SNP (Single Nucleotide Polymorphism) molecular marker combination related to high-oil character of peanut and application of SNP molecular marker combination

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker combination related to a peanut high-oil character and application of the SNP molecular marker combination, and belongs to the technical field of biological breeding, the SNP molecular marker combination provided by the invention consists of 538 SNP sites, and physical position information of the SNP site combination is determined based on comparison of a whole genome sequence of a peanut reference genome; and the version number of the whole genome sequence of the peanut reference genome is GCA004170445.1. The SNP molecular marker combination provided by the invention can be applied to molecular breeding work of peanuts, germplasm resources can be rapidly analyzed, and whether a breeding parent material contains high-oil allelic variation or not can be determined.

Description

Technical Field

[0001] The invention belongs to the technical field of biological breeding, and in particular relates to a SNP molecular marker combination related to high-oil trait of peanut and application thereof. Background Art

[0002] peanut( Arachis hypogaea L.) is one of the four major oil crops in my country, with an average oil content of about 51%. Some ultra-high oil resources with an oil content of more than 60% were found in the germplasm resources, indicating that there is still a lot of room for improvement in the oil content of peanut varieties. Therefore, improving the oil content of peanuts has become the primary goal of improving the quality of peanuts in my country. As one of the main oil crops in my country, the oil yield of peanuts is far lower than the market demand, resulting in an increasingly prominent contradiction between the supply and demand of peanut oil in my country and a serious shortage of raw materials for oil extraction. Therefore, breeding high-oil peanuts is still the preferred goal for improving the quality of peanuts in my country.

[0003] In the process of genetic improvement of peanut oil content, traditional breeding has played a certain role, but the progress is slow. Moreover, the oil content of peanut germplasm resources cannot be directly selected by traditional breeding methods, but molecular marker-assisted breeding technology is used. Especially with the sharp decline in the cost of high-throughput sequencing, SNP markers as the third-generation molecular markers are increasingly used in the identification and evaluation of phenotypes in assisted breeding.

[0004] At present, the breeding of high-oil peanuts still adopts the traditional phenotypic trait evaluation method, that is, identification is carried out by measuring the oil content in seeds. This requires seeds to be produced for identification, and cannot be carried out during the seedling or growth period. Therefore, it not only has a long cycle, but also requires a lot of land and labor costs. In addition, it is easily affected by the environment. With the rapid development of SNP marker technology, a SNP site combination related to the high-oil trait of peanuts, a liquid phase chip and its application are developed. By performing association analysis between the SNP site and the oil production trait of the flower, the genotype of the SNP site can be used to determine whether the plant belongs to a high-oil variety in the seedling or growth period, and non-high-oil peanut varieties can be eliminated in time, which is of great significance for improving the efficiency of molecular-assisted breeding and whole genome breeding of high-oil peanuts. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker combination related to the high-oil trait of peanuts, by screening out 538 SNP sites with high-oil traits, and designing a set of probes and gene chips that can be used to detect the molecular marker combination of the high-oil trait of peanuts, as well as a kit that can be used for the selection of high-oil traits in peanuts.

[0006] The present invention is achieved through the following technical solutions: To achieve the above purpose, the technical solution adopted by the present invention is as follows: The SNP molecular marker combination related to the high oil trait of peanut consists of 538 SNP sites, and the physical position information of the SNP site combination is determined based on the whole genome sequence alignment of the peanut reference genome; the physical position information of the 538 SNP sites is specifically shown in Table 1.

[0007] Table 1 Physical location information of 538 SNP loci Table 1 shows the physical location information of the 538 SNP sites selected by the present invention. The physical location information of the SNP sites in Table 1 is expressed as: chromosome_physical position. The detailed information of all SNP sites in Table 1 can be obtained by querying the whole genome sequence of the peanut reference genome with version number GCA_004170445.1, also known as ASM417044v1.

[0008] To achieve the technical purpose of the present invention, the second aspect of the present invention provides a SNP molecular probe combination related to the high oil trait of peanuts, which can be used to detect the SNP site combination described in Table 1 in the sample to be tested.

[0009] To achieve the technical purpose of the present invention, the third aspect of the present invention provides a gene chip for analyzing the high-oil trait of peanuts. The gene chip is a liquid phase chip, and the molecular probe combination as described above can be loaded on the liquid phase chip.

[0010] To achieve the technical purpose of the present invention, the fourth aspect of the present invention provides a reagent for analyzing the high-oil trait of peanuts, which may include the molecular probe combination as described above.

[0011] To achieve the technical purpose of the present invention, the fifth aspect of the present invention provides a kit for analyzing the high-oil trait of peanuts, which may include the molecular probe combination or gene chip as described above.

[0012] To achieve the technical purpose of the present invention, the sixth aspect of the present invention provides a method for analyzing the high-oil trait of peanuts. The method for analyzing the high-oil trait of peanuts may include detecting the peanut sample to be tested using the SNP molecular marker combination as described above, the SNP molecular probe combination as described above, the gene chip as described above, or the kit as described above.

[0013] To achieve the technical purpose of the present invention, the seventh aspect of the present invention further provides an application of a SNP molecular marker combination associated with the high oil trait of peanuts, and the use of the SNP molecular marker combination as described above, or the SNP molecular probe combination as described above, or the gene chip as described above, or the kit as described above, and the method for analyzing the high oil trait of peanuts as described above in detecting the high oil trait of peanuts; it can have any of the following uses: (1) application in the breeding of high oil peanuts; (2) application in the whole genome breeding of peanuts; (3) application in the evaluation of the high oil trait of peanuts; (4) application in the screening of varieties related to the high oil trait of peanuts; (5) application in the identification of varieties related to the high oil trait of peanuts; (6) application in the molecular assisted breeding of varieties related to the high oil trait of peanuts; (7) application in the protection of the germplasm resources of varieties related to the high oil trait of peanuts; (8) application in the improvement of the germplasm resources of varieties related to the high oil trait of peanuts.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The 538 SNP sites of the present invention are 8K variant sites selected from the whole genome according to uniform distribution as background markers, which are determined after careful screening and comparison of the whole genome sequence of the peanut reference genome. The SNP sites related to the high-oil trait of peanuts selected after screening and comparison can be applied to the molecular breeding of peanuts, and can realize rapid analysis of germplasm resources and determine whether the breeding parent material contains high-oil allele variation; 2. The gene chip and kit developed by the present invention are based on 538 carefully selected SNP sites of high oil trait. By optimizing a large number of SNP sites, the detection cost is reduced on the basis of improving the detection accuracy, and it is more suitable for large-scale gene screening. At the same time, it can not only detect the high oil gene with high oil yield trait quickly, but also perform genotyping for peanut populations with a large number of individuals without resequencing, so that the detection cost of a single sample and a single site is as low as 0.02 yuan, which greatly reduces the detection cost and has the advantage of low cost. In addition, it has a variety of application scenarios, for example, it can also be used for the prospect selection of high oil trait backcrossing, the evaluation and identification of high oil peanut haplotypes, and the prediction of peanut oil content in the whole genome; 3. Compared with whole genome resequencing and simplified genome sequencing, the present invention has the advantages of short detection cycle, relatively less data to be processed, and shorter cycle required for data analysis, which can ensure that researchers and breeders can get results in a relatively short period of time; 4. The gene chip of the present invention adopts GBTS targeted sequencing genotype detection technology, which can be customized to add new, personalized genes or background site markers according to the specific needs of customers. There is no requirement for the initial detection sample volume. It has the characteristics of wide platform adaptability, high sample delivery flexibility, high labeling flexibility, short detection cycle, and high result accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings: Figure 1 The distribution map of 538 SNP sites of the present invention in the peanut reference genome is shown. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0017] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to one of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid obscuring the present invention, well-known structures, materials or methods are not specifically described.

[0018] The present invention has no special limitation on the method for determining the oil content and plant height, and any conventional determination method in the art can be used. In the following examples, the method for determining the oil content is to use a near infrared instrument Foss2500 to scan the peanut seed sample, set the wavelength scanning range to 570-1098nm, collect the spectrum every 2nm, the optical path of the sample is 30nm, scan each sample three times and take the average value, and detect the oil content of the peanut seeds.

[0019] Example 1 Selection of SNP molecular marker combination related to high oil content in peanut: In this example, the nucleotide sequences of the published oil genes in Arabidopsis and rice were downloaded from NCBI and compared to the whole genome sequence of the first cultivated peanut Arachis aconitifolia reference genome deciphered by the company, version number GCA_004170445.1 (ASM417044v1), to form a SNP site dataset.

[0020] The collected SNP sites were analyzed with the high-oil trait of peanut. Among the genotyping results of 300 peanut germplasm resources and the oil content data of these germplasm resources, sites with rare allele frequency <5%, deletion rate >20% and heterozygosity rate >10% were filtered out. The sites were basically evenly distributed on the genome, and 8K background SNP sites were selected. Whole-genome association analysis was used to select the genes with the longest alignment and the highest consistency from the alignment results as homologs of peanut oil genes, and high-oil SNP sites were screened out. Finally, after removing duplicate sites from all candidate sites, 538 SNP sites related to the high-oil trait of peanut were obtained. The physical location information of these SNP sites is shown in Table 1. The distribution of the 538 SNP sites obtained on chromosomes was analyzed, and the distribution results obtained are shown in Table 1. Figure 1 shown.

[0021] Example 2 Acquisition of Nucleotide Probe Combinations: Based on the 538 SNP sites obtained in the example, GenoBaits Probe Designer software was used to perform site evaluation, ie, probe design, on peanut oil homologous genes and sites selected according to the uniform distribution principle.

[0022] The evaluation principles are: 1) The probe sequence length is 100 bp.

[0023] 2) The GC content of the probe sequence is 30%-80%.

[0024] 3) The number of homologous regions of the probe in the peanut genome is ≤5.

[0025] 4) The probe region does not contain SSR and unknown base N.

[0026] 5) 2x probes were designed for each locus, and a total of 1076 probes were obtained to cover the SNP loci of 538 candidate genes. These probes were used as liquid phase chip candidate probes for the breeding of high-oil trait peanut varieties for subsequent testing and evaluation.

[0027] Example 3 A liquid phase chip, according to the positions of the 538 SNP sites and their flanking sequences in Example 2, primers are designed and probes are synthesized by Shijiazhuang Boridi Biotechnology Co., Ltd. using targeted capture sequencing technology to obtain a liquid phase chip capable of detecting the high-oil trait of peanuts, which is a combination of nucleotide probes that can capture the SNP regions of the 538 candidate genes in Example 2.

[0028] Example 4 The method for performing genotype detection on the 538 SNP sites in Example 1 using the liquid phase chip developed in Example 3 comprises the following steps: Step 1: Genomic DNA extraction Young peanut leaf tissues were selected, and nucleic acid was extracted using the plant genomic DNA extraction kit produced by Shijiazhuang Boridi Biotechnology Co., Ltd., and the concentration of the extracted DNA was accurately quantified using Qubit.

[0029] Step 2: DNA fragmentation and end repair PCR amplification system: 300 ng DNA; 2.6 μL end repair enzyme, 4 μL buffer; add nuclease-free water to make up the system to 20 μL.

[0030] PCR amplification program: 37℃ 20min; 72℃ 20min.

[0031] Step 3, introducing a linker connection system into the system of step 2 by PCR; PCR system: 4 μL adapter; 2 μL ligase; 8 μL buffer; add nuclease-free water to 20 μL.

[0032] PCR amplification program: 22°C 60min.

[0033] Step 4, purifying the ligation system product; 4.1 Add magnetic beads to the PCR product and shake evenly; 4.2 After standing at room temperature for 5 minutes, briefly centrifuge; 4.3 Place the PCR tube on a magnetic rack until the solution becomes clear; 4.4 Carefully remove the supernatant, making sure not to aspirate the magnetic beads.

[0034] 4.5 Keep the PCR tube / plate in the magnetic rack, add 100 μL 80% ethanol, and let it stand at room temperature for 30 seconds; 4.6 Remove the supernatant and keep the PCR tube / plate in the magnetic rack at room temperature until the ethanol is completely evaporated.

[0035] Step 5: Perform PCR amplification using peanut genomic DNA as a template to obtain a DNA library.

[0036] PCR amplification system: Add 10 μL of the mixture of PCR enzyme and buffer to the product in step 4; 2 μL of sequencing barcode; and add nuclease-free water to make up the system to 20 μL.

[0037] PCR amplification program: 98°C 2 min 1 cycle; (98°C 30 s, 65°C 30 s, 72°C 40 s) 5 cycles; 72°C 4 min 1 cycle.

[0038] Step 6: Purify the obtained DNA library.

[0039] 6.1 Add 20 μl GenoPrep DNA Clean Beads to the product obtained in step 6 and vortex to mix. Avoid creating bubbles during the vortex. 6.2 After standing for 5 minutes, briefly centrifuge; 6.3 Place the PCR tube / plate on a magnetic rack until the solution is clear, which is expected to be at room temperature for at least 3 minutes; 6.4 Remove the supernatant, making sure not to aspirate the magnetic beads.

[0040] 6.5 Keep the PCR tube / plate in the magnetic rack, add 100 μL 80% ethanol and place at room temperature for 30 seconds.

[0041] 6.6 Remove the supernatant and keep the PCR tube / plate in the magnetic rack at room temperature until the ethanol evaporates.

[0042] 6.7 Take the PCR tube or plate out of the magnetic rack, add 35 μL Eultion Buffer, shake and mix, let stand for 5 minutes, and briefly centrifuge. 6.8 Place the PCR tube on a magnetic rack until the solution becomes clear; 6.9 Transfer the supernatant to a new 0.2 ml low-binding PCR tube / plate.

[0043] Step 7: The purified DNA libraries were mixed in equal amounts.

[0044] Step 8: The mixed library is concentrated and added to the hybridization system, and hybridization capture is performed using the prepared probe; PCR amplification system: 1.5-2.5 μg of mixed library; 300 ng of probe; add nuclease-free water to make up the system to 16 μl.

[0045] PCR amplification program: 95℃ 10min (hot cover temperature 105℃); 65℃ 2-4h (hot cover temperature 75℃).

[0046] Step 9: Elute to remove unbound DNA 9.1 Step 9 After hybridization is completed, open the lid of the PCR instrument, open the lid of the PCR tube, and transfer 16 μL of hybridization capture solution to the prepared magnetic beads.

[0047] 9.2 Vortex for 10 seconds to mix thoroughly and centrifuge immediately.

[0048] 9.3 Place the PCR tube in the PCR instrument, incubate at 65°C for 45 min, with the heated cover at 75°C.

[0049] 9.4 Every 12 minutes, oscillate for 5 seconds and centrifuge immediately.

[0050] 9.5 Add 100 μL of elution buffer preheated at 65℃ to each PCR tube.

[0051] 9.6 Vortex briefly for 5 seconds and centrifuge for 5 seconds.

[0052] 9.7 Place the PCR tube on the magnetic rack until the magnetic beads are completely separated from the solution.

[0053] 9.8 Remove the supernatant with a pipette, retain the magnetic beads, and place the PCR tube in a PCR machine at 65°C.

[0054] 9.9 Add 150 μL of GenoBaits 1X Stringent Wash Buffer preheated at 65℃, and slowly pipette up and down 10 times to mix the beads thoroughly. After the last set of samples is mixed, place them on the PCR instrument for 2 minutes.

[0055] 9.10 Place the PCR tube on the magnetic rack until the magnetic beads are completely separated from the solution, and quickly remove the supernatant with a pipette.

[0056] 9.11 Remove the PCR tube from the magnetic stand, add 150 μL of room temperature elution buffer I, and shake for 2 minutes.

[0057] 9.12 Place the PCR tube on a magnetic stand until the magnetic beads are completely separated from the solution. Remove the supernatant with a pipette.

[0058] 9.13 Remove the PCR tube from the magnetic stand, add 150 μL of room temperature elution buffer II, and shake for 1 min.

[0059] 9.14 Place the PCR tube on a magnetic stand until the magnetic beads are completely separated from the solution. Remove the supernatant with a pipette.

[0060] 9.15 Add 150 μL of room temperature elution buffer III and shake for 30 seconds.

[0061] 9.16 Place the PCR tube on a magnetic stand until the magnetic beads are completely separated from the solution. Remove the supernatant with a pipette.

[0062] 9.17 Remove the tube containing the captured DNA beads from the magnetic rack and add 20 μL of nuclease-free water. Use a pipette to slowly pipette up and down 10 times to ensure that all the beads are resuspended.

[0063] Step 10: Enrich the library.

[0064] PCR system: DNA enriched on magnetic beads in step 9; 15 μL of enzyme mixture; 1.2 μL of primers; and nuclease-free water to make up the system to 30 μL.

[0065] PCR amplification program: 1 cycle at 98°C for 45 s; 13 cycles of (98°C for 15 s, 60°C for 30 s, 72°C for 30 s); 72°C for 1 min.

[0066] Step 11: Purify the enriched product to complete the preparation of the sequencing library 11.1 Place the PCR tube on a magnetic rack until the solution becomes clear; 11.2 Transfer the supernatant to a new PCR tube; 11.3 Add 45 μl of magnetic beads to each reaction and vortex to mix.

[0067] 11.4 After standing at room temperature for 5 minutes, briefly centrifuge; 11.5 Place the PCR tube on a magnetic rack until the solution becomes clear; 11.6 Remove the supernatant with a pipette; 11.7 Keep the PCR tube on the magnetic rack and add 100 μL 80% ethanol. Incubate at room temperature for 30 seconds; 11.8 Remove the supernatant with a pipette; 11.9 Keep the PCR tube on the magnetic rack at room temperature until the ethanol evaporates; 11.10 Move the PCR tube from the magnetic stand to room temperature, add 35 μL of elution buffer, shake and mix. Let stand at room temperature for 5 minutes, and then centrifuge briefly.

[0068] 11.11 Place the PCR tube on a magnetic rack until the solution becomes clear; 11.12 Transfer the supernatant to a new 0.2 mL low-adsorption tube, making sure not to absorb the magnetic beads. The purified DNA library is stored at -20°C until sequencing is required.

[0069] Step 12: Equal amounts of the sequencing libraries were mixed and high-throughput sequencing was performed using a BGI T7 sequencer; Step 13: Split the original sequencing bases according to the barcodes of different materials, filter the low-quality sequencing data, align them back to the peanut reference genome, and perform variation mining; Step 14: extract and obtain the corresponding genotype detection file based on the obtained SNP information.

[0070] Example 5 Application of liquid phase chip in genotype detection With known high-oil resources (oil content 59.1%) and low-oil resources (oil content 42.4%) as parents, a population of 128 offspring recombinant inbred lines (RIL) individuals was constructed by hybridization and selfing. Using the liquid phase chip in Example 3, the 128 recombinant inbred lines (RILs) were genotyped with reference to the genotype detection process in Example 4. Through data analysis, the gene chip described in Example 3 was used to perform functional gene detection on germplasm resources and breeding parent materials, and 39 individuals with dominant genotypes in 538 SNP sites were obtained. The test results showed that the proportion of high-oil peanut individuals in the sample accounted for about 30.5% of the total population, indicating that the liquid phase chip can efficiently realize the genotyping of peanut samples.

[0071] The oil content of the 39 high-oil peanuts screened out above was tested, and the oil content was found to be in the range of 55.6% to 60.3%, with an average oil content of 57.7%. The average oil content of the remaining 89 samples screened was 46.8%. It can be seen that the oil content of the high-oil peanuts screened out by the liquid phase chip of the present invention is significantly different from that of the unscreened peanuts, confirming that the liquid phase chip described in the present invention can successfully screen out high-oil peanuts.

[0072] Example 6 Application of liquid microarray in whole genome selection breeding In this embodiment, a method for breeding high-oil peanuts using 538 SNP loci associated with high-oil traits of peanuts is disclosed, which specifically includes the following steps: Step a, randomly selecting 10 peanut samples with full grains, good skin color, high uniformity and clear texture from the 39 germplasm resources with dominant genotypes in 538 SNP loci selected in Example 5 as parent materials, constructing hybrid combinations with parent materials with other excellent traits (high yield, disease resistance, early maturity, drought resistance and hard work resistance, etc.), and carrying out high-oil peanut breeding to obtain progeny peanut samples as the experimental group; randomly selecting 10 peanut samples with full grains, good skin color, high uniformity and clear texture from the remaining 89 peanut germplasm resources screened in Example 5 as parent materials, constructing hybrid combinations with the parent materials with the above-mentioned other excellent traits, and carrying out peanut breeding, and the obtained progeny peanut samples are used as the control group.

[0073] Step b, using the liquid gene chip described in Example 3 to perform genotype detection on the progeny peanut samples obtained in step a, determine which of the progeny samples belong to high-oil peanuts, and detect the oil content of the progeny peanut samples, and obtain the oil content of the experimental group is in the range of 54.2% to 59.7%, with an average oil content of 56.1%, while the oil content of the control group is in the range of 43.6% to 52.8%, with an average oil content of 47.9%. It can be seen that the oil content of the progeny obtained by cross breeding of high-oil peanuts screened by the liquid chip of the present invention is significantly higher than that of the non-high-oil peanuts screened, confirming that the liquid chip of the present invention can not only screen high-oil peanuts in whole genome selection breeding, but also the progeny peanuts obtained after cross breeding with the high-oil germplasm resources screened by it still have high-oil traits, thereby realizing the auxiliary selection of molecular markers in peanut breeding.

[0074] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A SNP molecular marker combination associated with high oil content in peanuts, characterized in that: The SNP molecular marker combination consists of 538 SNP sites, and the physical location information of the SNP site combination is determined based on the whole genome sequence alignment of the peanut reference genome; The physical location information of the 538 SNP sites is specifically shown in Table 1; The version number of the whole genome sequence of the peanut reference genome is GCA_004170445.

1.

2. A SNP molecular probe combination associated with high oil content in peanuts, characterized in that: The molecular probe combination includes a probe for detecting the SNP site combination as claimed in claim 1, wherein the physical location information of the SNP site combination is determined based on a whole genome sequence alignment of a peanut reference genome; The version number of the whole genome sequence of the peanut reference genome is GCA_004170445.

1.

3. A gene chip for analyzing high oil content in peanuts, characterized in that: The gene chip is a liquid phase chip, and the molecular probe combination according to claim 2 is loaded on the liquid phase chip.

4. A reagent for analyzing the high oil content of peanuts, characterized in that: Comprising the molecular probe combination according to claim 2.

5. A kit for analyzing high oil content of peanuts, characterized in that: It includes the molecular probe combination according to claim 2 or the gene chip according to claim 3.

6. A method for analyzing high oil content in peanuts, characterized in that: The method comprises using the SNP molecular marker combination described in claim 1 or the SNP molecular probe combination described in claim 2 or the gene chip described in claim 3 or the kit described in claim 5 to detect the peanut sample to be tested.

7. An application of a combination of SNP molecular markers related to high oil content in peanuts, characterized in that: The use of the SNP molecular marker combination according to claim 1 or the molecular probe combination according to claim 2 or the liquid phase chip according to claim 3 or the kit according to claim 5 in detecting the high oil trait of peanuts has any of the following uses: (1) Application in breeding of high-oil peanuts; (2) Application in peanut whole genome breeding; (3) Application in the evaluation of high oil content in peanuts; (4) Application in screening peanut varieties related to high oil content; (5) Application in the identification of peanut varieties related to high oil content; (6) Application in molecular-assisted breeding of peanut varieties related to high oil content; (7) Application in the protection of germplasm resources of peanut varieties with high oil content; (8) Application in the improvement of peanut germplasm resources related to high-oil traits.

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