finally selected as the first choice for germplasm resource identification.

[0078] Example 3 The SNP molecular markers used to identify the germplasm resources of *Smilax glabra* include any one or any combination of 384 SNP molecular markers. The physical locations of the 384 SNP molecular markers were determined based on the reference genome alignment of *Smilax glabra*. The 384 SNP molecular marker sites are evenly distributed on the chromosome of *Smilax glabra*. The 384 SNP molecular marker sites are numbered MilSNP001-MilSNP384, as shown in Table 1. The specific probe sequences corresponding to each SNP site are shown in Table 2.

[0079] Example 4 The detection method for SNP molecular markers used to identify *Smilax glabra* germplasm resources is implemented according to the following steps: Step 1: Extract genomic DNA from the leaf tissue of the submitted sample and store it for later use; Step 2: After the DNA from Step 1 is broken, end-repaired, adapter-ligated, and the adapter product is purified, a sequencing library DNA is obtained. Step 3: After hybridizing the library DNA obtained in Step 2 with the above-mentioned 384 SNP probes, the unbound DNA is removed by elution to obtain the hybridized DNA. Step 4: Perform PCR amplification on the DNA obtained after hybridization in Step 3; Step 5: Sequencing the product obtained in Step 4 to obtain sequencing data; Step 6: Compare the sequencing data from Step 5 with the *Echinocactus grusonii* reference genome to obtain genotyping data.

[0080] Example 5 Application of SNP molecular markers for identifying *Smilax glabra* germplasm resources in *Smilax glabra* breeding or assisted breeding.

[0081] Breeding or assisted breeding includes any one or more of the following: assisted selection of major genes, molecular assisted breeding, whole genome selection breeding, identification of *Euphorbia hirta* varieties, construction of genetic maps, gene mapping, species evolution analysis, and identification of germplasm resources.

[0082] Example 6 The breeding method for *Smilax glabra* includes the following steps: using the above-mentioned *Smilax glabra* SNP molecular markers to detect the DNA of the sample to be tested, and selecting suitable *Smilax glabra* for subsequent breeding.

Claims

1. A SNP molecular marker for identifying *Smilax glabra* germplasm resources, characterized in that, It includes any one or any combination of 384 SNP molecular markers. The physical locations of the 384 SNP molecular markers are determined based on the reference genome alignment of *Echinopsis lanceolata*. The 384 SNP molecular marker sites are evenly distributed on the chromosome of *Echinopsis lanceolata*. The 384 SNP molecular marker sites are numbered MilSNP001-MilSNP384.

2. A specific probe for detecting the genotype of the 384 SNP loci described in claim 1, characterized in that, The specific probe sequence corresponding to each SNP site is as follows: 。 3. The detection method for SNP molecular markers used to identify *Smilax glabra* germplasm resources according to claim 1, characterized in that, The specific steps are as follows: Step 1: Extract genomic DNA from the leaf tissue of the submitted sample and store it for later use; Step 2: After the DNA from Step 1 is broken, end-repaired, adapter-ligated, and the adapter product is purified, a sequencing library DNA is obtained. Step 3: After hybridizing the library DNA obtained in Step 2 with the 384 SNP probes, the unbound DNA is eluted to obtain the hybridized DNA. Step 4: Perform PCR amplification on the DNA obtained after hybridization in Step 3; Step 5: Sequencing the product obtained in Step 4 to obtain sequencing data; Step 6: Compare the sequencing data from Step 5 with the *Echinocactus grusonii* reference genome to obtain genotyping data.

4. The application of the SNP molecular markers for identifying *Smilax glabra* germplasm resources as described in claim 1 in the analysis of genetic diversity of *Smilax glabra* varieties.

5. The application of the SNP molecular markers for identifying *Smilax glabra* germplasm resources as described in claim 1 in the identification of *Smilax glabra* varieties and kinship.

6. The application of the SNP molecular markers for identifying *Smilax glabra* germplasm resources as described in claim 1 in the evaluation and improvement of *Smilax glabra* germplasm resources.

7. The application of the SNP molecular markers for identifying *Smilax glabra* germplasm resources as described in claim 1 in genome-wide association analysis and genome-wide selection breeding of *Smilax glabra*.

8. The application of the SNP molecular markers for identifying *Smilax glabra* germplasm resources as described in claim 1 in *Smilax glabra* breeding or assisted breeding.

9. The application according to claim 8, characterized in that, The breeding or assisted breeding includes any one or more of the following: assisted selection of major genes, molecular assisted breeding, whole genome selection breeding, identification of *Euphorbia hirta* varieties, construction of genetic maps, gene mapping, species evolution analysis, and identification of germplasm resources.

10. A method for breeding *Smilax glabra*, characterized in that, The process includes the following steps: using the SNP molecular marker of *Smilax glabra* as described in claim 1 to detect the DNA of the sample to be tested, and selecting suitable *Smilax glabra* for subsequent breeding.

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