SNP (Single Nucleotide Polymorphism) marker related to branch number of base part of sweet potato and application of SNP marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) marker related to a sweet potato basal part branch number and application thereof, and belongs to the technical field of molecular breeding. The invention provides a group of SNP markers and SNP-KASP markers related to the number of branches of the base part of the sweet potato, the SNP markers are located at the 23703203 site of the 12 # chromosome of the sweet potato, the base polymorphism is T / C, and evaluation of the number of branches of the base part of the known sweet potato variety and screening of sweet potato germplasm resources with multiple branches can be realized at the DNA level. The identification method provided by the invention is simple and easy to operate, does not need to treat the sweet potatoes, can be carried out at any period of the sweet potato growth period, greatly shortens the overground part character screening period of the sweet potatoes, improves the breeding efficiency and improves the branch number of the base part of the sweet potatoes.

Description

Technical Field

[0001] The present invention belongs to the field of molecular breeding, and particularly relates to an SNP marker related to the number of basal branches of sweet potato and its application. Background Art

[0002] Sweet potato [Ipomoea batatas (L.) Lam.] is a hexaploid crop with wide uses and can be used as food, feed, industrial raw material and new energy. The yield and quality traits of the underground roots of sweet potato are closely related to the growth of the above-ground part, promoting and restricting each other. Research shows that the basal branches of sweet potato are an important reference for breeding high-yield varieties. During the sweet potato breeding process, varieties with more basal branches and relatively shorter vine lengths are beneficial to both the field management and harvesting of sweet potato and to increasing the yield of potato tubers. Therefore, strengthening the research on the number of basal branches of sweet potato is of great significance for revealing the genetic laws of important traits of sweet potato and improving the breeding efficiency of sweet potato varieties.

[0003] The number of basal branches of sweet potato is a quantitative trait controlled by multiple genes, and its phenotype is the result of the combined action of genotype and environment. With the development of molecular biology, based on molecular marker technology, by constructing a molecular linkage map of sweet potato and then locating the quantitative trait loci (QTL) of related traits, is an important direction for current sweet potato molecular breeding. In recent years, the research on sweet potato QTL mapping has mainly focused on quality traits such as starch content, dry matter content and β-carotene content. There are few major genes discovered for the phenotypic traits of the above-ground part of sweet potato and lack of molecular markers. The lack of gene resources available for breeding of multi-basal branch traits in the above-ground part remains the main problem faced by current sweet potato breeding work. Summary of the Invention

[0004] The purpose of the present invention is to provide a KASP molecular marker related to the number of basal branches of sweet potato.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the application of a reagent for detecting an SNP marker in sweet potato breeding, and the SNP marker is Chr12_23703203, located at the 23703203rd position of chromosome 12 of sweet potato, and the base polymorphism is T / C.

[0007] Preferably, the SNP marker Chr12_23703203 is associated with the number of basal branches of sweet potato;

[0008] For the base polymorphism T / C, there are three genotypes: CC, TT and TC;

[0009] Among them, the CC genotype is the homozygous type with the SNP locus being C, showing multiple basal branch numbers, and the branch number is greater than or equal to 25;

[0010] The TT genotype is the homozygous type with the SNP locus being T, showing few basal branch numbers, and the basal branch number is less than or equal to 15.

[0011] The present invention provides an SNP-KASP marker containing the said SNP marker, and the nucleotide sequence is as shown in SEQ ID NO.1.

[0012] The present invention provides a primer set for detecting the said SNP-KASP marker, which consists of upstream primer A and / or upstream primer B and downstream primer C;

[0013] The said upstream primer A is as shown in SEQ ID NO.2:

[0014] The said upstream primer B is as shown in SEQ ID NO.3:

[0015] The said downstream primer C is as shown in SEQ ID NO.4.

[0016] Preferably, a FAM fluorescent label is added to the 5' end of the said upstream primer A;

[0017] A VIC fluorescent label is added to the 5' end of the said downstream primer B.

[0018] The present invention provides a kit for detecting the said SNP-KASP marker, including the above primer set.

[0019] The present invention provides the application of the said SNP-KASP marker or the said primer set or the said kit in sweet potato breeding.

[0020] Preferably, the application in sweet potato breeding refers to the application in screening or identifying the trait of multiple basal branch numbers of sweet potato.

[0021] The present invention also provides a method for screening or identifying the trait of multiple basal branch numbers of sweet potato. The steps are as follows: using the genomic DNA of the leaves of the sweet potato to be tested as a template, performing KASP with the said primer set, and performing gene typing according to the fluorescence signal. If the genotype is CC, it is a sweet potato with the trait of multiple basal branch numbers.

[0022] Preferably, when the fluorescence signal shows red, the genotype of the sweet potato to be tested is CC;

[0023] When the fluorescence signal shows blue, the genotype of the sweet potato to be tested is TT;

[0024] When the fluorescence signal shows green, the genotype of the sweet potato is TC.

[0025] The present invention discloses a group of SNP markers and SNP-KASP markers related to the number of basal branches of sweet potato, which can be used to identify the number of basal branches of sweet potato. The SNP marker is located at position 23703203 on chromosome 12 of sweet potato, and the base polymorphism is T / C, named Chr12_23703203(T / C). The SNP-KASP marker includes the above SNP locus. The markers provided by the present invention can evaluate the number of basal branches of known sweet potato varieties and screen germplasm resources with multiple basal branches at the DNA level. The genotyping and identification method provided by the present invention is simple and easy to operate, without the need to process sweet potato, and can be carried out at any stage of the growth period of sweet potato, greatly shortening the screening cycle of above-ground traits of sweet potato, improving the breeding efficiency, and improving the number of basal branches of sweet potato.

[0026] The present invention can be used to quickly and accurately identify or assist in identifying the trait of the number of basal branches of sweet potato, can conduct early screening on sweet potato materials, and can be used for molecular marker-assisted breeding of sweet potato. Applying the substances in the present invention to the molecular marker-assisted selection of the trait of the number of basal branches of sweet potato can quickly screen out sweet potato varieties (germplasms) with multiple basal branches, thereby accelerating the process of breeding the above-ground part of sweet potato, and has important theoretical significance and economic value in the research of exploring sweet potato germplasm resources and breeding sweet potato varieties with multiple basal branches. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0028] Figure 1 It is the genotyping result of the sweet potato natural population based on the Chr12_23703203 marker in Example 1;

[0029] Figure 2 It is the genotyping result of Chr12_23703203 in 52 sweet potato varieties. Detailed Embodiments

[0030] During the application process of the primer set, the primer set can be labeled with a label or not. The label refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labels include but are not limited to dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, optionally, can be charge-neutral. The label can include a nucleic acid sequence or a protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

[0031] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they cannot be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] 1.1 Discovery of SNP markers for sweet potato basal branching genes

[0034] Two hundred and fourteen sweet potato natural populations for GWAS analysis and QTL mapping were planted in Xuzhou, Jiangsu from June to mid-June 2023-2024. A completely randomized block design was adopted, with 3 replicates, 3 rows per plot, row length of 2 m, row spacing of 90 cm, and 10 plants per row. The number of basal branches of sweet potato was investigated 90 days after planting. Six individual plants were randomly selected from each row to record the number of basal branches.

[0035] Extract the genomic DNA of young leaves of sweet potato plants, measure the DNA concentration with a NanoDrop2000c spectrophotometer, adjust the DNA sample to a standard concentration of 50 ng / μL, and then detect the DNA quality with 0.8% agarose gel. DNA with qualified quality was used for SNP mining.

[0036] 1.2 Convert SNP markers to SNP-KASP markers and design primer sets for detecting the markers

[0037] Sequences of 214 samples were downloaded from the NCBI database (accession number PRJNA857483). The PLINK software was used to perform quality control on the genotyping results. SNPs with a minor allele frequency (MAF) > 0.05 and missing data < 5% were selected for genome-wide association study (GWAS). The mixed linear model (CMLM) was used for GWAS analysis. According to the default settings, the threshold for significant P-values was set to 1.0e-5. SNPs that could be stably detected under multiple environments were screened out, located at position 23703203 on chromosome 12 of sweet potato, named Chr12_23703203, and named KASP_Chr12_23703203 after being converted into SNP-KASP markers. Its nucleotide sequence (90bp) is (SEQ ID NO.1): 5’-GAGAGGATGGAACTTATCTACATTTG[T / C]ACATTCTTATCATCTAA TATAGTGGTGGGCTACTGGGCTCACAGTGTTGGCAAGTCTTAAGTT-3’

[0038] The 27th nucleotide Y (Y is T or C) in the KASP_Chr12_23703203 sequence is the screened SNP locus Chr12_23703203, showing polymorphism of T or C. There are three genotypes at this SNP locus: CC, TT or TC genotypes, where the CC genotype is the homozygous type with C at the SNP locus, the TT genotype is the homozygous type with T at the SNP locus, and the TC genotype is the heterozygous type with T and C at the SNP locus. The genotyping results of 214 sweet potato natural populations are as Figure 1 shown.

[0039] A primer set for detecting SNP-KASP markers based on KASP technology was designed, abbreviated as the KASP primer set, as shown in Table 1. The KASP primer set consists of two upstream primers (upstream primer A and upstream primer B) and one downstream primer C (universal primer).

[0040] Table 1 SNP-KASP marker primer set

[0041]

[0042] Preparation of KASP marker primer working solution:

[0043] KASP primers were designed according to KASP_Chr12_23703203. The polymorphism of this SNP locus is the base difference between T / C. The target sequence containing the SNP locus is shown in SEQ ID NO.1;

[0044] Primer FAM-A is a primer with a FAM fluorescent tag sequence added to the 5' end of upstream primer A (SEQ ID NO.5), and it amplifies the fragment of KASP_Chr12_23703203 with C together with primer C. The fluorescence signal (red light) of the group can be read using a microplate reader or a real-time fluorescence quantitative PCR instrument;

[0045] Primer VIC-B is a primer with a VIC fluorescent tag sequence added to the 5' end of downstream primer B (SEQ ID NO.6), and it amplifies the fragment of KASP_Chr12_23703203 with T together with primer C. The fluorescence signal (blue light) of the FAM group can be read using a microplate reader or a real-time fluorescence quantitative PCR instrument;

[0046] Heterozygous TC shows green fluorescence.

[0047] Example 2

[0048] The experimental materials were 214 sweet potato varieties, and the natural population varieties were planted in Xuzhou, Jiangsu in mid-June of 2023 and 2024. A completely randomized block design was adopted, with 3 replicates, 3-row plots, 2 m in row length, 90 cm in row spacing, and 10 plants per row. The number of basal branches of sweet potato was investigated 90 days after transplanting. Fifty-two sweet potato varieties with different numbers of basal branches were selected, and 6 individual plants were selected from each row to record the number of branches.

[0049] Fifty-two sweet potato varieties were genotyped using the Chr12_23703203 marker, and the specific method is as follows;

[0050] (1) PCR amplification system and procedure. Genomic DNA of sweet potato leaves was extracted and dissolved in 200 μl of TE. DNA quality was detected by 1% agarose gel electrophoresis. The extracted DNA was required to have no obvious impurities, clear bands, and no degradation. After measuring the concentration of DNA, it was uniformly diluted to 50 ng / μl. Using the diluted genomic DNA of sweet potato as a template, PCR amplification was carried out. 12 μl of each of the upstream primer A (100 μM) and upstream primer B (100 μM) described in Table 1 and 30 μl of downstream primer C (100 μM) were respectively pipetted, and the reaction system was supplemented to 100 μl with sterile ultrapure water to prepare the primer working solution for KASP marker, which was stored at 4 °C for later use.

[0051] The PCR amplification system was: 1.5 μl of template DNA, 0.0417 μl of primer working solution, 0.75 μl of 2×KASP Master Mix (LGC, product number: KBS-1050-112), and the reaction system was supplemented to 3 μl with sterile ultrapure water.

[0052] The PCR reaction procedure was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing for 20 s (the annealing temperature for the first time was 61°C, and the temperature decreased by 0.6°C for each cycle), a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 1 min, a total of 26 cycles; extension at 72°C for 3 min, and preservation at 4°C.

[0053] (2) Genotyping.

[0054] After the PCR reaction was completed, a fluorescence signal reader (Omega, BMGLABTECH, Germany) and a fluorescence detection system (Araya) were used to convert the fluorescence signal into an analyzable value to read the fluorescence data of the reaction products. The fluorescence scanning results were graphically displayed using the ggplot package in R software. Samples with the C base type carried FAM fluorescence and were distributed near the x-axis; samples with the T base type carried VIC fluorescence and were distributed near the y-axis; samples with no detected signal were distributed near the origin.

[0055] Fluorescence plate reading was performed on an ABI7500 fluorescence quantitative PCR instrument at a temperature of 35°C for 30 s, and the fluorescence value at the end was used for genotyping. If a blue fluorescence signal was shown, the KASP_Chr12_23703203 genotype of the sweet potato to be tested was TT (i.e., the homozygous type with T at Chr12_23703203 in the sweet potato genome); if a red fluorescence signal was shown, the KASP_Chr12_23703203 genotype of the sweet potato to be tested was CC (i.e., the homozygous type with C at Chr12_23703203 in the sweet potato genome); if a green fluorescence signal was shown, the KASP_Chr12_23703203 genotype of the sweet potato was TC (i.e., the heterozygous type with T and C at Chr12_23703203 in the sweet potato genome), and the results were as Figure 2 shown in Table 1.

[0056] Table 1 Genotype detection results and basal branch numbers of 52 sweet potato varieties

[0057]

[0058]

[0059] By Figure 2As can be seen from Table 1, among the 52 sweet potato varieties, 28 varieties showed the TT genotype (blue), 23 varieties showed the TC genotype (green), 1 variety showed the CC genotype (red), and black represented the negative control. The T-test statistical test showed that the gene effect of KASP_Chr12_23703203 reached a significant difference (P<0.05). The number of basal branches of sweet potatoes with the CC genotype was significantly higher than that of sweet potatoes with the TT genotype. Most of the TT genotype varieties had less than 15 branches, while the average number of branches of the TC genotype sweet potato varieties was higher than that of the TT genotype, but the performance was unstable.

[0060] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of a reagent for detecting SNP markers in sweet potato breeding, characterized in that: The SNP marker is Chr12_23703203, located at position 23703203 of chromosome 12 of sweet potato, and the base polymorphism is T / C.

2. The use according to claim 1, characterized in that The SNP marker Chr12_23703203 is associated with the number of basal branches in sweet potato; The base polymorphism T / C has three genotypes: CC, TT and TC; Among them, the CC genotype is the homozygous type of the SNP site C, which is manifested by the number of multiple base branches; The TT genotype is a homozygous type in which the SNP site is T, and is characterized by a small number of basal branches.

3. A SNP-KASP marker comprising the SNP marker for use according to claim 1 or 2, characterized in that: The nucleotide sequence of the SNP-KASP marker is shown in SEQ ID NO.

1.

4. A primer set for detecting the SNP-KASP marker according to claim 3, characterized in that: The primer set consists of an upstream primer A and / or an upstream primer B and a downstream primer C; The upstream primer A is shown in SEQ ID NO.2: The upstream primer B is shown in SEQ ID NO.3: The downstream primer C is shown as SEQ ID NO.

4.

5. The primer set according to claim 4, characterized in that: Adding a FAM fluorescent label to the 5' end of the upstream primer A; A VIC fluorescent tag was added to the 5' end of the downstream primer B.

6. A kit for detecting the SNP-KASP marker according to claim 3, characterized in that: The kit comprises the primer set according to claim 5.

7. Use of the SNP-KASP marker according to claim 3, the primer set according to claim 5, or the kit according to claim 6 in sweet potato breeding.

8. The use according to claim 7, characterized in that The application of the sweet potato breeding refers to the application in screening or identifying the multi-basal branch number trait of the sweet potato.

9. A method for screening or identifying the multi-basal branch number trait of sweet potato, characterized in that: The following steps are involved: The leaf genomic DNA of the sweet potato to be tested is used as a template, KASP is performed with the primer set of claim 5, and genotyping is performed according to the fluorescent signal. The genotype is CC, which means the sweet potato has the multi-basal branch number trait.

10. The method according to claim 9, characterized in that When the fluorescent signal is displayed in red, the genotype of the sweet potato to be tested is CC; When the fluorescent signal is displayed in blue, the sweet potato genotype to be tested is TT; When the fluorescent signal is displayed as green, the genotype of the sweet potato is TC.

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